CN111961684B - Method for improving disease resistance of wheat by inhibiting expression of TaVQ5 gene in wheat - Google Patents

Abstract

The invention discloses a method for improving disease resistance of wheat by inhibiting expression of TaVQ5 gene in wheat. The invention adopts CRISPR/Cas9 technology, edits TaVQ5 gene at fixed point, knocks out wheat TaVQ5 gene by causing frameshift mutation, and obtains new wheat germplasm with obviously improved powdery mildew and banded sclerotial blight resistance. The invention improves the powdery mildew and banded sclerotial blight resistance of wheat, innovates disease-resistant germplasm resources of wheat, has important significance for new variety cultivation, environmental sanitation and grain safety, and has important application and popularization values.

Description

Method for improving disease resistance of wheat by inhibiting expression of TaVQ5 gene in wheat

Technical Field

The invention relates to a method for improving disease resistance of wheat by inhibiting expression of TaVQ5 gene in the wheat in the field of biotechnology.

Background

Wheat (triticum aestivum l) is one of the most important grain crops in the world, provides basic protein and carbohydrate for human beings, enhances the disease and insect resistance of wheat varieties, improves the yield and quality characteristics, and is an important target for improving wheat.

Wheat powdery mildew is an epidemic disease caused by wheat powdery mildew (Blumeria graminis f.sp.tritici), and is one of the main diseases in wheat production. Wheat powdery mildew can occur and cause damage in seedling stage and adult stage, the wheat powdery mildew damages organs of the overground part of wheat plants, mainly leaves, and can also damage stalks and ears when the disease is serious, and the yield loss can be 5-34% in the circulation year.

The sheath blight of wheat is mainly caused by the invasion of the basal part of wheat straw by Rhizoctonia (Rhizoctonia cerealis). After infection, the base of the stalk is rotten, the lodging resistance of the wheat is reduced, and dead seedlings and withered white ears occur in severe cases, so that the yield loss is caused.

A large number of researches show that the cultivation and planting of disease-resistant varieties are the most economic, effective, safe and reliable way for preventing and treating wheat powdery mildew and banded sclerotial blight, and the disease resistance identification, the resistance source screening and the disease-resistant new gene excavation of the varieties are the basis of the research for improving the disease resistance of wheat.

The traditional crossbreeding takes long time and has large workload, and the shape directional improvement is difficult to realize; it is difficult to perform precise site-directed mutagenesis by physical, chemical, biological and other mutagenesis means. In recent years, genome site-directed editing technologies represented by CRISPR/Cas9 technology have become a new means for plant breeding and direct study of gene functions. The basic principle is that a designed sgRNA (single-guide RNA) is utilized to mediate Cas9 nuclease to perform specific recognition and targeted cleavage on a target site, and an intracellular error-prone repair mechanism is utilized to introduce mutation. The method has the advantages of simple operation, short experimental period and low cost, can get a novel germ plasm resource without transgenosis by throwing away a vector sequence in the later stage, becomes a technology for efficiently and conveniently obtaining a specific gene mutant, and has important significance for germ plasm resource innovation and gene function research.

VQ proteins are a class of plant-specific proteins, all of which have a conserved VQ-motif, FxxxVQx (L/V/F) TG, where x is any amino acid. In 2002, VQ proteins were first discovered in arabidopsis thaliana, and subsequently, such proteins have also been discovered and identified in plants such as wheat, cotton, maize, grape, soybean, and the like. Studies of VQ function have found that it is involved not only in regulating various life processes of plants, but also in plant responses to biotic and abiotic stresses. Due to the large wheat genome and allohexaploids, the identification and biological functions of wheat VQ members remain to be further studied.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the resistance of wheat to powdery mildew and/or banded sclerotial blight.

In order to solve the above technical problems, a first object of the present invention is to provide a method for improving disease resistance of wheat, comprising the steps of: inhibiting the expression of TaVQ5 gene in receptor wheat to obtain target wheat with disease resistance higher than that of the receptor wheat; the disease resistance is resistance to powdery mildew and/or banded sclerotial blight; the TaVQ5 gene is a gene for coding a TaVQ5 protein; the protein is the following A1), A2) or A3):

A1) the amino acid sequence is the protein of the amino acid sequence shown in any one of sequence 3, sequence 5 or sequence 7 in the sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in any one of a sequence 3, a sequence 5 or a sequence 7 in a sequence table, has more than 80% of identity with the protein shown in A1), and is related to plant powdery mildew resistance and/or banded sclerotial blight resistance;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

In the method, the sequence 3 in the sequence table is composed of 221 amino acid residues, the sequence 5 in the sequence table is composed of 226 amino acid residues, and the sequence 7 in the sequence table is composed of 224 amino acid residues.

In the above methods, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above method, the 80% or greater identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above method, the TaVQ5 gene may be specifically a gene represented by E1 or E2 as follows:

e1, wherein the coding sequence (ORF) of the coding strand is a DNA molecule of any one of a sequence 2, a sequence 4 and a sequence 6 in a sequence table;

e2, the nucleotide sequence is any one DNA molecule of sequence 2, sequence 4 and sequence 6 in the sequence table.

In the method, the inhibition of the expression of the TaVQ5 gene in the wheat is realized by performing gene editing on the TaVQ5 gene in the wheat. The gene editing is realized by means of a CRISPR/Cas9 system.

In the method, the CRISPR/Cas9 system comprises a plasmid containing Cas9 and sgRNA, and the target sequence of the sgRNA can be the 1 st to 20 th sites of the sequence 8 in the sequence table.

In the method, the encoding DNA of the sgRNA is 6915-7017 th site of the sequence 1 in the sequence table.

In a specific embodiment of the invention, the recombinant vector of the CRISPR/Cas9 system comprises the recombinant vector pCXUN-Cas 9-gRNA; the recombinant vector pCXUN-Cas9-gRNA has encoding DNA of sgRNA consisting of 103 nucleotides, and the corresponding DNA molecule is 6915-7017 th site of a sequence 1 in a sequence table.

In the method, the target wheat is wheat which meets the following conditions: one or two or three of the A, B and D genomes are mutated at the target region.

In order to solve the above technical problems, the present invention also provides an agent against powdery mildew and/or banded sclerotial blight, the active ingredient of which is a substance that inhibits the expression of the gene encoding the TaVQ5 protein, reduces the abundance of the TaVQ5 protein, and/or knockouts the gene encoding the TaVQ5 protein.

In the above agent for resisting powdery mildew and/or banded sclerotial blight, the substance contains the following F1), F2) or F3):

F1) sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the gene;

F2) generating a DNA molecule that targets a sgRNA of the gene, a DNA molecule that generates an siRNA that targets the gene, a DNA molecule that generates an shRNA that targets the gene, a DNA molecule that generates an miRNA that targets the gene, or a DNA molecule that generates an antisense RNA that targets the gene;

F3) an expression vector that produces sgRNA targeting the gene, an expression vector that produces siRNA targeting the gene, an expression vector that produces shRNA targeting the gene, an expression vector that produces miRNA targeting the gene, or an expression vector that produces antisense RNA targeting the gene.

The active ingredients of the agent for resisting powdery mildew and/or banded sclerotial blight can also contain other biological components or/and non-biological components, and other active ingredients of the agent can be determined by a person skilled in the art according to the disease resistance effect of plants.

The invention also provides a TaVQ5 protein, wherein the TaVQ5 protein is a protein of A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of the amino acid sequence shown in any one of sequence 3, sequence 5 or sequence 7 in the sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in any one of a sequence 3, a sequence 5 or a sequence 7 in a sequence table, has more than 80% of identity with the protein shown in A1), and is related to plant powdery mildew resistance and/or banded sclerotial blight resistance;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

The invention also provides a nucleic acid molecule encoding a TaVQ5 protein, which is a gene shown as E1 or E2 as follows:

e1, the coding sequence is DNA molecule of any one of sequence 2, sequence 4 and sequence 6 in the sequence table;

e2, the nucleotide sequence is any one DNA molecule of sequence 2, sequence 4 and sequence 6 in the sequence table.

The inventor of the invention utilizes a new gene TaVQ5 which is separated and cloned from wheat in the laboratory and is related to jasmonic acid signal conduction, adopts CRISPR/Cas9 technology to edit the TaVQ5 gene at a fixed point, and knocks out the TaVQ5 gene by causing frameshift mutation, thereby obtaining a new wheat germplasm with obviously improved resistance to powdery mildew and banded sclerotial blight. Wheat powdery mildew and banded sclerotial blight are main diseases of wheat, and not only cause the yield reduction of wheat, but also cause the quality reduction of the wheat. The invention improves the powdery mildew and banded sclerotial blight resistance of wheat, innovating disease-resistant germplasm resources of wheat, and has important significance for new variety cultivation, environmental sanitation and grain safety. The invention has great application and popularization values.

Drawings

FIG. 1 shows a part T in example 2 of the present invention₀Transfer TaVQ5 gene plant toDetecting an electrophoretogram of a vector sequence after BsrI enzyme digestion, wherein M is 2k Marker, 2, 34, 40, 55, 74, 216, 217, 219, 220, 255-1, 255-2, 255-3, 255-4 and 255-5 are T₀The plants were tested by passage, WT/-was negative control, WT/+ was positive control.

FIG. 2 shows a part T in example 2 of the present invention₀The sequencing result diagram of a plant transformed with TaVQ5 gene, wherein TaVQ5-WT-A is the A genome sequence of TaVQ5 gene of wild type Zhengmai 7698; TaVQ5-WT-B is the B genome sequence of the TaVQ5 gene of wild type Zheng wheat 7698; TaVQ5-WT-D is the D genomic sequence of the TaVQ5 gene of wild type Zheng wheat 7698; TaVQ5-34-A is T₀The A genome sequence of the TaVQ5 gene substituting for TaVQ 5-34; TaVQ5-34-B is T₀The B genome sequence of the TaVQ5 gene of the TaVQ 5-34; TaVQ5-34-D is T₀The D genome sequence of the TaVQ5 gene of the TaVQ 5-34; TaVQ5-74-A is T₀The A genome sequence of TaVQ5 gene of TaVQ 5-74; TaVQ5-74-B is T₀The B genome sequence of the TaVQ5 gene of the generations of TaVQ 5-74; TaVQ5-74-D is T₀The D genome sequence of the TaVQ5 gene of the generations of TaVQ 5-74; TaVQ5-255-A is T₀The A genome sequence of TaVQ5 gene substituting for TaVQ 5-255; TaVQ5-255-B is T₀The B genome sequence of TaVQ5 gene substituting for TaVQ 5-255; TaVQ5-255-D is T₀The D genome sequence of the TaVQ5 gene of the generations of TaVQ 5-255. The gRNA target sequence is underlined; bold underline indicates inserted bases; "-" indicates deletion of one base; "-/-" indicates the deletion of multiple bases; "wt" means the same as wild type zheng mai 7698; "+ 1" indicates an addition of one base; "-7" indicates deletion of 7 bases, and so on.

FIG. 3 shows a part T in example 2 of the present invention₁A TaVQ5 transgenic plant is used for detecting an electrophoretogram of Cas9/gRNA/hptII, wherein M is 2Kmarker, wt is wild-type Zhengmai 7698, and 1-20 randomly selected 20T₁The generation TaVQ5-34 plants, "+" is a positive control and "-" is a blank control.

FIG. 4 shows T in example 3 of the present invention₃Sequencing result chart of plant transformed with TaVQ5 gene. The gRNA target sequence is underlined; bold underline indicates inserted bases; "-" indicates deletion of one base; "-// -"indicates deletion of a plurality of bases; "+ 1" indicates an addition of one base; "-7" indicates deletion of 7 bases, and so on. TaVQ5-WT-A is the A genome sequence of TaVQ5 gene of wild type Zheng Mai 7698; TaVQ5-34-2-9-B is TaVQ5-34-2-9 (belonging to T)₃Generation of TaVQ5-34), and so on.

FIG. 5 shows T in example 3 of the present invention₃The resistance identification result of wheat sheath blight of a plant transformed with TaVQ5 gene, wherein CK is 7698 of control wild type Zheng wheat; the significance analysis result of each plant compared with CK is analyzed by t-test, which represents 0.01<P<0.05; denotes 0.001<P<0.01; denotes P<0.001。

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Zheng Mai 7698, a wheat variety in the following examples, which is susceptible to powdery mildew, is described in non-patent documents "Guo G, Lei M, Wang Y, Song B, Yang J, et al, Accumulation of As, Cd, and Pb in pure wheat crops growth in associated resources and associated Health assessment [ J ]. Journal of Environmental Research and Public Health,2018,15(11): 2601", publicly available from the institute of crop science of Chinese agricultural sciences to repeat the experiments of this application and is not applicable for other uses.

Powdery mildew bacterium E09 in the following examples is described in non-patent document "Chengtianling. Shanxi province wheat variety resistance identification and evaluation for powdery mildew disease.2019. Jiangsu agricultural science", which is publicly available from the institute of crop science of Chinese academy of agricultural sciences to repeat the experiments of the present application and is not applicable for other uses.

The disease grade standard of wheat powdery mildew (see Chengtianling, identification and evaluation of resistance of wheat varieties in Shanxi province to powdery mildew, 2019, Jiangsu agricultural science) is specifically shown in Table 1.

TABLE 1 wheat powdery mildew disease grade Standard

Wherein, 0 grade is immunity, 1-2 grade is high resistance, 3-4 grade is medium resistance, 5-6 grade is medium feeling, 7-8 grade is high feeling, and 9 grade is extreme feeling.

The rhizoctonia solani R0301 in the following examples is described in the non-patent document, "Ervoniphaera, improvement of the identification method of the resistance to rhizoctonia solani in the seedling stage of wheat and screening of the disease-resistant variety, 2019, the report on plant pathology", which is publicly available from the research institute of crop science of Chinese academy of agricultural sciences to repeat the application experiment and cannot be used for other purposes.

The disease grade standard of wheat sharp eyespot (Wangfuyu, identification and evaluation of resistance of wheat main cultivar of Shandong province to sharp eyespot 2016, modern agricultural science) is shown in Table 2.

TABLE 2 sheath blight disease class criteria for wheat

Sheath blight disease grade of wheat (IT)	Sheath blight disease of wheat
		Level
0	The scab is not generated in the leaf sheath and the stem;
		level 1	Is developed from the leaf sheathNon-invasion of stalk
Stage
	2	1/4 for invasion of lesion into insufficient stem periphery
Grade
	3	1/4-1/2 of lesion spots invading stem periphery
4 stage			1/2-3/4 of plant disease spots invading stem periphery
	Grade
5		The plant has lesion on leaf sheath and stem, and withered booting ear or withered white ear

Wherein, 0 grade represents immunity, 1 grade represents resistance, 2 grade represents resistance, 3 grade-4 grade represents feeling, and 5 grade represents high feeling.

Disease Index (DI) [ (Σ number of diseased plants at each stage × corresponding disease stage)/(total number of plants × highest disease stage) ] × 100.

Example 1 preparation of recombinant plasmid

A recombinant plasmid pCXUN-Cas9-gRNA was artificially synthesized. The recombinant plasmid pCXUN-Cas9-gRNA is a circular plasmid. The nucleotide sequence of the recombinant plasmid pCXUN-Cas9-gRNA is shown as a sequence 1 in a sequence table. In the sequence 1, from the 5' end, the 115-position 367 nucleotide is reversely complementary with the NOS terminator, the 392-position 4522 nucleotide is reversely complementary with the coding gene of the Cas9 protein, the 4544-position 6533 nucleotide is reversely complementary with the Ubi promoter, the 6552-position 6914 nucleotide is the U6 promoter, and the 6915-position 7017 nucleotide is the coding gene of the sgRNA (wherein the 6915-position 6934 nucleotide is the target sequence recognition region). The recombinant plasmid pCXUN-Cas9-gRNA expresses sgRNA, the target sequence of the sgRNA is positioned in a wheat TaVQ5 gene, and the specific target sequence is shown as a sequence 8 in a sequence table.

In wheat cDNA: the coding region of the TaVQ5 gene in the A genome (corresponding to the A chromosome group) is shown as a sequence 2 in the sequence table (protein shown as a sequence 3 in the coding sequence table); the coding region of the TaVQ5 gene in the B genome (corresponding to the B chromosome group) is shown as a sequence 4 in the sequence table (protein shown as a sequence 5 in the coding sequence table); the coding region of the TaVQ5 gene in the D genome (corresponding to the D chromosome group) is shown as a sequence 6 in the sequence table (coding protein shown as a sequence 7 in the sequence table).

Example 2 Gene-edited wheat obtained by biolistic method Using sgRNA

One, genetic gun mediated wheat genetic transformation

1. And taking the young embryo of Zhengmai 7698 which is a wheat variety about 14 days after pollination as an explant. Inoculating the explant to hypertonic culture medium, and culturing at 22-25 deg.C in dark for 1-2 days.

Hypertonic culture medium: the MS minimal medium is added with sucrose, and the concentration of the sucrose is 180 g/L.

2. The recombinant plasmid pCXUN-Cas9-gRNA is used as DNA to be transformed, a PDS-1000/He gene gun of BIO-RAD company is adopted to bombard the immature embryo in the step 1 (Psi900, 27.5cmHg column), the bombarded immature embryo is transferred to an induction culture medium, and dark culture is carried out for 2-3 weeks at the temperature of 22-25 ℃.

The induction culture medium is a culture medium which takes an MS culture medium as a basic culture medium and is added with Vc (vitamin C), 2,4-D (2, 4-dichlorophenoxyacetic acid) and cane sugar, the concentration of Vc in the induction culture medium is 100mg/L, the concentration of 2,4-D in the induction culture medium is 0.5mg/L, the concentration of cane sugar is 120g/L, and the pH value of the culture medium is 5.8.

3. Taking the explant which is subjected to the step 2, and sequentially carrying out regeneration and strong seedling culture to obtain T₀Plants 345 were generated. The specific method comprises the following steps:

regeneration culture: the resistant calli after induction culture were transferred to regeneration medium and cultured under light for 3-6 weeks at 16h day/8h night, 24 ℃.

The regeneration culture medium adopts MS culture medium as basic culture medium, and 2,4-D (2, 4-dichlorophenoxyacetic acid) and CuSO are added₄And Zeatin, wherein the concentration of 2,4-D is 0.05mg/L, CuSO₄The concentration of (2) was 5mmol/L, and the Zeatin concentration was 5 mg/L. The medium contained hygromycin Hpt at a concentration of 15mg/L, and the pH of the medium was 5.8.

Strong seedling culture: green seedlings were transferred to strong seedling medium and cultured under light for 3 weeks at 16h day/8h night, 24 ℃.

The strong seedling culture medium takes 1/2MS culture medium as a basic culture medium, 25g of cane sugar is added, and the pH value of the culture medium is 5.8.

Second, detection of fixed point editing

1. For T₀Identification of the plant

The test plants were: t obtained in step one of 345 plants₀The generation plant, Zheng Mai 7698 (as reference plant).

1.1, extracting genome DNA of the leaf of 345 test plants as a template, and carrying out PCR amplification by using genome specific primers A, B and D of TaVQ 5. The three pairs of genome specific primers are respectively as follows:

primer set (A genome-specific primer) consisting of AF1 and AR1

AF1：5’-CGACCCCACACCCACCCTGA-3’；

AR1：5’-ATGTCCGCGCCGCCGCCGCCGTAGT-3’；

Primer set (B genome-specific primer) consisting of BF1 and BR1

BF1：5’-ACCCGCGCCAGCCGTCGGTTCG-3’；

BR1：5’-CTGCTGCAGGGACGAGGTGACA-3’；

Primer pair consisting of DF1 and DR1 (D genome-specific primer)

DF1：5’-GGCGAGCCCGACAGGACGGT-3’；

DR1：5’-CGAACGACATCACGGCCGAGG-3’。

TaVQ5 gene PCR reaction system: 5. mu.L of Fast Pfu buffer, 5. mu. L, dNTP 2. mu.L of PCR stimulant, 200ng of template and ddH for upstream and downstream primers (10. mu.M) of 0.5. mu. L, Pfu 0.5.5. mu. L, DNA, respectively₂Make up to 25. mu.L of O. The reaction conditions were as follows: 95 ℃ 2min, 95 ℃ 20s, A genome 69 ℃ 20s/B genome 67 ℃ 20s/D genome 62 ℃ 20s, 72 ℃ 20s, 35 cycles.

1.2, carrying out enzyme digestion screening on the product of the PCR reaction by using Bsr I, carrying out amplification by combining with a vector sequence (Cas9/Hpt/TaU6, and respectively carrying out amplification by using primers of Cas9, Hpt and TaU 6) to preliminarily detect a transgenic plant and a genome editing plant, and selecting a single clone for sequencing the PCR product which is not cut to determine the genotype of the editing plant.

Identifying a Cas9 gene by adopting a primer pair consisting of Cas9-F and Cas 9-R;

Cas9-F：5’-TCGACAAGAAGTACTCCATCGGC-3’；

Cas9-R：5’-CAAGAGAGAGGGCGATCAGGTTG-3’。

identifying the sgRNA gene by adopting a primer pair consisting of TaU6-F and TaU 6-R;

TaU6-F：5’-CTGACAGTTCTGGTGCTCAAC-3’；

TaU6-R：5’-AAAGCACCGACTCGGTGCCA-3’。

and identifying the hptII gene by adopting a primer pair consisting of Hpt-F and Hpt-R.

Hpt-F：5’-GAGGGCGTGGATATGTCCTG-3’；

Hpt-R：5’-ATTGACCGATTCCTTGCGGT-3’。

And (3) PCR reaction system: 5. mu.L of Fast Pfu buffer, 5. mu. L, dNTP 2. mu.L of PCR stimulant, 200ng of template and ddH for upstream and downstream primers (10. mu.M) of 0.5. mu. L, Pfu 0.5.5. mu. L, DNA, respectively₂Make up to 25. mu.L of O. The reaction conditions were as follows: 95 ℃ for 2min, 95 ℃ for 20s and an annealing temperature of 56 ℃. The PCR product was digested with Bsr I enzyme at 37 ℃ for 30 min. BsrI restriction system: PCR product 5. mu.L, BsrI enzyme 0.3. mu.L, Bufer 1. mu.L, ddH₂O 3.7μL。

If the PCR amplification product of DNA of the plant is only one and is identical with the nucleotide sequence of the PCR amplification product of Zheng wheat 7698, the plant is wild type. If the PCR amplification products of DNA of a plant are two, one is identical to the nucleotide sequence of the PCR amplification product of Zheng wheat 7698, and the other is mutated (mutation includes deletion, insertion or substitution of one or more nucleotides) compared with the nucleotide sequence of the PCR amplification product of Zheng wheat 7698, the plant is heterozygous. If the PCR amplification products of DNA of the plant are two kinds, both of them are mutated (mutation includes deletion, insertion or substitution of one or more nucleotides) compared with the nucleotide sequence of the PCR amplification product of Zheng Mai 7698, the plant is biallelic mutant. If the PCR amplification product of DNA of a plant is one and mutation (mutation including deletion, insertion or substitution of one or more nucleotides) occurs compared with the nucleotide sequence of the PCR amplification product of Zheng Mai 7698, the plant is a homozygous mutant. If the nucleotide sequence of the PCR amplification product of the plant DNA is more than three, the plant is chimeric. Plants of heterozygous, biallelic, homozygous, and chimeric types are collectively referred to as edited plants.

Of the 345 plants, 15 were identified as positive plants, of which 11 plants with edited genomes, 1 containing no vector sequence, were the product of transient expression cleavage of CRISPR/Cas9, and 3 containing vector sequence but not edited. The results of BsrI digestion and vector sequence detection are shown in FIG. 1.

Part T₀The results of sequencing the generation-edited plants are shown in FIG. 2.

Part T₀The genotype of the generation editing plants based on the genome of the target sequences a, B, D, the type of mutation based on the target sequences, the case of carrying the Cas9 gene, the case of carrying the sgRNA gene and the case of carrying the hptII gene are shown in table 3.

TABLE 3

Note: i represents an insertion, i1 represents an insertion of 1 nucleotide, and so on; d represents deletion, d7 represents deletion of 7 nucleotides, and so on; wt represents wild type; before and after "/" represent two chromosomes, respectively; for example, i1/wt represents i1 for the target sequence in the D genome and the wild type for the other chromosome. Y represents that the identification result is positive, and N represents that the identification result is negative.

2. For T₁Identification of the plant

Get T₀Plant generation TaVQ5-34, T₀Plant generation TaVQ5-74, T₀Carrying out inbreeding and harvesting T on the generation plants TaVQ5-255 respectively₁Seed generation and T cultivation₁Seed generation to obtain T₁And (5) plant generation.

Part T₁The generation plants detect Cas9/gRNA/hptII according to the step 1, and the result isSee fig. 3.

According to the method of step 1, for each T₁The generation plants are identified, and the results of all the identifications are shown in table 4.

TABLE 4

Note: the meanings of the symbols are as given in Table 1. 3i1 represents the homozygous mutation for strain i1 in 3, the heterozygous mutation for strain i1/wt in 10i1/wt in 10, and so on.

The results show that T₀The homozygous strain with site-directed mutagenesis of the TaVQ5 can stably inherit T₁Generation, T by strict selfing of Diallelic mutant lines of TaVQ5 edited at fixed points₁The segregation situation conforms to Mendelian inheritance rule at T₁No new types of variation were found in the passage lines. At T₁Editing strains with target regions mutated and without carrying vector sequences can be obtained.

3. Off-target analysis of CRISPR/Cas9

According to online forecasting software (http://crispr.dbcls.jp/) Inputting a TaVQ5 gene sequence, selecting a Wheat (Triticum aestivum L.) genome, IWGSC1.0+ popseq (Nov,2014) library, and predicting the possible existing off-target sites of the gRNA, wherein the result shows that the selected gRNA does not predict the existence of the off-target sites.

Example 3 detection of Properties

First, material acquisition

Plant editing trial: TaVQ5-34, TaVQ5-74 and TaVQ5-255 obtained in example 2 were selfed to obtain T₁Plant generation, selecting the T₁Selfing the plant to obtain T₂Seed generation, for each T following the procedure of example 2₂Identifying the plant generation from T₂Selecting homozygous mutation type based on target sequence from generation plants, and selfing plant not carrying vector sequence (TaVQ5-34-2-9, TaVQ5-74-2-11, TaVQ5-74-2-19, TaVQ5-74-6-13, TaVQ5-74-8-3, TaVQ5-74-9-5, TaVQ5-255-4-7, TaVQ5-255-4-16) to obtain T₃The generation seed is T₃Substitute for non-carrier vector sequenceThe plant of (1). Will originate from T₀T of generation plant TaVQ5-34₃The plant not carrying the carrier sequence is named as T₃The generation TaVQ5-34 will originate from T₀T of generation plant TaVQ5-74₃The plant not carrying the carrier sequence is named as T₃The generation TaVQ5-74 will originate from T₀T of generation plant TaVQ5-255₃The plant not carrying the carrier sequence is named as T₃To TaVQ 5-255. T is₃TaVQ5-34, T substitute₃TaVQ5-74, T substitute₃The plant genotypes of the generations of TaVQ5-255 are shown in FIG. 4. By T₃TaVQ5-34, T substitute₃TaVQ5-74, T substitute₃The TaVQ5-255 generation was used as a test editing plant for the next disease resistance identification.

Second, detection of character

The test plant is T₃TaVQ5-34, T substitute₃TaVQ5-74, T substitute₃TaVQ5-255 substitute, Zheng wheat 7698 is used as wild control plant.

1. Wheat powdery mildew resistance assay

(1) Under field conditions, 50 test plants were normally grown per strain. Induced and control plants were planted simultaneously.

(2) When the tested plants grow to the jointing stage, the Erysiphe cichoracearum E09 is inoculated on the induced plant by sweeping.

(3) In the filling stage of wheat, the condition of the patient is recorded by adopting 0-9 grade standard investigation, the specific disease grade standard is shown in table 1, and the identification result is shown in table 5.

TABLE 5

Note: CK is wild type Zheng Mai 7698.

The identification result shows that wild type Zhengmai 7698 plant shows the powdery mildew, T₃TaVQ5-34, T substitute₃TaVQ5-74, T substitute₃The plants of the generations of TaVQ5-255 all show the powdery mildew resistance, which indicates that the powdery mildew resistance of the wheat with the TaVQ5 gene knockout gene is improved to a certain level.

2. Wheat sheath blight resistance assay

(1) Under field conditions, 50 test plants were normally grown per strain. Control plants were planted simultaneously.

(2) And (3) inoculating rhizoctonia solani R0301 by adopting a toothpick inoculation method in the growth jointing stage of the tested plant.

(3) In the filling stage of wheat, 5 grades of disease grading standards are adopted for grading the disease, the disease index is calculated, specifically shown in table 2, and the identification result is shown in fig. 5.

The results show that the TaVQ5 gene knockout mutant T is compared with the wild type Zheng wheat 7698 plants₃TaVQ5-34, T substitute₃TaVQ5-74, T substitute₃The resistance of the plants of the TaVQ5-255 generation to the wheat sharp eyespot is obviously improved, which shows that the ability of knocking out the TaVQ5 gene to the wheat sharp eyespot is improved to a certain level.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

<120> method for improving disease resistance of wheat by inhibiting expression of TaVQ5 gene in wheat

<130> GNCSY201697

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15756

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaattcgagc tcggtacccc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 60

aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcccgatcta 120

gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt 180

ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaacccat ctcataaata 240

acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga aattatatga 300

taatcatcgc aagaccggca acaggattca atcttaagaa actttattgc caaatgtttg 360

aacgatcggg gaaattcgga tccccaatac ttcaatcgcc gccgagttgt gagaggtcga 420

tgcgtgtctc gtagaggcct gtgatagact ggtggatgag ggtggcgtcg agaacctcct 480

tggtagaggt gtagcgcttg cggtcgatgg tggtgtcgaa gtacttgaag gcggctggag 540

cgccgaggtt ggtgagggtg aagaggtgga tgatgttctc ggcctgctcg cgaattggct 600

tatcgcggtg cttgttgtag gcgctgagca ccttatcgag gttggcatcg gcgaggatca 660

cgcgcttgga gaactcggag atctgctcga tgatctcgtc gaggtagtgc ttgtgctgct 720

cgacgaacag ctgcttttgc tcgttgtcct ctggggagcc cttgagcttc tcgtagtggg 780

aggcgaggta gaggaagttc acgtacttgg acgggagagc aagctcgttg cccttctgaa 840

gctcgccagc agaggcgagc attctcttgc ggccgttctc aagctcgaag aggctgtact 900

tcgggagctt gatgatgagg tccttcttca cctccttgta gcccttggcc tcgaggaagt 960

cgattgggtt cttctcgaag ctgctgcgct ccatgatcgt gatgcccagc agctccttga 1020

cggacttgag cttcttgctc ttgcccttct cgaccttggc aaccacgagc acagagtagg 1080

ccacggtcgg agaatcgaag ccgccatact tcttcgggtc ccagtccttc ttgcgggcga 1140

tcagcttgtc ggagttgcgc tttgggagga tggactcctt ggagaagccg ccggtctgaa 1200

cctcggtctt cttcacgatg ttcacttgcg gcatggagag caccttgcgc actgtggcga 1260

aatccctgcc cttgtcccac acgatctcgc ctgtctcgcc gtttgtctcg atgagcggcc 1320

tcttcctaat ctcgccgttg gcgagcgtga tctcggtctt gaagaaattc atgatgttgg 1380

agtagaagaa gtacttggcg gtcgccttgc cgatctcttg ctcggacttg gcgatcatct 1440

tgcgcacgtc gtacaccttg tagtcgccgt acacgaactc ggactcgagc tttgggtact 1500

tcttgatgag ggctgtgccc accacggcat tgaggtaggc gtcgtgggcg tggtggtagt 1560

tgttgatctc gcgcaccttg tagaactgga agtccttgcg gaagtcggac acgagcttgg 1620

acttgagggt gatgaccttc acctcgcgga tgagcttgtc gttctcgtcg tacttggtgt 1680

tcatgcggga gtcgaggatc tgggccacgt gctttgtgat ctggcgtgtc tcgacgagct 1740

ggcgcttgat gaagccggcc ttatcaagct cggaaaggcc gcctctctcg gccttggtga 1800

ggttgtcgaa cttcctctgg gtgatgagct tggcgttgag gagctggcgc cagtagttct 1860

tcatcttctt gacgacctct tcggacggca cgttatcgga cttgcccctg ttcttgtcgg 1920

agcgggtgag caccttgttg tcgatggagt cgtccttcag gaaggactgc ggcacaatat 1980

ggtccacgtc gtagtcggag aggcggttga tgtccagctc ttggtccacg tacatgtcgc 2040

ggccgttctg gaggtagtag aggtagagct tctcgttctg gagctgggtg ttctcgactg 2100

ggtgctcctt gaggatctgg gagcccagct ccttaatgcc ctcctcgatc ctcttcatgc 2160

gctcgcggga gttcttttgg cccttctgtg tggtctggtt ctcgcgggcc atctcgatca 2220

cgatgttctc tggcttgtgc ctgcccatca ccttcaccag ctcgtccacc accttcacgg 2280

tctggagaat gcccttcttg atagccgggg agccggcgag attggcgata tgctcatgga 2340

gggaatcgcc ttggccggac acctgggcct tttggatgtc ctccttgaag gtgagggagt 2400

cgtcgtggat gagctgcatg aagttgcggt tggcgaagcc gtcggacttg aggaagtcga 2460

ggatcgtctt gccggactgc ttgtcgcgga tgccgttgat gagcttccta gagagcctgc 2520

cccagccggt atagcgcctg cgcttcagct gcttcatcac cttgtcgtcg aagaggtggg 2580

cgtatgtctt gaggcgctcc tcgatcatct cgcggtcctc gaagagggtg agggtgagca 2640

cgatgtcctc gaggatgtcc tcgttctcct cgttgtcgag gaagtccttg tccttgataa 2700

tcttgaggag gtcgtggtag gtcccgaggg aggcattgaa cctatcctcg acgccggaga 2760

tctcgacgga gtcgaagcac tcgattttct tgaagtagtc ctccttgagc tgcttcacgg 2820

tcaccttgcg gttggtcttg aacagcaggt cgacgatggc cttcttttgc tcgccgctaa 2880

ggaaagctgg cttcctcatc ccctcggtca cgtacttcac cttggtcagc tcgttgtaca 2940

cggtgaagta ctcgtagagg agtgagtgct tcgggagcac cttctcgttc gggaggttct 3000

tgtcgaagtt ggtcatgcgc tcgatgaaag actgggcaga ggcgccctta tccaccacct 3060

cctcgaagtt ccagggggtg attgtctcct cggactttct ggtcatccag gcgaacctgg 3120

agttgcccct ggcgagcggg cccacgtagt acgggatgcg gaaggtgagg atcttctcaa 3180

tcttctcgcg gttgtccttg aggaacgggt agaagtcctc ttgcctgcgg aggatagcat 3240

gaagctcgcc gaggtggatc tggtgcggga tggagccatt atcgaaggtg cgctgcttgc 3300

ggaggaggtc ctctctattg agcttcacga gcagctcctc ggtgccgtcc atcttctcga 3360

ggatcggctt gatgaacttg tagaactcct cttgagaagc gccgccatcg atgtagccgg 3420

cgtagccgtt cttggactgg tcgaagaaga tctccttgta cttctctggg agctgctgtc 3480

tcacgagggc cttgaggagt gtgaggtcct ggtggtgctc gtcgtacctc ttgatcatgg 3540

aggcggagag tggggccttg gtgatctcgg tgttcaccct gaggatgtcg ctgaggagga 3600

tggcgtcgga gagattcttg gcggcgagga acagatcggc gtactgatcg ccaatctggg 3660

cgaggagatt gtcgaggtcg tcgtcgtagg tgtccttgga aagctggagc ttggcgtcct 3720

cggcgaggtc gaagttggac ttgaagttcg gggtgaggcc aagagagagg gcgatcaggt 3780

tgccgaagag gccattcttc ttctcgcccg gaagttgggc gatcagattc tcgagcctgc 3840

gggacttaga gagcctggca gagagaatag ccttggcgtc aacgccagag gcgttgatcg 3900

ggttctcctc gaacagctgg ttgtaggtct gcacgagctg gatgaacagc ttgtccacat 3960

cggagttgtc cgggttgagg tcgccctcga tgaggaagtg gcccctgaac ttgatcatgt 4020

gggcgagggc gaggtagatg agcctgaggt cggccttatc ggtggagtcg acgagcttct 4080

tgcggaggtg gtagatggtc gggtacttct cgtggtaggc cacctcatcc acgatgttgc 4140

cgaagatcgg atggcgctcg tgcttcttgt cctcctcgac gaggaagctc tcctcgagcc 4200

tgtggaagaa gctgtcgtcc accttggcca tctcgttgga gaagatctct tggaggtagc 4260

agatgcggtt cttgcgcctg gtgtacctgc gtctagcggt cctcttgagc cttgtagcct 4320

cggctgtctc gccagagtcg aacagcaggg cgccgatgag attcttcttg atggagtggc 4380

ggtcggtgtt gccgaggacc ttgaacttct tggacggcac cttgtactcg tcggtgatca 4440

cggcccagcc aacagaattg gtgccgatgt cgaggccgat ggagtacttc ttgtcgacct 4500

tgcgcttctt ctttggggcc atagtattgg ggatcccccg ggctgcagaa gtaacaccaa 4560

acaacagggt gagcatcgac aaaagaaaca gtaccaagca aataaatagc gtatgaaggc 4620

agggctaaaa aaatccacat atagctgctg catatgccat catccaagta tatcaagatc 4680

aaaataatta taaaacatac ttgtttatta taatagatag gtactcaagg ttagagcata 4740

tgaatagatg ctgcatatgc catcatgtat atgcatcagt aaaacccaca tcaacatgta 4800

tacctatcct agatcgatat ttccatccat cttaaactcg taactatgaa gatgtatgac 4860

acacacatac agttccaaaa ttaataaata caccaggtag tttgaaacag tattctactc 4920

cgatctagaa cgaatgaacg accgcccaac cacaccacat catcacaacc aagcgaacaa 4980

aaagcatctc tgtatatgca tcagtaaaac ccgcatcaac atgtatacct atcctagatc 5040

gatatttcca tccatcatct tcaattcgta actatgaata tgtatggcac acacatacag 5100

atccaaaatt aataaatcca ccaggtagtt tgaaacagaa ttctactccg atctagaacg 5160

accgcccaac cagaccacat catcacaacc aagacaaaaa aaagcatgaa aagatgaccc 5220

gacaaacaag tgcacggcat atattgaaat aaaggaaaag ggcaaaccaa accctatgca 5280

acgaaacaaa aaaaatcatg aaatcgatcc cgtctgcgga acggctagag ccatcccagg 5340

attccccaaa gagaaacact ggcaagttag caatcagaac gtgtctgacg tacaggtcgc 5400

atccgtgtac gaacgctagc agcacggatc taacacaaac acggatctaa cacaaacatg 5460

aacagaagta gaactaccgg gccctaacca tggaccggaa cgccgatcta gagaaggtag 5520

agaggggggg ggggggagga cgagcggcgt accttgaagc ggaggtgccg acgggtggat 5580

ttgggggaga tctggttgtg tgtgtgtgcg ctccgaacaa cacgaggttg gggaaagagg 5640

gtgtggaggg ggtgtctatt tattacggcg ggcgaggaag ggaaagcgaa ggagcggtgg 5700

gaaaggaatc ccccgtagct gccgtgccgt gagaggagga ggaggccgcc tgccgtgccg 5760

gctcacgtct gccgctccgc cacgcatttc tggatgccga cagcggagca agtccaacgg 5820

tggagcggaa ctctcgagag gggtccagag gcagcgacag agatgccgtg ccgtctgctt 5880

cgcttggccc gacgcgacgc tgctggttcg ctggttggtg tccgttagac tcgtcgacgg 5940

cgtttaacag gctggcatta tctactcgaa acaagaaaaa tgtttcctta gtttttttaa 6000

tttcttaaag ggtatttgtt taatttttag tcactttatt ttattctatt ttatatctaa 6060

attattaaat aaaaaaacta aaatagagtt ttagttttct taatttagag gctaaaatag 6120

aataaaatag atgtactaaa aaaattagtc tataaaaacc attaacccta aaccctaaat 6180

ggatgtacta ataaaatgga tgaagtatta tataggtgaa gctatttgca aaaaaaaagg 6240

agaacacatg cacactaaaa agataaaact gtagagtcct gttgtcaaaa tactcaattg 6300

tcctttagac catgtctaac tgttcattta tatgattctc taaaacactg atattattgt 6360

agtactatag attatattat tcgtagagta aagtttaaat atatgtataa agatagataa 6420

actgcacttc aaacaagtgt gacaaaaaaa atatgtggta attttttata acttagacat 6480

gcaatgctca ttatctctag agaggggcac gaccgggtca cgctgcactg caggaattcg 6540

atatcaagct tgaccaagcc cgttattctg acagttctgg tgctcaacac atttatattt 6600

atcaaggagc acattgttac tcactgctag gagggaatcg aactaggaat attgatcaga 6660

ggaactacga gagagctgaa gataactgcc ctctagctct cactgatctg ggtcgcatag 6720

tgagatgcag cccacgtgag ttcagcaacg gtctagcgct gggcttttag gcccgcatga 6780

tcgggctttt gtcgggtggt cgacgtgttc acgattgggg agagcaacgc agcagttcct 6840

cttagtttag tcccacctcg cctgtccagc agagttctga ccggtttata aactcgcttg 6900

ctgcatcaga cttgcgccgt agatgcccgc ccaggtttta gagctagaaa tagcaagtta 6960

aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttggt 7020

accctgcatg ggagaggcgg tttgcgtatt ggtttaaaca tagctaaact atcagtgttt 7080

gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa taacggatat 7140

ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg 7200

gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt gcagtcggct 7260

tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc taagttacgc 7320

gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa 7380

agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc gccgccgctg 7440

gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac caacgggccg 7500

aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc accaggcgcg 7560

accgcccgga gctggccagg atgcttgacc acctagccct ggcgacgttg tgacagtgac 7620

caggctagac cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca 7680

ggaggccggc gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc 7740

cggccgcatg gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat 7800

cgaccgcacc cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg 7860

ccctaccctc accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg 7920

caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact 7980

tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga 8040

cgcattgacc gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc 8100

atgaaaccgc accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg 8160

gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg 8220

catgaaatcc tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc 8280

gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg 8340

cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac 8400

gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa 8460

cccatctagc ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc 8520

cccagggcag tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg 8580

gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag 8640

tgatcgacgg agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact 8700

tcgtgctgat tccggtgcag ccaagccctt acgacatatg ggcaaccgcc gacctggtgg 8760

agctggttaa gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt 8820

cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg 8880

agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg 8940

ccggcacaac cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc 9000

tggccgctga aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa 9060

aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt 9120

ggccagcctg gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga 9180

tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc 9240

tgaatacatc gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt 9300

tagcggctaa aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat 9360

gccccatgtg tggaggaacg ggcggttggc caggcgtaag cggctgggtt gtctgccggc 9420

cctgcaatgg cactggaacc cccaagcccg aggaatcggc gtgacggtcg caaaccatcc 9480

ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg 9540

caggccgccc agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg 9600

gccgctgatc gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt 9660

aggaagccgc ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg 9720

ggcacccgcg atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac 9780

cgacgagctg gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca 9840

gggccggccg gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat 9900

ctaaccgaat ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc 9960

cgtccacacg ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa 10020

gacgacctgg tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg 10080

aagaaggcca agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc 10140

tacaagatcg taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat 10200

tggatgtacc gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat 10260

tactttttga tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca 10320

ggcaaggcag aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga 10380

gagttcaaga agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag 10440

tacgatttga aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac 10500

ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt 10560

gccctagcag gggaaaaagg tcgaaaaggt ctctttcctg tggatagcac gtacattggg 10620

aacccaaagc cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt 10680

gggaaccggt cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc 10740

taaaactctt taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct 10800

ggccagcgca cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta 10860

cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta 10920

cggccaggca atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc 10980

ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 11040

gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg 11100

tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag 11160

cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg 11220

caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc 11280

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 11340

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 11400

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 11460

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 11520

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 11580

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 11640

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 11700

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 11760

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 11820

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 11880

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 11940

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 12000

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 12060

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 12120

gtcatgcatt ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc 12180

caatcaggct tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata 12240

tcctccctga tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt 12300

ctcccaagat caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc 12360

aggtcgccgt gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac 12420

agctcgcgcg gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc 12480

agatcgttat tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag 12540

ggacaatccg atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata 12600

atcttttcag ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca 12660

ctcatgagca gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc 12720

agctttcctt ccagccatag catcatgtcc ttttcccgtt caacatcata ggtggtccct 12780

ttataccggc tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat 12840

accttagcag gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt 12900

ttcaattccg gtgatattct cattttagcc atttattatt tccttcctct tttctacagt 12960

atttaaagat accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg 13020

cattctaaaa ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt 13080

ataacatagt atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct 13140

gtcatcgtta caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag 13200

cttagttgcc gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac 13260

ggctctcccg ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt 13320

gccgagctgc cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa 13380

caaattgacg cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa 13440

cgccgaatta attcggggga tctggatttt agtactggat tttggtttta ggaattagaa 13500

attttattga tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc 13560

aacacatgag cgaaacccta taggaaccct aattccctta tctgggaact actcacacat 13620

tattatggag aaactcgagc ttgtcgatcg acagatccgg tcggcatcta ctctatttct 13680

ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc 13740

catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc 13800

cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt 13860

caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc cggagtcgtg 13920

gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag 13980

ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg 14040

cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc 14100

cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct 14160

catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat 14220

acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc 14280

cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat 14340

ccatagcctc cgcgaccggt tgtagaacag cgggcagttc ggtttcaggc aggtcttgca 14400

acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgcta aactccccaa 14460

tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat 14520

ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat 14580

cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt 14640

cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatat 14700

ctcattgccc cccggatctg cgaaagctcg agagagatag atttgtagag agagactggt 14760

gatttcagcg tgtcctctcc aaatgaaatg aacttcctta tatagaggaa ggtcttgcga 14820

aggatagtgg gattgtgcgt catcccttac gtcagtggag atatcacatc aatccacttg 14880

ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc 14940

atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt tatcgcaatg 15000

atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt gacagatagc 15060

tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag tctcaatagc 15120

cctttggtct tctgagactg tatctttgat attcttggag tagacgagag tgtcgtgctc 15180

caccatgtta tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 15240

cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 15300

aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 15360

tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 15420

taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 15480

cttggagtag acgagagtgt cgtgctccac catgttggca agctgctcta gccaatacgc 15540

aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 15600

gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 15660

ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 15720

caatttcaca caggaaacag ctatgaccat gattac 15756

<210> 2

<211> 666

<212> DNA

<213> wheat (Triticum aestivum)

<400> 2

atgggcgaca ccggcgcgaa catgggccac tgggcgggca tctacggcgt cggcggcaat 60

ggggccgcgg cggcggaggg cagcgtggtg acggtgtcga gcccgacgtc ggggggctcg 120

ggcggcggga gccccacgag gtcggcgccc ggggtcgagg ggggccgcgt cggcaagccg 180

gcgcggcgga ggtcccgcgc ctcgcggcgg gcgcccgtca cgctgctcaa caccgacacc 240

accaacttcc gcgccatggt gcagcagttc accggcatcc cctccggccc ctacggcccg 300

gccggcgcgg gcggcgggcc cgtgatcagc ttcggcgcgg gcggtggcga ctacggcctc 360

ggcggcggca tgccggtgcg cccgtcgccg agctcggccg tgatgtcgtt cgaccacctc 420

ggccaccacc gcccgtccgc cgccacgtcg tccctgcagc agcagcagca tcagcagagc 480

cagctgttcc ggccgcagca gcagcaacag tacgccgact acggcggcgg cggcgcggac 540

atgtcgttcc tgcacgggtt cgagtcgtcg gcggaggaca gactgctgct gcagagcata 600

caggcggcgc agatgctgcc ccggccggcc tccactaaca cccccaatgg ctacaacttc 660

ggatga 666

<210> 3

<211> 221

<212> PRT

<213> wheat (Triticum aestivum)

<400> 3

Met Gly Asp Thr Gly Ala Asn Met Gly His Trp Ala Gly Ile Tyr Gly

1 5 10 15

Val Gly Gly Asn Gly Ala Ala Ala Ala Glu Gly Ser Val Val Thr Val

20 25 30

Ser Ser Pro Thr Ser Gly Gly Ser Gly Gly Gly Ser Pro Thr Arg Ser

35 40 45

Ala Pro Gly Val Glu Gly Gly Arg Val Gly Lys Pro Ala Arg Arg Arg

50 55 60

Ser Arg Ala Ser Arg Arg Ala Pro Val Thr Leu Leu Asn Thr Asp Thr

65 70 75 80

Thr Asn Phe Arg Ala Met Val Gln Gln Phe Thr Gly Ile Pro Ser Gly

85 90 95

Pro Tyr Gly Pro Ala Gly Ala Gly Gly Gly Pro Val Ile Ser Phe Gly

100 105 110

Ala Gly Gly Gly Asp Tyr Gly Leu Gly Gly Gly Met Pro Val Arg Pro

115 120 125

Ser Pro Ser Ser Ala Val Met Ser Phe Asp His Leu Gly His His Arg

130 135 140

Pro Ser Ala Ala Thr Ser Ser Leu Gln Gln Gln Gln His Gln Gln Ser

145 150 155 160

Gln Leu Phe Arg Pro Gln Gln Gln Gln Gln Tyr Ala Asp Tyr Gly Gly

165 170 175

Gly Gly Ala Asp Met Ser Phe Leu His Gly Phe Glu Ser Ser Ala Glu

180 185 190

Asp Arg Leu Leu Leu Gln Ser Ile Gln Ala Ala Gln Met Leu Pro Arg

195 200 205

Pro Ala Ser Thr Asn Thr Pro Asn Gly Tyr Asn Phe Gly

210 215 220

<210> 4

<211> 681

<212> DNA

<213> wheat (Triticum aestivum)

<400> 4

atgggtgaca ccggcgcgaa catgggccac tgggcgggca tctacggcgt cggcggcaat 60

ggggccgcga cggccgaggg cagcgtggtg acggtgtcta gcccgacgtc cgggggctcg 120

ggcggcggga gccccacgag gtcggcgccc ggggtcgagg gaggccgcgt cggcaagccg 180

gcgcggcgga ggtcccgcgc gtcgcgccgg gcgcccgtca cgctgctcaa caccgacacc 240

accaacttcc gcgccatggt gcagcagttc accggcatcc cctccggccc ctacggcccg 300

gctggcgcgg gcggcgggcc cgtgatcagc ttcggcgcgg gcggcggcga ctacggcctc 360

ggcggcggca tgccggtgcg cccgtcgccg agctcggccg tgatgtcgtt cgaccacctc 420

ggccaccacc gcccgtccgc tgtcacctcg tccctgcagc agcagcagca gcagcagagc 480

cggctgttcc ggccgcagca gcagcagcag cagtacggcg actacggcgg catgcacgga 540

ggcggcggcg cggacatgtc gttcctgcac gggttcgagt cgtcggccga ggacaggctg 600

ctgctgcaga gcatccaggc ggcgcagatg ctgccccgcc cggcctccac taacaccccc 660

aatggctaca acttcggatg a 681

<210> 5

<211> 226

<212> PRT

<213> wheat (Triticum aestivum)

<400> 5

Met Gly Asp Thr Gly Ala Asn Met Gly His Trp Ala Gly Ile Tyr Gly

1 5 10 15

Val Gly Gly Asn Gly Ala Ala Thr Ala Glu Gly Ser Val Val Thr Val

20 25 30

Ser Ser Pro Thr Ser Gly Gly Ser Gly Gly Gly Ser Pro Thr Arg Ser

35 40 45

Ala Pro Gly Val Glu Gly Gly Arg Val Gly Lys Pro Ala Arg Arg Arg

50 55 60

Ser Arg Ala Ser Arg Arg Ala Pro Val Thr Leu Leu Asn Thr Asp Thr

65 70 75 80

Thr Asn Phe Arg Ala Met Val Gln Gln Phe Thr Gly Ile Pro Ser Gly

85 90 95

Pro Tyr Gly Pro Ala Gly Ala Gly Gly Gly Pro Val Ile Ser Phe Gly

100 105 110

Ala Gly Gly Gly Asp Tyr Gly Leu Gly Gly Gly Met Pro Val Arg Pro

115 120 125

Ser Pro Ser Ser Ala Val Met Ser Phe Asp His Leu Gly His His Arg

130 135 140

Pro Ser Ala Val Thr Ser Ser Leu Gln Gln Gln Gln Gln Gln Gln Ser

145 150 155 160

Arg Leu Phe Arg Pro Gln Gln Gln Gln Gln Gln Tyr Gly Asp Tyr Gly

165 170 175

Gly Met His Gly Gly Gly Gly Ala Asp Met Ser Phe Leu His Gly Phe

180 185 190

Glu Ser Ser Ala Glu Asp Arg Leu Leu Leu Gln Ser Ile Gln Ala Ala

195 200 205

Gln Met Leu Pro Arg Pro Ala Ser Thr Asn Thr Pro Asn Gly Tyr Asn

210 215 220

Phe Gly

225

<210> 6

<211> 675

<212> DNA

<213> wheat (Triticum aestivum)

<400> 6

atgggcgaca ccggcgcgaa catgggccac tgggcgggca tctacggcgt cggcggcaat 60

ggggccgcgg cggcggaggg cagcgtggtg acggtgtcga gcccgacgtc cgggggctca 120

ggcggcggga gccccatgag gtcggcgccc ggggtcgagg ggggccgcgt cggcaagccg 180

gcgcggcgaa ggtcccgcgc ctcgcgccgg gcgcccgtca cgctgctcaa caccgacacc 240

accaactttc gcgccatggt gcagcagttc accggcatcc cctccggccc ctacggcccg 300

gccggcgcgg gcggcgggcc cgtgatcagc ttcggcgcgg gcggcggcga ctacggcctc 360

ggcggcggca tgccggtgcg cccgtcgccg acctcggccg tgatgtcgtt cgaccacctc 420

ggccaccacc gcccgtccgc cgccacgtcg tccctgcagc agcagcagca gagccagctc 480

ttccggccgc agcagcagca gcagtacggc gactacggcg gcatgcacgc cgggggcggc 540

ggcgcggaca tgtcgttcct gcacgggttc gagtcgtcgg cggaggacag gctgctgctg 600

cagagcatac aggcggcgca gatgctgccc cggccggcct ccactaacac ccccaatggc 660

tacaacttcg gatga 675

<210> 7

<211> 224

<212> PRT

<213> wheat (Triticum aestivum)

<400> 7

Met Gly Asp Thr Gly Ala Asn Met Gly His Trp Ala Gly Ile Tyr Gly

1 5 10 15

Val Gly Gly Asn Gly Ala Ala Ala Ala Glu Gly Ser Val Val Thr Val

20 25 30

Ser Ser Pro Thr Ser Gly Gly Ser Gly Gly Gly Ser Pro Met Arg Ser

35 40 45

Ala Pro Gly Val Glu Gly Gly Arg Val Gly Lys Pro Ala Arg Arg Arg

50 55 60

Ser Arg Ala Ser Arg Arg Ala Pro Val Thr Leu Leu Asn Thr Asp Thr

65 70 75 80

Thr Asn Phe Arg Ala Met Val Gln Gln Phe Thr Gly Ile Pro Ser Gly

85 90 95

Pro Tyr Gly Pro Ala Gly Ala Gly Gly Gly Pro Val Ile Ser Phe Gly

100 105 110

Ala Gly Gly Gly Asp Tyr Gly Leu Gly Gly Gly Met Pro Val Arg Pro

115 120 125

Ser Pro Thr Ser Ala Val Met Ser Phe Asp His Leu Gly His His Arg

130 135 140

Pro Ser Ala Ala Thr Ser Ser Leu Gln Gln Gln Gln Gln Ser Gln Leu

145 150 155 160

Phe Arg Pro Gln Gln Gln Gln Gln Tyr Gly Asp Tyr Gly Gly Met His

165 170 175

Ala Gly Gly Gly Gly Ala Asp Met Ser Phe Leu His Gly Phe Glu Ser

180 185 190

Ser Ala Glu Asp Arg Leu Leu Leu Gln Ser Ile Gln Ala Ala Gln Met

195 200 205

Leu Pro Arg Pro Ala Ser Thr Asn Thr Pro Asn Gly Tyr Asn Phe Gly

210 215 220

<210> 8

<211> 20

<212> DNA

<213> wheat (Triticum aestivum)

<400> 8

ctgggcgggc atctacggcg 20

Claims (7)

1. A method for improving disease resistance of wheat is characterized by comprising the following steps: inhibiting receptor in wheatTaVQ5Expressing the gene to obtain target wheat with disease resistance higher than that of the receptor wheat; the disease resistance is resistance to powdery mildew and/or banded sclerotial blight; the above-mentionedTaVQ5The gene is a gene for coding TaVQ5 protein; the TaVQ5 protein is the protein of A1) or A2) as follows:

A1) the amino acid sequence is the protein of the amino acid sequence shown in any one of sequence 3, sequence 5 or sequence 7 in the sequence table;

A2) a1) at the N-terminus or/and C-terminus.

2. The method of claim 1, wherein: the above-mentionedTaVQ5The genes are the genes shown as E1 or E2:

e1, the coding sequence of the coding chain is a DNA molecule of any one of sequence 2, sequence 4 and sequence 6 in the sequence table;

e2, the nucleotide sequence is any one DNA molecule of sequence 2, sequence 4 and sequence 6 in the sequence table.

3. The method of claim 2, wherein: said inhibiting recipient wheatTaVQ5The gene expression is carried out in wheatTaVQ5Gene editing is realized.

4. The method of claim 3, wherein: the gene editing is carried out by using a CRISPR/Cas9 system; the CRISPR/Cas9 system comprises a plasmid for expressing sgRNA, wherein the target sequence of the sgRNA is the 1 st-20 th site of the sequence 8 in the sequence table, and the coding DNA of the sgRNA is the 6915 th-7017 th site of the sequence 1 in the sequence table.

5. The method of claim 4, wherein: the target wheat is the wheat which meets the following conditions: one or two or three of the A, B and D genomes are mutated at the target region.

The application of TaVQ5 protein in preparing a reagent for resisting powdery mildew and/or banded sclerotial blight, which is characterized in that: the active ingredient of the agent is a substance that inhibits the expression of a gene encoding the TaVQ5 protein of claim 1, reduces the abundance of the TaVQ5 protein, and/or knockouts the gene encoding the TaVQ5 protein.

7. The use of claim 6, wherein: the substance contains the following F1), F2) or F3):

F1) sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the gene;

F2) generating a DNA molecule that targets a sgRNA of the gene, a DNA molecule that generates an siRNA that targets the gene, a DNA molecule that generates an shRNA that targets the gene, a DNA molecule that generates an miRNA that targets the gene, or a DNA molecule that generates an antisense RNA that targets the gene;

F3) an expression vector that produces sgRNA targeting the gene, an expression vector that produces siRNA targeting the gene, an expression vector that produces shRNA targeting the gene, an expression vector that produces miRNA targeting the gene, or an expression vector that produces antisense RNA targeting the gene.

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