Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a rice Wx mutant protein and nucleic acid or gene thereof.
The technical problem to be solved by the present invention is to provide an expression cassette, a recombinant vector or a cell.
The technical problem to be solved by the present invention is to provide a primer pair for identifying said gene or nucleic acid.
The technical problem to be solved by the invention is to provide the application of the rice Wx mutant protein, nucleic acid or gene, primer pair, the expression cassette, recombinant vector or cell in the aspects of plant quality improvement, strain/variety identification, strain/variety creation and/or breeding.
The technical problem to be solved by the invention is to provide a breeding method for creating quality-improved rice with transparent appearance and properly reduced amylose content by using gene editing. The invention firstly utilizes the gene editing technology to edit the Wx gene of the rice variety, creates a new soft rice allele and rejects a T-DNA exogenous sequence to obtain a new material which has a transparent appearance, properly reduces the amylose content, improves the quality and can be stably inherited, generally only about 2 years are needed, and compared with chemical mutagenesis and conventional transformation breeding, the breeding period is at least shortened by 2-6 years. Therefore, the gene editing molecular breeding has the advantages of accuracy, high efficiency and the like which are not possessed by conventional breeding, and has wide application prospect.
The technical problem to be solved by the present invention is to provide a method for identifying plants obtained by said method.
The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows: a rice Wx mutant protein, wherein the amino acid sequence of the Wx mutant protein comprises the following mutations: which corresponds to the amino acid sequence of rice Wx, is mutated at amino acid 159 and/or at amino acid 178.
Specifically, the 159 th amino acid is mutated from glycine to lysine for the first time, and the 159 th amino acid has the quality improvement characteristics of transparent appearance, proper reduction of amylose content and the like. The 159 th amino acid mutation of the present invention may include 19 types of mutations such as glutamic acid, aspartic acid, tryptophan, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, serine, threonine, cysteine, methionine, asparagine, glutamine, arginine, histidine, and a stop codon. Whether other variations or premature termination of the amino acids described above affect granular starch synthase activity, physiological function, and quality improvement properties has yet to be confirmed by further studies.
Specifically, the invention reports for the first time that the 178-bit amino acid is mutated from threonine to isoleucine, and has the quality improvement characteristics of transparent appearance, proper reduction of amylose content and the like. The mutation at amino acid 178 of the present invention may further include 19 types of mutations such as glycine, lysine, glutamic acid, aspartic acid, tryptophan, alanine, valine, leucine, proline, phenylalanine, tyrosine, serine, cysteine, methionine, asparagine, glutamine, arginine, histidine, and a stop codon. Whether other variations or premature termination of the amino acids described above affect granular starch synthase activity, physiological function, and quality improvement properties has yet to be confirmed by further studies.
The rice Wx mutant protein comprises the following components:
(a) the amino acid sequence is shown as SEQ ID NO: 2 or SEQ ID NO: 4 is shown in the specification; or
(b) And (b) the protein which is derived from the protein (a) and has granular starch synthetase activity, wherein the amino acid sequence in the protein (a) is substituted and/or deleted and/or added with one or more amino acids.
The present disclosure also includes nucleic acids or genes encoding the mutant proteins.
Wherein the nucleic acid or gene comprises:
(a) encoding said mutant protein; or
(b) A nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) under stringent conditions and encodes a protein having granular starch synthase activity; or
(c) The nucleotide sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 3, respectively.
The invention also comprises a primer pair for identifying the gene or the nucleic acid, wherein the primer pair is Wx1 and/or Wx2 and/or Wx9 and/or Wx10, and the sequence of the primer pair Wx1 is shown as SEQ ID NO: 19 and SEQ ID NO: 20, the sequence of the primer pair Wx2 is shown as SEQ ID NO: 20 and SEQ ID NO: 21, the sequence of the primer pair Wx9 is shown as SEQ ID NO: 20 and SEQ ID NO: 28, the sequence of the primer pair Wx10 is shown as SEQ ID NO: 20 and SEQ ID NO: as shown at 29.
The present disclosure also includes expression cassettes, recombinant vectors or cells containing the nucleic acids or genes.
The invention also comprises the rice Wx mutant protein, nucleic acid or gene, and the application of the primer pair, the expression cassette, the recombinant vector or the cell in the aspects of plant quality improvement, strain/variety identification, strain variety creation and/or breeding.
The present invention also comprises a method for obtaining a quality-improved plant with a transparent appearance and a suitably reduced amylose content, comprising the following steps:
1) allowing the plant to comprise said nucleic acid or gene; or
2) Allowing the plant to express the rice Wx mutant protein.
The invention also provides a breeding method for creating the quality-improved rice with transparent appearance and properly reduced amylose content by using gene editing, which comprises the following steps:
1) wx gene cloning and target site design for gene editing
2) Constructing a single-base editing vector containing a target fragment;
3) the rice having the Wx mutant protein, or the nucleic acid or the gene has a transparent appearance and a suitably reduced amylose content.
4) Mutation of target Gene into T 0 T obtained by selfing transgenic plants 1 Foreign DNA (T-DNA) fragment elimination of the generation plant;
5) T-DNA-knocked-out T 1 And identifying the genotype of the generation plant and obtaining the target gene homozygous mutant plant.
Wherein, the construction method of the single-base editing vector containing the target fragment in the step 2) comprises the following steps:
A) preparing a target joint fragment: is connected withddH for primer 2 Dissolving O into mother liquor, mixing and diluting the complementary paired joint primers, performing high-temperature denaturation, transferring to room temperature, cooling, and finishing annealing to obtain a target joint fragment;
B) target adaptor fragment ligation to intermediate vector: carrying out enzyme digestion by using a pHUE411 intermediate vector and a restriction enzyme BsaI to obtain a BsaI single-enzyme digestion pHUE411 vector; adopting BsaI single enzyme-digested pHUE411 vector, the target joint fragment and T4 ligase to carry out ligation reaction, carrying out escherichia coli transformation, and sequencing to obtain an intermediate vector plasmid containing the target joint fragment.
C) Constructing a final vector: carrying out double enzyme digestion by adopting a pH-nCas9-PBE binary expression vector, restriction enzymes PmeI and AyrII to obtain a double enzyme digested pH-nCas9-PBE vector; carrying out double enzyme digestion on an intermediate vector containing a target joint fragment, PmeI and AvrII to obtain an sgRNA expression cassette containing the target joint fragment and a corresponding promoter; performing ligation reaction by using a double-enzyme-digested pH-nCas9-PBE vector, a sgRNA expression cassette and T4 ligase, performing escherichia coli transformation, and sequencing to obtain a single-base editing vector finally containing a target fragment.
Wherein, the quality-improved rice with transparent appearance and properly reduced amylose content in the step 3) is obtained by the following steps: transferring the single base editing vector containing the target segment obtained in the step 2) into agrobacterium EHA105 and carrying out genetic transformation to obtain T 0 Generation of transgenic plants, T obtained with primer pairs Wxb-PCR-F and Wxb-PCR-R 0 And (3) carrying out amplification and sequencing on the transgenic plant to identify the genotype so as to obtain the plant with the mutant protein, the nucleic acid or the gene.
Wherein the T-DNA fragment of the step 4) comprises hygromycin phosphotransferase gene HPT and nuclease gene nCas9, and the T-DNA fragment is deleted by mutating the target gene 0 T obtained by inbreeding transgenic plants 1 The HPT gene and nCas9 gene of the generation plant are simultaneously detected and repeated for many times, and T not carrying the two genes is obtained by screening 1 The generation single plant is the target plant.
Wherein the HPT gene detection method comprises mutating T with target gene 0 T obtained by selfing transgenic plants 1 The genome DNA of the generation plant is taken as a template, hyg283-F and hyg283-R are taken as primers to carry out PCR amplification, and meanwhile, the nCas9 gene detection method is realized by the T with the mutation of a target gene 0 T obtained by selfing transgenic plants 1 And (3) carrying out PCR amplification by taking the genome DNA of the generation plant as a template and nCas9-F and nCas9-R as primers, wherein the HPT gene and the nCas9 gene are not detected simultaneously, which indicates that the T-DNA is successfully eliminated.
Wherein, the step 5) of obtaining the target gene homozygous mutant plant is to remove the T-DNA obtained in the step 4) 1 And (3) taking genome DNA of the generation plant as a template, taking Wxb-PCR-F and Wxb-PCR-R as primers to carry out PCR amplification and sequencing, and screening and obtaining the plant homozygous for the mutant protein and the nucleic acid or the gene.
The present disclosure also includes a method of identifying a plant obtained by said method, comprising the steps of:
1) determining whether the amylose content of the plant is reduced; and/or
2) Determining whether the RVA spectrum viscosity index disintegration value of the plant rice is increased or not; and/or
3) Determining whether the RVA spectrum viscosity index recovery value of the plant rice is reduced or not; and/or
4) Determining whether the RVA spectrum viscosity index peak time of the plant rice is reduced or not; and/or
5) Determining whether the appearance of the plant polished rice is transparent.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1) the invention firstly utilizes the single base editing technology to edit the coding region of the Wx gene, and the coding region is screened at T through the offspring 2 The new material which eliminates T-DNA, has transparent appearance, properly reduces amylose content, improves quality and can be stably inherited can be obtained. Compared with breeding such as chemical mutagenesis, cross transformation and the like, the gene editing and directionally improving molecular breeding technology has the advantages of rapidness, accuracy, high efficiency and the like, utilizes gene function markers to select genotypes, or directly utilizes other mature rice varietiesThe method converts the background material and obtains the corresponding material, thereby greatly improving the breeding efficiency and greatly accelerating the breeding process.
2) The content of the amylose of the rice material obtained by the breeding of the gene editing technology is distributed between 9.8 +/-0.2-12.9 +/-0.1 percent, and is reduced by 1.5-4.6 percent compared with 14.4 +/-0.2 percent of the wild type, the reduction value of the content of the amylose is appropriate, the trend is obvious, the effect is obvious, and the palatability and the taste quality can be effectively improved.
3) The rice disintegration value (BDV) of the rice material obtained by breeding through the gene editing technology is distributed between 943cP and 1327cP, compared with 862cP of a wild type, the BDV is increased by a proper value and has an obvious trend; the rice recovery value (CSV) of the rice material obtained by the breeding of the gene editing technology is distributed between 1010cP and 1070cP, compared with 1250cP of a wild type, the CSV has a proper value reduction value and an obvious trend; the rice peak time (PeT) of the rice material obtained by the breeding of the gene editing technology is distributed between 5.87min and 6.13min, and compared with the wild type 6.2min, the PeT is reduced by a proper value and has an obvious trend. The rice RVA spectrum viscosity characteristic optimization effect represented by the 3 indexes is remarkable, and the quality of the cooking taste can be effectively improved.
4) The rice material obtained by breeding by the gene editing technology has good appearance transparency, and the polished rice shows a better transparent state as the wild type when the water content is 10.1%; the soft rice compared with Nanjing 9108 has a cloudy appearance and poor transparency.
5) The invention develops the specific distinction between the wild type (GG at 475- m6 AA at base positions 475-476) of the gene, Wx1 and Wx 2; based on the base variation of the wild type and the mutant at the 533-534 th site of the Wx gene, the method has been developed to specifically distinguish the wild type (the 533-534 th base of the Wx gene is CC) from the mutant (Wx) m10 Base 533-534 was TT) as molecular markers Wx9 and Wx 10. The molecular marker can be used for molecular marker-assisted selective breeding.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not indicated, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The background material selected by the invention is Su-Ming 118 (purchased from Jiangsu Su-Ming variety Limited company), which is a new late-maturing Zhongjing variety bred by grain crop research institute of agricultural academy of sciences of Jiangsu province, has a total growth period of about 155 days, is suitable for planting in Suzhong and Ningyangyang hilly areas of Jiangsu province, has excellent comprehensive agronomic characters, has been popularized and applied in large area in production, and is deeply popular in the market. The Su-cultivated 118 plants are compact in type, strong in tillering force, light green in leaves, good in group uniformity, good in lodging resistance, good in color change in the mature period, about 14% in amylose content, and transparent in rice appearance. The invention carries out fixed-point single base editing on the Wx gene of Su-Ke-Gu 118 by a single base gene editing technology based on CRISPR/Cas9, obtains a plurality of mutants which have transparent appearance, properly reduced amylose content, gradient distribution (soft rice) appearance and the like and improved cooking and taste quality, and meets the urgent needs of the market and the common people on high-quality soft fragrant rice varieties.
Example 1: obtaining genetically transformed plants
1. Su-reclamation 118Wx gene cloning and target site design
With reference to the CTAB method of Murray et al, genomic DNA of Sookland 118 was extracted (Murray M G, et al, Nucleic Acids Research, 1980, 8 (19): 4321-4326). With primers Wx-F: CCCTAGCCACCCAAGAAA (SEQ ID NO: 5), Wx-R: CACCCAGAAGAGTACAACATCA (SEQ ID NO: 6) was subjected to PCR amplification of the genomic DNA, and the amplification product was sent to Yinzi Weiji (Shanghai) trade laboratory Co., Ltd for sequencing. Sequencing results Blast comparison analysis is carried out in NCBI (https:// blast.ncbi.nlm.nih.gov/blast.cgi) database, and the Wx gene coding region sequence of Sutom 118 is found to be the same as that of the reference genome rice Nipponbare.
According to the Wx gene sequence of Suzhou 118, a target site wxb #9 was designed on exon 4 predicted by the CRISPR-GE website (http:// skl.scau.edu.cn/targettdign /): CGACTCCACGCTTGTAGCAA (SEQ ID NO: 7), design a target site wxb # 24: AAGACCGGTGAGAAGATCTA (SEQ ID NO: 8), the positions of the two target sites on the gene are shown schematically in FIG. 1. According to previous studies, it was shown (Liu LL, et a1., Plant Mol Biol, 2009, 71: 609-626), that the two target sites are both located in the core catalytic domain of the starch synthase encoding the protein, and that single base variation within the respective editing windows is expected to form new effective mutations, and rice mutants with transparent appearance and reduced amylose content in different degrees are obtained.
2. CRISPR/Cas9 gene editing vector construction
The gene editing vector construction is carried out according to the following steps by referring to the report method of Zong et al (Zong Y, et al, nat. Biotechnol, 2017, 35, 438-440):
(1) target joint preparation
According to the designed target site wxb #9 and wxb #24 sequences and respective reverse complementary sequences, after adding a linker sequence at the 5' end, 4 primers are synthesized by Nanjing Ongzhike Biotechnology Co., Ltd, and the specific sequences are as follows:
wxb#9-F:5’-ggcgCGACTCCACGCTTGTAGCAA-3’(SEQ ID NO:9):
wxb#9-R:5’-aaacTTGCTACAAGCGTGGAGTCG-3’(SEQ ID NO:10);
wxb#24-F:5’-ggcgAAGACCGGTGAGAAGATCTA-3’(SEQ ID NO:11);
wxb#24-R:5’-aaacTAGATCTTCTCACCGGTCTT-3’(SEQ ID NO:12);
adapter primers wxb #9-F and wxb #9-R were applied as ddH 2 Dissolving O into 100. mu.M mother liquor, adding 1. mu.l of each mother liquor into 98. mu.l of ddH 2 And mixing and diluting the O to 1 mu M, performing high-temperature denaturation, transferring to room temperature, and cooling to finish annealing to obtain the double-stranded target joint fragment wxb # 9-F/R.
Adapter primers wxb #24-F and wxb #24-R were applied as ddH 2 Dissolving O into 100. mu.M mother liquor, adding 1. mu.l of each mother liquor into 98. mu.l of ddH 2 And mixing and diluting the O to 1 mu M, performing high-temperature denaturation, transferring to room temperature, and cooling to finish annealing to obtain the double-stranded target joint fragment wxb # 24-F/R.
(2) Target point adaptor fragment connecting intermediate carrier
First, the intermediate vector pHUE411(Xing HL et a1., BMC Plant Biol, 2014, 14, 327) was digested with the restriction enzyme BsaI purchased from ThermoFisher in the following manner:
After 1h of digestion at 37 ℃, agarose gel electrophoresis and gel tapping are carried out, and the BsaI single enzyme digestion vector is recovered for standby by using an OMEGA DNA recovery kit.
And secondly, connecting the target joint fragment prepared by natural annealing in the previous step into the BsaI single-enzyme digested pHUE411 vector by adopting T4 ligase. T4 ligase was purchased from Takara, Japan, and the reaction system (10. mu.L) was as follows:
after brief centrifugation, the mixed system was subjected to a water bath at 16 ℃ for 4 hours. The ligation products were transformed into E.coli DH5a competent cells (Beijing Tiangen) by heat shock. The transformed cells of 1/20 were spread evenly on LB solid medium containing 100mg/L ampicillin. After culturing for 16h at 37 ℃, picking out the strain, and sending the strain to Nanjing Kingsrei Biotech company for sequencing. The correctly inserted and sequenced plasmids were obtained and named pHUE411-wxb #9-F/R and pHUE411-wxb #24-F/R, respectively.
(3) Final vector construction
The final vector pH-nCas9-PBE is a plant binary expression vector, which is developed by the Highenxia researchers team of the institute of genetics and developmental biology of the Chinese academy of sciences (Zong Y, et al, nat. Biotechnol, 2017, 35, 438-440), firstly, the vector is subjected to double enzyme digestion by restriction enzymes PmeI and AvrII, the restriction enzymes used are purchased from ThermoFisher, and the enzyme digestion system is as follows:
After 1h of cleavage at 37 ℃, agarose gel electrophoresis and gel cutting were carried out, and the vector after double cleavage of PmeI and AvrII was recovered and used by using an OMEGA DNA recovery kit.
Secondly, the pHUE411-wxb #9-F/R and pHUE411-wxb #24-F/R vectors obtained in the previous step are subjected to double digestion by restriction enzymes PmeI and AvrII respectively, the restriction enzymes used are purchased from ThermoFisher, and the digestion system is as follows:
after 1h of enzyme digestion at 37 ℃, agarose gel electrophoresis and gel tapping are carried out, and the sgRNA expression cassettes of original fragments of OsU3-wxb #9-F/R or OsU3-wxb #24-F/R are obtained by carrying out double enzyme digestion on PmeI and AvrII by using an OMEGA DNA recovery kit and are respectively recovered and reserved.
Finally, the above recovered original fragment was ligated into pH-nCas9-PBE after PmeI and AvrII double digestion by T4 ligase, T4 ligase was purchased from Takara, Japan, and the reaction system (10. mu.L) was as follows:
after a short centrifugation, the mixed system is bathed in water at 16 ℃ for 4 h. The ligation product was transformed into E.coli DH 5. alpha. competent cells (Beijing Tiangen) by heat shock. All the transformed cells were spread evenly on LB solid medium containing 50mg/L kanamycin. After culturing at 37 ℃ for 16h, colonies were picked and sequenced by Nanjing Kingsry Biotech, using sequencing common primer M13-F. The obtained correctly sequenced plasmids were named pH-nCas9-PBE-wxb #9 and pH-nCas9-PBE-wxb #24, respectively.
3. Acquisition of genetically transformed plants and genotyping
The 2 plasmids were transformed into Agrobacterium EHA105, respectively. Rice reclamation 118 (purchased from Jiangsu Su reclamation cultivars, Ltd.) was transformed by a conventional Agrobacterium-mediated method. Obtaining positive T transferred into pH-nCas9-PBE-wxb #9 0 Generating 8 plants to obtain positive T transferred into pH-nCas9-PBE-wxb #24 0 6 plants are generated. To identify the base variation of the editing target site, T obtained as described above was extracted 0 Generating transgenic line leaf genome DNA, and performing PCR by using a primer Wxb-PCR-F: CAAGCAGCAGCGGTCGG (SEQ ID NO: 13) and Wxb-PCR-R: TTGAAGTATGGGTTGTTGTTGAGG (SEQ ID NO: 14), and the amplification product was sent to Weiji (Shanghai) trade Co., Ltd for sequencing.
The sequencing result shows that: positive T of transfer pH-nCas9-PBE-wxb #9 0 The identification of the corresponding target sites of the generation plants (parts) is shown in FIG. 2A, and the strain number B1-68 is 4The GG mutation from 75 to 476 is homozygous mutation of AA; the strain with the number of B2-42 is a double allelic mutation in which 475-; the strain numbered B6-29 was homozygous for mutation of 475-476 GG to AA.
Positive T of pH-nCas9-PBE-wxb #24 0 The identification condition of the corresponding target sites of the generation plants (parts) is shown in FIG. 2B, the strain numbered B2-21 is a double allelic mutation of which the 533-534 site CC mutation in one allelic gene is TT and the 533-534 site CC mutation in the other allelic gene is GT.
Example 2: mutant exogenous DNA (T-DNA) knockout, genotype re-identification and screening of homozygous plants
The pH-nCas9-PBE-wxb #9 and pH-nCas9-PBE-wxb #24 vectors of the directionally-precise single-base editing Wx gene constructed by the invention are binary T-DNA vectors, the T-DNA related by the invention mainly comprises a hygromycin phosphotransferase HPT gene and an nCas9 nuclease gene, and for a rice genome, exogenous DNA represented by the two genes needs to be removed for the following reasons: 1) the hygromycin phosphotransferase HPT gene mainly serves as a screening marker in the genetic transformation process, and the correspondingly coded hygromycin protein is a type of antibiotic; 2) the nCas9 gene has the main function of completing the site-specific cutting of a target gene target site, and secondary editing possibly caused by continuously remaining in a plant; 3) random insertion of T-DNA may also cause unexpected gene mutation, which is not favorable for stable character; 4) the social sensitivity of the transgene problem is high, and the public acceptance degree of T-DNA knockout materials is high.
In the process of agrobacterium-mediated transformation of Sutom 118, the T-DNA sequence can be randomly inserted into chromosome in a single copy or multiple copy form, and because rice is a relatively strict self-pollinated homologous diploid plant, and the T-DNA insertion site is generally not linked with the target site thereof, on the technical level, a plant not carrying T-DNA can be obtained by separation and identification in the later generation generated by transgenic plant selfing, and even if linkage, a material not carrying T-DNA can be screened by genetic exchange recombination. In order to obtain such plants which do not carry T-DNA, the present inventionA total of 4 strains of T were obtained by comparing B1-68, B2-42, B6-29 and B2-21 identified in example 1 1 Carrying out PCR detection on HPT gene and nCas9 gene of generation plant at the same time, repeating for 3 times, and screening T not carrying the two genes, namely T which is regarded as rejecting T-DNA 1 Generation individual, in which the primer hyg 283-F: TCCGGAAGTGCTTGACATT (SEQ ID NO: 15) and hyg 283-R: GTCGTCCATCACAGTTTGC (SEQ ID NO: 16) by PCR amplification of the HPT gene; with primer nCas 9-F: CGGCTACGCTGGGTACATC (SEQ ID NO: 17) and nCas 9-R: TTCTCGTTGGGCAGGTTCTT (SEQ ID NO: 18) was used to PCR amplify the nCas9 gene. Furthermore, during the addition process in the presence of the above-mentioned nCas9 protein (e.g. T in the present invention) 0 -T 1 Generations), the possible case of alteration of the type of mutation by secondary editing, requires a further genotype identification in the single strain or progeny of the T-DNA knockout (using methods and primers as described in example 1) to confirm that the mutant genotype has not been secondarily edited and is stably inherited. Each individual was specifically implemented as follows:
the B1-68 strain identified in example 1 was grown in an incubator and selfed to obtain T 1 And (5) seed generation. 107 seeds (T) were selected 1 Generation) to break dormancy, soak seeds, sowing in seedling trays and placing in a culture room, when the seedlings grow to have two leaves and one core, taking leaves to extract DNA, carrying out PCR detection by using the Hpt gene coding hygromycin and the nCas9 gene coding nCas9 nuclease (the method and the used primers are shown above in the embodiment), repeating the experiment for three times, and carrying out co-screening to obtain 10 single plants which can not be amplified to a target fragment, namely the single plants are regarded as T-DNA knockout single plants, wherein the proportion is 9.35%, and the T-DNA is supposed to be inserted into the rice genome in a multi-copy mode. Selecting one strain and naming it as 118-1-1, tracking the editing situation of the single target site (the genotype identification method and primers are shown in example 1), the result is shown in FIG. 3A, 118-1-1 is 475- 0 ) Consistent, indicating no secondary editing occurred. The single plant is planted in an incubator and selfed to obtain T 2 Generation of seed, still continuing the 118-9-15 nomenclature, wait for T 2 After breaking the dormancy of the seeds, soaking the seeds, sowing and planting the seeds in an incubator to verify the carrying condition of the T-DNAThe experiment is repeated three times, and the same previous generation is found, which shows that the strain T 1 -T 2 The generation did have knocked out T-DNA (FIG. 4); furthermore, the single plant was followed for target site editing and the same generation was found, indicating that the expression was determined from T o -T 2 The generation mutation genotype is stably inherited, namely GG at 475-476 th site of the Wx gene is mutated into AA, so that 159 th site amino acid is mutated into lysine from glycine. The nucleotide sequence of the Wx gene of the 118-1-1 mutant is shown as SEQ ID NO.1, the amino acid sequence of the encoded Wx protein is shown as SEQ ID NO.2, and the new allele obtained by cloning is named as Wx m6 。
The same method and steps are used for carrying out generation addition, deletion T-DNA and genotype re-identification to obtain T of B2-42 2 T of generation-stable, homozygous mutant line 118-3-2, B6-29 2 The generation-stable and homozygous mutant line 118-5-1. 118-3-2 and 118-5-1 are both T-DNA knockout lines (FIG. 4), the mutation types of target sites are 475- m6 The genes are the same, the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence of the encoded Wx protein is shown as SEQ ID NO. 2. Furthermore, T of B2-21 was obtained 2 The generation-stable homozygous mutant line 118-9-15 is also a T-DNA knockout line (FIG. 4), and the mutation type of the target site mutation is 533-534 base CC to TT homozygous mutation, which results in the mutation of amino acid 178 from threonine to isoleucine. The nucleotide sequence of Wx gene of 118-9-15 mutant is shown as SEQ ID NO.3, the amino acid sequence of Wx protein coded by the mutant is shown as SEQ ID NO.4, and the cloned new allele is named as Wx m10 。
Wx identified by the invention m6 The 475-476 site GG of the gene is mutated into AA, and the 159 site amino acid is mutated into the lysine mutation from glycine, Wx m10 The mutation of the 533-534 th base CC to TT and the resulting mutation of the 178 th amino acid from threonine to isoleucine are reported for the first time.
Meanwhile, the invention firstly utilizes the gene editing technology to carry out the Wx gene editing on the rice varietyDue to single base editing, a new material which is stable in heredity and can remove T-DNA can be obtained within 2 years, and the new material specifically comprises the following components: construction of Wx Gene Single-base editing vector and genetic transformation of conventional japonica Rice (Su-reclaimed 118 in this example) for 4 months, T 0 Genotype identification of generation plant, 5 months of plant growth and fruiting, T 1 The generation plant eliminates T-DNA, the homozygous genotype is screened, and the plant grows and fruit for 5 months, T 2 The generation plant T-DNA detection, the homozygous genotype identification, the plant growth and the plant fruit are carried out for 5 months, and only about 19 months are needed in total, so that the new material (namely the T-DNA in the embodiment) which has the target genotype, is stable in heredity, eliminates the T-DNA and is propagated by adding generations can be finally obtained 3 Generations 118-1-1, 118-3-2, 118-5-1, and 118-9-15). Therefore, compared with chemical mutagenesis and conventional transformation breeding, the technology can accurately and efficiently create new soft rice alleles, and the breeding period of obtaining a new homozygous material is at least shortened by 4-6 years.
Example 3 phenotypic analysis of mutants
The Cooking and Cooking Quality (ECQ) is a direct factor influencing the selection of consumers, and is the most important evaluation index in the rice Quality constitution. Although China has gone out of the national standard of sensory evaluation method for rice cooking edible quality (GB/T15682-2008), the rice quality cannot be accurately identified by a manual tasting mode because the influence of subjective factors cannot be completely eliminated. Since starch is a major component of rice endosperm, the composition and structure of starch are the most important factors affecting rice ECQ. This example utilizes the 4T's obtained in example 2 2 Seed (T) from homozygous mutants 118-1-1, 118-3-2, 118-5-1 and 118-9-15 with a knockout generation of T-DNA 3 Generation) were performed to determine some physicochemical indices of starch, and thus rice ECQ was objectively evaluated.
1. Determination of amylose content
The previous researches show that AC is an important character for determining the quality of the cooked taste of rice, is not only related to the viscosity and the softness of the rice, but also is closely related to a plurality of starch physicochemical parameters such as viscosity, gelatinization and retrogradation. The AC assay was carried out with reference to the Ministry of agriculture release standard NY147-88, 4 reference standard samples (AC: 1.5%, 10.6%, 16.4% and 25.6%) purchased from the Rice research institute of China. As shown in FIG. 5, the results are 12.9. + -. 0.1% for 118-1-1, 11.9. + -. 0.1% for 118-3-2, 11.9. + -. 0.1% for 118-5-1 and 9.8. + -. 0.2% for 118-9-15. Compared with 14.4 +/-0.2% of the wild type, the AC of 118-1-1, 118-3-2 and 118-5-1 with the same genotype is reduced by 1.5-2.5%, and the AC of 118-9-15 is reduced by 4.6%.
2. Rice RVA spectrum viscosity determination
There is a close relationship between the RVA profile characteristic of rice and the quality of cooked flavour of rice, measured using a rapid viscosity analyzer (RVA Super 4, NEWPORT SCIENTIFIC, Australia) with parameter settings according to the American Association of cereals AACC61-01 and 61-02 protocols. Previous studies have shown that rice with good taste quality tends to have a large disintegration value (BDV), rice with relatively non-retrogradation of the chills tends to have a small recovery value (CSV), and in addition, peak time PeT refers to the time taken for the sample to reach peak viscosity, generally the smaller the PeT, the better the expansion and breakage of the starch. Results Table 1 shows that BDV of 118-1-1, 118-3-2, 118-5-1 and 118-9-15 are 943cP, 946cP, 1128cP and 1324cP, respectively, which are all significantly larger than 862cP of wild type; the CSV of 118-1-1, 118-3-2, 118-5-1 and 118-9-15 are 1070cP, 1049cP, 1067cP and 1010cP, respectively, all significantly less than 1250cP of wild type; PeT of 118-1-1, 118-3-2, 118-5-1 and 118-9-15 is respectively 6.13min, 6.07min, 6min and 5.87min, which are all obviously smaller than 6.2min of the wild type. This data shows that the 4 mutants have better taste quality compared to the wild type, the cold meal is relatively non-retrograded, and the starch granules have better bulking and breakage during cooking. The overall variation of the characteristic values of the RVA is shown in figure 6A.
The data were analyzed for correlation in a step-by-step manner, and it was shown that BDV and AC showed very significant negative correlation, CSV and AC showed very significant positive correlation, and PeT and AC showed very significant positive correlation in each sample (fig. 6B).
TABLE 1 RVA spectral characteristics
3. Thermodynamic characteristics
The rice gelatinization and the quality of the cooked food flavor have a close relationship, the rice gelatinization with high gelatinization temperature needs higher cooking temperature, and thermodynamic parameters measured by a DSC instrument can accurately reflect the gelatinization characteristics of the rice. The thermodynamic properties of the starch granules were analyzed using a differential heating value scanner DSC (Q2000, TAInstructions, USA). As shown in Table 2, the temperatures To, Tp at each gelatinization stage were maximum values in wild-type WT, Tc was maximum value in 118-1-1, and To, Tp and Tc were minimum values in 118-3-2, but the overall difference was not large; the enthalpy of gelatinization Δ Hgel is greatest in 118-9-15 and smallest in WT, with no major difference remaining. The above results indicate that the gelatinization characteristics are random among the tested samples, and the gelatinization temperature of 118-1-1 is the largest and consistent with PaT measured by RVA instrument.
TABLE 2 thermodynamic Properties
4. Polished rice transparency observation
The transparency is one of the main indexes for measuring the appearance quality of rice, and the invention utilizes the wild type threo reclamation 118 (non-soft rice, transparent) and the Nanjing 9108 (Wx) mp Genotype soft rice, cloud) polished rice was used as a control, and the appearance of the polished rice was observed. Rice is hulled by a huller (SY88-TH, Korean Bilong) to obtain coarse rice, refined by a small-sized refined rice machine (BLH-3120, Burley constant, Taizhou) to obtain refined rice, and water content of the refined rice is measured by a water analyzer (Mettler, Switzerland) to ensure consistent water content of the tested sample. As a result, as shown in fig. 7, the rice of sovereign 118 showed transparency even at a water content of 10.1%, but the southern japonica 9108 showed cloudiness and poor transparency; the 4 mutants showed the same behavior as those of the reclamation of Suzhou 118 and still showed a transparent state. Indicating that the gene contains two new alleles (Wx) m6 ,Wx m10 ) Although the amylose content of the 4 mutants is reduced to different degrees, particularly the amylose content of the mutant 118-9-15AC is reduced to 9.8 percent, the mutants still have appearanceBut can maintain better transparency.
Example 4 Co-separation and preliminary functional analysis
1. Coseparation analysis
To verify that the AC reduction phenotype in the mutant is due to a mutation in the gene of interest, a co-segregation analysis was performed genetically. Using T obtained in example 2 2 Homozygous mutant 118-5-1(118-1-1, 118-3-2 and 118-5-1 with the same genotype and 118-5-1 as representative) and 118-9-15 plants with generation T-DNA knockout are respectively hybridized with high-generation stable breeding material 93042(AC 15.6 +/-0.2 percent), F 1 After selfing, 112 and 131F were obtained, respectively 2 Individuals were screened for genotype, the methods and primers used for genotype identification were as described in example 1, and then wild-type and homozygous mutant genotype individuals were selected for phenotypic analysis (with AC as a representative index), and the AC test method was as described in example 3.
The results showed that 118F were created in 118-5-1 and 93042 crosses 2 In the individual plants, 35 individual plants of wild type genotypes are obtained, and all ACs are more than 14%; obtaining homozygous Wx m6 23 individuals with mutant genotypes, all ACs<12% indicating Wx m6 And the AC-reduced phenotype.
131F created by hybridization between 118-9-15 and 93042 2 31 individuals of wild-type genotypes are obtained from the individual plants, and all ACs are more than 14%; obtaining homozygous Wx 10 24 individuals with mutant genotypes, all ACs<12% indicating Wx m10 And the AC-reduced phenotype.
The above results confirm that the AC reduction phenotype in the mutant is indeed due to mutation of the gene of interest.
2. Wild type Wx and Wx m6 、Wx m10 Detection of mutein Activity and correlation analysis with AC
As described above, the rice Wx gene is the major gene controlling amylose synthesis and encodes granular starch synthase. Generally, Wx enzyme activity is positively correlated with AC, and a material with reduced AC will have a corresponding decrease in enzyme activity. To verify this hypothesis, the enzymatic activity was carried out according to the method of the predecessor Detection (Liu DR et al, Plant Sci, 2013, 210, 141-150). As shown in FIG. 8A, the wild type Wx protease activity was the highest, 1011.3. + -. 80.3nmol/min/g, and the enzymatic activity of the mutant proteins was significantly reduced, among which Wx in 118-1-1, 118-3-2 and 118-5-1 m6 The activity of the compounds is 712.1 + -54.1, 688.3 + -40.0 and 913.3 + -63.0 nmol/min/g, respectively, Wx in 118-9-15 m10 The activity of (A) was shown to be the lowest, 439.4. + -. 30.1 nmol/min/g.
A step correlation analysis of the data indicated that Wx enzyme activity and AC appeared to be very significantly positively correlated in each sample (fig. 8B).
3. Three-dimensional structure prediction
Wild type, Wx, were paired using an online website (http:// www.sbg.bio.ic.ac.uk/phyre2/) m6 And Wx m10 The three-dimensional structure of the mutein is predicted. Wx compared to wild-type protein m6 Mutation of glycine to lysine at position 159 in the mutein did not alter the beta turn conformation at this site (FIGS. 9A-B); wx compared to wild-type protein m10 The mutation of threonine 178 to isoleucine in the mutein did not change the random coil conformation at this site (FIGS. 9C-D). The above results indicate that the phenotype of reduced mutant AC, reduced mutant protease activity is not due to conformational changes in the secondary structure of the protein caused by single amino acid variation. Presumably, the cause of the phenotype is: 1)159 and 178 amino acids are positioned in the core catalytic domain of the starch synthetase from 84 to 345, and the mutation affects the catalytic capability of the protein; 2) the 159 th and 178 th amino acids are close to the 33-36 th (Lys-Ser-Gly-Gly) amino acid residue, and the site is considered as a binding site of the ADP-glucose during the synthesis of starch and glycogen, and the mutation can cause the reduction of the binding capacity of the binding site; 3) the 159 and 178 amino acids may be phosphorylation sites, and the mutation causes a decrease in phosphorylation ability, resulting in a decrease in enzyme activity.
Example 5: application of Wx mutant protein and gene thereof in rice breeding
1、Wx m6 And Wx m10 Functional marker development and application
Molecular marker assisted selection is advantageously providedAnd (5) the breeding process is fast. Wx of the invention m6 And Wx m10 The gene is continuous two base mutations, enzyme digestion target point markers can be designed in a targeted manner, but the process is relatively complicated, allele specific PCR is developed, genotypes with high/low amylose content can be distinguished through two PCR, and the operation is simple, convenient and quick. The invention aims at the wild type and the mutant in Wx m6 Base variation at the 475-476 th site of the gene, wild type and mutant in Wx m10 The base variation of the 533-fold 534 locus of the gene utilizes the allele specific PCR principle to design 6 groups of primers (Table 3): against wild type and Wx m6 Wx 1-Wx 6 of genes against wild type and Wx m10 Wx 7-Wx 12 of the gene. Wherein Wx 1-Wx 12 share a downstream primer Wx-1R, and the upstream primers are respectively Wx-1F-Wx-12F. In order to further improve the specificity of the primer, base mismatch is introduced at the 3' end of the partial primer: the third last base at the 3' end of the Wx-3F and Wx-4F primers is mismatched into G from T; the third last base at the 3' end of the primers Wx-5F and Wx-6F is mismatched into C by T; the penultimate base at the 3' end of the primers Wx-13F and Wx-14F is mismatched into G from A; the penultimate base at the 3' end of the primers Wx-15F and Wx-16F is mismatched from A to C.
Through multiple rounds of screening and optimization of PCR reaction conditions, as shown in FIG. 10, it is found that the primer pairs Wx1 and Wx2 both have good amplification efficiency and specificity, and can be used for distinguishing wild type Wx from mutant Wx m6 Primer pairs for genotypes and heterozygous genotypes; the primer pair Wx9 and Wx10 both have good amplification efficiency and specificity, and can be used for distinguishing wild type Wx from mutant Wx m10 Genotype and heterozygous genotype primer pairs. The optimal reaction system for PCR is: 1 μ L of wild type or mutant DNA template, 7.5 μ L of 2 XPCR Taq enzyme mix, 0.75 μ L of upstream primer (2pM), 0.75 μ L of downstream primer (2pM), ddH 2 O5. mu.L. The PCR reaction program is 94 ℃ for 5 min; 30s at 94 ℃, 30s at 56 ℃, 30s at 72 ℃ and 33 cycles; 5min at 72 ℃; storing at 4 ℃.
TABLE 3 molecular markers for detection of mutant genes
Note: the bases indicated by lower case letters are mismatched bases.
The rice variety is detected by using Wx1+ Wx2 and Wx9+ Wx10 markers. As shown in FIGS. 11A-B, it was found that 118-1-1, 118-3-2 and 118-5-1 were not band-amplified by Wx1 but band-amplified by Wx2 in the test sample, and wild type Sokoku 118, 118-9-15 and the remaining japonica or indica varieties (Nippon, Nanjing 9108, Huanghuazhan, 9311, Huai rice No. 5, Henong Jing No. 8, Nanjing 51, Neizu No. 7, Suxiu 867, Wu Yu Jing No. 27, Wu Yu Jing No. 29, Xu Rice No. 8, Xu Rice No. 9, and Yan Rice No. 16) were band-amplified by Wx1 but not band-amplified by Wx2, indicating that Wx1+ Wx2 were band-specific for detecting Wx m6 The variation of GG to AA at the 475-476 bases of the gene can be used for molecular marker-assisted selection breeding.
Furthermore, as shown in FIGS. 11C-D, 118-9-15 could not be band-amplified by Wx9 but could be band-amplified by Wx10, and wild type Suzhou reclamation 118, 118-1-1, 118-3-2, 118-5-1 and the rest of japonica or indica varieties could be band-amplified by Wx9 but could not be band-amplified by Wx 10. It shows that Wx9+ Wx10 can specifically detect Wx m10 Mutation of the 533-534 bases of the gene from CC to TT can be used for molecular marker-assisted selective breeding.
To validate the application of Wx1+ Wx2, Wx9+ Wx10 in sub-marker assisted selection breeding, 112 Fs created by 118-5-1 and 93042 crosses obtained in example 4 using Wx1+ Wx2 2 The results of the tests carried out on the individuals showed that the genotype detected and resolved by the marker was completely matched with the genotype detected by the sequencing method (see example 1) (including 35 wild-type individuals, 60 heterozygous individuals and 23 homozygous Wx) m6 Mutant individuals). Furthermore, 131F s created by hybridization of 18-9-15 and 93042 obtained in example 4 using Wx9+ Wx10 2 The detection was carried out in individuals and the results showed that the genotype detected and resolved using this marker was completely matched to the genotype detected using the sequencing method (see example 1) (31 wild-type individuals, 76 heterozygous individuals and 24 homozygous Wx) m10 Mutant individuals). The above results show thatThe markers Wx1+ Wx2 and Wx9+ Wx10 developed by the invention can be used for precise breeding of quality improvement breeding, and F is 2 The generation screening is carried out on homozygous genotypes with transparent appearance, reduced AC and improved taste quality, and further early generation selection can be carried out.
2. Directly uses the rice variety with excellent agronomic characters as background material to carry out transformation
Jinjing 818 is a conventional japonica rice variety bred by the rice institute in Tianjin, and is planted in Jingjin Tang japonica rice area. The variety is a non-soft rice variety with about 16% of AC, and the AC of the variety can be reduced by using the gene editing method described by the invention, and the taste quality can be improved on the premise of not influencing the appearance quality. The actual Jinjing 818 is from the germplasm resource platform of agricultural science institute of Jiangsu province.
1) The vector, transformation method and genotype identification method used are described in example 1. Finally, positive T transferred into pH-nCas9-PBE-wxb #9 is obtained 0 Generating 9 plants to obtain positive T transferred into pH-nCas9-PBE-wxb #24 0 6 plants are generated.
2) Mutant T-DNA knock-out, homozygous addition and re-genotype identification methods are described in example 2. As shown in FIGS. 12A-B, screening to obtain homozygous Wx m6 Mutant type, T-DNA knockout T 2 The generation lines are 5: 818-4-13, 818-5-8, 818-6-8, 818-10-2 and 818-4-15; obtaining homozygous Wx m10 Mutant type, T-DNA knockout T 2 1 generation line: 818-9-7.
3) And (4) performing phenotype identification on the mutant. AC. RVA viscosity measurements and polished rice transparency observations are given in example 3. As in fig. 12C, wild type AC was 14.1 ± 0.3% maximum, and each mutant was significantly reduced, with 818-9-7 being 7.6 ± 0.1% minimum. As in fig. 12D, the wild-type BDV had a minimum of 975cP, with each mutant significantly elevated, with 818-9-7 up to 1540 cP. As in FIG. 12E, the wild type CSV was at maximum 1295cP, and each mutant was significantly reduced, with 818-9-7 at minimum 912 cP. As in fig. 12F, wild-type PeT was 6.27min maximum, and each mutant was significantly reduced, with 818-9-7 min minimum of 5.6 min. As shown in fig. 12G, when the water content is 13.5%, the Jing 818 polished rice still appears transparent, but the Nanjing 9108 appears cloudy and has poor transparency; the 6 mutants showed the same appearance as jin-jing 818 and still showed a transparent state.
4) The enzyme activity was measured as described in example 4. The results are shown in FIG. 12H, where the activity of wild-type Wx protease is highest, 788.92 + -16.53 nmol/min/g, and the enzymatic activity of the mutant protein is significantly reduced, among which Wx in 818-9-7 m10 The activity of (D) was minimal, being 407. + -. 30.01 nmol/min/g.
The results show that Wx is directly created in the Jinjing 818 background m6 And Wx m10 The mutant can reproduce target excellent characters and quickly obtain related plant materials.
The above embodiments show that the Wx gene is single base edited by gene editing technique, and then screened in T 2 The generation can obtain a new material which eliminates T-DNA, reduces AC and improves the taste quality without influencing the appearance quality and has stable heredity. Compared with breeding means such as chemical mutagenesis, hybridization and matching and the like, the method for directionally improving the molecular breeding technology by gene editing has the advantages of rapidness, accuracy, high efficiency and the like, and by combining gene function marker genotype selection or directly using other mature rice varieties as background materials for transformation and obtaining corresponding materials, the breeding efficiency can be greatly improved, and the breeding process is greatly accelerated (Table 4).
TABLE 4 comparison of Gene editing Breeding methods with conventional breeding methods
Sequence listing
<110> institute of genetics and developmental biology, academy of agricultural sciences, academy of Chinese sciences, Jiangsu province
<120> Wx mutant protein based on gene editing technology and application of gene thereof in plant breeding
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gccggcggcg acgcgacgtc gctcagcgtg acgaccagcg cgcgcgcgac gcccaagcag 180
cagcggtcgg tgcagcgtgg cagccggagg ttcccctccg tcgtcgtgta cgccaccggc 240
gccggcatga acgtcgtgtt cgtcggcgcc gagatggccc cctggagcaa gaccggcggc 300
ctcggtgacg tcctcggtgg cctcccccct gccatggctg cgaatggcca cagggtcatg 360
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gcgagctacc tgaagaacaa ctaccagccc aatggcatct acaggaatgc aaaggttgct 780
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<212> PRT
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Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser
85 90 95
Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met
100 105 110
Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln
115 120 125
Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala Glu Ile Lys Val Ala
130 135 140
Asp Arg Tyr Glu Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val
145 150 155 160
Asp Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val Trp Gly
165 170 175
Lys Ile Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys
180 185 190
Asp Asn Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala
195 200 205
Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr
210 215 220
Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu
225 230 235 240
Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile Tyr Arg Asn
245 250 255
Ala Lys Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe
260 265 270
Ala Phe Glu Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser
275 280 285
Ser Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys
290 295 300
Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr
305 310 315 320
Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly
325 330 335
Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val
340 345 350
Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr Ile
355 360 365
Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys Ala Leu Asn
370 375 380
Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile
385 390 395 400
Pro Leu Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp
405 410 415
Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile
420 425 430
Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser
435 440 445
Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn
450 455 460
Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp Val Leu Ala Val
465 470 475 480
Pro Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg
485 490 495
Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr
500 505 510
Val Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp
515 520 525
Cys Lys Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr Leu
530 535 540
Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val
545 550 555 560
Arg Asn Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn
565 570 575
Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly
580 585 590
Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala
595 600 605
Pro
<210> 5
<211> 18
<212> DNA
<213> Wx-F(Artificial Sequence)
<400> 5
ccctagccac ccaagaaa 18
<210> 6
<211> 22
<212> DNA
<213> Wx-F(Artificial Sequence)
<400> 6
cacccagaag agtacaacat ca 22
<210> 7
<211> 20
<212> DNA
<213> wxb#9(Artificial Sequence)
<400> 7
cgactccacg cttgtagcaa 20
<210> 8
<211> 20
<212> DNA
<213> wxb#24(Artificial Sequence)
<400> 8
aagaccggtg agaagatcta 20
<210> 9
<211> 24
<212> DNA
<213> wxb#9-F(Artificial Sequence)
<400> 9
ggcgcgactc cacgcttgta gcaa 24
<210> 10
<211> 24
<212> DNA
<213> wxb#9-R(Artificial Sequence)
<400> 10
aaacttgcta caagcgtgga gtcg 24
<210> 11
<211> 24
<212> DNA
<213> wxb#24-F(Artificial Sequence)
<400> 11
ggcgaagacc ggtgagaaga tcta 24
<210> 12
<211> 24
<212> DNA
<213> wxb#24-R(Artificial Sequence)
<400> 12
aaactagatc ttctcaccgg tctt 24
<210> 13
<211> 17
<212> DNA
<213> Wxb-PCR-F(Artificial Sequence)
<400> 13
caagcagcag cggtcgg 17
<210> 14
<211> 24
<212> DNA
<213> Wxb-PCR-R(Artificial Sequence)
<400> 14
ttgaagtatg ggttgttgtt gagg 24
<210> 15
<211> 19
<212> DNA
<213> hyg283-F(Artificial Sequence)
<400> 15
tccggaagtg cttgacatt 19
<210> 16
<211> 19
<212> DNA
<213> hyg283-R(Artificial Sequence)
<400> 16
gtcgtccatc acagtttgc 19
<210> 17
<211> 19
<212> DNA
<213> nCas9-F(Artificial Sequence)
<400> 17
cggctacgct gggtacatc 19
<210> 18
<211> 20
<212> DNA
<213> nCas9-R(Artificial Sequence)
<400> 18
ttctcgttgg gcaggttctt 20
<210> 19
<211> 20
<212> DNA
<213> Wx-1F(Artificial Sequence)
<400> 19
ttccattgct acaagcgtgg 20
<210> 20
<211> 21
<212> DNA
<213> Wx-1R(Artificial Sequence)
<400> 20
tcatcatgga ttccttcgaa g 21
<210> 21
<211> 20
<212> DNA
<213> Wx-2F(Artificial Sequence)
<400> 21
ttccattgct acaagcgtaa 20
<210> 22
<211> 20
<212> DNA
<213> Wx-3F(Artificial Sequence)
<400> 22
ttccattgct acaagcgggg 20
<210> 23
<211> 20
<212> DNA
<213> Wx-4F(Artificial Sequence)
<400> 23
ttccattgct acaagcggaa 20
<210> 24
<211> 20
<212> DNA
<213> Wx-5F(Artificial Sequence)
<400> 24
ttccattgct acaagcgcgg 20
<210> 25
<211> 20
<212> DNA
<213> Wx-6F(Artificial Sequence)
<400> 25
ttccattgct acaagcgcaa 20
<210> 26
<211> 22
<212> DNA
<213> Wx-7F(Artificial Sequence)
<400> 26
atttcaggtt tggggaaaga cc 22
<210> 27
<211> 22
<212> DNA
<213> Wx-8F(Artificial Sequence)
<400> 27
atttcaggtt tggggaaaga tt 22
<210> 28
<211> 22
<212> DNA
<213> Wx-9F(Artificial Sequence)
<400> 28
atttcaggtt tggggaaagg cc 22
<210> 29
<211> 22
<212> DNA
<213> Wx-10F(Artificial Sequence)
<400> 29
atttcaggtt tggggaaagg tt 22
<210> 30
<211> 22
<212> DNA
<213> Wx-11F(Artificial Sequence)
<400> 30
atttcaggtt tggggaaagc cc 22
<210> 31
<211> 22
<212> DNA
<213> Wx-12F(Artificial Sequence)
<400> 31
atttcaggtt tggggaaagc tt 22