Title of Invention

METHOD FOR MAKING BENTAZON AND SULFONYLUREA HERBICIDE RESISTANT AND SENSITIVE TRANSGENIC PLANTS

Abstract The present invention provides a kind of rice endogenous bentazon and sulfonylurea herbicide resistant gene (Cyp81A6 gene), and its functional conservative variants, the biological activity subfragments or derivatives with the same function. It also provides a kind of method to prevent the selfing mixtures during hybrid seed production. It also provides the novel means of the directional genetic manipulation and the improvement of biological traits.
Full Text Form 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)
1. TITLE OF THE INVENTION:-


















2. APPLICANTS:-

"A BENTAZON
AND
SULFONYLUREA
HERBICIDE-RESISTANT GENE CYP81A6 OF RICE"



1. Name
Nationality Address
2. Name
Nationality Address

ZHEJIANG UNIVERSITY
Chinese (CN)
268 Kaixuan Road, Hangzhou, Zhejiang Province, 310029,
P.R.China (CN)
WUHAN FORTUNE SCIENCE AND
TECHNOLOGY CO., LTD.
Chinese (CN)
T 1 Nanhu-Yaoyuan, Hongshan District, Wuhan City,
Hubei Province, 430064, P.R.China (CN)

3. PREAMBLE OF THE DESCRIPTION:- The following specification
particularly describes the invention and the manner in which it is to be performed.

The rice bentazon and sulfonylurea herbicide resistant gene Cyp81A6
TECHNICAL FIELD
The present invention belongs to the technical field of genetic engineering, more particularly, it relates to mapping, isolation, and cloning of a novel rice gene that is resistant to bentazon and sulfonylurea, the two different kinds of herbicides used in paddy field. In addition, the present invention also provides uses of this new gene that is resistant to bentazon and sulfonylurea in improving some important agronomical character of various crops including rice, preventing selfing mixtures during the hybrid seed production. The present invention also relates to conduction of other locus/site-directed genetic manipulation based on use of the herbicide resistant gene of present invention.
TECHNICAL BACKGROUND
By utilization of heterosis of rice, China succeeded in developing of hybrid rice and this made the rice production increased substantially in total. Currently, China is large scaly exploring two-line system hybrid rice after success in utilization of the three-line heterosis based on nucleo-cytoplasmic interaction male sterility. The hybrid seeds of the two-line system are produced by photo- and thermo- sensitive genie male sterile line. But the sterility of this male sterile line is easily affected by environmental temperature, especially the midsummer low temperature that may cause its fertility restored. This thus results in a potential risk: when seed production of two-line system meets low temperature, the harvest seeds may comprise of false hybrid seeds (selfing seeds from male sterile line) mixed in the real hybrid seeds. Once presence of this mixture of the hybrid seeds with selfings from female parent and fail to eliminate them, it will causes a great loss to seed or field production. Guangxi in 1989 and Hunan in 1999 suffered such big loss right because of these.
The existing data proved that the seed purity declined 1 percent and yield of hybrid rice would reduce 75 kg per hectare. This is why the seed standard published by Chinese Ministry of Agriculture stipulates that the purity of hybrid seeds has to stand over 98%. Not only the photo- and thermo-sensitive male sterile line produced the two-line hybrid seeds have the selfing contamination problems, but so do the hybrid seeds when produced by incomplete male sterile lines, such as their sterility


governed by nucleic major gene/s that have modifier gene/s participated in, or resulted from artificial chemical emasculation, or from environment sensitive nucleo- cytoplasmic interaction, or from artificially developed aneuploid. Many attempts have been made to eliminate such selfing mixture problems and thus to have the crop heterosis utilization establish on a more reliable foundation. However, in view of the characteristic of herbicide tolerant/ resistant being widely used in the modern crop breeding program, not a few of scientists also attempt to utilize the wild-type rice that have the resistance to both bentazon and sulfonylurea to solve the above mentioned selfing mixture problems during the hybrid seed production.
There are two main categories of selective herbicides applied to rice. The former one is a benzothiadiazinone contact herbicide, such as bentazon, and its effective component can be absorbed through roots and leaves of crop. It kills the overwhelming dicot plants and sedges in most gramineous species excluding leguminous while it is harmless to rice. The herbicide mechanism of this herbicide is to inhibit Hill reaction in Photosynthesis. But the endogenous gene that is resistant to such herbicide of bentazon has not yet been cloned from plants so far. The later one is sulfonylurea-like herbicides explored by DuPont Company, which represent a new category of super effective herbicides characterized in high selectivity, broad spectrum, low poisons, and interior absorption. Among which, the tribenuron-ethyl, and bensulfuron-methyl and their complex formula are the most widely used herbicides in paddy field in China at present. The most notable characteristic of the sulfonylurea herbicides is the high activity, which makes their on-use dosage usually within 5-100 gram per hectare. The sulfonylurea-like herbicides are the acetolactate synthase (ALS) inhibitors, which have special effect to many annul or perennial weeds, especially the broadleaf weeds and are already widely used to eliminate the weeds growing in the field of rice, wheat, soybean, corn, canola, and lawn and other non-cultivated land. The DuPont Company has explored several sulfonylurea-resistant genes. One of these genes is SURB-Hra cloned from a tobacco ALS mutant. The SURB-Hra expresses resistance is because the mutated ALS is insensitive to the sulfonylurea. This gene has been applied to various crops including cotton and soybean (US5013659 .US5084086 .US5141870 .US5378824 , US5605011); Another sulfonylurea resistant gene developed by DuPont Company is the P450 sul gene isolated from soil bacteria. The mode of action of this gene is to


accelerate the metabolism of sulfonylurea to non-toxic. DuPont has made great efforts on the studies of this P450 gene and its application (see patent US5349127 for relevant information). In a patent (WO9708327) document, Japanese Nissan Chemical Corporation also publicized an ALS gene that was isolated from the cDNA of Kochia coparia, a kind of dicotyledonous broadleaf plant, having the function to make the transgenic plant resistant to sulfonylurea.
At present, two major approaches are applied to develop herbicide tolerant or resistant crops: the first one is by use of traditional physical/chemical mutagenesis to obtain the crop mutant capable to resistant or tolerant to herbicides; the second one is through recombinant DNA technology to introduce herbicide tolerant or resistant gene/s into the existing species to create the new materials tolerant or resistant to herbicides. Among which, the latter approach is the most widely used method. Currently, to enhance the crop herbicide tolerance or resistant, there are also two strategies involving in use of this recombinant DNA technology : first is to modify the herbicide target protein and make it insensitive to herbicide or over expressed to let plant still capable to normally metabolize the herbicide after absorption; second is to introduce in an new enzyme or enzyme system, such as P450 monooxygenase (Wang Guanlin and Fang Hongjun, 1998), to degrade or detoxify the absorbed herbicide before it functions.
Wild-type rice is naturally resistant to bentazon and sulfonylurea herbicides. Mori, a Japanese scholar and Zhang Jiwen et al from Hubei Academy of Agricultural Sciences made two recessive bentazon-sensitive-lethal mutants Norin 8m (Mori, 1984) and 8077S (Zhang Jiwen and Wu Xiaozhi, 1999) using y-ray radiation to treat Norin 8 and W6154, respectively. Based on these, Zhang et al (2001) further developed a selfing seed removal technology system to ensure the hybrid seed purity by use of the recessive mutant locus to tag the thermo-sensitive male sterile line. Since such a germplasm source plays an important role in the seed purity security and ensuring system of the two-line hybrid rice, e.g. use of on-8077S mutant locus tagged two-line's male sterile line could largely reduces the risk of seed production of the two-line hybrid rice, it is thus highly recognized by rice breeders and seed enterprises. However, since this hybrid seed purity-ensuring system functions only after seed-harvesting, even though its effect is not bad, it is hardly approved by seed administration department because what the technology


ensured is the non-purified seeds and these are not according with the government-published seed purity standard before sale. Therefore, there is a need for creating a new mechanism to remove mixtures and to ensure the purity. However, the problem is that the gene controlling this trait has not yet been cloned for many years. It thus makes people have no way to conduct manipulation and further utilization of this trait, which has already become a major technical obstacle in this field.
Based on above situations, the present inventor used two existing bentazon sensitive lethal mutants as materials to conduct fine mapping of their mutant loci and finally cloned their common wild-type alleles through deepgoing investigation. Further on the base of this, the inventors developed several useful methods and techniques. These include a method for development of chemically supplemented emasculation and thermo-sensitive male sterile line, a genetic manipulation technique of double- or multi-sites targeted co- modification, a new approach for investigating biological function of plant genes, and a new technology for genetic improvement of plant traits. Therefore, this invention not only solves the selfing mixture problems during the hybrid seed production, but also provides the useful means with broad application perspective for investigating biological functions of genes and improving the biological traits genetically.
DETAILED DESCRIPTION OF THE INVENTION
An object of the present invention is to provide an already-isolated rice endogenous gene Cyp81A6, which is resistant to both bentazon and sulfonylurea herbicides (hereinafter referred to as Cyp81A6), and its functionally conservative variants, biologically active sub-fragments or derivatives with equal functions.
Another object of the present invention is to provide the cDNA sequence of the bentazon and sulfonylurea herbicide resistant gene, and its functionally conservative variants, biologically active sub- fragments or derivatives with equal functions.
Yet another object of the present invention is to provide a gene which is bentazon and sulfonylurea herbicide sensitive.
A further object of the present invention is to provide a recombinant vector which comprises Cyp81A6, its bentazon and sulfonylurea herbicide sensitive allele, or something with equal functions to both.
A still further object of the present invention is to provide polypeptides


encoded by Cyp81A6, bentazon and sulfonylurea herbicide sensitive allele or something with equal functions to both.
Another object of the present invention is to provide genetically engineered cells that comprise Cyp81A6, bentazon and sulfonylurea herbicide sensitive allele or something with equal functions to both, or comprise the polypeptide encoded by Cyp81A6, or something with equal functions to both.
Yet a further object of the present invention is to provide a method which prevents the selfing mixtures during hybrid seed production.
Still another object of the present invention is to provide a new method for genetic manipulation directionally.
One more object of the present invention is to provide a new method for improving plant traits.
These and other objects and features of the invention will become more fully apparent when the following detailed descriptions are read in conjunction with the accompany drawings.
First of all, the present inventor conducted fine mapping of the bentazon and sulfonylurea herbicide sensitive lethal mutant loci identified in both 8077S and Norin 8m, and then isolated its resistant allele Cyp81A6 and the DNA fragment of promoter that regulate this gene from wild-type indica rice. This gene can be utilized to improve the characteristic of the resistance to bentazon and sulfonylurea herbicides for most of the soybean-excluded dicotylandon and cyperaceae weed plants.
The bentazon and sulfonylurea herbicide resistant gene provided by the present invention comprises a kind of nucleotide sequence selected from the following groups consist of:
(1) a nucleotide sequence having the nucleotide sequence shown in SEQ ID
NO.l;
(2) The nucleotide fragments or derivatives thereof, which have the equal functions as the nucleotide sequences of position 1949 through 4216 of SEQ ID NO.l;
(3) a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO.2;
(4) The nucleotide fragments or derivatives thereof, which have the equal


functions as the nucleotide sequences of position 54 through 1595 of SEQ ID NO.2;
(5) a nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO.2 under the stringent conditions.
Preferably, the said isolated rice endogenous bentazon and sulfonylurea herbicide resistant gene comprises the nucleotide sequence as shown in SEQ ID NO.l; the cDNA of the said isolated rice endogenous bentazon and sulfonylurea herbicide resistant gene comprises the nucleotide sequence as shown in SEQ ID NO.2.
The present invention provided polypeptides encoded by the rice endogenous bentazon and sulfonylurea herbicide resistant gene comprise the polypeptide of the amino acid sequence encoded by one of the nucleotide sequences selected from the following groups consist of:
(1) a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO.l;
(2) a nucleotide fragments or derivatives thereof, which have the equal functions as the nucleotide sequences of position 1949 through 4216 of SEQ ID NO.l;
(3) a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO.2;
(4) a nucleotide fragments or derivatives thereof, which have the equal functions as the nucleotide sequence of position 54 through 1595 of SEQ ID NO.2;
(5) a nucleotide sequence which can hybridize with the nucleotide sequence as shown in SEQ ID NO.l or SEQ ID NO.2 under the stringent conditions;
The amino acid sequence comprises the amino acid sequence shown in SEQ ID NO.3 is preferred;
The present invention also provides a genetic manipulation method of co-modification of the target sequences simultaneously at double or multiple sites of genome in the living cells. The characteristics of this method take the nucleotide sequence that can be used as selectable marker after modification as the first modification target and take the key base of the target gene of living cells as additional modification target. Use co-introduction technology to introduce double or multiple RNA-DNA chimeric oligonucleotides-RCOs designated for targeted modification of different target nucleotide sequences into the recipient cells, so that


they can simultaneously modify or mutate both target sites as mentioned above. Then, further utilizes the modified phenotype of the above mentioned nucleotide sequence that can be used as selectable marker after modification to conduct indirect selection of genotype resulted from endogenous gene target after targeted modification or mutation.
In the above genetic manipulation methods, the nucleotide sequence which can be used as selectable marker after targeted modification includes but not limits to the mutated or non-mutated herbicide resistant/sensitive gene, antibiotics resistant gene, biological or chemiluminescence gene, enzyme gene etc. The technicians in this field may select them freely according to general knowledge. Among which, the herbicide resistant/sensitive gene is the prior choice, especially those possess the nucleotide sequence as shown in SEQ ID NO.l or SEQ ID NO.2 or its sub-fragments or derivatives which have the same function, or those have a deletion of the 2455th base C or the 4006th base G in the nucleotide sequence shown in SEQ ID NO.l, or those has a deletion of the 560th base C or the 1385th base G in the nucleotide sequence shown in SEQ ID NO.2.
Present research indicated that the isolated-bentazon and sulfonylurea
herbicide resistant gene encodes a cytochrome P450 protein, which has been
formerly designated as CYP81A6 ( http://
drnelson.utmen.edu/cytochromep450.html) according to the international standard classification and nomenclature system. This protein contains the conservative motifs present in common P450 proteins and its amino acid sequence is listed in SEQ ID NO. 3. At present, although the several herbicide resistant P450 genes has been cloned from plants, such as the CYP71A11 and CYP81B2 (by Yamada etc, 2000) in tobacco, CYP71A10 (by Siminszky etc, 1999) in soybean, CYP73A1 (by Pierrel etc, 1994) and CYP76B1 (by Didierjean etc, 2002) in Chinese sorghum, as well as the CYP71B1 (by Lamb etc, 1998) in hlaspi arvensae. But the similarities between these P450 genes and CYP81A6 gene are all less than 40%, and they all cannot degrade the bentazon and sulfonylurea herbicides. These results reveal that the CYP81A6 gene is a novel category of herbicide resistant gene. Therefore, this invention refers to use of this novel herbicide resistant gene that is operatedly linked either to itself promoter or to other constitutive or tissue specific promoter and development of novel herbicide resistant plant lines by introduction of it into the


bentazon and sulfonylurea sensitive species, those of broadleaf plants except leguminous or cyperaceae weeds.
As shown in the present invention, the expression of DNA fragment is in a constitutive manner. Therefore, be able to utilize the antisense RNA or RNAi of this gene in connecting with the anther specific promoter, such as Osg6B and RA39 etc, then by introduction of it into the thermo-sensitive male sterile lines of the rice (not limited to rice) to let this gene unable to express in the anthers, so that able to use the sulfonylurea herbicide to kill the pollens and create a new chemically supplemented emasculation and thermo-sensitive male sterile line.
Besides, the present invention identified two bentazon and sulfonylurea herbicide sensitive Cyp81A6 single-base deletion mutant sites provide modifiable targets for gene-targeted mutation. Therefore, be able to utilize the artificially designated RCOs molecules for targeted repair of Cyp81A6 mutant sites to recovery their resistant to bentazon and sulfonylurea herbicides and thus to meet with the selection purpose. With the aid of these two mutant genes, be able to design and introduce two or more kinds of RCO molecules aiming at different targets simultaneously. Among which, one molecule is used to directionally repair the single-base deletion site of Cyp81A6 gene and for resuming its resistance to bentazon and sulfonylurea herbicides for creating selectable marker; and another one or more are used to directionally mutate the target endogenous genes and then according to the phenotype or biochemitype differences presented between the mutants and wild controls deduce their exact biological functions or conduct pure line selection based on the mutated target genes to obtain the desired new varieties or lines with improved agronomic traits. Besides, it is also able to indirectly select the genotypes of the target genes based on the selectable modification phenotype of other gene locus or a co-introduced foreign selectable marker gene.
In addition, there is possibility for make use of the above technology or other DNA homologous recombination technology or physical or chemical mutagenesis technology to mutate the Cyp81A6 gene either directionally or randomly and thus to change its functions and create a new bentazon and sulfonylurea herbicide sensitive lethal mutant, which is capably used for selfing seed-removing and hybrid seed purity-ensuring. Besides, the cloned wild-type allele can endow most of the broadleaf plants except leguminosae or cyperaceae weeds with the character of


bentazon and sulfonylurea herbicide resistance after genetic transformation.
The Cyp81A6 coding sequence is widely preserved in plants of different species. Therefore, the primer and the probe at a length about 8 nucleotides or more copied from the rice Cyp8JA6 and its derivative sequence can be used for isolation and cloning of the homologous genes of other species in gramineae family. Use of the above method is able to clone the gene that has high similarities with rice Cyp81A6. Fuse this gene sequence to the appropriate plasmid vector and then introduce it into the genome of the plant sensitive to bentazon and sulfonylurea herbicides, so that be capable to generate transgenic lines with the resulted bentazon and sulfonylurea herbicide resistant.
The meanings of following terms used in this description and Claim are familiar to and frequently used by the skilled in the art. Demonstrative brief description to some terms is as follows.
The term "Nucleotide sequence" used herein refers to single-nucleotide, nucleotide and multi-nucleotide and their segments or parts, or even genome or synthesized DNA or RNA. They can be either single-chain or double-chain, representing the sense chain or antisense chain.
"Sub-fragments with equal functions", "biological active sub-segments with equal functions" refer to a part or sub-sequence of the isolated DNA fragments, hereinto, no matter whether these segments or sub-sequences encode functionally active proteins, they preserve the ability to change gene's expression pattern or generate certain herbicide resistance. For example, the above-mentioned fragments can be used for the designation of chimeric gene or antisense inhibition of native gene. "Derivatives with equal functions", "functionally conservative mutants" mean the entire, or more, or partial sequences of the isolated DNA fragments. Among which, no matter whether these fragments encode active protein, they all preserve the ability to change gene's expression pattern or generate certain herbicide resistance, and can be used for designing of chimeric gene or antisense inhibition of native gene.
"Mutant" used herein refers to a kind of amino acid sequences or polynucleotide sequences that possess one or few amino acid residue or nucleic acid base changes. The mentioned changes include the deletion, insertion or substitution etc of amino acid residue/s or nucleotide base(s in the amino acid sequence or
10

nucleotide sequence. The "mutant" mentioned in this invention possesses conservative change, in which the changed amino acid sequence has the structural or chemical property similar to the original amino acid sequence. The mutant of such polynucleotide can be generated either naturally or artificially. These nucleotide mutants include substitution, deletion and insertion etc. As known in this field, the allelic variant is a replacement form of polynucleotide, it can be the substitution, deletion and insertion of one or more nucleotides, but does not essentially change the functions of its encoded polypeptide.
The term "amino acid sequence" as used herein, refers to an oligopeptide, polypeptide, peptide or protein sequence, or a fragment of any of these. The polypeptide or protein as used herein, are not meant to limit them to the complete native amino acid sequence associated with the recited polypeptide or protein molecule.
"Homology" can be determined electronically, e. g., by using the MEGALIGN program (Lasergene software package, DNASTARTnc. Madison Wis. ). TheMEGALIGN program can create alignments between two or more sequences according to different methods, e. g., the cluster method. (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237- 244). Percent identity between nucleic acid sequences can also be calculated by the cluster method, or by other methods known in the art, such as the Jotun Hein method. (See, e. g., Hein, J. (1990) Methods in Enzymology 183: 626-645.)
The term "Stringent conditions" used herein refers to: (1) Molecular hybridization and strip/wash off under lower ionic strength and higher temperature, such as strip off under 0.2xSSC, 0.1% SDS, 60D, or (2) Add denaturant upon hybridization, such as 50%(v/v) formamide, 0.1% bovine serum/0.l%Ficoll, 42D etc; or (3) The identity between two sequences is at least 95% but the hybridization will take place only when the identity is over 97%. And, the polypeptide-encoding polynucleotide capable for hybridization has the same biological functions as the polypeptide-encoding nucleotide shown in SEQ ID NO.l.
"Vector" used in the present invention refers to the bacterial plasmids, bacteriophage, yeast plasmid, or plant cells' virus, etc. The applicable vectors in this invention include Agrobacterium tumefaciens vector, E.coli plasmid vector and virus vector etc. Anyway, as long as it is capable to replicon and stably inherited in


the body of host, any plasmid and vector can be used to construct the recombined expression vector of this invention.
"Cells of host" used herein refers to the genetic engineering host cells into which the nucleotide sequence of this invention may be introduced or that contain the recombinant vectors of the nucleotide sequence of this invention. These cells include the mustard, solanaceae, cyperaceae, convolvulaceae, malvaceae, and linaceae plant cells etc.
The "sulfonylurea herbicide" is a category of herbicides with super-high efficiency, broad spectrum, low poison and high selectivity. Its biological activity is 100-1000 times higher than those of the traditional herbicides. It can be absorbed by the plant's root, stem and leaf. It acts on acetolactate synthase and inhibits the biosynthesis of branched-chain amino acids leucine, isoleucine and valine, thus leading to preventing biosynthesis of proteins that are required by plant. Consequently, the growth of the sensitive plant is stopped. What the Cyp81A6 gene in this invention resists includes but not limit to the sulfonylurea herbicides listed in Table 1.
Table I. Main sulfonylurea herbicides for paddy-field weed prevention
General name Developed by Dosage(g/ha)
Metsulfuron-methyl DuPont 3.0-7.5
Tribenuron-ethyl DuPont 9.0-18.0
Bensulfuron-methyl DuPont 20.0-30.0
Japanese Nissan
Pyrazosulfuron-methyl 20.0-50.0
Chemical Corporation.
In addition, the nucleic acid sequence of this invention or the transforming host cells that contain the recombinant vector of nucleic acid sequence of this invention can be processed by the routine techniques that the technicians in this field are familiar with. When the host is prokaryotic organism, such as E.coli, the CaCb method, electroporation method etc can be applied. When the host is eukaryotic cell/s, Agrobacterium tumefacien, biolistic, direct DNA transformation method, calcium phosphate coprecipitation method, microinjection method or liposome package etc can be selected to use.


BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. The position of rice bentazon sensitive lethal mutant site bel of this invention and its co-separated PCR-RFLP markers DPI and DP2 on molecule marker genetic linkage map of the chromosome 3.
Figure 2. Allelism test of bentazon sensitive lethal sites in Norin 8m and 8077S. a: the plants before bentazon treatment, left: Norin 8m; right: 8077S; middle: the Fl hybrid of Norin 8m and 8077S. b: the plants one week after the bentazon treatment. The applied concentration of bentazon is 1250mg/l.
Figure 3. Flow chart of PCR-RFLP analysis to verify the cyp81A6-\ single-base deletion site in 8077S. Note the marks in the figure for the mutation site, primer sequence for PCR specific amplification, the manually introduced Bgll digestion site through primer designing, as well as the restriction length polymorphism between PCR products from the target sequences with or without manually introduced Bgll digestion site.
Figure 4. PCR-RFLP analysis results of mapping population. M: lOObp DNA Ladder (Takara); 1-5: five DNA mixture samples (46 plants/sample) from F2 mapping population (recessive sensitive lethal homozygous lines); 6: 93-11; 7: Peiai 64m.
Figure 5.. Flow chart of PCR-RFLP analysis to verify the cyp81A6-2 single-base deletion site in Norin 8m. Note the marks in the figure for the mutation site, primer sequence for PCR special amplification, the original Nael restriction site on wild-type Cyp81A6 sequence corresponding to cyp81A6-2 single-base deletion site, as well as the restriction length polymorphism between PCR products from the target sequences with or without manually introduced Nael digestion site.
Figure 6. Bensulfuron-methyl selection results of Cyp81A6- transformed 8077S calli. Left: transgenic; right: non-transgenic control.
Figure 7. Bentazon test results of Cyp81A6 transgenic 8077S seedlings. Left: negative control of 8077S; middle: transgenic seedling; right: positive control of W6154S.
Figure 8. Structure of Cyp81A6 gene. Black rectangle: exon of the gene; grey rectangle: 5'- and 3'-UTR of the gene; real line in the middle: intron of the gene. Their lengths are marked on top of the figure respectively. Those marked at the bottom of the figure are the initiation codon ATG, termination codon TGA as well


as splice point sequences GT and AG.
Figure 9. The target sequence of Wax" gene and the RNADNA Chimeric Oligonucleotide RCOl, which is designed accordingly.
Figure 10. The target sequence of cyp81A6-\ gene and the RNADNA Chimeric Oligonucleotide RC02, which is designed accordingly.
Figure 11. The target sequences of Cyp81A5, Cyp81A6, Cyp81A7, Cyp81A8 genes and the RNA-DNA Chimeric Oligonucleotide RC03, which is designed accordingly.
Figure 12. The target sequence of Cyp81A8 gene and the RNA-DNA Chimeric Oligonucleotide RC04, which is designed accordingly.
Figure 13. The target sequence of cyp81A6-2 gene and the RNADNA Chimeric Oligonucleotide RC05, which is designed accordingly.
Figure 14. The target sequence of rice P450 gene (the GenBank accession number is B1147A04) whose functions are unidentified and the RNADNA Chimeric Oligonucleotides RC06, which is designed accordingly.
Figure 15. The pHPH plasmid map which carries hygromycin phosphotransferase gene.
Figure 16. pAANTIl plasmid structural map of Cyp81A6 antisense RNA driven by Actin I promoter.
Figure 17. PCR confirmation results of Cyp81A6 antisense RNA-transformed plant originally derived from Minghui 63 restorer line. M: DL2000 (Takara) molecule weight marker; 1-2: transgenic plant; 3: wild-type control plant; 4: plasmid control.
Figure 18. The bentazon test results of Cyp81A6 antisense RNA transformed plant originally derived from Minghui 63 restorer line. Left: negative control of bentazon sensitive mutant; middle: antisense RNA-transformed plant; right: positive control of wild-type Minghui 63. The applied concentration of bentazon treatment is 1250mg/L.
Figure 19. pOANTIl plasmid structural map of Cyp81A6 antisense RNA driven by the tissue specific promoter Osg6B.
SEQ ID NO.l: comprising Cyp81A6 coding sequence and Cyp81A6 promoter.
SEQ ID NO.2: the full length cDNA sequence of Cyp81A6 gene.
SEQ ID NO.3: the amino acid sequence encoded by Cyp81A6 gene.


The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. Generally, the implementation examples without indications of the experimental conditions and testing methods shall be operated under general conditions such as those mentioned in Molecular Cloning: A Laboratory Manual, (Third edition by Sambrook, 2001 Cold Spring Harbor Laboratory Press,) or according to the manufactory instructions known in the prior art.
Examples
Example 1: Fine mapping and cloning of target gene
1. Fine mapping of bentazon sensitive lethal gene bel in indica mutant 8077S
1.1 Mapping population
The mapping population used in this Example was consisted of F2 recessive individual plants. While constructing, firstly, make use of Peiai 64 backcrossing line that carries the recessive bel locus (hereinafter referred to as Peiai 64S) to cross with 93-11, a wild-type thermo-sensitive male sterile restorer line (bred by Agricultural Science Research Institute of Li-Xia-He District, Jiangsu Province) for obtaining Fl. Then, reproduce F2 offspring selfing from Fl generation, with the population up to 1,000 plants in total. When the seedlings grow to 3-4 leaves after sowing, treat all F2 plants by cutting leaves one by one (cut about 1cm from the leaf apex) and smearing with bentazon (the 25% bentazon aqueous produced by Jiangsu Sword Agrochemicals Co., Ltd.) at a concentration of 1250mg/L, three leaves per plant. Then, select 231 individual plants homozygous for recessive bel locus as the mapping population of this example based on the sensitivity of treated leaves to bentazon.
1.2 DNA extraction


Total DNA was extracted from fresh leaves collected from both parental plants of 93-11 and Peiai 64m as well as F2 offspring of 231 bentazon-sensitive lethal homozygates, respectively, using CTAB method as described previously by McCouch etc (1988). The chemicals used for this purpose were all purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd. (hereinafter referred as Shanghai Sangon).
1.3 SSR analysis and fine mapping of bel gene
The SSR amplification reaction system is: 50 ng template DNA, lxPCR reaction buffer solution, 1.87mM Mg2+, 0.2mM dNTP, 1.0 U rTaq enzyme [TaKaRa Biotechnology (Dalian) Co., Ltd., namely Takara Biotech, hereinafter referred to as "Takara"], and forward and reverse primers, 0.2 uM for each. The total volume of reaction per tube is 20ul. The amplification was programmed as follow: initial denaturation step at 95 D for 3 min was followed by 35 cycles of 94 D for 1 min, 60D for 1 min and 72 D for 1 min with a final extension at 72 D for 5 min and then stored forever at 10D. The resulted PCR products were separated with 3.5% agarose gel (from Shanghai Sangon). After that, the gels were stained with EB (from Shanghai Sangon) and pictures were taken by UVP imaging system (Germany).
In the previous work, the inventors had primarily mapped the bel locus
existing in 8077S onto the long arm of rice chromosome 3 using SSR markers, with
the defined genetic distance about 7.1cM from one of them, namely RM168 (Zhang
et al 2002). In order to further fine map this locus, the present inventors redesigned
and synthesized a set of new SSR markers located between RM168 and the end of
long arm of chromosome 3 based on genomic sequence analysis ( http :
//btn.genomics.org.cn/rice) with the aid of SSR primer search software SSRHunter
1.3. These SSR markers include 7a (forward primer:
5'-GTCAGAGCAAGGTCGGAGAG-3'; reverse primer:
5-TCGGTGATCATTGTCATTTG-3), 3a (forward primer:
5-TGTTTTCTTTTTCGCTGTGTG-3'; reverse primer: 5-GCAA
GCCTTTTTGCGTATTC-3') and 8a (forward primer: 5'-
GCTTCCCTCTCCTTCCACTT-3'; reverse primer:
5-CTTGTGAGTGAGTGGTGACG-3), etc (The primer sequences were all synthesized by Shanghai Sangon). Among which, the 3a and 7a are located on the same BAC clone AC084282. Use of 8a marker, four out of 231 the extreme


individual plants with pure recessive locus were detected as recombinants from single-exchange, and other four of such individual plants were detected for 7a marker. These results further verified that the bel was located between these two SSR markers. In addition, only one single-exchange recombinant plant was detected by use of 3a marker near to 8a, this thus indicating that the bel was located in a shorter region from 3a to 7a. The fine map (Figure 1) of bel locus is constructed using MAPMARKER3.0. The genetic distance from marks 3a and 7a to the bel locus were calculated no longer than 0.1 cM and 0.4cM, respectively.
2. Deduction, isolation, and cloning of candidate genes
2.1 Deduction and sequence analysis of candidate genes
The results of sequence analysis showed that there are totally 18 putative genes between the SSR markers 3a and 7a, which includes a cluster of four cytochrome P450 genes (the accession no in GenBank as AAK63940.1, AAK63920.1, AAK63922.1 and AAK63925.1 respectively), which are named formally as Cyp81A5, Cyp81A6, Cyp81A7 and Cyp81A8 (http://drnelson.utmen. edu/cytochrome p450.html) according to the international standard classification and nomenclature system. The previous reports showed that the P450 gene in rice microsome was involved in detoxification of bentazon (Haack etc, 1994). Furthermore, Deng and Hatzios (2003) isolated and purified a P450 protein with a molecular weight of 60kDa from the seedlings of rice and they confirmed that it plays an important role in degradation metabolism of herbicide BSM (bensulfuron-methyl). The data present above all implied that there was an association between the bel and P450 and we thus considered that cluster of four P450 genes as mentioned above as candidate genes.
To further define the candidate genes more precisely, we synthesized four sets of specific primers (Table 2) according to the genome sequence of four P450 genes, respectively. Each of the above deduced candidate genes was amplified from both wild control W6154S and mutant 8077s, respectively, using high fidelity pyrobest™ polymerase (Takara), and then directly sequenced by Perkin Elmer AMI 377 (Shanghai GeneCore Bio Technologies Co., Ltd., hereinafter referred to as Shanghai GeneCore). The sequence analysis showed that no differences were observed in Cyp81A5, Cyp81A7, and Cyp81A8 except that a single base G deletion was appeared in Cyp81A6 derived from mutant 8077S as compared with the wild control W6154S


(the 4006 base of the sequence shown in Sequence Table SEQ ID NO.l). We therefore took the Cyp81A6 as the sole candidate gene.
The primers used for PCR amplification and sequencing of the above mentioned four genes (the sequence is synthesized by Shanghai Sangon) were: Cyp81A5: Pl-la and Pl-lb, Pl-2a and Pl-2b, Pl-3a and Pl-3b; Cyp81A6: P2-la and P2-lb, P2-2a and P2-2b, P2-3a and P2-3b, P2-4a and P2-4b, P2-5a and P2-5b, P2-6a and P2-6b; Cyp81A7: P3-la and P3-lb, P3-2a and P3-2b, P3-3a and P3-3b; Cyp81A8: P4-la and P4-lb, P4-2a and P4-2b, P4-3a and P4-3b. The sequence of each primer is listed in Table 2.
2.2 Allelism test of the bel between japonica and indica rice As mentioned above, the Japanese scholar Mori obtained a bentazon sensitive lethal mutant Norin 8m by y-ray radiation treatment of Norin 8 (Mori , 1984). Classical genetic analysis indicated that this mutant was controlled by a pair of recessive alleles. In order to verify whether the bentazon sensitive loci in both 8077S and Norin 8m are allelic, we made a cross between 8077S and Norin 8m and obtained the Fl hybrid and its selfed F2 population containing 800 plants. After sowing, when the seedlings grew to 9-10 leaves, sprayed bentazon onto the surface of leaves of both the parents and their derived Fl plants at a concentration of 1250mg/L. All the treated plants withered and died one week later (Figure 2). These results primarily verified that the bentazon sensitive loci in the two mutants are allelic to each other. Moreover, all of the treated 800 F2 plants from sefled Fl plants were also withered and died too, thus further confirming the above results. To distinguish the loci originated from 8077S and that originated from Norin 8m, we named them as cyp81A6-l and cyp81A6-2, respectively.
Together with high fidelity pyrobest™ polymerase (Takara), oligonucleotide primers listed in Table 2, which specific to CYP81A6 have been used to amplify the wild allele Cyp81A6 and the mutant cyp81A6-2 from wild type Norin 8 and mutant Norin8m, respectively, and then the products were sequencing in Shanghai (Perkin Elmer AMI 377, Shanghai GeneCore). The results revealed that the cyp81A6-2 from the mutant Norin 8m had a single C deletion as compared with the wild allele of Cyp81A6. This single C deletion was at the 2455th base of the sequence as shown in SEQ ID NO. 1. These data again verified the result of allelic test.


Table 2: Specific primers used in PCR amplification

Primer name Primer sequence (5'-3')
Pl-la GCTGTGCGTATCCAATGAAG
Pl-lb TCAGGGAGAGCTCGAACAG
Pl-2a CTCATGTCGGGGCTCATC
PI-2b TAGCTTTCTCCCGATTGACC
Pl-3a TTCATGACCCAGACGAAAAA
Pl-3b ATGAGTTTGCCCTGGAGATG
P2-la TGAGAAGACCAAGGCAGGAG
P2-lb GGCAACAAATCGACACACG
P2-2a GGCTGCCTCCTCCTCTCT
P2-2b TGAGGATCGAGAGTCCGAGA
P2-3a AATAATCGCCCAACGATTGA
P2-3b GGAGACAATCCAGGCATCTC
P2-4a GATCGCATCTGCGTTTCAG
P2-4b GATGAGCCCCGACATGAG
P2-5a CCTCATGTCGGGGCTCAT
P2-5b CGCACCAATGAGAGAATTCAG
P2-6a AAATCTTAGTTCCACCCTCTTGC
P2-6b TCGTCCTGGAGATGCAAAC
P3-la TGCGTAATACAACTTACTATTTCCGTA
P3-lb GAACAGCCTCCGCTTCAG
P3-2a ATGGTGCAGAGGATGTACCG
P3-2b TTCAAATTAAGCGTTCAAAATTCA
P3-3a ACCCCTTTTCCTCTTTCGTG
P3-3b GATGAAGCCTACCTGGTGGA
P4-la CCTCAAGGCTCAAGCATCAT
P4-lb GAACAGCCTCCGATTCAGC
P4-2a ACATGGTGCGGAGGATGTA
P4-2b TGGTTTCTGATCAAGCGTTTT
P4-3a AGGCATGTTTCGAATTGTACTT
P4-3b AACTTTATTCCCTGCTACACAGC
2.3 PCR-RFLP analysis
To prevent the sequence errors that might be resulted from the sequencing


analysis, we further tested and verified the single-base deletion of cyp81A6-\ and cyp81A6-2 in two mutants by PCR-RFLP analysis. During the experiment, we first compared and analyzed the wild and mutating sequence flanking the cyp81A6-\ single-base deletion site using WEBCUTTER 2.0 software to check whether there was any restriction enzyme cutting sites that was changed or newly generated after mutation. To the mutant of cyp81A6-\ without such site, we directionally mutated bases G and A nearby the upstream of the deletion site to two C bases by substitution through primer designing to create a new artificial Bgli cutting site (GCCNNNNNGGC). The further testing data showed that the amplified products containing this fragment of the modified sequences from wild control material could not be acted as recognition and digestion site by Bgli since it had an extra base G as compared to the mutated allele. After digestion with Bgli enzyme, only one belt at a length of 251bp appeared on the gel; for the mutated allele, however, the PCR amplified products could result in two belts after digestion with the same enzyme. Among which, one belt had length of 24bp and anther one had length of 227bp (Figure 3). Furthermore, applied the same PCR-RFLP primers to amplify DNA samples from 93-11 and Peiai 64m as well as five bulks of DNA mixtures (46 plants per bulk) derived from F2 mapping population. The obtained amplification products were then purified by PCR-specific purification kit (Takara) and subjected to Bgli digestion and the resulted belt-types from all of the F2 DNA bulk samples were exactly the same as that of parental DNA sample extracted from Peiai64m (Figure 4). These results thus confirmed that the polymorphism 2?g/I-PCR-RFLP marker artificially introduced to the cyp81A6-\ deletion site was indeed co-segregated with cyp81A6 gene. We therefore named this marker as DPI (Figure 1).
As for the cyp81A6-2 mutant, use the same WEBCUTTER 2.0 software to analyze restriction sites between its wild-type and mutant DNA sequences and the single-base deletion of C in the mutant site was found just occurred in Naei (GCCGGC) enzyme recognition site, this thus no longer cut by the Naei enzyme. However, another Naei enzyme digestion site located at 50bp of its upstream (within the PCR amplification scope) was still maintained. The further PCR-RFLP analysis (see also Figure 5 for technical flow) indicated that the PCR amplification products obtained from wild Norin 8 and purified by Takara PCR-specific purification kit were cut into three belts (21bp, 50bp and 151bp) by this Naei


(Takara) enzyme, whereas the PCR amplification products from mutant Norin8m subjected to the same purification procedure could not be cut by Nael enzyme on the single base deletion site, thus producing only two belts with the sizes of 21bp and 200bp (Figure 5). Similar to the PCR-RLP analysis made on cyp81A6-\, these results also confirmed that the polymorphism Mael-PCR-RFLP marker originally existing in the cyp81A6-2 deletion site was really co-segregated with Cyp81A6 gene. We therefore named this marker as DP2 (Figure 1). Moreover, these results further verified the sequencing results mentioned above.
2.4 Cloning of candidate gene Cyp81A6 and complementary confirmation of its biological functions
The full length sequence of candidate gene Cyp81A6 was obtained through
lump-sum amplification using LA TaqTM polymerase reagent kit purchased from
Takara Company. The long fragment PCR specific primer pairs (forward primer:
5-CAAACTTCCAACTTTCCCGTCACCTTCACT-3'; reverse primer:
5 -CCGCGGGTCACCGAGC AGAAAGCCCTTCCT CCCAAGTTAGAA-3',
synthesized by Shanghai Sangon) used in this experiment were designated at a start from 124bp before and end with 4145bp after BamHI enzyme digestion site located at 5'-end of Cyp81A6 gene, respectively, according to the DNA sequence publicized by indica rice genome database (http : //btn.genomics.org.cn/rice). According to the designing, these primers have a BstEll enzyme cutting site attached to the 3'-end of the primers, which is convenient for production of cohesive end and ligation during cloning. The amplified fragment with the total size of 431 lbp by this pair of primers is consisted of those parts: 124bp upstream sequence of the BamHI digestion site, 132lbp promoter sequence, 232lbp sequence of leader region plus exon plus intron , 272bp 3'-UTR sequence, followed by 288bp genome sequence (including the 7bp BstEll recognition site and 5bp protection base). The amplified fragments was then ligated to TA vector (Takara) by T4-DNA ligase of bacteriophage for further repeated sequencing analysis (Perkin Elmer AMI 377, Shanghai GeneCore) and the obtained clones that were confirmed to contain the correctly amplified exons were then selected and cut off from the TA vector by both BamHI and BstEU(Takara) enzymes. This double-enzyme digested fragment was then ligated to the genetic transformation vector pCAMBIA1301, which was cut by the same pair of enzymes. After that, choose the correct insertion-containing plasmid and introduce it into the


EHA105 strains of Agrobacterium tumefaciens by electroporation method. Use the resulted positive EHA105 strain to transform the 8077S genome for complementary confirmation of the biological functions of the cloned Cyp81A6 gene. The transformed calli were screened in the selection medium complemented with 50mg/L hygromycin (ABI, USA) for three rounds and then with 4.2uM/L Bensulfonyluron- methyl (Sigma) (Figure 6). The positive calli resistant to both selection agents were then transferred onto regeneration medium supplemented with 50mg/l hygromycin for green seedling differentiation. The regenerated green plantlets were then subjected to the PCR analysis and the positive ones identified with the transgene were then further verified by the bentazon test at a concentration of 1250mg/l. As a result, all the transformants recovered the resistance to bentazon (Figure 7 shows the result of one of the plants). These results thus confirmed that the cloned Cyp81 A6 was indeed to have function of resistance to both bentazon and sulfonylurea herbicides.
2.5 Structure Characteristics of the Cyp81A6
The structure features of Cyp81A6 include: the 5'-UTR of 53 bp prior to the translation initiation codon (the sequence from 1896 to 194 8bp as shown in SEQ ID NO.l), the coding region of 2268bp (the sequence from 1949 to 4216 bp as shown in SEQ ID NO.l), and the 3-UTR of 272bp after the termination codon (the sequence from 4217 to 4488 bp as shown in SEQ ID NO.l). The coding region of this gene is composed of two exons plus one intron (see Figure 8). The length of two exons is 924 bp (the sequence from 1949 to 2872bp as shown in SEQ ID NO.l) and 618bp (the sequence from 3599 to 4216 bp as shown in SEQ ID NO.l), respectively. The length of the intron is 726bp (the sequence from 2873 to 3598bp as shown in SEQ ID NO.l).
The Cyp81A6 encodes a novel cytochrome P450 protein (see SEQ ID.NO.3 for its sequence). It has four conserved domains shared by the majority of P450 proteins, namely the heme-binding domain of Phe-x-x-Gly-x-Arg-x-Cys-x-Gly which is located at C-terminal. The I helix of Ala/Gly- Gly-x-Asp/Glu-Thr-Thr/ Ser located at 150 amino acid residues upstream of heme-binding domain and this conserved domain plays an important role in oxygen activation. The meander area of Pro-Glu/Asp-Arg/His-Phe/Trp located between the heme- binding domain and I helix and the proline-rich hinge located at the N-terminal (Werch-Reichhart etc. ,


2000). In fact, it is just because of existence of these conserved domains the conservative tri-dimensional structures of the majority of cytochrome P450 proteins was capable to be maintained.
Example 2: The targeted mutation and genetic improvement of rice Wx gene
Two wild-type alleles Wxa and Wxb in rice cultivars are widely distributed on the Wx locus. Among which, Wxa is the feature of indica type rice and its expression activity on the RNA and protein level is 10 times stronger than the Wxb,s. Its high level of expression results in high amylose content and makes the cooked rice hard and loose with a bad taste. While Wxb mainly exists in the japonica rice. Its low level of expression results in typical japonica-type amylose content at a middle level. Its rice after cooking is usually soft and delicious. The previous investigation indicated that the major difference between these two alleles of Wx" and Wxb on the expressive activity is that there is a G to T substitution in the 5'-splicing site of the leading intron of the latter (Cheng Shijun etc. 2001). This substitution results in decrease of splicing efficiency of the leading intron in the pre-mRNA of Wxb, and thus causes the reduction of amount of both mature mRNA and its translated granule-bound starch synthas (GBSS). This finally reflects by the reduction of synthesis amount of the amylose.
Based on the sequence flanking the 5' splicing site of leading intron of Wx gene and the sequence flanking the single base deletion of Cyp81A6, separately design muton molecule that can mutate the G base of the Wx splicing site (see figure 9 RCOl) and repairer molecule that can repair single-base deletion mutant of cyp81A6-\ (see figure 10 RC02). Use the particle gun to co-introduce them into the genome of 8077S mutant simultaneously according to ratio of 1 repairer: 3 mutants. Use the sulfonylurea herbicide to screen the repairer of the cyp81A6-\ single-base deletion mutant and the co-modified mutant of wild Wx gene. After that, use the designed specific primers (forward primer: 5-CTCTCTC ACCATTCCTTCAG-3', reverse primer: 5'-AGCCTAACCAA ACATAACGA-3') to conduct the PCR amplification followed by Accl (Takara) enzyme digestion analysis of the target sequence of the co-modified mutant. The results confirmed the successful mutation of Wx gene. Based on this, a new line with the mutated Wx gene was capable to develop through homozygous selection followed by the field test.


Example 3: Use the double RCOs-mediated co-modification technology to investigate the biological function of rice CYP81A5, CYP81A7 and CYP81A8 and other unknown genes.
In theory, the gene targeted modification technology is precisely proceeded to aim at target gene locus. As it is known that, the encoded products of Cyp81A5-> Cyp81A 7 and Cyp81A8 genes are a category of cytochrome P450 monooxygenase proteins and the rice P450 is a huge gene family. For instance, only one subspecies of indica type rice has 454 p450 family members. This gene family possesses a highly conserved heme binding motifs (F-X-X-G-X-R-X-C-X-G) on the protein sequence, especially their core residue of cystine, which is already confirmed to be an extremely important determinant to the biological function of the P450 gene. Therefore, it can be used as an ideal mutation target.
From the existing rice P450 database (http://drnelson.utmen. edu/ cytochromep 450.html), it is clear that the amino acid sequences of the heme binding domain of rice CYP81A5s CYP81A7 and CYP81A8 and other three cytochrome P450 are FGMGRRRCPGETLA, FGMGRRK CPGETMA, and FGMGR RRCPGEMLA, respectively. Based on the nucleotide sequence and Cyp81A6 single base deletion mutation sequence information of these motif, we designed a muton molecule that could make mutation of the codon for the key amino acid residue of cystine (C) or other residues of the motif ( see figure 11 RCO 3 and figure 12 RCO 4 ) and the repairer molecule that could carry on the repair for the single base deletion of cyp81A6-2 (see figure 13 RC05). Use the particle gun to co-introduce them into the genome of Norin8m mutant simultaneously according to the ratio of 1 repairer: 3 mutons or so. Apply the sulfonylurea herbicide in the selection medium to screen the repairer of cyp81A6-2 single-base deletion mutant and the co-modifier of wild-type Cyp81A5s Cyp81A7 and Cyp81A8. After that, confirm the repairer and modifier by specific-primer-mediated PCR analysis and sequencing test of their target DNA sequence. Compare the differences of phenotypes or biochemitypes between positive co-modifier of Cyp81A5 , Cyp81A7 and Cyp81A8 with the wild-type and deduce their biological functions.
In the above experimental procedures, the purpose of conducting repair at the cyp81A6-2 single base deletion locus is to provide an indirect selection for the mutation of other genes. Therefore, theoretically speaking, as long as a genetic


locous such as sulfonylurea target enzyme acetolactate synthase (ALS) (Okuzaki and Toriyama 2004 ) , which is capable for selection after modification, can be used for this purpose as the modification target.
Example 4: Studies on biological function of the unknown P450 genes in rice with the aid of selection effect of exogenous marker gene and modification effect of RCOs molecule
The exogenous selectable marker gene such as anti- antibiotic marker gene, bioluminescence or chemiluminescence marker gene, carbon source metabolism key enzyme gene, herbicide resistant gene originating from bacteria, animal or other plants and GUS reporter gene etc. can provide selection effect to the mutant of target gene modified by co-introduced RCOs with them. Here we present a case study in which using hygromycin phosphotransferase (hph) gene as selectable marker. Design a RCOs molecule to target a P450 gene with unknown function (see figure 14 RC06) and at the same time ligate the selectable hph gene into plasmid vector to generate a expression construct, namely pHPH (see figure 15). Then use the conventional particle bombardment mediated co-transformation method (Tu et al, 1998) to co-introduce both of these plasmid pHPH and RCOs into the recipient genome, and indirectly select the RCO modified P450 mutant based on the _.. hygromycin resistance expressed by the co-introduce hph gene. Afterwards, confirm the hph transgene and the p450 putative mutants by specific-primer-mediated PCR analysis and sequencing test of the relative target DNA sequence. Compare the phenotypes or biochemical differences between the confirmed p450 mutants with the wild-type and deduce their biological functions.
Example 5: The impression effects of anti-sense RNA against the rice endogenous Bel gene.
Design anti-sense RNA (RNAi) sequences (see the 1939th to 2439th nucleotide sequence of SEQ ID NO.l) in according with the coding sequence of the cloned rice Cyp81A6 gene. Fuse these antisense RNA (RNAi) sequence to the rice constitutive expression promoter such as Actinl and then insert them into the binary vectors of pAANTll (see figure 16). Use this vector to transform wild-type rice such as Minghui 63 using the modified procedures of Agrobacterium-mediated transformation method. After that, use the specific primers to carry on the PCR


amplification analysis of transgenic To generation plants for molecular confirmation. The results revealed that all of transgenic plants presented the fragments in according with those detected in the plasmid control lane, this thus confirming integration of the exogenous antisense RNA fragment into recipient genome of Minghui 63 (see figure 17). Furthermore, for phenotype confirmation, we use 1250mg/L of bentazon to smear the leaves of the PCR positive transgenic plants (3 pieces of leaf/plant). All of the treated leaves and plants were faded and died 36 hours later (see figure 18). These results verified that antisense RNA was indeed to effectively impress the expression of the rice endogenous Bel gene.
Example 6: Development of chemically supplemented emasculation and thermo-sensitive male sterile line
Fuse the antisense RNA fragments that their impression effects were confirmed effective to the rice tapetal and pollen specific expression promoter such as Osg6B or RA39 and then insert them into the binary vectors (see figure 19). Afterwards, use this vector to transform the photoperiod- and thermo-sensitive genie male sterile line Peiai 64S that has been widely used in rice production in China at present using the modified procedures of Agrobacterium-mediated co-transformation method. Let this antisense RNA to specifically inhibit the expression of the endogenous Cyp81A6 gene in the tapetal cell and pollen grain of thermo-sensitive male sterile line. On this base, it is able to develop the novel chemically supplemented emasculation and thermo-sensitive male sterile line through strict pure line selection and field test. Applying this novel male sterile line in the hybrid seed production, the mixed selfing seeds caused by unusual low midsummer temperatures can be easily examined after germination and their-derived seedlings when grown in the seedling bed can be completely killed by simply spraying sulfonylurea herbicide. The purpose of the male sterile line selfing seeds removing and its hybrid seed purity ensuring is thus reached.
Example 7: Exploitation and application of a new category of herbicide-resistant selectable marker
Fuse the entire or just coding sequence of the bentazon and sulfonylurea herbicide resistant gene Cyp81A6 to the constitutive expression promoter such as CaMV35S, Ubi-1, Actin 1 etc and the nos terminator and insert the recombinant


DNA sequence into the binary vectors to replace the currently commonly used hygromycin or kanamycin resistant gene or GUS reporter gene etc. Introduce this recombinant resistant gene into the genome of 8077S, a bentazon and sulfonylurea herbicide-sensitive male sterile parental line of two-line hybrid rice. The resulted transgenic calli can continue to grow on the culture medium supplemented with sulfonylurea herbicide of BSM (on the left of figure 6), while the non-transgenic calli derived from wild type rice stop to grow on such culture medium (on the right of figure6). These results indicate that the Cyp81A6 is capably used as selectable marker gene.
Example 8: Development of bentazon and sulfonylurea herbicide resistant transgenic plant.
Fuse the entire sequence of the bentazon and sulfonylurea herbicide resistant gene Cyp81A6 to the constitutive expression promoter such as 35S% Ubi-1 > Actinl etc and the nos terminator and insert this recombinant DNA sequence into the binary vector, which was further transformed into the Agrobacterium strain. Directly use the resulted Agrobacterium strain and chemicals of sulfonylurea herbicide for genetic transformation and resistant callus selection. The transgenic plant that is verified by molecular analysis and phenotypic characterization can be developed into bentazon and sulfonylurea herbicide resistant transgenic line through strict pure line selection and field test. Introduction of the entire sequence of CYP81A6 gene into the mutant genome of 8077S could obtain transgenic new line highly resistant against bentazon and sulfonylurea herbicide (on the left of figure 7).
Example 9: Isolation of Cyp81A6 analog/s from genome of other crops or plant species
There are four conservative motifs or domains among different members of cytochrome P450 gene family in plant genomes, these including heme-binding domain which plays a key role to catalysis, the N-terminal hydrophobic regions important to membrane binding, the proline/glycine-rich area responsible for the correct assembling of protein, and I helix of 150 amino acid residues located at the upstream of heme binding region (Werch-Reichhart etc. , 2000). Therefore, the designated specific primers based on these highly conserved region (such as forward direction primer: 5'-GCAGGAA CAGAGACAACC-3', reverse direction:


5'-CACCTCCGCCT CCCCATC-3') is possibly used to amplify the genome of gramineae or legume except for rice to isolate the core sequences with high homology. Then, based on this, isolate the 5'- and 3'-end flanking sequences around this core fragment by means of 5'- and 3'-RACE. So up to here, the full length sequence of the target gene that has high homology to that of rice P450 gene/s and originated from other species or family is isolated.
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We Claim:-
1. A bentazon and sulfonylurea herbicide resistant gene, which comprises a
nucleotide sequence selected from the groups consisting of:
( 1 ) a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO.:
l;
( 2 ) a nucleotide sequence fragments or derivatives thereof, which have the same function as the nucleotide sequence of position 1949 through 4216 of SEQ ID NO.:
l;
( 3 ) a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO.: 2;
( 4 ) nucleotide sequence fragments or derivatives thereof, which have the same function as the nucleotide sequence of position 54 through 1595 of SEQ ID NO.: 2;
( 5 ) a nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.: 1 or SEQ ID NO.2 under the stringent condition.
2. A bentazon and sulfonylurea herbicide resistant gene, which having the
nucleotide sequence shown in SEQ ID NO.: 1 or SEQ ID NO.: 2.
3. A polypeptide encoded by bentazon and sulfonylurea herbicide resistant gene,
wherein the bentazon and sulfonylurea herbicide resistant gene comprises a
nucleotide sequence selected from the groups consisting of:
( 1 ) a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO.:
l; ( 2 ) a nucleotide sequence fragments or derivatives thereof, which have the same
function as the nucleotide sequence of position 1949 through 4216 of SEQ ID NO.l;
( 3 ) a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO.: 2;
( 4 ) nucleotide sequence fragments or derivatives thereof, which have the same function as the nucleotide sequence of position 54 through 1595 of SEQ ID NO.: 2;
( 5 )a nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.: 1 or SEQ ID NO.: 2 under the stringent condition.
4. A polypeptide encoded by a bentazon and sulfonylurea herbicide resistant gene,
wherein said polypeptide has the amino acid sequence shown in SEQ ID NO.3.


5. A recombinant vector, wherein said the recombinant vector comprises the nucleotide sequence according to claim 1 or 2 and the regulatory elements essential for transcription or expression of said nucleotide molecule.
6. The recombinant vector according to claim 5, wherein said recombinant vector comprises the nucleotide sequence according to claim 1, which operatively linked to the transcriptional regulatory elements.
7. The recombinant vector according to claim 5, wherein said the regulatory elements essential for transcription or expression include promoter, terminator, enhancer, MAR sequence or 5' upstream regulatory sequence.
8. The recombinant vector according to claim 5, wherein said vector is an expression vector.
9. A plant cell, wherein said the plant cell comprises the polypeptide according to claim 1 or 3.

10. The plant cell according to clam 9, which includes the cyperaceae ruderal plant cell or the cell of almost all broad leaf plants except for legume.
11. The use of nucleotide sequence according to claim 1 or the recombinant vector according to any one of claims 5 to 8 as a selectable marker of herbicide resistant character.
12. A bentazon and sulfonylurea herbicide sensitive gene, which comprises a
nucleotide sequence selected from the groups consisting of:
( 1 ) a nucleotide sequence obtained by deleting the 2455th base C or 4006th base G from the nucleotide sequence shown in the SEQ ID NO.: 1 ;
( 2 ) a nucleotide sequence obtained by the deleting the 560th base C or 1385th base G from the nucleotide sequence shown in the SEQ ID NO.: 2 ;
( 3 ) a nucleotide sequence that can hybridized with nucleotide sequence as


mentioned in above (1) or (2) under the stringent conditions.
13. A recombinant nucleic acid molecules, which comprises the artificial antisense RNA or RNAi fragments, wherein said antisense RNA or RNAi fragments comprise the nucleotide sequence of any one of claim 1, 2 or 12.
14. The use of recombinant nucleic acid molecules according to claim 13 in developing the chemically supplemented emasculation and thermo-sensitive male sterile line.
15. A recombinant vector, which comprises the nucleotide sequence of claim 12 and the regulatory element essential for transcription or expression of the nucleotide sequence.
16. The recombinant vector according to claim 15, wherein said regulatory elements essential for transcription or expression include promoter, terminator, enhancer, MAR sequence or 5'upstream regulatory sequence.
17. A plant cell, which comprises the nucleotide sequences of claim 12, or
recombinant vectors according to claim 15 or 16.
18. The use of nucleotide sequence according to claim 12 or the recombinant vector according to claim 15 or 16 as a negative selectable marker.
19. A genetic manipulation method that can be used to conduct co-modification of the target sequences at two or multiple loci of the genome in the living cell simultaneously and directionally, wherein the method include the following steps: using the nucleotide sequence that can function as the selectable marker after modification as the first modifying target, furthermore using the key base pair of target endogenous gene in the living cells as the additional modifying target,
co-introducing the double or multi-RNADNA chimeric oligonucleotide molecules (RCOs) that are designated against the above-mentioned different modifying target sequences into the recipient genome to further conduct co-modification of the


abovementioned double- or multi-target loci simultaneously and directionally by the particle gun-mediated co-introduction technology; and
selecting the modifying event occurred on the target endogenous gene in the same recipient genome according to the phenotype expressed by the first target sequence after modification.
20. The genetic manipulation method according to claim 19, wherein the nucleotide sequence that can be used as selectable marker includes the mutated or non-mutated herbicide resistance gene, antibiotic resistance marker gene, bioluminescence or chemiluminescence gene, carbon source metabolism key enzyme gene, the coding sequence or regulatory sequence originated from bacteria or GUS gene etc.
21. The genetic manipulation method according to claim 19, wherein the nucleotide sequence that can be used as selectable marker is the nucleotide sequence according to any one of claim 1, 2 or 12.
22. The genetic manipulation method according to claim 19, wherein the screenable phenotype can also be performed by the expression of exogenous gene introduced together with RCOs.
23. The genetic manipulation method according to claim 19, wherein the additional modifying target(s) is (are) the gene(s) with unknown function.
24. The genetic manipulation method according to claim 19, wherein the additional modifying target(s) is (are) the gene(s) with a known function.
25. A method for investigation of gene's function, wherein the method includes the step of applying the nucleotide sequence, polypeptide, recombinant vector, cell and method according to any one of claims 1-8, 12, 15-16 and 21-24.
26. A method for improvement of the biological traits, wherein the method include


Abstracts:-
The present invention provides a kind of rice endogenous bentazon and sulfonylurea herbicide resistant gene (Cyp81A6 gene), and its functional conservative variants, the biological activity subfragments or derivatives with the same function. It also provides a kind of method to prevent the selfing mixtures during hybrid seed production. It also provides the novel means of the directional genetic manipulation and the improvement of biological traits.


Documents:

148-MUMNP-2008-ABSTRACT(GRANTED)-(14-7-2011).pdf

148-mumnp-2008-abstract.doc

148-mumnp-2008-abstract.pdf

148-MUMNP-2008-CANCELLED PAGES(21-6-2011).pdf

148-MUMNP-2008-CLAIMS(AMENDED)-(09-11-2010).pdf

148-MUMNP-2008-CLAIMS(AMENDED)-(15-12-2010).pdf

148-MUMNP-2008-CLAIMS(AMENDED)-(21-06-2011).pdf

148-MUMNP-2008-CLAIMS(GRANTED)-(14-7-2011).pdf

148-MUMNP-2008-CLAIMS(MARKED COPY)-(09-11-2010).pdf

148-MUMNP-2008-CLAIMS(MARKED COPY)-(15-12-2010).pdf

148-MUMNP-2008-CLAIMS(MARKED COPY)-(21-06-2011).pdf

148-mumnp-2008-claims.doc

148-mumnp-2008-claims.pdf

148-MUMNP-2008-CORRESPONDENCE(1-6-2011).pdf

148-MUMNP-2008-CORRESPONDENCE(16-07-2008).pdf

148-MUMNP-2008-CORRESPONDENCE(22-9-2009).pdf

148-MUMNP-2008-CORRESPONDENCE(24-11-2010).pdf

148-MUMNP-2008-CORRESPONDENCE(25-5-2011).pdf

148-MUMNP-2008-CORRESPONDENCE(26-5-2011).pdf

148-MUMNP-2008-CORRESPONDENCE(IPO)-(14-7-2011).pdf

148-mumnp-2008-correspondence(ipo)-(17-12-2009).pdf

148-mumnp-2008-correspondence-others.pdf

148-mumnp-2008-correspondence-received.pdf

148-MUMNP-2008-DECLARATION(24-11-2010).pdf

148-mumnp-2008-description (complete).pdf

148-MUMNP-2008-DESCRIPTION(GRANTED)-(14-7-2011).pdf

148-MUMNP-2008-DRAWING(GRANTED)-(14-7-2011).pdf

148-mumnp-2008-drawings.pdf

148-MUMNP-2008-ENGLISH TRANSLATION(15-12-2010).pdf

148-MUMNP-2008-FORM 1(16-07-2008).pdf

148-MUMNP-2008-FORM 1(28-1-2008).pdf

148-MUMNP-2008-FORM 18(16-07-2008).pdf

148-MUMNP-2008-FORM 2(GRANTED)-(14-7-2011).pdf

148-MUMNP-2008-FORM 2(TITLE PAGE)-(28-1-2008).pdf

148-MUMNP-2008-FORM 2(TITLE PAGE)-(GRANTED)-(14-7-2011).pdf

148-MUMNP-2008-FORM 26(16-07-2008).pdf

148-MUMNP-2008-FORM 3(16-07-2008).pdf

148-MUMNP-2008-FORM 5(16-07-2008).pdf

148-MUMNP-2008-FORM 5(16-7-2008).pdf

148-MUMNP-2008-FORM 5(28-1-2008).pdf

148-mumnp-2008-form-1.pdf

148-mumnp-2008-form-2.doc

148-mumnp-2008-form-2.pdf

148-mumnp-2008-form-5.pdf

148-MUMNP-2008-OTHER DOCUMENT(15-12-2010).pdf

148-MUMNP-2008-OTHER DOCUMENT(24-11-2010).pdf

148-MUMNP-2008-PCT-IB-373(16-07-2008).pdf

148-MUMNP-2008-PCT-ISA-237(16-07-2008).pdf

148-mumnp-2008-pct-search report.pdf

148-mumnp-2008-power of attorney(16-7-2008).pdf

148-MUMNP-2008-REPLY TO EXAMINATION REPORT(09-11-2010).pdf

148-MUMNP-2008-REPLY TO EXAMINATION REPORT(15-12-2010).pdf

148-MUMNP-2008-REPLY TO HEARING(21-06-2011).pdf

148-MUMNP-2008-SEQUENCE LISTING(28-1-2008).pdf

148-MUMNP-2008-US DOCUMENT(21-06-2011).pdf


Patent Number 248430
Indian Patent Application Number 148/MUMNP/2008
PG Journal Number 29/2011
Publication Date 22-Jul-2011
Grant Date 14-Jul-2011
Date of Filing 28-Jan-2008
Name of Patentee ZHEJIANG UNIVERSITY
Applicant Address 268 KAIXUAN ROAD, HANGZHOU, ZHEJIANG 310029 P.R.,
Inventors:
# Inventor's Name Inventor's Address
1 TU, JUMIN HANGZHOU CITY, ZHEJIANG PROVINCE, 310029 P.R.,
2 ZHANG, JIWEN WUHAN CITY, HUBEI PROVINCE, 430064
3 PAN, GANG HANGZHOU CITY, ZHEJIANG PROVINCE,310029
4 ZHANG, XIANYIN HANGZHOU CITY, ZHEJIANG PROVINCE 310029
5 WU, XIAOZHI WUHAN CITY, HUBEI PROVINCE, 430064
PCT International Classification Number C12N15/29, A01P13/00
PCT International Application Number PCT/CN2005/000936
PCT International Filing date 2005-06-28
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 WO2005CN00936 2005-06-28 China