CN114891759A - Wx mutant protein based on gene editing technology and application of gene thereof in plant breeding - Google Patents

Wx mutant protein based on gene editing technology and application of gene thereof in plant breeding Download PDF

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CN114891759A
CN114891759A CN202210501884.3A CN202210501884A CN114891759A CN 114891759 A CN114891759 A CN 114891759A CN 202210501884 A CN202210501884 A CN 202210501884A CN 114891759 A CN114891759 A CN 114891759A
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杨杰
高彩霞
许扬
王芳权
张瑞
陈智慧
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Institute of Genetics and Developmental Biology of CAS
Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention discloses a rice Wx mutant protein, a mutant gene and application thereof, and also discloses a breeding method for creating quality-improved rice with transparent appearance and properly reduced amylose content by using gene editing. The invention utilizes gene editing technology for the first timeWxEditing gene with single base, screening the progeny, and determining the presence of T 2 The product can be obtained by eliminating T-DNA, has transparent appearance and properly reduced amylose contentImproved quality and stable inheritance. Compared with breeding such as chemical mutagenesis, cross breeding and the like, the gene editing directional improvement molecular breeding technology has the advantages of rapidness, accuracy, high efficiency and the like, combines gene function marker genotype selection, or directly utilizes other mature rice varieties as background materials to carry out transformation and obtain corresponding materials, can greatly improve the breeding efficiency and greatly accelerate the breeding process.

Description

Wx mutant protein based on gene editing technology and application of gene thereof in plant breeding
Technical Field
The invention belongs to the field of crop genetic breeding, new resource innovation of crop quality improvement and good taste crop breeding, and particularly relates to Wx mutant protein based on a gene editing technology and application of genes thereof in plant breeding.
Background
Rice (Oryza sativa L.) is an important grain crop in China and even in the world, and plays a significant role in guaranteeing grain safety.
The rice quality refers to the essential characteristics of rice in commodity circulation, including four main aspects of grinding quality, appearance quality, cooking and taste quality, nutrition quality and the like, wherein the appearance quality, the cooking and taste quality are particularly important. The seed endosperm is the main edible part of rice, and the main component of the endosperm is starch which generally accounts for 90% or more of the dry weight of the endosperm by chemical composition analysis, so that the cooking taste quality of rice depends on the physicochemical properties of starch to a great extent, and genes participating in the synthesis and regulation of the starch in the rice endosperm play an extremely important role in the formation of the rice quality. Through a great amount of research by researchers, people have deeply understood the genetic basis of rice cooking and taste quality characters, and the way of synthesizing rice endosperm starch, and involved genes or enzymes have been clarified in detail. Wherein, the enzymes involved in starch synthesis mainly comprise ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, starch branching enzyme, starch debranching enzyme and the like.
There are two main types of starch constituting rice, namely amylose (amylose) and amylopectin (amylopectin). Amylose is a glucose polymer linked by alpha-1, 4-glycosidic bonds, which is a linear macromolecule with few or no branches. Amylose Content (AC) is generally considered to be a determining factor in rice cooking, processing, and eating quality. The rice Wx gene encodes granular starch synthase (GBSS), which is a major gene controlling amylose synthesis and directly affects the amylose content in rice endosperm and pollen. The gene was cloned in 1990 and located in the short arm of chromosome 6, and in general japonica varieties, the gene consists of 14 exons and 13 introns and encodes a protein consisting of 609 amino acids. The different allelic variations of this gene determine the amylose content of rice, and up to now there are at least 8 published Wx alleles. In glutinous rice, Wx causes premature transcription termination due to deletion of 23bp of exon 2. In non-waxy varieties, Wx differentiates primarily into two allelic types, Wx-a and Wx-b. Wherein the amylose content of the rice carrying Wx-a is more than 25 percent, and the rice belongs to a high amylose type. Wx-b is mainly distributed in japonica rice varieties, the amylose content of the rice carrying the gene is moderate to low (15% -18%), compared with Wx-a, the variation of Wx-b is caused by the variation of G-T at the splicing site of the first intron, and the mutation reduces the splicing efficiency of precursor mRNA so as to reduce the amylose content of the rice. In addition, researchers have cloned Wx-in alleles, demonstrating that A-C variation at exon 6 results in a moderate level (18% -22%) of amylose content. In addition, 3 "soft rice genes" namely Wx-mq, Wx-mp and Wx-op were cloned sequentially (so-called soft rice, i.e., rice with AC between 8% and 12%, bright surface, soft texture, high elasticity, no hardness in cold rice and excellent taste quality), and Wx-mq had 2 mutations in exons 4 (arginine to histidine at amino acid sequence 158) and 5 (amino acid sequence histidine to histidine at amino acid sequence 191) compared with Wx-b, resulting in a decrease in amylose content to around 10%. The other allele Wx-mp has mutation in the 4 th exon (arginine at the 158 th position of the amino acid sequence is mutated into histidine), the position is the same as that of Wx-mq, the 5 th exon has no mutation, the amylose content of rice carrying the allele is about 10 percent, the other soft rice gene is caused by mutation of A-G of the 4 th exon (aspartic acid at the 165 th position of the amino acid sequence is mutated into glycine) of Wx-hp and Wx-hp derived from Yunnan Haopi, the former person uses agrobacterium tumefaciens glycogen synthase as a model to draw a three-dimensional structure diagram of Wx-hp, and the mutation is presumed to be positioned in an amylase catalytic domain of starch synthase to cause starch synthesis capacity reduction. In summary, we believe that artificially mutating (e.g., using gene editing techniques) individual amino acids of the core catalytic domain of starch synthase encoded by the Wx gene, particularly the related amino acids adjacent to amino acid residues 33-36 (Lys-Ser-Gly) in the 3-D model (the positions of the amino acid residues 33-36 are reported to be binding sites for ADP-glucose in starch and glycogen synthesis), may create new mutation types and provide material support for meeting the higher requirements of the current market for rice quality.
At present, soft rice japonica rice varieties in Jiangsu, Yunnan, Shanghai and other places, such as Nanjing 46, Nanjing 9108, Chujing 39, Shanghai soft 1212 and the like, have outstanding taste and quality, and are popular with consumers and widely planted. Many varieties obtain the national quality rice variety taste quality appraisal golden prize. However, although the soft rice japonica rice is popular and touted in the market in terms of taste, two major defects still exist:
1. the method can utilize the shortage of soft rice resources and the single genotype, and has low improvement efficiency by utilizing conventional breeding. Taking soft rice variety bred in Yangtze triangle as an example, the allelomorphic mutant gene Wx-mp of Kanto 194 from Japan, about AC 9 percent, is mainly used at present. The germplasm resource is used for cultivating soft rice varieties, and target genes are introduced into excellent variety backgrounds, mainly through hybridization, backcross, multiple cross, ladder hybridization and the like. For quality traits, backcross transformation is a common method, backcross for 4-6 generations is generally needed, at least 3-5 generations are needed in addition to selfing homozygosis and the like, single traits need to be improved and can play a role in production, at least 6-8 years are needed, the breeding period is long, and the urgent requirements of markets and common people on high-quality soft fragrant rice varieties are difficult to meet. If a new soft rice gene resource is to be mined, the method mainly depends on chemical mutagenesis at present, as is well known, the chemical mutation frequency is low, the early investment is large, 2-3 years are probably needed for obtaining one stable material by screening, meanwhile, the chemical mutagenesis can cause multiple gene mutations of wild materials, and the undesirable gene mutations need to be eliminated by hybridization improvement when the chemical mutagenesis is applied to production.
2. Most of soft rice and japonica rice varieties have poor appearance quality. When the water content of the rice is about 15%, most of the rice is cloudy and poor in transparency, is commonly called semi-glutinous rice or stiff rice, and seriously influences the commodity of the rice. Rice processing plants can increase the clarity of rice by increasing the moisture content above 17%, but often cause the rice to be intolerant to storage and even mildew. Therefore, the excavation and utilization of new soft rice gene resources and the change of the current situation that the appearance of the existing soft rice does not reach the standard are urgent needs for high-quality breeding.
The genome editing technology is a technology of site-directed modification, single base editing, targeted knockout or target gene insertion of a genome and a transcription product thereof by using a special nuclease. At present, the CRISPR/Cas9(CRISPR-associated protein 9) mediated genome editing technology is one of the mature, rapidly developed and widely applied in crop genetic improvement. Compared with traditional breeding and aiming at the defects existing in soft rice breeding, the gene editing breeding has the following advantages:
1. high efficiency and short period. As the agrobacterium-mediated genetic transformation efficiency of japonica rice varieties is higher, the previous experimental results show that 10T are obtained 0 Generation transformants are typically selected to be 3-5 homozygous target alleles edited simultaneouslyTransformants, i.e.at T 0 Phenotypes such as flavor, dark endosperm due to very low amylose content, early heading, etc. were observed. Because rice is a strict self-pollinating plant, exogenous DNA fragments can be removed by self-crossing separation, and the DNA fragments are expressed in T 0 T obtained by selfing and fructification of generation single plant 1 In the generation, about 5 individuals of the 100 individuals can be screened for foreign DNA represented by hygromycin, Cas9 gene, SgRNA + vector skeleton. 5 seedlings are bred, each seedling can generally collect 500 seeds, 400 seedlings can be bred by one seedling, and about 20 jin of conventional japonica rice seeds can be bred. That is, within three generations (-1.5 years), enough seeds are available for quality analysis (even in various trials such as trials, demonstrations, etc.). Compared with conventional breeding, the breeding period is greatly shortened. Furthermore, even at T 0 When clone variation occurs in the generation tissue culture process, the poor variation can be eliminated by hybridizing the genome edited material with the wild type, or enough T can be generated 0 In the case of transformants, the clonally variant plants can even be discarded.
2. The expected target gene can be accurately, directionally and massively created. In conventional breeding, only resources with target genes can be screened from variety resources, and then the existing varieties are improved by means of hybridization backcross and the like, generally speaking, the variety resource materials have poor agronomic characters, so the genetic improvement period is longer, while the genome editing technology can directly utilize rice varieties which have excellent agronomic characters and are popularized in large areas in production as background materials to edit the target genes, the target is clear, and the screening is efficient; in addition, in view of the current high-efficiency genetic transformation technology of rice, a specific phenotype (e.g. transparent appearance) in the variation of a specific target gene (e.g. Wx gene) can be screened in the background material in an unlimited amount theoretically, and compared with the search of natural variation materials or chemical mutation materials meeting the phenotypic condition, the strategy is more feasible and has obvious advantages. By taking the multi-gene editing system of the related genes of the heading stage constructed by the agricultural academy of sciences of Jiangsu province at present as an example, Wuyujing 24 and Nanjing 9108 which are popularized by Jiangsu province in large area are used as genetic transformation background materials, and new materials for removing exogenous marker genes of heading stages of more than 60 days, more than 70 days and more than 80 days are obtained, compared with wild types, the new materials are rich in variation types, the heading stage is advanced and distributed in a gradient manner, the planting range of high-quality varieties can be expanded, the improvement process of the high-quality varieties is accelerated, the breeding cost is greatly saved, and the vitality and the creativity of gene editing molecular breeding are highlighted.
3. The single-base editing system of the gene based on CRISPR/Cas9 can further realize single-base editing. In crop breeding and production, many important agronomic traits are caused by single nucleotide polymorphism or dominant gain-of-function point mutation, so that the application of a simple gene knockout strategy is very limited, the technical problem can be well solved by the appearance of a single base editing system, and the strategy can avoid extreme variation phenotype caused by the loss of biological functions of mutant proteins caused by gene knockout, even rice plants can not grow normally and finally can not be applied to breeding. The "weak mutation" (individual amino acid substitution rather than premature translation termination) resulting from single base editing does not result in complete loss of biological function of the encoded protein, and thus does not cause significant changes in important agronomic traits of the background material, and would be more conducive to the generation of types of variation that can be exploited for breeding and production.
In conclusion, by using the gene editing technology, the target site for designing and editing the exons 3 and 4 (located in the core catalytic domain of the encoded starch synthase) of the Wx gene is subjected to single base editing, a series of new materials which are transparent in appearance, low in amylose content and gradient in distribution can be efficiently created and screened, and the specific genotypes of the new materials can be further applied to breeding by using the materials and determining the specific genotypes of the new materials. Corresponding gene function markers can be developed, genotype selection is carried out in hybrid combined offspring prepared by the materials by means of molecular marker assisted selection, and target trait gene heterozygosis and homozygous genotypes are screened, so that the target gene homozygous process is accelerated. In addition, the rice variety with excellent agronomic characters and large-area popularization in production can be directly used as a background material to edit the target gene, and the needed genotype materials can be efficiently screened in a large scale. Aiming at single base mutation, enzyme cutting target point markers can be designed, but the process is relatively complicated, allele specific PCR is developed, and genotypes with high and low amylose contents can be distinguished through two times of PCR.
At present, technologists want to obtain new soft rice materials or new genes with the AC reduced to 8-12% and transparent appearance, and need to perform chemical or radiation mutagenesis and the like, so that the workload is large and the effect is not ideal. Meanwhile, conventional chemical mutagenesis and conventional transformation breeding are adopted, the breeding age is at least 6-8 years, the breeding period is long, and the urgent requirements of the market and the common people on high-quality soft fragrant rice varieties are difficult to meet.
Meanwhile, no relevant report is provided for the relevant research of creating a new soft rice allele by carrying out site-specific mutagenesis on the Wx gene of the rice variety by using a gene editing technology.
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 transparent appearance, properly reduced amylose content, improved quality and stable inheritance, generally only needs about 2 years, and the breeding period is at least shortened by 2-6 years compared with the chemical mutagenesis and conventional transformation breeding. 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, wherein the 159 th amino acid is mutated, and/or the 178 th amino acid is mutated.
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: ddH for adaptor 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 on a pH-nCas9-PBE binary expression vector, restriction enzymes PmeI and AvrII 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 Generating transgenic plants, and obtaining T by using primers Wxb-PCR-F and Wxb-PCR-R 0 And amplifying the transgenic plant, sequencing and identifying the genotype 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 selfing 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 nCas9 gene are not detected at the same time, 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 mutant protein homozygous plant and the nucleic acid or gene homozygous plant.
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 mutation, hybridization transformation and the like, the gene editing directional improved molecular breeding technology has the advantages of rapidness, accuracy, high efficiency and the like, genotype selection is carried out by utilizing gene function markers, or other mature rice varieties are directly utilized as background materials to be transformed and corresponding materials are obtained, so that the breeding efficiency can be greatly improved, and the breeding process can be greatly accelerated.
2) The content of the amylose of the rice 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 amylose content is proper, 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 been shown to be cloudy and has poor transparency.
5) The invention develops the specific discrimination of the wild type (GG at 475- m6 AA at bases 475-476 of the gene) molecular markers 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 of gene is TT) are marked with Wx9 and Wx 10. The molecular marker can be used for molecular marker-assisted selective breeding.
Drawings
FIG. 1 is a schematic diagram of the gene structure and target site.
FIG. 2T against Su reclamation 118 0 Identifying the target site sequence of the generation plant (part); a: wxb #9 target site; b: wxb #24 target site.
FIG. 3T against Su reclamation 118 1 Identifying the target site sequence of the T-DNA knockout plant; a: wxb #9 target site; b: wxb #24 target site.
FIG. 4T 2 Detecting results of HPT gene and Cas9 gene of the generation plant; a: an HPT gene; b: cas9 gene. M: DNA marker 2000 with the sizes of 2000, 1000, 750, 500, 250 and 100 from top to bottom; lane 1 is 118-1-1 strain and has been marked with a red arrow; lane 2 is 118-3-2 strain, and has been marked with a red arrow; lane 3 is 118-5-1 strain and has been marked with a red arrow; lane 11 is 118-9-15 line and has been marked with a red arrow; other lanes are other numbered strains of sovern 118 background; labeled P is a plasmid positive control; labeled E is the water control.
FIG. 5 determination of Amylose Content (AC).
FIG. 6 analysis of the correlation between the overall variation of each characteristic value of RVA and the disintegration value (BDV), the recovery value (CSV), the peak time (PeT) and AC respectively.
Figure 7 polished rice appearance observation.
FIG. 8 wild-type Wx and Wx m6 、Wx m10 Detecting the activity of the mutant protease and analyzing the correlation between the mutant protease and AC; a: enzyme activity detection; b: and (5) carrying out correlation analysis.
Fig. 9 three-dimensional structure prediction. A: a wild-type Wx protein; b: mutant Wx m6 A protein; c: a wild-type Wx protein; d: mutant Wx m10 A protein.
FIG. 10 development of functional markers; the DNA templates used for each lane are indicated in the figure. Lane M is DNA marker 2000 with bands of 2000, 1000, 750, 500, 250, and 100, respectively, from top to bottom; the primers used in the PCR for each lane are as follows: lanes 1 and 7 are Wx-1, lanes 2 and 8 are Wx-2, lanes 3 and 9 are Wx-3, lanes 4 and 10 are Wx-4, lanes 5 and 11 are Wx-5, lanes 6 and 12 are Wx-6, lanes 13 and 19 are Wx-7, lanes 14 and 20 are Wx-8, lanes 15 and 21 are Wx-9, lanes 16 and 22 are Wx-10, lanes 17 and 23 are Wx-11, and lanes 18 and 24 are Wx-12.
FIG. 11Wx1+ Wx2 and Wx9+ Wx10 functional marker test varieties; a: primers used for PCR were Wx 1; b: primers used for PCR were Wx 2; c: primers used for PCR were Wx 9; d: the primer used for PCR was Wx 10. Lane M is DNA marker 2000 with bands of 2000, 1000, 750, 500, 250, and 100, respectively, from top to bottom; lanes 1-19 represent Su-reclamation 118, 118-1-1 mutant, 118-3-2 mutant, 118-5-1 mutant, 118-9-15 mutant, Nipponbare, Nanjing 9108, Huanghuazhan, 9311, Huai rice No. 5, Channong No. 8, Nanjing 51, Neijing No. 7, Suxiu 867, Wuyujing No. 27, Wuyujing No. 29, Xudao No. 8, Xudao No. 9, and Ji rice No. 16, respectively.
FIG. 12 Jinjing 818 background T 2 Identifying the genotype and phenotype of the generation plant; a: wxb #9 target site genotype identification; b: wxb #24 target site genotype identification; c, determining Amylose Content (AC); d, BDV determination; e, CSV determination; f, PeT determination; g, observing the appearance of polished rice; h, protease ActivityAnd (6) detecting.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate 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 specified, 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 the reclamation of Suzhou 118 (purchased from Jiangsu reclamation seed industry Co., Ltd.), the variety is a new late-maturing Zhongjing variety bred by the food crop research institute of the agricultural academy of sciences of Jiangsu province, has about 155 days of the whole growth period, is suitable for planting in Suzhou province and Ningzhengyangyang hilly areas of Jiangsu province, has excellent comprehensive agronomic characters, is 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 the Suzhou reclamation 118 by using a single base gene editing technology based on CRISPR/Cas9, obtains a plurality of mutants with transparent appearance, properly reduced amylose content, gradient distribution (soft rice) and other appearance, improved cooking and taste quality, and meets the urgent need of the market and the common people for high-quality soft fragrant rice varieties.
Example 1: obtaining genetically transformed plants
1. Su-reclamation 118 Wx 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) by PCR amplification of the genomic DNA, the amplification product was sent to England Elite (Shanghai) trading 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 al, Plant Mol Biol, 2009, 71: 609-626), that the two target sites are both located in the core catalytic domain of starch synthase encoding the protein, and single base variation in the respective editing windows is expected to form new effective mutations, so as to obtain rice mutants with transparent appearance and reduced amylose content to different degrees.
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 wxb #9 and wxb #24 sequences of target sites and respective reverse complementary sequences, after adding a connector 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 al, BMC Plant Biol, 2014, 14, 327) was digested with the restriction enzyme BsaI purchased from ThermoFisher in the following manner:
Figure BDA0003635721700000121
after 1h of digestion at 37 ℃, agarose gel electrophoresis and gel tapping are carried out, and BsaI single enzyme digestion is carried out on the vector for later use 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:
Figure BDA0003635721700000122
after brief centrifugation, the mixed system was subjected to a water bath at 16 ℃ for 4 hours. The ligation product was transformed into E.coli DH 5. alpha. 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 plasmids obtained, correctly inserted and correctly sequenced, were designated 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:
Figure BDA0003635721700000131
after 1h of enzyme digestion at 37 ℃, agarose gel electrophoresis and gel tapping are carried out, and the vector after PmeI and AvrII double enzyme digestion is recovered and reserved 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:
Figure BDA0003635721700000132
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:
Figure BDA0003635721700000133
Figure BDA0003635721700000141
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 and genotyping of genetically transformed plants
The 2 plasmids were transformed into Agrobacterium EHA105, respectively. Rice reclamation 118 (purchased from Jiangsu reclamation cultivars, Inc.) 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. In order to identify the base variation of the editing target site, the T obtained above was extracted 0 Generating transgenic line leaf genome DNA, and carrying out 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 condition of the corresponding target sites of the generation plants (parts) is shown in FIG. 2A, and the strain with the number of B1-68 is homozygous mutation of 475-476 site GG into AA; the strain with the number of B2-42 is a biallelic mutation in which 475-; the strain numbered B6-29 was homozygous for AA by mutation of 475-476 GG.
Positive T of transfer pH-nCas9-PBE-wxb #24 0 The identification of the corresponding target sites of (part of) the generation plants is shown in FIG. 2B, the mutation of CC at position 533-534 of one allele to TT and the mutation of CC at position 533-534 of the other allele to GT are both allelic mutations in the line with the number B2-21.
Example 2: mutant exogenous DNA (T-DNA) knockout, genotype re-identification and screening of homozygous plants
The pH-nCas9-PBE-wxb #9 and the pH-nCas9-PBE-wxb #24 vectors of the directionally-precise single-base editing Wx gene, which are 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, the two genes are used as representative exogenous DNAs to be removed, and the reasons are as follows: 1) the hygromycin phosphotransferase HPT gene mainly has the function of being used 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 functions of completing the site-specific cutting of a target gene target site and being continuously retained in a plant to possibly cause secondary editing; 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 transgenic problem is high, and the public acceptance degree of the T-DNA knockout material 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 multi-copy mode, and because the rice is a relatively strict auto-pollinated homodiploid plant and the T-DNA insertion site is generally not linked with the target site, on the technical level, a plant not carrying T-DNA can be obtained by separation and identification from progeny generated by transgenic plant selfing, and even if the T-DNA sequence is linked, materials not carrying T-DNA can be screened by genetic exchange recombination. In order to obtain the above-mentioned plants not carrying T-DNA, the present inventors have obtained a total of 4 lines of T1-68, 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 a mutation type change due to secondary editing, requires re-identification of the genotype (using methods and primers as described in example 1) in the individual or progeny of the T-DNA knockout 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, seed soaking, sowing in seedling tray and placing in culture chamber, when seedling grows to two leaves and one heart, taking leaf to extract DNA, using hygromycin-coding Hpt gene and nCas9 gene coding nCas9 nuclease to make PCR detection (the method and the used primer are described in the above embodiment), repeating the experiment three times, co-screening to obtain 10 single plants which can not be amplified to the target fragment, namely, it is considered as T-DNA removed single plant, the proportion is 9.35%, and supposing that T-DNA is inserted into rice genome in multicopy mode. Selecting one strain and naming it as 118-1-1, tracking the editing condition of the target site of the single strain (see example 1 for genotype identification method and primers), 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 generation seeds, soaking the seeds, sowing and planting in an incubator, verifying the carrying condition of the T-DNA, repeating the experiment for three times, finding the same generation, and showing that the strain T is 1 -T 2 The generation did have knocked out T-DNA (FIG. 4); in addition, the single plant target site editing situation is tracked, the same generation is found, and the result shows that T is obtained from T 0 -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 the 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 Gene identity, nucleotide sequenceThe 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 (figure 4), and the mutation type of the target site is 533-534 base CC to TT homozygous mutation, which causes the 178 th amino acid to be mutated from threonine to isoleucine. The nucleotide sequence of the Wx gene of the 118-9-15 mutant is shown as SEQ ID NO.3, the amino acid sequence of the encoded Wx protein is shown as SEQ ID NO.4, and the new allele obtained by cloning 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 edit the Wx gene of the rice variety by single base, obtains a new material with stable heredity and T-DNA elimination within 2 years, and specifically comprises the following steps: 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 and 5 months of plant growth and seed setting 1 Eliminating T-DNA from plant, screening homozygous gene type, growing and setting for 5 months 2 The generation plant T-DNA detection, homozygous genotype identification and plant growth and fructification 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 additive generation and can be finally harvested 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
Cooking and Eating Quality (ECQ) is a direct factor affecting consumer choice and is thusThe rice quality constitutes the most important evaluation index. 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 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, as measured using a rapid viscosity analyzer (RVA Super 4, NEWPORT SCIENTIFIC, Australia) with parameter settings made in accordance with the American Association of cereal chemists 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 in cold rice tends to have a small recovery value (CSV), and in addition, the peak time PeT refers to the time taken for the sample to reach the peak viscosity, and generally the smaller the PeT, the better the swelling and breakage of the starch grains. 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 eigenvalues
Figure BDA0003635721700000181
3. Thermodynamic characteristics
The rice gelatinization and the quality of the cooked food flavor have close relation, 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 there is randomness of the gelatinization characteristics among the tested samples, and that the gelatinization temperature of 118-1-1 is the largest, consistent with PaT performance measured by the RVA instrument.
TABLE 2 thermodynamic Properties
Figure BDA0003635721700000182
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. Husking rice by a huller (SY88-TH, Korea double dragon) to obtain coarse rice, refining with a small-sized polished rice machine (BLH-3120, Burley constant, Taizhou) to obtain polished rice, and measuring water content of the polished rice with a water analyzer (Metteler, Switzerland) to ensure consistent water content of the 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 118 from soda, 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 mutants 118-9-15AC is reduced to 9.8 percent, the mutants still can keep better transparency in appearance.
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 tested for genotype and the methods and primers used for genotype identification were as described in example 1, and then individuals of wild type and homozygous mutant genotypes were selected for phenotypic analysis (with AC as a representative index) and tested for AC 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%, indicated 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%, indicated 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 enzyme activity assay was performed with reference to the method of predecessors (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 showed very significant positive correlation 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 knotThe results indicate that the phenotype of reduced mutant AC and 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 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
The molecular marker assisted selection is beneficial to accelerating the breeding process. Wx of the invention m6 And Wx m10 The gene is continuous two base mutations, enzyme cutting 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 and 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. 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 μ 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 deg.C.
TABLE 3 molecular markers for detection of mutant genes
Figure BDA0003635721700000211
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, Neizuan No. 7, Suxiu 867, Wu Yu Jing No. 27, Wu Yu Jing No. 29, Xuan Rice No. 8, Xuan Rice No. 9, and Yan Rice No. 16) were band-amplified by Wx1 but not band-amplified by Wx2, indicating that Wx1+ Wx2 were able to specifically detect Wx m6 The variation of GG to AA at the 475-476 bases of the gene can be used for molecular marker-assisted selective 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 Gene 533-site 534 alkaliThe variation 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, 112F 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 results obtained 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 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 results obtained by the sequencing method (see example 1) (including 31 wild-type individuals, 76 heterozygous individuals and 24 homozygous Wx) m10 Mutant individuals). The results show that the markers Wx1+ Wx2 and Wx9+ Wx10 developed by the invention can be used for accurate 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 make transformation
Jinjing 818 is a conventional japonica rice variety bred by the rice institute of 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 vectors, transformation methods and genotyping methods 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. Screening to obtain homozygous Wx, as shown in FIGS. 12A-B m6 Of the mutant type, TDNA-deleted 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 The generation line is 1: 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 moisture content was 13.5%, the jin jing 818 polished rice still appeared transparent, but the nan jing 9108 was cloudy and poor in 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 (A) was minimal, being 407. + -. 30.01 nmol/min/g.
The above results indicate that Wx was created directly in the Kingjing 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 matching and the like, the gene editing method has the advantages of rapidness, accuracy, high efficiency and the like, combines gene function marker genotype selection, or directly utilizes other mature rice varieties as background materials to carry out transformation and obtain 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
Figure BDA0003635721700000231
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
<160> 31
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1830
<212> DNA
<213> Wx-m1 mutant Gene (grain-bound mutant synthase)
<400> 1
atgtcggctc tcaccacgtc ccagctcgcc acctcggcca ccggcttcgg catcgccgac 60
aggtcggcgc cgtcgtcgct gctccgccac gggttccagg gcctcaagcc ccgcagcccc 120
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
gtgatctctc ctcggtacga ccagtacaag gacgcttggg ataccagcgt tgtggctgag 420
atcaaggttg cagacaggta cgagagggtg aggtttttcc attgctacaa gcgtaaagtc 480
gaccgtgtgt tcatcgacca tccgtcattc ctggagaagg tttggggaaa gaccggtgag 540
aagatctacg gacctgacac tggagttgat tacaaagaca accagatgcg tttcagcctt 600
ctttgccagg cagcactcga ggctcctagg atcctaaacc tcaacaacaa cccatacttc 660
aaaggaactt atggtgagga tgttgtgttc gtctgcaacg actggcacac tggcccactg 720
gcgagctacc tgaagaacaa ctaccagccc aatggcatct acaggaatgc aaaggttgct 780
ttctgcatcc acaacatctc ctaccagggc cgtttcgctt tcgaggatta ccctgagctg 840
aacctctccg agaggttcag gtcatccttc gatttcatcg acgggtatga cacgccggtg 900
gagggcagga agatcaactg gatgaaggcc ggaatcctgg aagccgacag ggtgctcacc 960
gtgagcccgt actacgccga ggagctcatc tccggcatcg ccaggggatg cgagctcgac 1020
aacatcatgc ggctcaccgg catcaccggc atcgtcaacg gcatggacgt cagcgagtgg 1080
gatcctagca aggacaagta catcaccgcc aagtacgacg caaccacggc aatcgaggcg 1140
aaggcgctga acaaggaggc gttgcaggcg gaggcgggtc ttccggtcga caggaaaatc 1200
ccactgatcg cgttcatcgg caggctggag gaacagaagg gccctgacgt catggccgcc 1260
gccatcccgg agctcatgca ggaggacgtc cagatcgttc ttctgggtac tggaaagaag 1320
aagttcgaga agctgctcaa gagcatggag gagaagtatc cgggcaaggt gagggccgtg 1380
gtgaagttca acgcgccgct tgctcatctc atcatggccg gagccgacgt gctcgccgtc 1440
cccagccgct tcgagccctg tggactcatc cagctgcagg ggatgagata cggaacgccc 1500
tgtgcttgcg cgtccaccgg tgggctcgtg gacacggtca tcgaaggcaa gactggtttc 1560
cacatgggcc gtctcagcgt cgactgcaag gtggtggagc caagcgacgt gaagaaggtg 1620
gcggccaccc tgaagcgcgc catcaaggtc gtcggcacgc cggcgtacga ggagatggtc 1680
aggaactgca tgaaccagga cctctcctgg aaggggcctg cgaagaactg ggagaatgtg 1740
ctcctgggcc tgggcgtcgc cggcagcgcg ccggggatcg aaggcgacga gatcgcgccg 1800
ctcgccaagg agaacgtggc tgctccttga 1830
<210> 2
<211> 609
<212> PRT
<213> Wx-m1 mutant protein (grain-bound stage synthsase)
<400> 2
Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe
1 5 10 15
Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe
20 25 30
Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu
35 40 45
Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60
Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly
65 70 75 80
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 Lys 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 Thr 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> 3
<211> 1830
<212> DNA
<213> Wx-m2 mutant Gene (grain-bound mutant synthase)
<400> 3
atgtcggctc tcaccacgtc ccagctcgcc acctcggcca ccggcttcgg catcgccgac 60
aggtcggcgc cgtcgtcgct gctccgccac gggttccagg gcctcaagcc ccgcagcccc 120
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
gtgatctctc ctcggtacga ccagtacaag gacgcttggg ataccagcgt tgtggctgag 420
atcaaggttg cagacaggta cgagagggtg aggtttttcc attgctacaa gcgtggagtc 480
gaccgtgtgt tcatcgacca tccgtcattc ctggagaagg tttggggaaa gattggtgag 540
aagatctacg gacctgacac tggagttgat tacaaagaca accagatgcg tttcagcctt 600
ctttgccagg cagcactcga ggctcctagg atcctaaacc tcaacaacaa cccatacttc 660
aaaggaactt atggtgagga tgttgtgttc gtctgcaacg actggcacac tggcccactg 720
gcgagctacc tgaagaacaa ctaccagccc aatggcatct acaggaatgc aaaggttgct 780
ttctgcatcc acaacatctc ctaccagggc cgtttcgctt tcgaggatta ccctgagctg 840
aacctctccg agaggttcag gtcatccttc gatttcatcg acgggtatga cacgccggtg 900
gagggcagga agatcaactg gatgaaggcc ggaatcctgg aagccgacag ggtgctcacc 960
gtgagcccgt actacgccga ggagctcatc tccggcatcg ccaggggatg cgagctcgac 1020
aacatcatgc ggctcaccgg catcaccggc atcgtcaacg gcatggacgt cagcgagtgg 1080
gatcctagca aggacaagta catcaccgcc aagtacgacg caaccacggc aatcgaggcg 1140
aaggcgctga acaaggaggc gttgcaggcg gaggcgggtc ttccggtcga caggaaaatc 1200
ccactgatcg cgttcatcgg caggctggag gaacagaagg gccctgacgt catggccgcc 1260
gccatcccgg agctcatgca ggaggacgtc cagatcgttc ttctgggtac tggaaagaag 1320
aagttcgaga agctgctcaa gagcatggag gagaagtatc cgggcaaggt gagggccgtg 1380
gtgaagttca acgcgccgct tgctcatctc atcatggccg gagccgacgt gctcgccgtc 1440
cccagccgct tcgagccctg tggactcatc cagctgcagg ggatgagata cggaacgccc 1500
tgtgcttgcg cgtccaccgg tgggctcgtg gacacggtca tcgaaggcaa gactggtttc 1560
cacatgggcc gtctcagcgt cgactgcaag gtggtggagc caagcgacgt gaagaaggtg 1620
gcggccaccc tgaagcgcgc catcaaggtc gtcggcacgc cggcgtacga ggagatggtc 1680
aggaactgca tgaaccagga cctctcctgg aaggggcctg cgaagaactg ggagaatgtg 1740
ctcctgggcc tgggcgtcgc cggcagcgcg ccggggatcg aaggcgacga gatcgcgccg 1800
ctcgccaagg agaacgtggc tgctccttga 1830
<210> 4
<211> 609
<212> PRT
<213> Wx-m2 mutant protein (grain-bound stage synthsase)
<400> 4
Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe
1 5 10 15
Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe
20 25 30
Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu
35 40 45
Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60
Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly
65 70 75 80
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

Claims (17)

1. A rice Wx mutant protein, wherein the amino acid sequence of the Wx mutant protein comprises the following mutations: the 178 th amino acid of the amino acid sequence of the rice Wx is mutated, and the amino acid sequence is shown as SEQ ID NO. 4.
2. The rice Wx mutant protein according to claim 1, wherein the amino acid sequence of the Wx mutant protein further comprises the following mutations: the 159 th amino acid of the amino acid sequence corresponding to rice Wx is mutated from glycine to lysine.
3. A nucleic acid or gene encoding the mutein of any one of claims 1 to 2.
4. The nucleic acid or gene of claim 3, having a nucleotide sequence as set forth in SEQ ID NO 3.
5. A primer pair for identifying the gene or the nucleic acid as claimed in claim 3 or 4, wherein the primer pair is Wx9 and/or Wx10, the sequence of the primer pair Wx9 is shown as SEQ ID NO. 20 and SEQ ID NO. 28, and the sequence of the primer pair Wx10 is shown as SEQ ID NO. 20 and SEQ ID NO. 29.
6. An expression cassette or recombinant vector comprising the nucleic acid or gene of claim 3 or 4.
7. Use of the rice Wx mutant protein of claim 1 or 2, the nucleic acid or gene of claim 3 or 4, the primer pair of claim 5, the expression cassette or recombinant vector of claim 6 for obtaining a rice plant with transparent appearance, improved quality of rice plants with reduced amylose content, transparent appearance, identification of rice lines/varieties with reduced amylose content, transparent appearance, creation and/or breeding of rice lines/varieties with reduced amylose content.
8. A method for obtaining a quality-improved plant with a transparent appearance and a suitably reduced amylose content, characterized in that it comprises the following steps:
1) allowing a plant to comprise the nucleic acid or gene of claim 3 or 4; or
2) Expressing in a plant the rice Wx mutant protein of claim 1 or 2; the plant is rice.
9. A breeding method for creating quality-improved rice with a transparent appearance and a properly reduced amylose content by using gene editing, characterized by comprising the steps of:
1）Wxcloning of genes and designing target sites for gene editing;
2) constructing a single-base editing vector containing a target fragment;
3) a rice having improved quality, which comprises the mutant protein of claim 1 or 2, the nucleic acid or gene of claim 3 or 4, has a transparent appearance and an appropriately reduced amylose content 0 Obtaining the generation plants;
4) mutation of target Gene into T 0 T obtained by selfing transgenic plants 1 Removing T-DNA fragments of the generation plants;
5) T-DNA-knocked-out T 1 And identifying the genotype of the generation plant and obtaining the target gene homozygous mutant plant.
10. A breeding method as claimed in claim 9, wherein when the 178 th amino acid of the amino acid sequence of rice Wx is mutated, the nucleotide sequence of the target site of gene editing of step 1) is shown as SEQ ID NO 8; when the 178 th amino acid and the 159 th amino acid of the amino acid sequence of the rice Wx are mutated simultaneously, the nucleotide sequence of the target site edited by the gene in the step 1) is shown as SEQ ID NO. 7 and SEQ ID NO. 8.
11. A breeding method according to claim 9, characterized in that the method for constructing the single-nucleotide editing vector containing the target fragment in step 2) is as follows:
A) preparing a target joint fragment: ddH for adaptor 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; performing a ligation reaction by using a BsaI single-enzyme-digested pHUE411 vector, a target joint fragment and T4 ligase, performing 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 on a pH-nCas9-PBE binary expression vector, restriction enzymes PmeI and AvrII to obtain a double enzyme digested pH-nCas9-PBE vector; carrying out double enzyme digestion on an intermediate vector plasmid 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.
12. A breeding method according to claim 9, characterized in that the method of obtaining in step 3) is as follows: 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 Amplifying and sequencing the transgenic plant to identify the genotype and obtain the plant with the mutant protein of claim 1 or 2 and the nucleic acid or gene of claim 3 or 4; the Wxb-PCR-F sequence is shown as SEQ ID NO. 13, and the Wxb-PCR-R sequence is shown as SEQ ID NO. 14.
13. A breeding method according to claim 9, characterized in that the T-DNA fragment of step 4) comprises the hygromycin phosphotransferase geneHPTAnd nuclease genenCas9。
14. A breeding method as claimed in claim 9, characterized in that the T-DNA fragments of step 4) are knocked out by mutating T of the target gene 0 T obtained by selfing transgenic plants 1 Plant generation plantIs/are as followsHPTGenes andnCas9the genes are simultaneously detected and repeated for many times, and T which does not carry the two genes is obtained by screening 1 The generation single plant is the target plant.
15. A method as claimed in claim 14, wherein the method is as set forth inHPTGene detection method by mutating T with target gene 0 T obtained by selfing transgenic plants 1 Taking genome DNA of the generation plant as a template, and taking hyg283-F and hyg283-R as primers to carry out PCR amplification, and meanwhile, carrying out PCR amplification on the genome DNA of the generation plantnCas9Gene detection method by mutating T with target gene 0 T obtained by selfing transgenic plants 1 Genome DNA of the generation plant is taken as a template, nCas9-F and nCas9-R are taken as primers for PCR amplification, and when none of the primers is detected at the same timeHPTGenes andnCas9genes show that T-DNA is successfully eliminated;
the sequence of hyg283-F is shown as SEQ ID NO. 15, the sequence of hyg283-R is shown as SEQ ID NO. 16, the sequence of nCas9-F is shown as SEQ ID NO. 17, and the sequence of nCas9-R is shown as SEQ ID NO. 18.
16. A breeding method according to claim 9, characterized in that the homozygous mutant plant for the target gene obtained in step 5) is obtained by knocking out T-DNA obtained in step 4) 1 Using genome DNA of the generation plant as a template, using Wxb-PCR-F and Wxb-PCR-R as primers to carry out PCR amplification and sequencing, and screening and obtaining a homozygous plant with the mutant protein of claim 1 or 2 and the nucleic acid or the gene of claim 3 or 4; the Wxb-PCR-F sequence is shown as SEQ ID NO. 13, and the Wxb-PCR-R sequence is shown as SEQ ID NO. 14.
17. Method for identifying plants obtained by the method according to claim 8, characterized in that it comprises the following steps:
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.
CN202210501884.3A 2020-01-08 2020-01-08 Wx mutant protein based on gene editing technology and application of gene thereof in plant breeding Pending CN114891759A (en)

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