|Title of Invention||
A PROCESS OF ISOLATING ADENYLATE KINASE NUCLEOTIDE SEQUENCE TO FOR ALLEVIATION OF ENVIRONMENTAL STRESS AND USES THEREOF
|Abstract||The present invention relates to the isolation of the Glucose 1 phosphate adenylate transferase gene ("AGP") from the RASI variety of rice seedlings under environmental stress conditions. The invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for salt stress inducible proteins, salt associated proteins, homologes associated with saline stress in plants and the resulting alteration of plant tolerance to environmental stress as well as osmotic stress like salt stress, a change in the plant recovery on exposure to environmental stress with its changed metabolism.|
possessing a low rate of crop rotation, it is desirable to additionally use plants other than sugar beet in the production of ologosaccharides.
An increase in the concentration of protein in seeds and tubers of a plant would be desirable if plant products of improved quality were obtained, like protein-enriched food or cattle fodder.
Soil salinity is one of the major causes of environmental stress and acts as a severe constraint on crop production, both, in dryland and irrigated areas. Over 10% of the worlds' arable land and 23% of the cultivated land comprises of various kinds of salt affected soils. It is further expected that the situation will further worsen on account of the cumulated soil built up especially in cultivated tracts of land due to irrigation.
Although nucleic acid sequences inducing salt inducible and salt responsive proteins have been isolated for certain species of plants, there still remains a need to improve plant tolerance to environmental stress. The objective of the present invention is to overcome or alleviate these deficiencies.
The individual or simultaneous enhancement or manipulation of the activities of the genes in plants may enhance or otherwise alter plant tolerance to environmental stress, may increase or otherwise modify the plant capacity to tolerate salt shocks, by increasing the spectrum of abiotic stress tolerance, by reducing plant damage caused by environmental stress like salinity, sodicity, dehydration and water deficient conditions, and cold.
ADP-glucose pyrophosphorylase (AGP) catalyses the conversion of ATP and, alpha.-glucose-1-phosphate to ADP-glucose and pyrophosphate. ADP-glucose is used as a glycosyl donor in starch biosynthesis by plants and in glycogen biosynthesis by bacteria. The importance of ADP-glucose pyrophosphorylase as a key enzyme in the regulation of starch biosynthesis was noted in the study of starch deficient mutants of maize (Zea mays) endosperm (Tsai and Nelson, 1966; Dickinson and Preiss, 1969). AGP enzymes have been isolated from both, bacteria and plants. Bacterial AGP consists of a heterotetramer, while plant AGP from photosynthetic and non-photosynthetic tissues is a heterotetramer composed of two different subunits. The plant enzyme us encoded by two different genes, with one subunit being larger than the other. This feature has been noted in a number of plants.
The cloning and characterisation of the genes encoding the AGP enzyme subunits have been reported for various plants. These include Sh2 cDNA (Bhave et al., 1990), Sh2 genomic DNA (Shaw and Hannah, 1992), and Bt2 cDNA (Bae et al., 1990) from maize; small subunit cDNA (Anderson et al., 1989) and genomic DNA (Anderson et al., 1991) from rice; and small and large subunit cDNAs from spinach leaf (Morell et al., 1987) and potato tuber (MuUer-Rober et al., 1990; Nakata et al., 1991). In addition, cDNA clones have been isolated from wheat endosperm and leaf tissue (Olive et al., 1989) mid Arabidopsis thaliana leaf (Lin et al., 1988).
This invention relates to the isolation of Glucose 1 phosphate adenylate transferase gene from the RASI variety of rice seedlings under environmental stress conditions. The polynucleotides of the present invention comprise of a Glucose 1 phosphate adenylate transferase encoding gene.
The nucleic acid or nucleic acid fragment may be of any suitable type and includes DNA such as cDNA or genomic DNA and RNA such as mRNA, which is single or double stranded, with the option of synthetic, non-natural or altered nucleotide bases and combinations thereof.
Isolated means and refers to the material that is removed from its natural environment. A naturally occurring nucleic acid present in a plant is not isolated but the same nucleic acid when separated from some or all of the coexisting materials in the natural system is isolated. Such nucleic acids could be part of a vector and/or composition and still be isolated in that such vector or composition is not part of its natural environment.
"Functionally active" in relation to nucleic acid means and signifies that the fragment or variant such as the analogue, derivative or mutant, encodes a polypeptide which can alter the plant tolerance to environmental stress thereby enhancing the capability of the plant to withstand plant shocks, modify the plant recovery after exposure to salt stress and amend the plant metabolism under salt stress. Such variants include naturally occurring allelic variants and non naturally occurring variants. Additions, deletions, substitutions and derivzatizations of one or more of the nucleotides are contemplated as long as the alterations do not result in loss of functional activity of the variant or fragment.
The nucleic acid or the nucleic acid fragments have been isolated. The nucleic acid or nucleic acid fragments may be used to isolate cDNAs and genes encoding homologues proteins from the same or other plant species.
The cDNAs or genomic DNAs may be isolated directly by using all or a portion of the nucleic acid or nucleic acid fragments of the present invention as hybridization probes to screen libraries from the desired plant. Specific oligonucleotide probes based upon the nucleic acid sequence of the present invention may be designed and synthesized. The entire sequence may be used directiy to synthesise the DNA probes by known methods like random primer DNA labeling, end labeling techniques or RNA probes making use of the available in vitro transcription systems and specific primers may be designed to amplify a part or all of the sequences of the present invention. The resulting amplification products may be labeled directly during or after amplification reactions and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency. Further short segments of the nucleic acid or nucleic acid fragments of the present invention may be used in amplification protocols to amplify a longer nucleic acid or its fragment encoding homologus genes from DNA or RNA. The polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid sequence of the present invention and the sequence of the other primer takes advantage of the presence of polyadeneyllic acid traits to the 3' end of the mRNA
precursor encoding plant genes. In the alternative, the second primer sequence may be based upon the sequence derived from the cloning vector, following the RACE protocol (Frohman et al. 1998).
In a further quintessence of this invention, there is provided a polypeptide recombinantly provided from a nucleic acid or its fragment.
Availability of the nucleotide sequence and deduced amino acid sequence facilitates immunological screening of the cDNA expression libraries. Synthetic peptides representing portions of the amino acid sequence may be synthesized. These peptides may be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins including the amino acid sequence. The antibodies may be used to screen the cDNA expression libraries to isolate full length cDNA clones of interest.
A genotype is the genetic constitution of an individual or group and variations in the genotype are important for commercial breeding programs to determine the parentage, in diagnostics and fingerprinting. A genetic marker identifies a specific locus or region in the genome. The more the genetic markers, the finer defined is the genotype. A genetic marker is significant when it is allelic between organisms and when based on nucleic acid sequence information, it can clearly establish the genotype of an individual and when the function encoded by such nucleic acid is known, such nucleotide sequence information including single nucleotide polymorphisms (SNPs), variations in single nucleotides between allelic forms of such nucleotide sequence may be used as perfect markers or candidate genes for the given trait. In this invention there is provided a substantially purified isolated nucleic acid or its fragment including a SNP from the nucleic acid or its fragment, which nucleic acid library may be of any suitable type and is preferably a cDNA library. The nucleic acid or its fragment may be isolated from a recombinant plasmid or amplified using polymerase chain reaction. In this invention there is provided, use of the nucleic acid or its fragment including SNPs and/or nucleotide sequence information, as molecular genetic markers.
The nucleic acid or its fragments and/or nucleotide sequence information may be used as a molecular genetic marker for a quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in the RASI variety of indica rice. The nucleic acid and/ or its fragment and/or the nucleotide information thereof may be used as molecular genetic markers in plant enhancement of withstanding plant tolerance to environmental and osmotic stress, the plant capacity to survive salt shocks, the plant recovery after exposure to salt stress, namely, tagging QTLs for tolerance to salinity, tolerance to drought etc. The sequence information revealing SNPs in allele variants of the nucleic acid or its fragments and/or nucleotide information may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in the RASI variety of indica rice.
In the present invention there is also included a construct including a nucleic acid or its fragment. The term construct refers to an artificially assembled isolated nucleic acid molecule which includes the gene of interest. A construct may also include a marker gene which includes the gene of interest and appropriate regulatory sequence.
In the present invention there is also provided a vector including a nucleic acid or its fragment. The term vector covers both, cloning and expression vectors. Vectors are mostly recombinant molecules containing nucleic acid molecules from various sources. In this invention, the vector may include a regulatory element like the promoter, a nucleic acid or its fragment and a terminator with the nucleic acid or its fragment being operatively linked with the terminator. Operatively lined indicates that the said regulatory element is capable of causing expression of the nucleic acid or its fragment in the plant cell with the regulatory element being upstream and terminator being downstream. The vector may be viral or non-viral and may be an expression vector. The vector may be an expression vector and include chromosomal, non chromosomal and synthetic nucleic acid sequences
A variety of promoters may be employed for the vectors and factors including the choice of the promoter include the tissue specificity of the vector, whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed.
The constructs and vectors of the present invention may be incorporated in a variety of plants including monocotyledons, dicotyledons and gymnosperms. The choice of the technique for incorporating the constructs and vectors of the present invention into plant cells will depend to a large extent on the type of plant to be transformed.
The present invention confers plant tolerance to environmental and osmotic stress, plant capacity to survive salt shocks, plant recovery after exposure to salt stress and alteration of plant metabolism under salt stress.
Documents cited are for reference purposes only and their inclusion is not an acknowledgment that they form part of the common general knowledge in the relevant art.
1. A nucleic acid sequence and/or a fragment of the same thereof encoding a salt inducible or salt responsive protein selected from Glucose 1 Phosphate Adenylate Transferase from the RASI variety of Indica rice or a functionally active fragment or variant thereof and use of the said nucleic acid and/or fragment thereof and/or nucleotide sequence information thereof and/or single nucleotide polymorphism thereof as a molecular genetic marker and applicable to all crops.
2. A claim as in claim 1, wherein the nucleic acid or its fragment thereof includes a salt like protein.
3. A construct including a nucleic acid or nucleic acid fragment according to claim 1.
4. A vector including a nucleic acid or nucleic acid fragment according to claim 1.
5. A vector according to claim 5 including a promoter, with the said promoter, nucleic acid or nucleic acid fragment being operatively linked.
6. A plant, plant cell, plant seed or other plant part including a construct according to claim 3.
7. A plant, plant cell, plant seed or other plant part including a vector according to claims 4 and 5.
8. A plant, plant cell, plant seed or other plant part according to claims 6 and 7.
9. A method of modifying plant tolerance to environmental and/or osmotic stress.
10. A method according to Claim 9 wherein the said environmental and/or osmotic stress includes salt stress.
11. A method of modifying the plant capacity to withstand the salt shock, according to claim 1.
12. A method of modifying plant recovery after exposure to salt stress according to claim 1.
13. A method of modifying plant metabolism to salt stress according to claim 1.
14. Use of nucleic acid or its fragment thereof and/or nucleotide sequence information thereof and/or single nucleotide polymorphisms thereof as a molecular genetic marker, according to claim 1.
15. A substantially isolated or purified polypeptide and functionally active fragments and variants thereof.
16. A claim as in claim 15 wherein the polypeptide includes an amino acid and/or its sequence thereof and its functionally active fragments and variants thereof.
17. A claim as in claim 1 wherein the invention can be applied to all crops.
The Controller of Patents AVPSTHA GENGRAINE TECHNOLOGIES PVT. LTD.
The Patent Office . DISCOVERER " 9th FLOOR
Chennai INTERNATIONAL TECHNOLOGY PARK
WHITEFIELD ROAD BANGALORE-560 066
|Indian Patent Application Number||772/CHE/2003|
|PG Journal Number||30/2010|
|Date of Filing||24-Sep-2003|
|Name of Patentee||AVESTHA GENGRAINE TECHNOLOGIES PVT LTD|
|Applicant Address||'DISCOVERER' 9TH FLOOR INTERNATIONAL TECH PARK WHITEFIELD ROAD BANGALORE 560 066|
|PCT International Classification Number||A01H5/00|
|PCT International Application Number||N/A|
|PCT International Filing date|