|Title of Invention||
"ANTHER SPECIFIC PROMOTERS AND USES THEREOF"
|Abstract||The present invention provides as isolated anther specific promoter nucleic acid molecule homologous to the Ta39 promoter of tobacco. Also contemplated are used of the nucleic acid molecule to direct expression of a heterologous nucleic acid molecule to anther and/or pollen of a plant.|
|Full Text||2 ANTHER SPECIFIC PROMOTERS AND USES THEREOF
The present invention relates to anther specific promoters and their use in the production of transgenic plants.
BACKGROUND OF THE INVENTION
The isolation and characterisation of tissue-specific genes allows the analysis of tissue development and the 10 identification of regulatory elements.
An anther-specific promoter is required to direct expression of heterogenous DNA to the anthers and/or pollen, for use, for example in the development of a male
15 sterility system. A number of regulatory sequences from the promoter region of anther or pollen-specific genes have been identified using promoter deletion analysis. Sequence similarities among tissue specific promoters are restricted to short sequence motifs. Promoters may share a
20 similar sequence but are also influenced by upstream regulatory elements that influence expression levels.
The tobacco TA39 gene is expressed in the anther tissue in tobacco (Goldberg et al., (1993) Plant Cell 5: 1217-1229). 2 5 Unreported studies by the inventors found that TA39
promoter is active only in anthers of Arabidopsis and canola.
It is an aim of the present invention to provide an 30 alternative to the TA39 promoter and to use this anther specific promoter in the production of transgenic plants engineered to have desirable characteristic. It is a further aim of the present invention to provide an anther
specific promoter for use in Arabidopsis, wheat, Canola and other crops. The invention is particularly important in relation to legumes, crop, cereal and native grasses, fruiting plants, and flowering plants as it provides means 5 for increasing yield.
SUMMARY OF THE INVENTION
Accordingly, in a first aspect, the present invention 10 provides an isolated anther specific promoter nucleic acid molecule comprising:
(a) a nucleotide sequence shown as SEQ ID NO: 1;
(b) a nucleotide sequence encoding a polypeptide
shown as SEQ ID NO: 2;
15 (c) a homologue or orthologue of a nucleotide
sequence of (a) and having at least 50% sequence identity with the nucleotide sequence of (a);
(d) a homologue or orthologue of the nucleotide
20 sequence of (b) and having at least 50%
sequence identity with the nucleotide sequence encoding the polypeptide shown as SEQ ID NO: 2;
(e) a nucleotide sequence complementary to the
25 nucleotide sequence of (a), (b), (c) or (d);
(f) a nucleotide sequence capable of hybridising
to a nucleotide sequence of (a), (b), (c) or
(d) under conditions of high stringency.
In a second aspect the invention provides an anther specific promoter nucleic acid molecule comprising:
(a) a fragment of a nucleotide sequence of the
first aspect; and/or
(b) a derivative of a nucleotide sequence of the
first aspect, wherein the fragment or
5 derivative is capable of directing expression
of a heterologous nucleic acid to which it is operably linked to anther and/or pollen tissue of a plant transformed with the nucleic acid molecule. 10
In a third aspect the invention provides an expression cassette comprising an anther specific promoter nucleic acid molecule according to the first or second aspects of the invention and a site for inserting a heterologous 15 nucleic acid molecule, such that the heterologous nucleic acid is operably linked to the promoter and is specifically expressed in anther and/or pollen tissue of a plant transformed with the nucleic acid molecule.
2 0 In a fourth aspect the invention provides a recombinant
plasmid comprising an anther specific promoter nucleic acid molecule according to the first or second aspects of the invention and a heterologous nucleic acid operably linked to the promoter. 25
In a fifth aspect the invention provides a plant cell or cell line transformed with the nucleic acid molecules according to the first or second aspects of the invention, the expression cassette according to the third aspect of
3 0 the invention or recombinant plasmid according to the
fourth aspect of the invention.
In a sixth aspect the invention provides a transgenic plant generated from the transformed cell according to the fifth aspect of the invention.
5 In a seventh aspect the invention provides a method for introducing into a plant a heterologous nucleic acid molecule which is to be specifically expressed in anthers and/or pollen, the method comprising the steps of:
(a) transforming a plant cell with the nucleic
10 acid molecules according to the first or
second aspects of the invention, the
expression cassette according to the third
aspect of the invention or the recombinant
plasmid according to the fourth aspect of the
15 invention; and
(b) generating a plant from the transformed plant
In an eighth aspect the invention provides a method of 20 specifically expressing a heterologous nucleic acid
molecule in anther and/or pollen of a plant, the method comprising the steps of:
(a) transforming a plant cell with the nucleic
acid molecules according to the first or
25 second aspects of the invention, the
expression cassette according to the third
aspect of the invention or the recombinant
plasmid according to the fourth aspect of the
30 (b) generating the plant from the plant cell.
In a ninth aspect the invention provides the use of the nucleic acid molecules according to the first or second
aspects of the invention, the expression cassette according to the third aspect of the invention or the recombinant plasmid according to the fourth aspect of the invention for specifically expressing a heterologous 5 nucleic acid molecule in anther and/or pollen of a plant.
The heterologous nucleic acid may be one that has the function of inhibiting the formation of anthers and/or pollen. Use of such nucleic acid in the seventh, eighth,
10 or ninth aspects of the invention allows the creation of male sterile plants. Alternatively, the heterologous nucleic acid may impart resistance to environmental stresses such as extremes of temperature, salinity, pests, infection or provide other desirable properties.
The invention also relates to propagation material of the transgenic plants of the sixth aspect of the invention, e.g. fruits, seeds, tubers, root-stocks, seedlings, cuttings etc.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 provides a sequence alignment of TA3 9 (top) and At39 protein sequences from a BLAST search. The two 25 proteins show 3 0% identity in amino acid sequence and a further 10% of the amino acids are similar (shown as +). Ten gaps were introduced to provide the best possible alignment.
30 Figure 2 shows RT-PCR analysis of floral RNA using tubulin primers. Lane 1. 100 bp DNA ladder (Promega). Lane 2. The 500 bp tubulin product amplified from cDNA generated with the tubulin reverse primer. Lane 3. The 500 bp
tubulin product amplified from cDNA generated with the poly(T) primer.
Figure 3 shows the cDNA clone of the At3 9 gene. Using cDNA 5 obtained from RNA as the template for the PCR with At3 9 gene-5' and 3' primers.
Lane 1. lOObp DNA ladder.
Lane 2 and 3. The 200 bp At3 9 gene fragment
10 Figure 4 shows the At39 gene nucleotide and protein sequence.
A. The At39 gene sequence contains 365 nucleotides, and
encodes a protein containing 8 9 amino acids. The
intron is shown in lowercase letters and comprises 96
15 base pairs. The deduced amino acid sequence is represented in single letter code. The At3 9 gene-specific primers are shown with arrows.
B. The hydropathicity plot of the At3 9 protein.
20 Figure 5 shows the At3 9 promoter nucleotide sequence
(SEQ ID NO. 1), showing the region - 1850 bp upstream of the translational start site (shown in italics). The putative regulatory or tissue-specific motifs are indicated in bold. The location of promoter-specific
25 primers is indicated by underlining. The restriction
sites, Eco Rl, Bel 1, Ssp 1, Sphl, Hind 111 and BamHl are shown in lower case.
Figure 6 shows the strategies attempting to clone the At3 9 3 0 promoter region into a high copy number vector.
a. The 1.9 kb At3 9 promoter fragment obtained from PCR using At39prom-5' and At39prom-3' primers.
b. The digested 1.9 kb At3 9 promoter cloned into the Bam
HI and Hind 111 restriction sites in pBluescript.
c. The 1.7 kb At3 9 promoter digest with Bel 1 and Bam HI
cloned into pl9UC.
5 d. The blunt ended 1.9 At3 9 promoter PCR product inserted into the PCR cloning site in pPCR script plasmid.
Figure 7 shows the cloning strategy to produce the At3 9 promoter expression vector. 10 A. The 1.9 kb At39 promoter region showing the
restriction sites and translational start site (ATG).
B. The 3.8 5 kb pDrive plasmid showing the PCR product
C. The 1.9 kb At3 9 promoter inserted into the pDrive PCR
15 product cloning site to create the 5.75 kb pDrive/At39
D. The promoterless pB1101.3 binary vector.
E. The 1.9 kb At39 promoter digested from pDrive with Bam
HI and Hind 111 and cloned into pBllO1.3 to produce
20 the 14.1 kb pBl/At39 plasmid.
Figure 8 shows identification of At3 9 promoter inserts cloned into the pDrive plasmid. Lane 1. A / Hind 111 ladder.
2 5 Lane 2. The pDrive plasmid without an insert.
Lane 3 and 5. The pDrive plasmid clones (Bl and B2) digested with Hind 111.
Lane 4 and 6. The pDrive plasmid clones digested with Bam HI.
3 0 Lane 7. The pDrive plasmid (B2) digested with Eco Rl.
Figure 9 shows sequence alignment of the At3 9 promoter insert from the pDrive plasmid (top) and the original promoter sequence (bottom).
5 Figure 10 shows cloning the At3 9 promoter into the pBl expression vector.
Lane 1. X / Hind 111 ladder.
Lane 2. The At39 promoter fragment amplified during PCR
using At3 9prom-5' and At3 9prom-3' primers from genomic 10 DNA.
Lane 3. The empty pBl 101.3 vector digested with Bam HI
and Hind 111.
Lane 4. The pBl/At39 promoter plasmid digested with Bam
HI and Hind 111. 15
Lane 5. Eco Rl digestion of the pBl/At39 promoter
Figure 11 shows sequence of the At3 9 promoter and GUS gene 20 junction in the pBl plasmid to ensure the reporter gene will function correctly. The pBl/At39 plasmid (top) contains a linker region from the vector. The GUS translation start site is shown at +1080.
25 Figure 12 shows PCR analysis to verify Agrobacterium tumefaciens contains the pBl/At3 9 promoter plasmid. Lane 1. The X / Hind 111 ladder.
Lane 2. PCR with At3 9prom-5' and At3 9prom-3' primers. Lane 3. PCR with pBl-GUS forward and reverse primers. 30
Figure 13 shows confirmation the plants were transformed with the pBl/At39 promoter construct. Using PCR analysis
of plant material with the pBl-GUS forward and reverse primers.
Lane 1. The X -EcoRl/Hind 111 ladder.
Lane 2. Control using non-transformed tissue from a 5 wildtype plant.
Lane 3-7. Transgenic plant lines 1-5 containing the
Lane 8. A plant line without the promoter construct.
Lane 9. Control using pBl/At39 plasmid DNA. 10
Figure 14 shows At39 promoter-GUS expression in florets.
A. Flowers from plant line #3. GUS expression (blue) is
evident in anthers.
B. Flowers from plant line #5. GUS expression is evident
15 in the anthers and sepals.
2 5 Lane 3
Figure 15 shows Triticum aestivum Ta39 promoter expression. A 3 50bp DNA band was amplified in lanes 4, 5 & 6. The PCR in these lanes was performed on cDNA samples 20 prepared from three different flower tissues, and suggests that Ta39 is expressed in anther, gynocium and lemma and short awn cDNA.
Template was wheat leaf cDNA (2 week old plants).
Template was wheat leaf cDNA (6 week old plants).
Template was wheat anther cDNA.
Lane 6 Lane 7
Template was wheat gynocium cDNA.
Template was wheat lemma & short awn cDNA.
Template was wheat stem cDNA.
Template was wheat young root cDNA (4 days after
3 0 Lane 8
Lane 9: Positive control.
Lane 10: Negative control, no template added to this PCR
DETAILED DESCRIPTION OF THE INVENTION
The inventors have isolated promoters from Arabidopsis 5 thaliana, Triticum aestivum, and canola which are
primarily expressed in anther tissue. These promoters are particularly important in relation to legumes, crop, cereal and native grasses, fruiting plants, and flowering plants as they may provide means for increasing yield.
Arabidopsis has become the model system used for genetic analysis in plant molecular biology. It is ideal because of its small size, short life cycle and small genome. Self-fertilization results in large amounts of seed being
15 produced, and efficient transformations systems are
available. Importantly, Arabidopsis and Canola belong to the crucifer family and share significant genetic homology. As an appropriate promoter has been identified in Arabidopsis an ortholog is likely to exist in Canola.
20 If a Canola promoter cannot be identified, the Arabidopsis anther-specific promoter could be used instead.
The nucleotide sequence of the Arabidopsis At39 gene was obtained from the Genbank database following a BLAST
25 search, which was performed to identify a protein
homologous to the tobacco TA39 protein (Goldberg, supra). The At39 gene encodes a protein that shares 3 0% homology with the TA39 protein. The nucleotide sequence of At3 9 is provided as SEQ ID NO: 1 and the amino acid sequence of
3 0 the At3 9 polypeptide is provided as SEQ ID NO: 2. The TA39 polypeptide sequence is provided as SEQ ID NO: 3.
The nucleotide sequence of the Triticum aestivum Ta39 genes were obtained from the Genbank database following a search to identify proteins with sequence homology to the Arabidopsis thaliana At39 gene. The Ta39 genes encode 5 proteins with 64% and 50% sequence identity to the tobacco At39 polypeptide and more than 90% sequence identity to the TA3 9 protein. The sequence of a Ta39 nucleic acid molecule is provided as SEQ ID NO: 12 and the corresponding Ta3 9 amino acid sequence is provided as 10 SEQ ID NO: 13.
Tissue-specific promoters are important tools for research and may have useful applications in agricultural practices. Such applications include the development of a
15 male sterility system that can be used to breed hybrid crops. The At39 promoter may be used to regulate gene expression in the anther region of crops such as Canola. Promoters are commonly interchangeable between a variety plant species. As with the tobacco TA39 promoter, the
20 At39 promoter is active and is anther-specific in Brassica species.
As defined herein "isolated" means substantially free from material present in nature in the plant from which the 25 nucleic acid molecule is derived, that is in an
environment different from that in which the compound naturally occurs.
"Isolated" is meant to include compounds that are within 3 0 samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.
"Anther specific" is used herein to describe cDNAs, genomic DNAs and messenger RNAs which are associated with anther tissue. Such a promoter nucleic acid molecule directs expression almost exclusively to the male 5 reproductive tissues, i.e. the anther and pollen, rather than to all plant cells. Such promoters may not solely direct expression to the anther or pollen, but will direct expression to the anther or pollen to a greater degree than to other cells or tissues. In the case of promoter
10 DNA sequences, anther specific describes a regulatory sequence which directs the transcription of associated coding sequences so that when assayed through northern blot hybridisation, the mRNA corresponding to the heterologous sequence is present in anther and/or pollen
15 cells or tissues in concentrations at least 10 times more than to other plant cells or tissues, preferably at least 20 times, more preferably at least 50 times and most preferably at least 100 times more to the anther and/or pollen cells or tissues than to other plant cells or
Anther tissue describes the tissue of the male reproductive organs in a plant, be it fully developed or partially developed. The definition of anther tissue used 25 herein is intended to include all structures making up the anther, that is the epidermis, endothecium, middle layer and tapeturn.
Because anther and pollen tissue are both involved in the 3 0 male sexual function of a plant, a nucleic acid molecule may be considered to be "anther specific" for the purpose of the present invention if it is expressed specifically in pollen as well as in anther tissues.
Pollen is the haploid male gamete in flowering plants and carries the sperm cells required for fertilisation of the ovules. These tiny grains develop within the anther and 5 are released as the anther matures by a process referred to as dehiscence.
As defined herein a "promoter" is the minimal nucleic acid molecule that specifically binds RNA polymerase to 10 determine where transcription begins. Transcription is the production of RNA from the DNA template.
A promoter is the minimum sequence sufficient to drive transcription. "Promoter" is also meant to encompass those
15 promoter elements sufficient for promoter-dependent gene expression controllable for cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the native gene. As the promoters of the present invention
20 are anther specific they can be used to direct expression to the anther of heterologous nucleic acid operably linked to the promoter.
"Promoter elements" as used herein refers to sub-domains 25 within the promoter that confer tissue-specific
expression, enhance expression, or inhibit expression. A promoter can contain a multiplicity of promoter elements. Furthermore, some elements can appear more than once within a single promoter. Examples of such elements are 30 E-box motifs, RY-repeat elements, AT-rich regions, ACGT-core elements, Opaque-2-like elements, and conserved gymnosperm-like regions. Additional examples of promoter elements can be found in U.S. Pat. No.: 5,723,751 to Chua;
U.S. Pat. No. 5,608,149 to Barry et al.; U.S. Pat. No. 5,589,615 to De Clercq et al.; U.S. Pat. No. 5,589,583 to Klee et al.; U.S. Pat. No. 5,677,474 to Rogers; U.S. Pat. No. 5,487,991 to Vandekerckhove et al.; and U.S. Pat. No. 5 5,53 0,194 to Knauf et al. Typically, a TATA box is found on the 3'-end of the series of promoter elements.
Examples of specific promoter elements are provided below and in relation to the Figures. However, one of skill in
10 the art will appreciate that a specific promoter element sequence provided can be modified while still maintaining activity. For example a base in an RY-repeat element can be changed without the RY-repeat element losing its functionality within the overall promoter sequence.
"Nucleic acid molecule" as used herein refers to an oligonucleotide, polynucleotide, nucleotide and fragments or portions thereof, as well as to peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof,
2 0 and to DNA or RNA of genomic or synthetic origin which can
be single- or double-stranded, and represent the sense or antisense strand. Where "nucleic acid" is used to refer to a specific nucleic acid sequence "nucleic acid" is meant to encompass polynucleotides that encode a polypeptide 25 that is functionally equivalent to the recited
polypeptide, e.g., polynucleotides that are degenerate variants, or polynucleotides that encode biologically active variants or fragments of the polypeptide, including polynucleotides having substantial sequence similarity or
3 0 sequence identity relative to the sequences provided
The terms "nucleotide sequence" and "nucleic acid sequence" are used herein interchangeably.
"Polypeptide" as used herein refers to an oligopeptide, 5 peptide, or protein. Where "polypeptide" is recited herein to refer to an amino acid sequence of a naturally-occurring protein molecule, "polypeptide" and like terms are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the
10 recited protein molecule, but instead is meant to also encompass biologically active variants or fragments, including polypeptides having substantial sequence similarity or sequence identify relative to the amino acid sequences provided herein.
A "homologue" is defined as a nucleic acid molecule sharing the same function as another nucleic acid molecule. Homologues are generally determined by sequence identity or similarity as defined by alignment using
2 0 algorithms such as that in the Advanced BLAST2 service
provides by EMBL.
"Orthologues" are nucleic acid or amino acid sequences that share a common ancestral sequence, but that diverged 25 when a species carrying that ancestral sequence split into two species. Orthologous sequences are usually also homologous sequences.
In a preferred embodiment the homologues or orthologues
3 0 encode or are cysteine rich peptides. In such peptides
the cysteine-rich regions (roughly 12 cysteine residues over a 60 residue peptide) are highly homologous with more than 50% identity. However, the promoter nucleotide
sequences of the genes encoding such peptides can vary greatly.
Homologous sequences are generally those with a percentage 5 sequence identity of at least 50% at nucleotide or amino acid level according to BLAST analysis. Sequences that have identity of at least 50%, 60%, 70%, 80% and at least 90% that are functionally active are said to be homologous sequences.
"Percent (%) sequence identity" with respect to the nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the specific nucleotide
15 sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence
20 identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring
25 alignment, including any algorithms needed to achieve
maximal alignment over the full length of the sequences being compared. For purposes herein, however, % nucleotide sequence identity values are generated using the WU-BLAST-2 computer program (Altschul et al., Methods
30 in Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parameters are set to the default values. Those not set to default values, i.e., the adjustable parameters, are set with the following values: overlap
span = 1, overlap fraction = 0.12 5, word threshold (T) = 11, and scoring matrix = BLOSUM62. For purposes herein, a % nucleotide sequence identity value is determined by dividing (a) the number of matching 5 identical nucleic acid residues between the nucleotide sequence of the promoter of interest having a sequence derived from the promoter and the comparison nucleotide sequence of interest (i.e., the sequence against which the promoter sequence of interest is being compared which may 10 be a promoter variant) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the promoter of interest.
Percent nucleic acid sequence identity may also be 15 determined using the sequence comparison program NCBI-
BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)) . The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of 20 those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25 25 and scoring matrix = BLOSUM62.
"A homologue" as defined herein means a nucleic acid molecule which encodes an active promoter as defined below. In addition, a "homologue" has at least about 50% 3 0 nucleic acid sequence identity with the nucleotide
sequence shown as SEQ ID NO: 1 or the nucleotide sequence encoding the polypeptide having SEQ ID NO: 2 as disclosed herein, or any fragment thereof. Ordinarily, a homologue
will have at least about 50% nucleic acid sequence identity, more preferably at least about 51% nucleic acid sequence identity, more preferably at least about 52% nucleic acid sequence identity, more preferably at least 5 about 53% nucleic acid sequence identity, more preferably at least about 54% nucleic acid sequence identity, more preferably at least about 55% nucleic acid sequence identity, more preferably at least about 56% nucleic acid sequence identity, more preferably at least about 57%
10 nucleic acid sequence identity, more preferably at least about 58% nucleic acid sequence identity, more preferably at least about 59% nucleic acid sequence identity, more preferably at least about 60% nucleic acid sequence identity, more preferably at least about 61% nucleic acid
15 sequence identity, more preferably at least about 62%
nucleic acid sequence identity, more preferably at least about 63% nucleic acid sequence identity, more preferably at least about 64% nucleic acid sequence identity, more preferably at least about 65% nucleic acid sequence
20 identity, more preferably at least about 66% nucleic acid sequence identity, more preferably at least about 67% nucleic acid sequence identity, more preferably at least about 68% nucleic acid sequence identity, more preferably at least about 69% nucleic acid sequence identity, more
25 preferably at least about 70% nucleic acid sequence
identity, more preferably at least about 71% nucleic acid sequence identity, more preferably at least about 72% nucleic acid sequence identity, more preferably at least about 73% nucleic acid sequence identity, more preferably
30 at least about 74% nucleic acid sequence identity, more preferably at least about 75% nucleic acid sequence identity, more preferably at least about 76% nucleic acid sequence identity, more preferably at least about 77%
nucleic acid sequence identity, more preferably at least about 78% nucleic acid sequence identity, more preferably at least about 79% nucleic acid sequence identity, more preferably at least about 8 0% nucleic acid sequence 5 identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more
10 preferably at least about 85% nucleic acid sequence
identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably
15 at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least
20 about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97%
25 nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence of SEQ ID NO:1 or the nucleic acid sequence encoding the polypeptide
3 0 sequence shown as SEQ ID NO: 2.
Persons skilled in the art would readily be able to determine if a homologue, orthologue, fragment or variant
of the sequence provided according to SEQ ID NO: 1 functions as an anther specific promoter and accordingly falls within the scope of the claims. An example of a test to see if a sequence acts as an anther specific 5 promoter in accordance with the present invention the sequence under test is fused in frame with the GUS reporter gene in a binary vector. Agrobacterium containing the putative promoter/GUS binary vector is used to transform plant tissues from which plantlets are
10 regenerated. Tissues from the transgenic plants at various stages of development are assayed for GUS expression using X-GLUC as substrate, whereby GUS expression in anther tissue shows that the sequence under test is an anther specific promoter and falls within the
15 scope of the invention.
Complementary as used herein in relation to nucleic acid molecule "complementary" to the nucleic acid sequence of (a), (b) or (c) is intended to encompass those sequences 2 0 that are capable of hybridising under high stringency conditions to the nucleic acid molecules defined.
"Hybridisation" in relation to nucleic acids is the forming of a hybrid of two single complementary strands of
2 5 nucleic acid to form a double strand.
"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe
3 0 length, washing temperature, and salt concentration. in
general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the
ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher 5 the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see
10 Ausubel et al., Current Protocols in Molecular Biology,
Wiley Interscience Publishers, (1994) and Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) .
Reference herein to "high stringency conditions" may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium
20 dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium
25 citrate at 42°C; or (3) employ 50% formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, SxDenhardt's solution, sonicated salmon sperm DNA (50 u/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2xSSC
30 (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.lxSSC containing EDTA at 55°C.
A "fragment" is defined as a portion or domain of the full length sequence provided according to the present invention, which fragment maintains the capacity to direct expression of heterologous nucleic acid to which it is 5 operably linked to anther or pollen tissue in plants.
Experiments to ascertain if a fragment maintains the ability of the full length sequence to direct expression of heterologous nucleic acid to which it is operably 10 linked to anther or pollen tissue in plants are provided in the Examples section.
Ordinarily, the anther specific promoter fragment is at
least about 30 nucleotides in length, often at least about
15 60 nucleotides in length, more often at least about 90
nucleotides in length, more often at least about 120
nucleotides in length, more often at least about 150
nucleotides in length, more often at least about 180
nucleotides in length, more often at least about 210
20 nucleotides in length, more often at least about 240
nucleotides in length, more often at least about 270
nucleotides in length, more often at least about 3 00
nucleotides in length, more often at least about 450
nucleotides in length, more often at least about 600
25 nucleotides in length, more often at least about 900
nucleotides in length, more often at least about 1000
nucleotides in length, more often at least about 1200
nucleotides in length, more often at least about 1400
nucleotides in length, more often at least about 1600
30 nucleotides in length, more often at least about 1800
nucleotides in length, more often at least about 1850
nucleotides in length, or more.
"Derivatives" of nucleic acid molecules or proteins or peptides as defined herein encompass those molecules comprising non-naturally occurring residues or those naturally occurring residues that have been modified by 5 chemical or other means.
"Derivatives" as used herein in relation to nucleic acid molecules, proteins and peptides are also intended to encompass single or multiple nucleotide or amino acid 10 substitutions, deletions and/or additions as well as
parts, fragments, portions, homologues and analogues of the nucleic acid molecule or protein or peptide.
A "transformed" cell is a cell into which a nucleic acid 15 molecule has been introduced by molecular biology
techniques. As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with a viral vector, transformation with a 20 plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
In this context "heterologous" nucleic acid means that the 25 nucleic acid introduced into transformed cells is a
nucleic acid not naturally occurring in the cells in this form. On the one hand, it may be nucleic acid which does naturally not at all occur in these transformed cells or nucleic acid which, even if it does occur in these cells, 3 0 is integrated at other genetic positions as exogenous nucleic acid and is therefore situated within another genetic environment.
The heterologous nucleic acid whose expression is to be directed specifically to anther tissue may be any nucleotide sequence that is desirable to be introduced. For example, it may be desirable to provide a transgenic 5 plant in which the anthers express a gene which confers resistance to pathogens, insects and pests, or a gene which confers resistance to stresses such as extremes of temperature, for example a frost resistance gene. Frost resistance genes include dehydrin genes, genes coding for
10 CBF transcription factors and genes in the CBF regulon (CBF-targeted genes). Genes coding for protease inhibitors and Bt toxins may be included to provide resistance to pathogens, insects and pests. In a particularly preferred embodiment the heterologous
15 nucleic acid is antisense to a gene(s) involved in pollen development, such that transformation of a cell with the vector according to one aspect of the invention turns off expression of the gene(s) involved in pollen development, thereby producing male sterile plants.
Preferably the male sterile plants may be produced by RNA interference utilising antisense nucleic acid molecules against one or more genes involved in pollen development, such as BnMYB103 as described in our co-pending
"Antisense nucleic acid molecules" as described herein defines sequences that are complementary to a gene of interest or part thereof. Such antisense nucleic acid 3 0 molecules, may bind to the endogenous gene and block
prevent expression of the functional gene in a plant cell. Antisense techniques generally use short 10 to 20 oligonucleotide fragments which hybridise to essential
parts of the gene thereby blocking its expression. Such essential regions of the gene may include regions within the 5' regulatory region such as enhancer and promoter regions and may also include the transcription start site. 5
In another embodiment, the present invention contemplates a method of inducing or otherwise facilitating male sterility in a plant, said method comprising operably linking a cytotoxic nucleic acid molecule to the anther
10 specific promoter according to one aspect of the
invention, such that upon expression of the promoter, the cytotoxic nucleic acid molecule is expressed to produce a product that inactivates, kills or otherwise renders substantially non-functional male gametes in said
15 transformed plant.
The plant may be a monocotyledonous or dicotyledonous plant. The invention is particularly important in relation to legumes, crop, cereal and native grasses,
20 fruiting plants, and flowering plants as it provides means for increasing yield. Preferred plants according to the present invention include, but are not limited to, the Brassicaceae and other Solanaceae species such as potato and the cole vegetables cabbage, kale, collards, turnips,
25 rutabaga, kohlrabi, Brussels sprouts, broccoli and cauliflower, the mustards and oilseeds, crucifers, brocoli, canola, tomato, grain legumes, wheat, barley, maize, tobacco, rice, and the like. A particularly preferred model system for research is Arabidopsis.
3 0 Particularly preferred plants are canola and wheat.
An "expression cassette" according to the present invention is a nucleic acid molecule made up of at least
the anther specific promoter and a site for inserting heterologous nucleic acid such that the expression of the heterologous nucleic acid in a transformed cell is driven by the anther specific promoter. 5
The expression cassette will preferably comprise at least one restriction enzyme site to facilitate insertion of the heterologous nucleic acid.
10 The expression cassette preferably comprises the anther
specific promoter operably linked to heterologous nucleic acid.
In practice, the expression cassette used to transfect the 15 plant nucleus will generally additionally comprise various control elements. Such control elements may include a ribosome binding site (RBS), positioned at an appropriate distance upstream of a translation initiation codon to ensure efficient translation initiation. 20
Expression cassettes envisaged according to the present invention include those comprising an anther specific promoter and at least one heterologous nucleic acid fragment or gene. 25
A person skilled in the art will be readily able to determine suitable expression cassettes.
Preferably most or all of the constituents of the 3 0 expression cassette are operably linked.
A "recombinant" nucleic acid is one having a sequence that is not naturally occurring or having a sequence made by an
artificial combination of two otherwise separated sequences. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the manipulation of isolated segments of nucleic acids, 5 e.g., by genetic engineering techniques.
The anther specific promoter and heterologous nucleic acid may be used to transformed a cell by any means known in the art. Preferably the expression cassette is provided in a vector.
A "vector" is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include one or more nucleic acid sequences, such as an origin of replication, that permit the vector
15 to replicate in a host cell. A vector also may include one or more selectable marker genes and other genetic elements known in the art.
The term "vector" as used herein is intended to encompass 20 any carrier for nucleic acid, including plasmids and phage.
As used herein a "transgenic plant" refers to a plant that contains recombinant genetic material ("transgene") not
25 normally found in a wild-type plant of the same species. Thus, a plant that is generated from a plant cell or cell line into which recombinant DNA has been introduced by transformation is a transgenic plant, as are all offspring of that plant containing the introduced transgene (whether
3 0 produced sexually or asexually).
As used herein a "cell line" is a population of cells which has been maintained in a culture for an extended
The present invention provides transformed cells comprising a nucleic acid molecule or fragment thereof 5 according to one aspect of the invention or an expression cassette or a plasmid according to other aspects of the invention. By means of methods known to the skilled person the transgenic plant can be generated from a transgenic plant cell. Thus, the plants obtained from the
10 transgenic plant cells of the invention are also the subject-matter of the present invention. The present invention also extends to plants which contain the above-described transgenic plant cells. The transgenic plants may in principle be plants of any desired species as
15 previously defined.
For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a 2 0 corresponding meaning.
It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of
2 5 these documents forms part of the common general knowledge
in the art, in Australia or in any other country.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to
3 0 the invention as shown in the specific embodiments without
departing from the spirit or scope of the invention as broadly described. The present embodiments are,
therefore, to be considered in all respects as illustrative and not restrictive.
Unless otherwise defined, all technical and scientific 5 terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention,
10 suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Embodiments of the present invention will now be described in the following non-limited examples.
1. Identification of TA39 gene orthologue in Arabidopsis
The tobacco TA3 9 protein amino acid sequence was used to identify an orthologous protein in Arabidopsis thaliana. A BLAST search was performed using the National Centre for Biotechnology Information database
10 (www.ncbi.nlm.nih.gov/BLAST/). Several proteins were found showing similarities to the TA39 protein. The protein most similar in length, and with the greatest amount of sequence similarity was chosen for further investigation. The Arabidopsis thaliana gene, designated At39 encodes a
15 protein that shows 30% identity with the TA39 protein and a further 10% of the amino acids were not identical, but are similar in structure allowing for conservative substitutions (Figure 1). Oligonucleotide primers specific for the coding and promoter regions of the At39
2 0 gene were designed.
2. Tissue-specificity of the At39 gene
RT-PCR analysis was used to ascertain the abundance of the 25 Arabidopsis At39 gene transcripts in various tissues and to determine if the At39 gene has a similar expression pattern to the TA39 gene.
The RNA used for the RT-PCR reactions was isolated from 30 leaf, flower and root tissues harvested from wildtype
A.thaliana plants (ecotype Columbia). RNA products were visualized on a 1% agarose gel stained with ethidium bromide.
Controls were required to ensure an equivalent amount of RNA was used from each tissue. Several RT-PCR reactions were performed using tubulin primers to quantify the 5 amount of RNA used in each reaction. The tubulin reverse primer was used to generate the cDNA template from RNA samples. However, the RT-PCR only amplified a tubulin product from flower RNA and not from leaf or root RNA. Another primer, poly-T (PE Biosystems) was used to obtain
10 the cDNA template. However, once again tubulin primers
amplified a product from the flower RNA but not from the root or leaf RNA. In figure 2, lanes 2 and 3 show the 500 bp tubulin product amplified cDNA generated from floral RNA.
The RT-PCR was repeated several times with slight variations each time in an attempt to obtain tubulin products from leaf and roots, but to no avail.
20 3. Identification of At39 gene orthologue in Triticum aestivxm and its tissue specificity
The Arabidopsis thaliana At39 amino acid sequence as identified in Example 1 was used to identify orthologous
25 proteins in Triticum aestivium. A BLAST search was performed using the Grain Gene database (http://wheat.pw.usda.gov). Several proteins were found showing sequence similarity to the At3 9 protein. The proteins most similar in length, and with the greatest
3 0 amount of sequence similarity, were chosen for further investigation.
The Triticum aestivum genes, designated Ta39-1 (SEQ ID NO: 12)and Ta39-2 encode proteins that show 64% and 50% sequence identity respectively with the At3 9 protein and have about 80% sequence identity to the tobacco TA39 gene. 5 The deduced amino acid sequences of the Ta39-1 (SEQ ID NO:13)and Ta39-2 genes show more than 90% sequence identity to the TA3 9 protein from tobacco.
Oligonucleotide primers (SEQ ID Nos 14 and 15) specific 10 for the coding and promoter regions of the Ta39-1 and Ta39-2 genes were designed.
RT-PCR analysis was used to ascertain the abundance of the Ta39-1 and Ta39-2 gene transcripts in various tissues and 15 to determine if the Ta39-1 and Ta39-2 genes have a similar expression pattern to the At39 gene from Arabidopsis.
The RNA used for the RT-PCR reactions was isolated from anther, spiklet (flower), leaf, stem and root tissues 20 harvested from T.aestivum. RNA products were visualized on a 1% agarose gel stained with ethidium bromide. The tubulin reverse primer was used to generate the cDNA template from RNA samples. As shown in Figure 15, the RT-PCR amplified a tubulin product from spiklet and anther
2 5 RNA. No product was amplified from leaf, stem or root RNA.
4. Cloning the At39 gene and promoter region
The cDNA produced from flower tissue during the RT-PCR
3 0 reaction of Example 2 was used to amplify the Arabidopsis
At39 gene. A cDNA clone of the At39 gene was obtained from a PCR using the At3 9 gene 5' and 3' primers. The PCR
product was run on a 1% agarose gel (Figure 3). The 200 bp cDNA clone of the At39 gene is shown in lanes 2 and 3.
The At39 gene fragment was isolated from the gel and the 5 purified cDNA was sequenced to confirm that the desired gene had been cloned. The sequence was also used to determine the size and location of the intron. The At39 gene sequence is shown in figure 4. The gene is 365 bp in length and contains a 96 bp intron. The deduced peptide
10 sequence reveals the gene codes for a protein 89 amino
acids long. Several differences were observed between the cloned At39 gene sequence and the sequence obtained from the Genbank database. The cloned gene encodes a protein of 89 amino acids rather than 58. There were also
15 discrepancies associated with the position of the intron. The exon begins at position +178 rather than +174. The promoter region of the At39 gene was cloned from genomic DNA extracted from Arabidopsis thaliana (ecotype Columbia). The PCR reaction was performed using At39 prom-
2 0 5' and 3' primers. The PCR product was run on a
1% agarose gel to confirm the presence of the 1.9 kb promoter fragment. The nucleotide sequence of the At39 promoter obtained from the Genbank database is shown in figure 5, the binding positions of the promoter-specific 25 primers are represented. A number of short sequences are also highlighted these are putative regulatory element from other anther/pollen-specific promoters, which are considered important for tissue-specificity.
3 0 5. Construction of the At39 promoter expression vector
The At39 promoter fragment was digested with Bam HI and Hind 111 restriction enzymes and ligated into pBluescript
vector (Stratagene). The resulting plasmid was transformed into electro-competent E.coli (DH5a) cells, and selected using ampicillin resistance and blue/white screening. The success of the cloning was determined with restriction 5 digestion to determine if the plasmid contained the
insert. After numerous unsuccessful attempts, cloning the At39 promoter into pBluescript was never achieved. Other cloning strategies were considered. The promoter insert was digested with Bel 1 and Bam HI restriction enzymes to
10 produce a smaller fragment 1200 bp in size, and ligated
into the pl9UC vector (2.5 kb). Stratagene's pPCR script was also utilised, which is a pre-digested, blunt ended vector designed for the direct cloning of PCR products. The only requirement is that the PCR products must be
15 blunt-ended to remove A-overhangs. Using this vector avoids any problems encountered due to incomplete restriction digests of the insert or vector DNA. After many attempts, none of these cloning techniques were successful. The strategies attempting to clone the At39
20 promoter into pBluescript, pl9UC and pPCR script are outlined in figure 6.
The QIAGEN pDrive cloning kit, consisting of the pDrive cloning vector (3.85 kb) designed for the direct cloning
25 of PCR products, was used in a new cloning strategy
(Figure 7). The At39 promoter PCR product was ligated into pDrive. The resulting plasmid was transformed into electro-competent Esherichia coli (DH5a) cells and selected using ampicillin resistance and blue/white
3 0 screening. Restriction digests with BamHl and Hindi 11
confirmed the cloning success by releasing the fragment from the vector.
The first positive clone was verified with a triple restriction digest using Bam HI, Hind 111 and Sphl but the expected fragments sized 3.85kb, 1.7 kb and 0.2 kb were not obtained. Another triple digest using Sspl instead of 5 Sphl produced the expected band sizes 3.85kb, 1.1kb and 0.8 kb visible on a 1% agarose gel. This confirmed the At39 promoter had been cloned, but the failure of Sph 1 to digest the insert suggests the sequence may contain errors. The pDrive/At3 9 plasmid was sequenced using the
10 universal M13 reverse primer. The sequence revealed the
At39 promoter insert contained a base substitution at the Sph 1 restriction site and other significant errors. As the first At39 promoter clone was incomplete other pDrive white colonies were screened using restriction
15 digestion with a single enzyme, either Bam HI or Hind 111. The single enzyme digestion releases the insert from the plasmid due to presence of the restriction site on the end of the insert and one in the plasmid. Indicating each end of the At39 promoter fragment is intact. Figure 8 shows
20 the successful digestion of two pDrive clones Bl and B2. Lane 2 represents the pDrive vector without the insert. Lanes 3 and 5 show the pDrive vector digested with Hind 111, while 4 and 6 show the pDrive vector digested with Bam HI. The restriction digest of the pDrive/At39 (B2)
25 plasmid with Eco Rl is shown in lane 7. The At39 promoter insert contains the correct number of restriction sites producing the expected sized fragments, namely 1.8kb, 1.3kb and 0.4 kb. This verified the insert is correct and was subsequently used for cloning.
The pDrive/At39 plasmid was sequenced using the M13 reverse primer to ensure the clone did not contain errors in the sequence. Figure 9 shows the sequence alignment of
the pDrive/At3 9 clone and the original At39 promoter sequence. The At39 promoter clone matches closely with the original sequence, proving the errors found in the first clone were not generated during the PCR reaction. 5 The At39 promoter insert was isolated from the pDrive/At3 9 vector following a double digestion using Bam HI and Hindi11, and cloned into the pBI101.3 binary vector. The resulting plasmid was transformed into electro-competent E.coli (DH5cO cells, and transformants were selected using
10 kanamycin resistance. A control transformation was also performed, involving the self-ligated pBI101.3 vector without any insert DNA transformed into E. coli electro-competent cells. Due to the different cohesive ends the vector should not self-ligate and colonies cannot grow.
15 If the control transformation produces a large number of colonies it indicates the restriction digestion of the vector was incomplete and it is unlikely that the At39 promoter insert has been incorporated into the binary vector.
The successful cloning of the At39 promoter inserts into the pBI vector were confirmed with restriction digestion using Bam HI, Hind 111 and Eco Rl (Figure 10). The At39 promoter PCR product (lane 2) acts as a control to compare
25 the size of the cloned At39 promoter. The pBI101.3 vector digested with Bam HI and Hind 111 is shown without an insert in lane 3, while lane 4 shows the 1.9 kb At39 promoter insert released from the vector. Lane 5 represents the pBl/At39 plasmid digested with Eco Rl. The
30 expected band sizes were present, namely 12.2 kb, 2.4 kb and 1.3 kb fragments.
To ensure the At39 promoter was cloned into the vector in the correct reading frame to regulate GUS expression, the junction between the At39 promoter and GUS gene was sequenced. Figure 11 shows a portion of the sequence 5 alignment comparing the plasmid and original At39 promoter sequences. The sequences are identical, except for the complimentary region due to the binding position of the reverse primer. The pBl/At3 9 promoter sequence (shown on the top) contains extra bases from the linker region on 10 the pBI101.3 vector. The GUS gene translation start site (ATG) starts at position 1078 bp.
6. Transformation of Arabidopsis thaiiana
15 The pBI/At3 9 promoter binary vector was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, using rifampicin, gentamycin and kanamycin selection. The presence of the transformed plasmid was confirmed by PCR analysis. The PCR products
20 were run on a 1% agarose gel (Figure 12). The PCR product obtained with the At39 promoter-specific primers is shown in lane 2, and the PCR product amplified with the PBI-GUS primers is shown in lane 3. The PBI-GUS primers bind each side of the multiple cloning sites in the pBI vector.
25 Agrobacterium containing the pBI/At39 promoter plasmid was used to transform Arabidopsis thaliana (ecotype Landsberg erecta) using vacuum infiltration. The transformed plants yielded a large number of seeds, which were subsequently harvested and germinated with kanamycin selection and
30 timentin to kill the Argobacterium. The seedlings with kanamycin resistance were subjected to PCR analysis to confirm transformation had occurred (Figure 13). As the At39 promoter-specific primers would amplify the
endogenous At39 promoter, they were not suitable to confirm the promoter construct had been incorporated into the plant genome. Hence, the PCR was performed using the PBI-GUS primers. Lane 2 represents the PCR using leaf 5 tissue from a wildtype non-transformed plant. Lanes 3-7 show the 2 kb fragment amplified from leaf tissue harvested from five transformed plant lines. Lane 9 is a control, the fragment was produced using pBl/At39 plasmid DNA. The bands are all identical in size demonstrating 10 plant lines 1-5 had been transformed with the promoter construct. Plant line 6 (lane 8) did not amplify the fragment, indicating this plant is not transgenic.
7. Analysis of GUS expression pattern
The five transgenic lines were analysed for GUS expression. Due to the small size of the plants only a limited amount of tissue could be collected. A leaf and a single floret were removed from each line. Line 3
2 0 contained a number of flowers so the whole bolt was removed to encourage secondary flowering. Line 5 was infected with fungal contamination and was dying, so the whole plant was used for GUS staining.
2 5 The plants were incubated in the GUS substrate for 6 hours, but strong GUS expression was observed in the anther region after two hours (Figure 14). GUS expression was located in the anther tissue for each plant line, however lines 2 and 5 also contained GUS expression in the
30 sepals. GUS expression was not observed in any other tissue.
The project has identified a novel promoter from 5 Arabidopsis that is primarily expressed in anther tissue. A number of putative regulatory elements that direct tissue specificity were also identified.
1. The At39 promoter
The nucleotide sequence of the Arabidopsis At39 gene was
obtained from the Genbank database following a BLAST search, which was performed to identify a protein homologous to the tobacco TA39 protein. The At39 gene 15 encodes a protein that shares 30% homology with the TA39 protein.
The promoter region of the At39 gene was amplified using PCR and cloned into an expression vector containing the
20 GUS reporter gene. Promoter-GUS fusions resulted in GUS
expression in anthers, demonstrating that At39 is strongly expressed in the anther region of Arabidopsis plants. Anther-specific promoters commonly contain short regulatory sequences that are sufficient to direct tissue-
25 specific expression, and upstream regions that are
important for higher levels of activity. Key features are conserved among tissue-specific promoters. These include consensus sequences common to some anther-specific promoters, however an element regulating anther-specific
3 0 expression is yet to be identified that is common to all anther-specific promoters.
The At39 promoter contains a number of sequences in common with other anther-specific promoters. The AAATGA motif, originally identified in tobacco, occurs twice in the At39 promoter at position -1339 and -440. This motif is also 5 shared with PO149 an anther-specific promoter from
alfalfa. The CCACAAAAA sequence at position -240 is also shared with the chiA gene from petunia, and is highly conserved in other chalcone flavanone isomerase (chi) promoters. The TGAACG sequence located at -900 is present
10 in the nopaline synthase (nos), the cauliflower mosaic virus 3 5S, and the pollen-specific Brassica napus Bp 19 promoters. The TATATATA site at position -12 3 0 is also present in the A9 gene from Arabidopsis. However, the At39 promoter does not contain elements identical to the 52/56
15 or 56/59 boxes from the tomato LAT gene promoters.
The At39 and TA39 protein sequences share limited homology, and the promoter regions are even less similar. Nevertheless, the two genes exhibit similar expression
20 patterns in anthers. There are some short sequences that are shared between the two promoter regions. The shared sequence TTGATATCCTT occurs at position -1450 in the At39 promoter. This region is a considerable distance upstream of the translational start site and is unlikely to be
25 involved in tissue-specific gene expression. Other short sequences containing single base changes occur throughout the promoter regions. The TAA(C/G)TTTG sequence occurs at position -1128, TA(C/T)AAT at position -1096, GTCCT(C/G)AA at position -915, TT(G/C)TGA at position
3 0 -747, T(A/T)TTGT at position -739, and ATGCAGATTT at position -500. These sequences occur a considerable distant upstream of the coding region and might not play a regulatory role. Both promoters contain high proportions
of A and T residues, which may account for the short sequence similarities.
The TA39 promoter is primarily expressed in the tapetum 5 layer of anthers. The actual site of At39 expression is not determined, as cross-sections of the anther have not yet been prepared. The At39 gene may be expressed throughout the anther or in a specific region such as the tapetum, middle layer or locule. GUS expression was
10 detected in other tissue in only two transgenic lines. In these plants GUS expression occurred in the sepals as well as anthers. Only a limited number of plant lines were analysed for GUS expression patterns because the plants were required to be at the flowering stage of development.
15 GUS expression in the sepal may be related to the
developmental stage of the flower at the time of staining, or be caused by other factors such as a positional effect with respect to integration of the transgene. Whether At39 expression occurs in sepals can be best ascertained
20 using in situ hybridisation.
The At39 gene encodes an 89 amino acid protein with a molecular weight of 9.75 kDa, while the TA39 protein encodes a 110 amino acid protein with a molecular weight
25 of 11.9 kDa. The functions of the two proteins are not known, although they may be involved in pollen development. The mRNA from anther-specific genes may be pre-synthesised and stored in the pollen grains until it is required for processes involved in fertilisation. The
3 0 function and mechanisms regulating the proteins encoded by many anther-specific genes are not known.
The At39 protein contains a large proportion of lysine (11%), cysteine (13%) and serine (11%) . These three amino acids constitute 35% of the protein. However, obvious sequence repeats are not present. A hydropathicity plot 5 revealed the At39 protein contains a hydrophobic region in the first 20 amino acids while the rest of the protein is hydrophilic (Figure 4B). The hydrophobic region may represent a signal sequence, suggesting the protein is secreted.
On the Genbank database (accession no. AB015475), the At39 gene is categorised as gibberellin -regulated. A BLAST search of the database using the TA39 protein sequence identified a number of other genes also associated with
15 gibberellins. Gibberellin may act as a regulatory factor involved in anther-specific gene expression.
Tissue-specific promoters are important tools for research and may have useful applications in agricultural
20 practices. Such applications include the development of a male sterility system that can be used to breed hybrid crops. Potentially the At39 promoter can be used to regulate gene expression in the anther region of crops such as Canola. To determine if the At39 promoter retains
25 tissue-specificity in other plant species, it must be transformed into other species.
MATERIALS AND METHODS
1. Seed Surface Sterilization and Germination
Seed were collected, placed in eppendorf tubes and wet with 500 ul of 70% ethanol for 5 minutes. The ethanol was 5 removed and replaced with 500 ul seed sterilization
solution (a mixture of bleach, sterile water and 5% SDS at a ratio of 8:15:1 respectively). The tube was shaken and left for 10 minutes at room temperature, and then pulse spun to sediment the seeds. The supernatant was removed, 10 and the seeds were washed four times with sterile water.
Sterilised seeds were grown in 25 mm deep petri dishes containing 50 mL of germination media (GM: 0.5 g MES, 10 g sucrose, 4.6 g Murashige and Skoog Basal Salt mixture,
15 1 mL 1M KOH, 2g Phytagel and water to 1 L). Media was autoclaved prior to use, and 1 mL of filter sterilized lOOOx vitamin stock added before pouring plates. If selection was required 1 mL of 50mg/mL kanamycin was also added. Once the seed were sown the plates were sealed
2 0 with micropore tape and incubated in growth cabinets at 22°C under constant light.
2. RNA Extraction
25 RNA extraction was performed following the Progen Industries DRP3 separation reagent protocol.
Arabidopsis plant tissue was collected, and 100 mg was ground to a fine powder in the presence of liquid nitrogen 30 using a mortar and pestle. The tissue powder was
homogenized in 1 mL of DRP3 reagent, and incubated for 5 minutes at room temperature, then transferred to a 1.5 mL eppendorf tube. The sample was suspended in 200 ul
chloroform, mixed by vortexing for 20 seconds and incubated for 10 minutes at room temperature. The mixture was separated into phases by centrifugation (Biofuge) (11,300 rpm, 15 minutes at 4°C). The upper aqueous phase 5 containing the RNA was transferred to a new eppendorf
tube, 500 ul of isopropanol was added and the sample was incubated for 5 minutes at room temperature. The RNA was precipitated by centrifugation (11,300 rpm, 8 minutes at 4°C) and the supernatant was discarded. The RNA pellet was
10 washed in 1 mL of 75% ethanol by vortexing and
precipitated by centrifugation (8,800 rpm, 5 minutes at room temperature). The supernatant was removed and the pellet was air dried for 5 minutes before being dissolved in 50ul water. RNA samples were stored at -20°C.
3. Extraction of Genomic DNA from Plant Tissue using QIAGEN DNeasy®
The DNA extraction method followed the QIAGEN DNeasy Plant 20 mini kit protocol.
Plant leaves were collected into a 15 mL falcon tube and frozen in liquid nitrogen. The plant tissue (100 mg) was ground to a fine powder in the presence of liquid
25 nitrogen, using a mortar and pestle. The powder was
transferred to an eppendorf tube, 400 ul Buffer API and 4 ul RNase A stock solution was added. The suspension was mixed by vortexing and incubated at 6 5°C for 10 minutes to lyse the cells. To precipitate unwanted material, 130 ul
3 0 Buffer AP2 was added. The lysate was mixed and incubated on ice for 5 minutes. The lysate was added to the QIAshredder spin column sitting in a 2 mL collection tube. Without disrupting the pellet the flow-through was
transferred to a new eppendorf tube. The total volume was determined, 0.5 volume of Buffer AP3 and 1 volume of 95% ethanol was added and mixed by pipetting. The sample along with any precipitate was place into the DNeasy mini spin 5 column sitting in a collection tube and centrifuged (8000 rpm, 1 minute at room temperature), the flow through was discarded. The column was place into a new collection tube, and 500 ul Buffer AW was added to wash the column. The column was centrifuged (8000 rpm, 1 minute at room
10 temperature) the collection tube was emptied and the wash was repeated as above but was spun for 2 minutes to dry the membrane. The column was transferred to a new eppendorf tube, the DNA was eluted with 100 ul Buffer AE (preheated to 65°C) added directly onto the column
15 membrane. The tube was incubated for 5 minutes at room temperature before centrifugation (8000 rpm, 1 minute at room temperature). The DNA sample was stored at -2 0°C until required.
2 0 4. Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)
The two-step RT-PCR procedure was performed using Superscript II RNase reverse transcriptase (GibcoBRL) to
25 synthesise first strand cDNA. The RT reaction mixture was placed in a 0.6 mL thin-walled PCR tube and consisted of 5 ul of 5x First-strand Buffer, 1 ug of template RNA, 30 pmol of Tubulin reverse or Poly (T) primer (PE Biosystems), 1 ul Superscript II, 1 pi of 10 mM dNTP, 2 ul
30 of 0.1M DTT, 1 pi RNase Out inhibitor and sterile water to a total volume of 50 ul. The cDNA was synthesized at 55°C for 3 0 minutes in the MJ research minicycler PCR machine.
The cDNA was then used as template DNA in a PCR reaction see below.
5. Polymerase Chain Reaction (PCR)
PCR was initially used to amplify the DNA sequence of
interest for use in cloning procedures and later used to verify that the DNA insert had been successfully cloned or transformed into the plant genome. Hence the template DNA 10 used in PCR reactions was either genomic DNA, plasmid DNA isolated by miniprep or a 2mm2 piece of alkali prepared leaf tissue.
The reaction mix was prepared in 0.6 mL thin-walled PCR 15 tubes and consisted of 5 ul of lOx PCR Buffer, 2 ul of 10 mM dNTP's, 3 ul of template DNA, 1 pi of each primer (30pmol) 1 ul of Taq DNA polymerase and sterile water to total volume of 50 ul. When using a PCR machine without a heat lid, 40 ul mineral oil was added to the surface to 2 0 prevent evaporation.
The PCR tubes were placed in a MJ Research PCR minicycler and programmed for the following conditions: step 1: 94°C for 2 minutes, step 2: 55°C for 30 seconds, step 3: 72°C
25 for 1-2 minutes, step 4: 94°C for 30 seconds, step 5: 55°C for 30 seconds, step 6: 72°C for 1-2 minutes, step 7: Repeat steps 4-6 for 30 cycles, step 8: 72°C 10 minutes and finally kept at 4°C until the reactions were removed from the PCR machine. When the PCR reaction was completed,
30 10-15 ul of PCR product was run on a 1% agarose gel to confirm the presence of the amplified fragment. The remaining samples were stored at -20°C.
The same basic program was used for all PCR, however there were adjustments made depending on the success of the PCR. The primers tended to have different optimum annealing temperatures (steps 2 and 5) ranging from 50-60°C, 5 although 55°C was appropriate for most reactions. The
extension time also varied depending on the length of the PCR product. Occasionally the number of cycles was increased, but in most cases 30 cycles was adequate.
At39gene-5'(SEQ ID NO: 4) 5'
ATGAAATTCCCGGCTGTAAAAGTTCT 3' (2 6mer)
At39gene-3'(SEQ ID NO: 5) 5'
AGAAACAAAAGGTATTCACGGACTT 3' (2 5me r)
15 At3 9prom-5'(SEQ ID NO: 6) 5'
GCACAAGCTTGCTTATAAGCTACTCTTTGCC 3' (31mer)
At3 9prom-3'(SEQ ID NO: 7) 5'
TTCCGGATCCGAACTTTTACAGCCGGGAATTT 3' (3 2mer)
Canprom-5' (SEQ ID NO: 8) 5'
2 0 GCACAAGCTTGTATAGAGTAAATGAGCA 3' (2 8mer)
At3 9codereg-3'(SEQ ID NO: 9) 5'
TTCCGGATCCGGTTGAGAGTATGAACAAAGAA 3' (3 2mer)
NK2 (SEQ ID NO: 10) 5'
TTGAGAGCTCGTAGGAACAGAGCAC 3 ' (2 5mer)
25 NK1 (SEQ ID NO: 11) 5'
CTTGAGCTCGAAGAAATGGGTCGGATTCCATGTT 3' (34mer)
The restriction sites are shown: BamHl is underlined, Hindlll is in bold and Sacl is in italics. 30
The pair of primers used in the reaction depended on the purpose of the PCR. The At3 9 gene-specific primers were used to amplify the At39 gene. At3 9gene-5' binds at
position +1 (the ATG translation start site) in the At39 gene, the At39gene-3' primer binds at position +350 in the coding region of the gene, the binding positions are shown in figure 4. The At39 promoter-specific primers were used 5 to amplify the At39 promoter region used for cloning, the At39prom-5' primer binds at position -1850 in the 5' promoter region, and the At39prom-3' also binds at position +1 on the complimentary strand, the binding positions are represented figure 5. The At39-codereg 10 primer binds at position +50 in the coding region of the At39 gene shown in figure 4.
6. Testing the suitability of primers used for T-DNA insertion screening.
The primers used for screening the T-DNA insertion mutant library were tested under specific PCR conditions that were consistent with those used at the University of Wisconsin-Madison Knockout Facility.
The PCR reaction was performed according to the conditions outlined by the KO facility using TaKaRa Ex-Taq™. The PCR reaction included 4 ul 10X Ex-taq buffer, 4 ul dNTP, 1 ul of each primer (12 pmol), 2 ul Arabidopsis genomic DNA
25 (ecotype WS) and water to a total volume of 40 ul. The samples were placed in the PCR machine (MJ research minicycler) and heated to 96°C for a hot start, before 10 pi of hot start enzyme mix was added. The hot start mix consisted of 8.5 ul water, 1 ul Ex-taq buffer and 0.5 ul
30 Ex-taq polymerase. After the mix was added the PCR program continued with 36 cycles of 94°C for 15 seconds, 65°C for 30 seconds and 72°C for 2 minutes, followed by a final
extension time of 72°C for 4 minutes then kept at 4°C until removed from the machine.
Four reactions were performed using different primer 5 combinations, 1) Con-IA + Con-IB provided a standard to compare the effectiveness of the gene specific primers 2). Con-IA + Con-IB + JL-202 tested the compatibility of the control primers with the T-DNA left border primer 3) ScrnlO3-5' + ScrnlO3-3' tested the suitability of the 10 AtMYB103 gene specific primers 4) ScrnlO3-5' + ScrnlO3-3' + JL202 tested the compatibility of the gene specific primers with the T-DNA border primer.
15 SEQ ID NO: 12 - Con-IA: 5' CGTCTAGGTGGTTCAGTACCTGTTGAATG
SEQ ID NO: 13 - Con-IB: 5' TTTATCGAAGAAACATGTCGTTGAACCAG
SEQ ID NO: 14 - JL-202: 5' CATTTTATAATAACGCTGCGCACATCTAC 20 3' (29mer)
SEQ ID NO: 15 - ScrnlO3-5'5' GGCTAGTTTGTTATCCAAGTCGTTCTACC
SEQ ID NO: 16 - ScrnlO3-3'5' AGTTTTGTGTATGCGTTCAATAACCTTT
3' (28mer) 25
7. DNA Fragment Isolation using UltraClean™ 15
DNA fragments were isolated following the Mo Bio Laboratories UltraCleanl5 protocol. 30
The required DNA fragment or PCR product was run on a 1% agarose gel. Using a razor blade the desired band was cut from the gel and place into an eppendorf tube. The weight
of the gel band was determined, 0.5 volume of Ultra TBE Melt and 4.5 volumes of Ultra Salt was added. The tube was incubated at 55°C for 5 minutes or until the gel melted. The Ultra Bind was vortexed until homogenous then 6 pi was 5 added to the solution. The mixture was incubated for 5 minutes at room temperature, and mixed several times during the incubation. The solution was briefly centrifuged (13,000rpm 5 seconds at room temperature) and the supernatant was discarded. The pellet was washed with
10 1 mL of Ultra Wash solution by vortexing, and then
centrifuged (13,000 rpm, 5 seconds at room temperature). All traces of the supernatant were removed by pippetting. The pellet was resuspended in 15 ul of water and incubated for 10 minutes. The suspension was centrifuged (13,000
15 rpm, 1 minute at room temperature) and the DNA was transferred to a new tube, and stored at -20°C.
8. Sequence Analysis-Big Dye Terminator
2 0 The sequencing reactions were performed using the Big Dye Terminator Cycle sequencing ready reaction kit manufactured by PE Applied Biosystems.
The sequencing reaction consisted of 6 ul of Terminator 25 Ready Reaction Mix, 100 -500 ng of template DNA, 3.2 pmol of primer and sterile water to a total volume of 20 ul. The reagents were placed in a 0.6 mL thin-walled PCR tube and mixed well. The tubes were placed in the thermal cycler PCR machine (MJ research minicycler). The program 30 was set for the required conditions, step 1: 96°C for
30 seconds, step 2: 50°C for 15 seconds, step 3: 60°C for 4 minutes, step 4: repeated steps 1-3 for 25 cycles and finally held at 4°C until the samples were removed.
The sequencing reaction product was purified using ethanol precipitation. The reaction mixture was transferred to an eppendorf tube containing 2.0 ul 3M sodium acetate and 5 40 ul of 95% ethanol. The contents of the tube were
vortexed and place in -80°C freezer for 20 minutes. The DNA was precipitated by centrifugation (13,000 rpm, 15 minutes at 4°C), and the supernatant was removed. The pellet was washed with 200 ul 70% ethanol, then vortexed
10 and centrifuged (13,000 rpm, 10 minutes at 4°C). The
supernatant was removed by pippetting and the pellet was dried under vacuum in a Speedvac concentrator for 10 minutes. The sample was sent to the Microbiology Department at Monash University, Clayton for automated
9. Preparation of E.coli Competent Cells
E.coli cells (DH5a) were plated on 2YT plates without 2 0 antibiotics and incubated overnight at 3 7°C. A single colony was used to inoculate a 15 mL 2YT culture in a falcon tube and grown overnight at 3 7°C orbital shaker. The cells were collected by centrifugation (Interfuge) (3000 g, 15 minutes at room temperature), and the
2 5 supernatant was discarded. The cells were resuspended with
1 mL of sterile ice-cold water and transferred to an eppendorf tube. The cells were centrifuged (Biofuge) (13,000 rpm, 7 minutes at 4°C), the supernatant was discarded and the pellet was resuspended in 1 mL ice-cold
3 0 water, this process was repeated three times. Then the
cells were centrifuged (13,000 rpm, 5 minutes at 4°C), the supernatant was discarded. The cells were resuspended in 1 mL of ice-cold 10% glycerol, and harvested by
centrifugation (13,000 rpm, 3 minutes at 4°C). The supernatant was removed and finally the cells were resuspended in 500 ul ice cold 10% glycerol. The cell suspension was transferred to eppendorf tubes in 50 ul 5 aliquots, snap frozen in liquid nitrogen and stored at -70°C.
10. Restriction Digestion
To prepare various vectors and DNA inserts for cloning, a 10 number of restriction digest were performed, using a number of restriction enzymes (Boehringer-Mannheim) depending on the application and type of DNA or plasmid used in the reaction.
15 The digestion reaction contained the DNA of interest, 1 ul of each restriction enzyme (Boehringer-Mannheim) , 2 )jl of the corresponding restriction buffer and sterile water up to a total volume of 20 ul. The reaction was incubated at 3 7°C for 1-2 hours, and the entire reaction was run on a
20 1% agarose gel and then isolated using the UltraCleanl5
kit (4.7). A small portion of the digested DNA was run on a 1% agarose gel to visualize the amount of DNA recovered to determine the amount required for ligation.
2 5 Dephosphorylation:
The pB4.1.21 vector was kindly supplied by Trudi Higginson. This vector was digested with Sacl as above, but was dephosphorylated to reduce self-ligation. The dephosphorylation reaction was performed immediately after
3 0 the restriction digest was incubated so that the DNA only
needed to be isolated from the gel once, to reduce the amount of DNA lost during the purification process. Dephosphorylation was achieved by adding 1 ul of calf
intestinal phosphatase (CIP) (Boehringer-Mannheim) and 2 jil of lOx CIP buffer to the digest reaction and incubated at 37°C for 30 minutes. The reaction was then run on a 1% gel and isolated as above. 5
PCR amplified products were blunt ended before cloning into the pPRscript cloning vector, since A-overhangs on the PCR product can interfere with cloning. In the 10 reaction 1 pi of T4 polymerase buffer and 1 ul T4 DNA
polymerase (Promega) was added to the purified PCR product and incubated at 3 7°C for 3 0 minutes. The insert DNA was then used in a ligation reaction as described below.
A large number of ligations were performed to clone insert
2 0 DNA into vectors. The volume of vector and insert DNA
varied in each reaction depending of the amount visualised after isolation from the restriction digest, usually a 2-3 fold excess of insert DNA was present compared to the amount of vector DNA. The vector and insert DNA were added 25 to an eppendorf tube containing 1 ul of lOx ligation
buffer (Promega) and 1U T4 DNA ligase (Promega) along with sterile H20 to a total volume of 10 ul. Ligations were incubated at room temperature overnight.
3 0 In some instances the lOx ligation buffer was replaced
with 5 ul of 2x rapid ligase buffer (Promega) and the total volume was increased to 15 ul, the ligation reaction was incubated at room temperature for 10 minutes.
Ligations were used immediately for transformation or stored at -20°C.
QIAGEN PCR cloning kit ligation
5 Ligations involving the pDrive cloning kit were performed under the conditions specified by QIAGEN to ensure optimal results. The ligation reaction mixture consisted of 1 ul pDrive cloning vector (50 ng/ul), 2 ul PCR product (non-purified) , 5 ul ligation master mix and distilled water to 10 a total volume of 10 ul. The mixture was incubated at 16°C in a water bath for 2 hours. The ligation mixture was used immediately or stored at -20°C.
12. Transformation of E.coli Competent Cells
A 50 ul aliguot of electro-competent E.coli (DH5a) cells
were thawed on ice, and 2 ul of ligated plasmid (4.11) was added. This mixture was mixed by pippetting, and then left on ice for 1 minute. The cell mixture was transferred to
20 an ice-cold 0.1 cm electroporation curvette, and tapped gently to remove any air bubble. The outside of the curvette was wiped dry with a tissue and placed into the electroporator (BIORAD micropulser), the program was set for Ecol. An electric shock (1.8 kV) pulse was applied to
25 the cells. Immediately 100 ul of ice-cold 2YT medium was added to the curvette. The suspension was transferred to an eppendorf tube and placed in the 3 7°C incubator for 1 hour. The cells were spread onto 2YT plates containing the appropriate antibiotic to select for cells containing
30 the transformed plasmid. The plates were incubated at 37°C overnight.
In addition to antibiotic selection, blue/white colony-selection was also used for pBluescript and pDrive vectors to assist in the identification of positive colonies. For 5 blue/white screening, 20 ul of 50mg/mL X-Gal (5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside) was spread onto the plates and allowed to dry before the plating transformed cells.
10 13. Plasmid DNA Miniprep
A single bacterial colony from a transformation plate was used to inoculate 15 mL of 2YT medium containing the correct antibiotic to select for the plasmid. The 15 bacterial cultures were grown overnight at 37°C in an orbital shaker.
The culture was transferred to an eppendorf tube and centrifuged (13,000 rpm, 2 minutes at room temperature)
20 the supernatant was discarded. This process was repeated until 5 mL of culture was used. The bacterial cells were resuspended in 200 ul of Resuspension solution by vortexing. To lyse the cells, 200 ul of Cell lysis solution was added. After mixing, 200 ul of neutralisation
25 solution was added, the sample was mixed before centrifugation (13,000 rpm, 7 minutes at room temperature). The supernatant was transferred to a new eppendorf tube containing 1 mL of DNA purification resin. The solution was added to the minicolumn barrel and a
3 0 vacuum was applied drawing the solution through the
minicolumn. Applying 2 mL of column wash to the minicolumn washed the plasmid DNA. The minicolumn was placed into an eppendorf tube and briefly pulse spun to dry the resin and
remove any residual column wash. The minicolumn was transferred to a new eppendorf tube and 50 pi of water was added, after incubation for 10 minutes at room temperature, the DNA was eluted by centrifugation 5 (10,000 rpm, 1 minute at room temperature). The minicolumn was removed and the plasmid DNA is stored at -20°C. Adapted from Promega technical bulletin protocol.
14. Transformation of Agrobacterium tumefaciens electro-10 competent cells
A 50 pi aliquot of electro-competent Agrobacterium (GV3101) cells was thawed on ice. The pBI binary vector containing the insert was isolated and purified with
15 miniprep (see 12), 2 ul of plasmid DNA was added to the
thawed cells and incubated on ice for 1 minute. The sample was transferred to a 0.1cm ice-cold curvette ensuring no air bubbles were present. The outside of the curvette was wiped dry with a tissue and placed into the
20 electroporation machine (BIORAD micropulser). The program was set for AGR and an electric pulse (2.2kV) was applied to the bacterial cells. Immediately 100 pi of 2YT broth was added to the curvette and gently mixed. The suspension was transferred to an eppendorf tube and incubated at 25°C
25 for 2 hours. The cells were spread onto 2YT plates containing kanamycin, gentamycin, and rifampicin antibiotics to select for the plasmid. The plates were incubated at 25°C for two days until the Agrobacterium grew.
58 15. Genetic Transformation of Arabidopsis thaliana
Wildtype Arabidopsis thaliana (Columbia) seeds were sown in punnets containing potting mix. The potting mix was 5 held in place with plastic mesh secured by rubber bands. Plants were grown at 20°C with a 24 hour day cycle until they were ready for transformation. When the plants were beginning to flower the bolts were removed to encourage secondary shoots to increase the number of florets.
Colonies from A. tumefaciens (GV3101) transformed with the binary vector containing the gene of interest were used to inoculate 10 mL 2YT broth containing 50 ug/mL rifampicin, 25 ug/mL gentamycin and 50 ug/mL kanamycin. The cells were
15 grown overnight at 25°C in an orbital shaker. When thick, 3 mL was used to inoculate 3 00mL 2YT broth containing 50 ug/mL kanamycin and 25 ug/mL gentamycin in a 1 litre flask. The culture was grown overnight at 25°C in an orbital shaker. The culture was transferred into two
20 250 mL centrifuge bottles and centrifuged using a HS4 rotor Sorval RC) (5000 rpm, 15 minutes at room temperature). The supernatant was discarded and the pellet was resuspended in 200 mL of infiltration media (2.3 g Murashiage and Skoog Basal Salt mixture, 1 mL of 1000 x
25 vitamin stock, 50g sucrose, 100 ul of 100 ug/mL BAP and 1 mL Silwet). The suspension was poured into a container, which was placed into the vacuum desiccator. The pots of Arabidopsis plants were saturated with water prior to the transformation to prevent the Agrobacterium absorbing into
30 the soil.
The pots were placed upside down into the Agrobacterium solution in the container. The plants were place under
vacuum in the desiccator for 5 minutes, during this time the desiccator was periodically tapped to remove any air bubbles trapped amongst the plants. The vacuum was released quickly and the pots were removed and left on 5 their side to drain for 5 minutes. The pots were placed upright into a plastic tub covered in paper towel, and placed in the dark for 24 hours. The plants were uncovered and placed under constant illumination until the seeds were ready to harvest. The seeds were sterilized and 10 germinated on growth medium containing 50 ug/mL kanamycin (see 2).
16. Alkali Plant Tissue Preparation
15 Transformed plants with kanamycin resistance underwent PCR analysis to determine if the plant is transgenic. The plant tissue was prepared for PCR reactions following the protocol published by Klimkuk et al., (1993) Plant Journal 3:493-494.
A leaf from kanamycin resistant seedlings were collected and cut into small pieces (2mm2) with a sterile razor blade. The tissue pieces were placed into a 1.5 mL eppendorf tube containing 40 ul of 0.25 M NaOH and boiled
25 at 100°C for 1 minute in a water bath. The samples were
neutralized with 40 ul 0.25 M HCl and 20 ul 0.5 M Tris-HCl (pH 8.0), and boiled for a further 2 minutes. A piece of the leaf was transferred to a 0.6 mL PCR tube and analysed with PCR reactions (see 5) .
17. (b-Glucuronidase (GUS) Staining
Tissues samples from transgenic plants were placed into an 1.5 mL eppendorf tube and submersed in X-gluc solution 5 (0.5 mg/mL X-gluc in dimethylformamide, 50mM NaPO4; pH 7 and 0.05% Trition X-100). The samples were incubated at 3 7°C for several hours or overnight. The X-gluc solution was removed and replaced with 70% ethanol. The samples were incubated at room temperature for several hours or 10 until the tissue was void of chlorophyll. The GUS
expression pattern was analysed with light microscopy and photographed.
18. Southern Blotting onto a Nylon membrane
The PCR products obtained from the University of Wisconsin-Madison Knockout Facility were run on a 1.0% agarose gel and photographed with a ruler beside the gel to identify band positions later on.
The gel was rinsed in distilled water and placed into a glass dish containing 500 mL denaturation solution (1.5 M NaCl, 0.5 M NaOH) to make the DNA molecules single stranded. The dish was placed onto a platform rocker to
25 shake for 20 minutes, the solution was replaced with a
equal volume of fresh denaturation solution and shaken a further 2 0 minutes. The denaturation solution was removed. The gel was rinsed in distilled water, and 500 mL of neutralisation solution (1.5 M NaCl, 0.5 M Tris pH 7.0)
3 0 was added to the dish and left to shake a further 20 minutes. The solution was replaced with fresh neutralization solution and shaken a further 20 minutes.
The transferred pyramid was set up by placing a glass sheet across top of a plastic container partially filled with 20 x SSC transfer buffer (3M NaCl, 0.3M Na3 citrate.2H2O) . The wick consisted of three lengths of 3mm 5 whatman paper placed over the top of the glass plate extending over the edges down into the transfer buffer. The gel was placed onto the wick and air bubbles were removed by rolling a glass rod over the gel. Strips of glad wrap were placed around the edges of the gel to
10 ensure the transfer buffer flowed through the gel rather than around it. A piece of Amersham Hybond-N nylon membrane was cut to the size of the gel. The nylon membrane was place onto the gel with forceps to avoid touching the membrane. The top left hand corner was cut
15 off the membrane to remember the orientation of the gel. Air bubbles were removed by rolling a glass pipette over the surface. Five sheets of whatman paper cut to the same size as the nylon membrane were soaked in 20x SSC and placed onto of the nylon membrane. A stacked of paper
20 towel approximately 10 cm high were place on top. Finally a glass plate and a weight were placed on top to keep everything in place. The transfer was left to proceed overnight.
25 The transfer pyramid was dismantled. The paper towel and filter papers were removed. A pencil was used to mark the position of the wells. The nylon membrane was removed and washed in 2x SSC to remove any agarose for 1 minute. The membrane was place on a sheet of filter paper to dry
30 before being cross-linked in the Stratagene UV
Stratalinker 2400, this allows the membrane to be probed several times. The membrane was sealed in plastic wrap and stored at 4°C until probed.
19. Labeling 32P Probe using BIO-RAD megaprime kit
The probe was amplified using the PCR conditions and the 5 specific primers described in 4.6 and isolated from a gel using the Ultraclean kit (see 7 materials and methods).
After the DNA was isolated, 25 ng of template DNA was denatured at 95°C for 5 minutes. At room temperature 10 ul
10 labeling buffer, 5 ul reaction buffer, 2 pi enzyme and
distilled water to a total volume of 50 ul was added. The tube was well mixed and pulse spun in a micro-centrifuge to bring the content to the bottom of the tube. The radio-labeled dNTP, 5 ul [a-32P] dCTP activity 3000 Ci/mmol was
15 added and mixed by pippetting The tube was incubated at 37°C for 10 minutes, then 5 ul 0.2 M EDTA was added to stop the reaction.
The NICK® Spin Column was used to remove the unincorporated 20 32P-nucleotides. The column was inverted several times to re-suspend the gel the caps were removed to drain the column. The column was rinsed with 2 mL water and allowed to drain. The column was placed into a centrifuge tube and centrifuged (500 x g, 4 minutes at room temperature), the 25 contents of the tube was discarded. A 0.6 mL PCR tube was placed at the bottom of the centrifuge tube, ensuring the tip of the column is placed inside the PCR tube. The nick-translated sample was applied to the gel surface inside the column, and eluted by centrifugation (500 x g, 3 0 4 minutes at room temperature). The column was discarded
and the PCR tube containing the purified probe was removed and stored at -20°C until required for hybridization.
63 20. Hybridisation Analysis of Southern Blot
The nylon membranes were wet with 6x SSC. The membranes were rolled up and placed into a hybridization tube 5 containing 40 mL aqueous pre-hybridisation/hybridisation (APH) solution. The tube was placed into a hybridization oven (Mini Oven MKII) and incubated with rotation for 3 hours at 68°C. To prepare for hybridisation, the probe was denatured in a 100°C water bath for 10 minutes and 10 then place on ice.
After pre-hybridisation the APH solution was removed from the hybridisation tube and replaced with the same volume of APH solution pre-warmed to 68°C. The denatured probe 15 was added and the tube was place back into the
hybridisation oven and incubated with rotation at 68°C overnight.
After hybridization the APH solution was removed and 20 disposed of into radioactive waste container. The
membranes were then subjected to a series of washes at increasing levels of stringency. First 100 mL of 2x SSC/0.1% SDS solution was added to the tube and incubated with rotation at room temperature for 25 10 minutes, the solution was replaced and incubated a further 10 minutes.
This solution was removed and replaced with 100 mL 0.2x SSC/0.1% SDS and incubated with rotation for 10 minutes. The solution was changed, repeating the 3 0 incubation. For a moderate stringency wash the solution was replaced with 100 mL 0.2x SSC/0.1% SDS solution pre-warmed to 42°C, and a further two washes were repeated as described.
The radioactivity level was monitored with a gieger counter throughout the washing process. If the radioactivity was still high a final high stringency wash 5 was performed. The membranes were removed from the tube and placed into a glass-baking dish and covered with O.lx SSC/0.1% SDS solution pre-warmed to 68°C. The container was placed into a 68°C water-bath with shaker and incubated for 15 minutes. The final wash solution was 10 removed and the membranes were rinsed with 2x SSC and wrapped in plastic wrap.
In the darkroom, the membranes were placed into a cassette with a piece of autoradiograph film placed on top. The 15 cassette was closed and placed into a black plastic bag and stored at -80°C for two nights.
The cassette was removed from the freezer and allowed to return to room temperature. The autoradiograph film was
2 0 developed in the dark room by submersing the film in to developing fluid for 5 minutes. The film was rinsed in water then submersed in fixative solution for 5 minutes. The film was rinsed in running water for 5 minutes then left to dry.
Sequence Listing SEQUENCE LISTING
LA TROBE UNIVERSITY
GRAINS RESEARCH AND DEVELOPMENT CORPORATION
ANTHER SPECIFIC PROMOTERS AND USES THEREOF
Patentln version 3.3
gcacaagctt gcttataagc tactctttgc caacaaattc gcaacaatga tttctagaac 60
tataatcagt tgatggggga agaaaatgtt gaaagttgta caatgaatca agttaaagtt 120 aaaatacttt tttcccgatt ctctgcaggt tacatatatg tgtatataca cagtatgtga 180 atgatcaaaa agaagattta atatatctaa gcatgcagac aaaaacctat tgctaaaaag 240 atttctaaga accggagacc gtttaccaaa caaaatatga agttgaattc atcccatttg 300 tcactcgatt agacaagatt cgtcgaacga agatatctat taacgatcta ctaactttag 360 ttaaatcgtg acaaaacaca catcattcat atttgatagt gaataagtcg gtggtccatc 420 gttttaactt tgattgatat ccttaaaatt gatgcatagc tttaaacaac caatactttc 480 ttatggattg tttttcttcc aacttctcta agggttttat tttagaaaat tgattataag 540 tattaaatga aatctaagag aaaaaaaaaa aaaaaaagag gagaaagatg agaagttccc 600 atgcctttag attcggatta cgtgtgtcac tctttttata gctttaacgc gatggtcgct 660
Sequence Listing caacgtgaac gacattgtcc cactaagaaa aataatgatc atttcatgtg tattttttct 720
ttatcaaatt tttaaattat atatacatat ctaactttga taacaacaac aagaatctgt 780 aatacaatta tacaacggca cgcaaacagc agaattagta gatattcttt aaagcaaatt 840 taccatattt gtaacatttc tattagtatg atatgataca aaagtttgga acatgatttg 900 atagaagcta acgtcaattc catttcttta ataaatggta aaaggtatat aaacagagta 960 ttagtcctca aaaacattgt aaacatattg ttttaaaaca atttaccagt atatattgac 1020 aatagtttaa ctgaattgac gtgcaagtca atattattac cttattaggg ggcgttattg 1080 gttcttaatt tacaaggaat ttagatgatt tcaatcacat tctataaagt attttaaagt 1140 attgttagag agttttttat aatcttgttg attagttttt cataattttg taaagttttt 1200 caaacaatct ctctatttta ataatacttt tcatgacttt ccatgacttt attttgtgaa 1260 gaaaaatgta aaaagtcatg aaccaataac ataataattg aaatcattaa caatgagaaa 1320 tttttttgtt ttaattgaat aacacaaaac ttttaatgac ttgagtatga atccaataac 1380 ccaaaattta tgcagatttt agaatacttc ttataaatct taaatgaata acacaaaact 1440 ttaacatact tttaacaaat cttgattgaa taacaacaga ttctacatga cattttaaat 1500 cactaaaact cttttgaaat cataaaccaa taacaacccc ttagtttttt actatttgaa 1560 ttctgacgta cttttttatt agttgaattt ctataaatga gaaaacatta attatttctt 1620 aatctttgaa cttaagcccc acaaaaatct tataaattgg gacagatgga ctagataaca 1680 agcgtttcac ctactccaaa atttccctat aagtaactct ttttgtaacc tccttttctt 1740 cccaaaccat cactcctttt gcattgtgtg aaaccttcga gttttctctt catcttctca 1800 aagtaacaaa ctttctccaa acagattatt attaaaacaa tctcatcaag aactacgatg 1860
Sequence Listing 2 84 PRT Arabidopsis thaliana
Met Lys Phe Pro Ala Val Lys Val Leu lie lie Ser Leu Leu He Thr
15 10 15
Ser Ser Leu Phe He Leu Ser Thr Ala Asp Ser Ser Cys Asn Val Arg
20 25 30
Cys Ser Lys Ala Gly Arg Gin Asp Arg Cys Leu Lys Tyr Cys Asn He
35 40 45
Cys Cys Glu Lys Cys Asn Tyr Cys Val Pro Ser Gly Thr Tyr Gly Asn
50 55 60
Lys Asp Glu Cys Pro Cys Tyr Arg Asp Met Lys Asn Ser Lys Gly Thr
65 70 75 80
Ser Lys Cys Pro
lie His Val Leu Ala Leu Leu Leu Leu He Phe Ala Ser Thr Lys He
15 10 15
His His Ala Gin Gly Lys Ser He Thr Gly Pro Cys Val Val Ala Cys
20 25 30
Ser Lys Lys Thr lie Ala Cys Val Val Arg Cys Arg Phe Ala Thr Asp
35 40 45
Lys Cys Ser Gin Asp Cys Ala He Asp Ser lie His Cys Val Ser Ser
50 55 60
Cys Leu Leu Gin Asn Ser Ser Ser Pro Pro
4 26 DNA Synthetic
atgaaattcc cggctgtaaa agttct 26
5 25 DNA Synthetic
agaaacaaaa ggtattcacg gactt 25
6 31 DNA Synthetic
gcacaagctt gcttataagc tactctttgc c 31
7 32 DNA Synthetic
ttccggatcc gaacttttac agccgggaat tt 32
8 28 DNA Synthetic
gcacaagctt gtatagagta aatgagca 28
9 32 DNA Synthetic
ttccggatcc ggttgagagt atgaacaaag aa 32
ttgagagctc gtaggaacag agcac 25
Sequence Listing DNA Synthetic
cttgagctcg aagaaatggg tcggattcca tgtt 34
atgaagaagc ttcgcaccac cactgccacc accactctcg ctctcattct cctcctcgtc 60
ctcatagcag ccacgtccct ccgtgtcgcc atggctggat cagcgttctg cgacagcaag 120
tgcggggtga ggtgctccaa gacgggccgg cacgacgact gcctcaagta ctgcgggata 180
tgctgcgccg agtgcaactg cgtgccgtcg gggacagccg gcaacaagga cgagtgcccc 240
tgctaccgcg acaagaccac cggccacggc gcgcgcacga ggcccaagtg cccatgatcc 300
Met Lys Lys Leu Arg Thr Thr Thr Ala Thr Thr Thr Leu Ala Leu He
15 10 15
Leu Leu Leu Val Leu He Ala Ala Thr Ser Leu Arg Val Ala Met Ala
20 25 30
Gly Ser Ala Phe Cys Asp Ser Lys Cys Gly Val Arg Cys Ser Lys Thr
35 40 45
Gly Arg His Asp Asp Cys Leu Lys Tyr Cys Gly He Cys Cys Ala Glu
50 55 60
Cys Asn Cys Val Pro Ser Gly Thr Ala Gly Asn Lys Asp Glu Cys Pro
65 70 75 80
Cys Tyr Arg Asp Lys Thr Thr Gly His Gly Ala Arg Thr Arg Pro Lys
85 90 95
14 23 DNA Synthetic
tgcaactgcg tgccgtcggg gac 23
15 24 DNA Synthetic
aacaaggacg agtgcccctg ctac 24
1. An isolated anther specific promoter nucleic acid molecule comprising:
(a) a nucleotide sequence shown as SEQ ID NO: 1;
(b) a nucleotide sequence comprising the promoter
of a gene encoding a polypeptide shown as SEQ
ID NO: 2;
(c) a homologue or orthologue of a nucleotide
sequence of (a) and having at least 50%
sequence identity with the nucleotide sequence
(d) a homologue or orthologue of the nucleotide
sequence of (b) and having at least 50%
sequence identity with the nucleotide sequence
encoding the polypeptide shown as SEQ ID NO:
(e) a nucleotide sequence complementary to the
nucleotide sequence of (a), (b), (c) or (d);
(f) a nucleotide sequence capable of hybridising
to a nucleotide sequence of (a), (b), (c) or
(d) under conditions of high stringency.
2 . An isolated anther specific promoter nucleic acid molecule comprising:
(a) a fragment of a nucleotide sequence of claim
(b) a derivative of a nucleotide sequence of claim
1, wherein the fragment or derivative is
capable of directing expression of a
heterologous nucleic acid to which it is
operably linked to anther and/or pollen tissue
of a plant transformed with the nucleic acid molecule.
3. An expression cassette comprising an anther specific
promoter nucleic acid molecule according to claim 1 or
claim 2 and a site for inserting a heterologous nucleic
acid molecule, such that the heterologous nucleic acid is
operably linked to the promoter and is specifically
expressed in anther and/or pollen tissue of a plant
transformed with the nucleic acid molecule.
4. A recombinant plasmid comprising an anther specific
promoter nucleic acid molecule according to claim 1 or
claim 2 and a heterologous nucleic acid operably linked to
5. A plant cell or cell line transformed with the
nucleic acid molecules according to claim 1 or claim 2,
the expression cassette according to claim 3 or
recombinant plasmid according to claim 4.
6. A transgenic plant generated from the transformed
cell according to claim 5.
7. Propagation material of the transgenic plant
according to claim 6.
8. Propagation material of claim 7, including one or
more of a fruit, seed, tuber, root-stock, seedling, and a
9. A method for introducing into a plant a heterologous
nucleic acid molecule which is to be specifically
expressed in anthers and/or pollen, the method comprising the steps of:
(a) transforming a plant cell with the nucleic
acid molecules according to claim 1 or claim
2, the expression cassette according to claim
3 or recombinant plasmid according to claim 4,-
(b) generating the plant from the transformed
10. A method of specifically expressing a heterologous
nucleic acid molecule in anther and/or pollen of a plant,
the method comprising the steps of:
(a) transforming a plant cell with the nucleic
acid molecules according to claim 1 or claim
2, the expression cassette according to claim
3 or recombinant plasmid according to claim 4;
(b) generating the plant from the plant cell.
11. Use of the nucleic acid molecules according to claim
1 or claim 2, the expression cassette according to claim 3
or recombinant plasmid according to claim 4 for
specifically expressing a heterologous nucleic acid
molecule in anther and/or pollen of a plant.
12. An anther specific nucleic acid molecule according to
claim 2, expression cassette according to claim 3,
recombinant plasmid according to claim 4, method according
to claim 9 or claim 10, or use according to claim 11,
wherein the heterologous nucleic acid molecule inhibits
the formation of anthers and/or pollen, and/or imparts
resistance to environmental stress.
13. An anther specific nucleic acid molecule, expression
cassette, recombinant plasmid, method or use according
to claim 12, wherein the environmental stress is one or
more selected from the group consisting of temperature
extreme, salinity, pests, and infection.
14. An anther specific nucleic acid molecule, expression
cassette, recombinant plasmid, method or its use
substantially described herewith foregoing description,
examples and diagrams.
Dated this 09th day of January 2007.
The present invention provides as isolated anther specific promoter nucleic acid molecule homologous to the Ta39 promoter of tobacco. Also contemplated are used of the nucleic acid molecule to direct expression of a heterologous nucleic acid molecule to anther and/or pollen of a plant.
|Indian Patent Application Number||164/KOLNP/2007|
|PG Journal Number||43/2011|
|Date of Filing||15-Jan-2007|
|Name of Patentee||LA TROBE UNIVERSITY|
|Applicant Address||PLENTY ROAD, BUNDOORA, VICTORIA 3083|
|PCT International Classification Number||C12N15/29|
|PCT International Application Number||PCT/AU2005/000853|
|PCT International Filing date||2005-06-15|