CN114134155B

CN114134155B - MLO gene mutant and preparation method and application thereof

Info

Publication number: CN114134155B
Application number: CN202111203405.1A
Authority: CN
Inventors: 李超; 梅德圣; 石育钦; 胡琼; 孙梦丹; 陈帆; 成洪涛; 胡学志; 付丽
Original assignee: Wuhan Zhongyou Dadi Hope Seed Co ltd; Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Wuhan Zhongyou Dadi Hope Seed Co ltd; Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2021-06-29
Filing date: 2021-10-15
Publication date: 2023-09-12
Anticipated expiration: 2041-10-15
Also published as: CN114134155A

Abstract

The invention discloses an MLO gene mutant and a preparation method and application thereof, and belongs to the technical field of biology. The invention discloses a method for improving sclerotinia rot resistance of colza by using a gene editing technology, in particular to a method for simultaneously mutating 6 homologous copies of BnMO 6 genes by using a CRISPR/Cas9 gene editing technology to obtain a stably inherited material (called as BnMLO6 for short) with 6 homologous copies of BnMO 6 genes. The field and indoor observation results show that the rape bnmlo6 material not only shows stronger powdery mildew resistance, but also can improve the resistance of rape to sclerotinia. The invention reports that BnMOO 6 gene can improve the resistance of rape to sclerotinia rot and powdery mildew for the first time, and has wide application prospect.

Description

MLO gene mutant and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an MLO gene mutant and a preparation method and application thereof.

Background

Rape is the oil crop with the largest planting area in China, and ensuring the healthy development of the rape industry is an important basic stone for stabilizing the edible oil supply safety in China. Sclerotinia rot is a main disease in rape production, the annual average incidence rate is about 10% to 30%, the annual average yield loss caused by the sclerotinia rot is about 10% to 20%, and the annual yield reduction with serious harm can reach more than 80%. Powdery mildew is another important disease of rape, especially in the main rape planting area in Yangtze river basin, the rain is abundant, the large-scale outbreak of the powdery mildew of rape is easy to be caused, and the quality and the harvest index of the rape are seriously affected by the generated pathogenic bacteria spores.

Because of the lack of sclerotinia resistance source in rape germplasm resources, the molecular genetic improvement process of rape disease resistance is seriously hindered. The gene editing technology is a novel genome operation tool, and can simultaneously carry out careful analysis on gene functions of multiple homologous copies in polyploid crops. The mutant material produced simultaneously can remove the transgene element, obtain the mutation material which is stable in inheritance and does not contain the transgene element, and has a stronger application prospect.

MLO (Mildew resistance locus) is a family of genes with transmembrane structures. Earlier studies showed that there was a large difference in the number of family members of the MLO genes in different species, e.g. 15 MLO family members in the model plant arabidopsis, 8 in wheat, 39 in soybean and 17 in grape. The function between the different family members is also evident. AtMLO2 single gene mutation can obviously improve the resistance of arabidopsis to powdery mildew, and is a main negative regulatory factor of powdery mildew resistance; atMLO6 and AtMLO12 genes also have partial auxiliary functions on powdery mildew resistance. The MLO gene also shows obvious functional differentiation to other pathogenic bacteria. The three-gene mutation of Arabidopsis thaliana mlo2mlo6mlo12 has slightly increased resistance to anthracnose (Colletotrichum higginsianum), significantly increased resistance to gray mold (Botrytis cinerea), increased sensitivity to black spot (Alternaria alternata) and leaf spot (Alternaria brassicicola), and no significant change in resistance to Pityrosporum indicum (Serendipita indica) and downy mildew (Hyaloperonospora arabidopsidis). The function of the MLO gene varies from species to species. The barley mlo gene mutant has increased susceptibility to root rot caused by wheat root rot (Bipolaris sorokiniana) and erythema leaf spot caused by fungi (Ramularia collo-cygni); the CaMLO2 gene of the capsicum is induced to express by bacterial scab (Xanthomonas campetris pv.vesica), and the silencing of the CaMLO2 gene can improve the resistance of the capsicum to the bacterial scab. Thus, the number of members of the plant MLO gene family in different species, the disease resistance modulation response in which the different members are involved, the number of copies of a single gene in the genome, and the function between the different copies may all vary greatly.

Rape is an heterotetraploid crop, most genes typically have 2 and more copies, and the function between copies of these genes tends to be differentiated and redundant. The BnMO 6 is firstly identified from the genome of rape by utilizing a method of bioinformatics and molecular biology experiment combined with gene editing, and is involved in negative regulation of resistance of sclerotinia rot of rape. The mutation of the BnMO 6 gene can obviously improve the resistance of rape to sclerotinia rot and powdery mildew at the same time, and is first reported in rape through search.

Disclosure of Invention

In order to solve the technical problems, the invention provides an MLO gene mutant and a preparation method and application thereof.

According to the invention, through comparing and analyzing the expression quantity before and after the sclerotinia sclerotiorum inoculation of rape, 6 homologous copies of BnMOO 6 genes are found to be induced to be expressed by oil pathogenic bacteria, and presumably involved in the resistance reaction to the sclerotinia sclerotiorum, a CRISPR/Cas9 gene editing method is utilized, and a mutant strain with 6 copies of BnMOO 6 genes simultaneously edited is obtained. The mutant line resistance investigation result shows that 6 copies of BnaA03g60240D (MLO 6-A03), bnaCnng04240D (MLO 6-C03), bnaA01g22680D (MLO 6-A01), bnaC01g29530D (MLO 6-C01), bnaA09g13700D (MLO 6-A09) and BnaCnng31840D (MLO 6-C09) are simultaneously mutated, so that the resistance of rape to sclerotinia sclerotiorum and powdery mildew can be remarkably improved.

In order to achieve the above purpose, the invention adopts the following technical scheme:

one of the objects of the present invention is to provide a mutant MLO6 gene, named BnaA03G60240D (MLO 6-A03), bnaCnng04240D (MLO 6-C03), bnaA01G22680D (MLO 6-A01), bnaC01G29530D (MLO 6-C01), bnaA09G13700D (MLO 6-A09) and BnaCnng31840D (MLO 6-C09) in the rape darmor genome, respectively, which has a homozygous MLO6-A03 copy, 15 bases deleted such that the encoded amino acid sequence is deleted after 356 th site of-L-V-Q-P-G; MLO6-C03 copies are heterozygous mutations with 13 deleted alkali groups and 2 deleted alkali groups, and frame shift mutations are respectively caused after amino acid 357 and 356; MLO6-A01 copy is heterozygous mutation deleting 2 bases and single base G insertion, and frame shift mutation is caused after 365 amino acid sites; one strand of the MLO6-C01 copy lacks 12 bases, L is replaced by R at amino acid 357, and then four amino acids of-V-Q-P-G are lost; one strand of the MLO6-A09 copy lacks 3 bases and V at amino acid 356; the MLO6-C09 copy has 2 bases deleted from one strand, resulting in a frame shift mutation after amino acid 355.

Another objective of the present invention is to provide a polypeptide encoded by the mutant MLO6 gene.

It is a further object of the present invention to provide a construct comprising the mutant MLO6 gene described above.

It is a fourth object of the present invention to provide a recombinant cell obtained by transforming a recipient cell with the above-described construct.

The fifth object of the present invention is to provide a method for obtaining the above mutant MLO6 gene, comprising the steps of:

(1) BnMUO 6-sgRNA2 is designed, and the sequence is shown as SEQ ID NO. 30;

(2) Constructing sgRNA2 in step (1) into a vector plasmid backbone;

(3) Introducing the constructed gene editing vector into rape receptor material, and obtaining regenerated seedlings;

(4) Identification and mutation detection of transgenic plants.

Further, the vector plasmid backbone in the step (2) is pKSE401.

Furthermore, the step (3) of introducing the constructed gene editing vector into the rape receptor material adopts an agrobacterium-mediated rape hypocotyl genetic transformation method.

The sixth object of the present invention is to provide an application of the above mutant MLO6 gene in plant sclerotinia resistance.

Compared with the prior art, the invention has the following beneficial effects:

through the study, the CRISPR/Cas9 system is proved to be an effective tool for editing the complex genome of the brassica napus, and a plurality of homologous copies of the target gene of the brassica napus can be successfully knocked out. The knocking-out of the disease-sensing gene BnMLO6 in rape can excite spontaneous accumulation of callose in leaf cells and regulate ethylene and jasmonic acid disease-resistant signal paths. Therefore, the BnMOL6 gene possibly participates in a plurality of disease resistance signal paths in the rape, negatively regulates and controls the resistance to powdery mildew and sclerotinia, and provides a reference for research of utilizing the BnMOL6 gene to cooperatively regulate and control the resistance of a plurality of pathogenic bacteria of the rape. The disease-resistant material generated by the gene mutation obtained by research has important significance for the basic research and disease-resistant breeding of the disease resistance correlation of the rape.

Drawings

FIG. 1 is a graph showing the results of detecting the expression level of 6 copies of the BnMOO 6 gene in example 1.

FIG. 2 is a schematic representation of two targets, sgRNA1 and sgRNA2, in example 1.

FIG. 3 is a diagram showing the result of a portion of the PAGE electrophoresis typing of BnMOO 6-C01 copy gene in example 1.

FIG. 4 shows mlo6-212 individual T in example 1 ₀ And a generation BnMOL6 gene target point mutation result diagram.

FIG. 5a is T of field mlo6-212 in example 1 ₁ And (5) a graph of the powdery mildew infection results of the generation plants.

FIG. 5b is T of indoor mlo6-212 in example 1 ₁ And (5) a graph of the powdery mildew infection results of the generation plants.

FIG. 6 is mlo6-212T of example 1 ₁ And (5) observation and analysis result graphs of generation callose.

FIG. 7a is a graph showing the results of 24 hours after infection with Sclerotinia sclerotiorum in example 1.

FIG. 7b is a graph showing the results of example 1 after 36 hours of sclerotinia infection.

FIG. 7c is a graph of leaf plaque area analysis in example 1.

FIG. 8 is a diagram of the multiple genes in example 1 in wild type and mlo6-212 mutant T ₂ Expression change pattern after inoculation.

Detailed Description

In the following examples, the genetic transformation acceptor material was double 6 in Brassica napus, vector plasmid backbone pKSE401 was supplied by the university of agriculture Chen Jijun teacher laboratory, E.coli DH5a and Agrobacterium GV3101 competent purchased from Biotechnology only Co., ltd, sclerotinia was supplied by the institute of oil institute Liu Shengyi teacher group of agricultural sciences, primers were synthesized by Jin Kairui Bioengineering Co., ltd, and sequencing was completed by Oracle technologies Co., ltd.

Example 1

1. Test method

(1) Semi-quantitative RT-PCR

Taking medium double 6-leaf blades at different time after the inoculation and the sclerotium bacteria inoculation, extracting RNA by using an RNA extraction kit of Tiangen company, wherein the operation method is referred to a specification, and estimating the concentration of RNA by using a spectrophotometer. First strand cDNA was synthesized according to the protocol of the full-scale Jin Fanzhuai protocol. The first strand cDNA is used as a template, the BnMOO 6 gene is subjected to PCR amplification by using specific primers of homologous copies, the BnACtin gene is used as an internal reference, and 1% agarose gel electrophoresis is used for separation and identification, wherein the primer sequences are shown in Table 1.

TABLE 1 primer sequences

(2) sgRNA design and vector construction

Using the protein sequence of Arabidopsis MLO6 gene in rape genome databasehttps://www.genoscope.cns.fr/ brassicanapus/) In the above, six homologous copies of the canola MLO6 gene were identified, namely BnaA03g60240D (MLO 6-A03), bnaCnng04240D (MLO 6-C03), bnaA01g22680D (MLO 6-A01), bnaC01g29530D (MLO 6-C01), bnaA09g13700D (MLO 6-A09) and BnaCnng31840D (MLO 6-C09), respectively, and the corresponding nucleotide sequences thereof are shown in SEQ ID Nos. 53-58, respectively. Two sgRNAs were designed, bnMOL 6-sgRNA1 (SEQ ID NO.29: AAGCAGAGCTTATGCTGAT) and BnMOL 6-sgRNA2 (SEQ ID NO.30: CTTGTTCAGC CCGGTATC), using the sgRNA target design website (http:// CRISPR. Hzau. Edu. Cn/CRISPR2 /) at the conserved sequence positions of the third and tenth exons of the six gene copies, respectively. Two targets adoptIs constructed into a final vector for gene editing (see MaX, zhang Q, zhu Q, liu W, chen Y, qiaR, wang B, yang Z, li H, lin Y, xie Y, shen R, chen S, wang Z, chen Y, guo J, chen L, zhao X, dong Z, liu YG.A Robust CRISPR/Cas9 System for Convenient, high-Efficiency Multiplex Genome Editing in Monocot and Dicot plants.molecular Plant, 2)015,8:1274-84.)。

(3) Genetic transformation of rape

The constructed gene editing vector is introduced into rape receptor material by agrobacterium-mediated rape hypocotyl genetic transformation method, double No. 6, regenerated seedlings are obtained by means of Plant tissue culture, specific procedures are referred to Li C, hao M, wang W, wang H, chen F, chu W, zhang B, mei D, cheng H, hu Q.an effect CRISPR/Cas9 Platform for Rapidly Generating Simultaneous Mutagenesis of Multiple Gene Homoeologs in Allotetraploid Oilseed Rape.

(4) Identification and mutation detection of transgenic plants

Positive plants are screened by using caliamycin, leaves of regenerated plants are taken to extract DNA, NPT II gene specific primers are used for carrying out PCR amplification reaction, positive regenerated plants are identified, and 1% agarose gel electrophoresis is used for separation and identification.

Primers are designed at the difference sites at two sides of each copy target point of the BnMOO 6 gene, fragments containing the target points are amplified by PCR, 8% non-denaturing polyacrylamide gel electrophoresis (Polyacrylamide gel electrophoresis PAEG) is used, mutation plants are initially selected through electrophoresis banding typing, sequence mutation is detected through high-throughput sequencing, and related primer sequences are shown in table 1.

(5) Fluorescent staining observation of callose

Wild type and mutant leaves are taken at the early stage of bolting, stained with aniline blue and observed under a fluorescence microscope. The specific operation is as follows: the leaves were soaked in 95% alcohol for at least 24 hours until all tissues were transparent. After washing with 0.07M phosphate buffer (ph=9) for 2 hours, staining was performed in 0.07M phosphate buffer containing 0.01% aniline blue for 2 hours. Callose staining was followed by visualization under an optical microscope with an ultraviolet filter in the form of a spot under ultraviolet light. Six leaves were observed per material, each leaf randomly picking 5 fields of view.

(6) Identification of disease resistance

Powdery mildew resistance identification is directly planted and observed in Hanchuan transgenic base fields and indoor growth. Sclerotinia sclerotiorum inoculation identification method is described in He Liegan, song Laijiang, shang Jie, zhou Yinsheng, ma Huigang. Colza sclerotiorum resistance identification method is compared and disease resistant germplasm resource is selected. Jiangsu agricultural science, 2018,46:90-93. When the tested material grows to a five-leaf period, selecting plants with the same size, and selecting 1 healthy leaf with the same size and leaf position for standby. Each material was inoculated with 5 leaves, 2 inoculation points per leaf, and three replicates were made.

The mycelium of the strain for standby is taken out, inoculated on a PDA (Potato dextrose agar) flat plate, placed at 25 ℃ for 3d culture, and then punched into mycelium discs at the edge of a colony by a puncher with the diameter of 5 mm. Preparing a plurality of plastic trays, paving 2 pieces of filter paper on the bottom of each tray, adding a proper amount of distilled water into the tray, soaking the filter paper, and respectively paving spare blades on the wetted filter paper. Selecting mycelium discs, inoculating the mycelium discs onto rape leaves, taking 2 inoculating points in the middle of each leaf along the veins in an axisymmetric way, covering a sealing film for moisturizing, horizontally placing the rape leaves in a culture temperature chamber at 25 ℃, and measuring the diameter of 1 lesion by a crisscross method every 12 hours.

(7) Detection of plant defense-related gene expression level

Total RNA was extracted from leaves of the wild type and mlo6 mutants of Zhongshuang6, which were not inoculated and were inoculated at different times, and then the expression of genes after the nuclear dish was inoculated with the key marker response genes ERF1 (Ethylene response factor), ERF2 (Ethylene response factor 2), PR4 (Pathenesis-related 4) and ORA059 (Octadecaloid-responsive Arabidopsis AP2/ERF 59) by RT-PCR, and primers were shown in Table 2.

TABLE 2 defense-related Gene primers

2. Test results

(1) Expression analysis of BnMLO6 gene after inoculation of sclerotinia and construction of CRISPR/Cas9 gene editing vector

The detection of the expression level of the gene copies after inoculation by RT-PCR shows that the expression levels of 6 copies of the BnMOO 6 gene all show different rising trends after inoculation, and the result is shown in figure 1, wherein the rising of the expression level of A01/C01/A03/C03 copies is more obvious. Preliminary estimation of BnMOO 6 gene may be related to sclerotinia resistance of rape.

In order to further study the function of the gene, two targets sgRNA1 and sgRNA2 are designed in the conserved regions of the 3 rd and 10 th exons of the BnMUO 6 gene respectively, see FIG. 2, and the construction of CRISPR/Cas9 gene editing vector is completed.

(2)T ₀ Analysis of substitution gene mutation

For T ₀ And detecting NPTII marker genes of the generation plants, and screening transgenic positive plants. And in the 494 regenerated seedlings detected, the positive plants are 137, and the positive rate is 27.7%. After screening the difference bands in 137 positive plants by a PAGE electrophoresis method, 41 edited plants are obtained, the mutation efficiency of the vector is 29.9%, and the result of the PAGE electrophoresis typing of the BnMOO 6-C01 copy genes is shown in figure 3.

Of the 41 edited plants obtained by PAGE typing, 10 showed six copies with altered banding patterns (fig. 3), and there was a possibility of mutation. Sequencing analysis after PCR amplification was performed on the mutation sites of the 10 individual strains, and found that at the sgRNA2 target point, 2 strains were each edited for two and four copies of the gene, and 3 strains were each edited for five and six copies.

Among 3 strains in which all 6 gene copies were edited, the mutation of mlo6-212 was best seen in FIG. 4, in which the A03 copy was homozygous, deleting 15 bases, so that the encoded amino acid sequence was deleted at 356 th site. The C03 copy is a heterozygous mutation with 13 deleted bases and 2 deleted bases, which results in frame shift mutations after amino acid 357 and 356, respectively. The A01 copy is heterozygous mutation deleting 2 bases and single base G insertion, and all the heterozygous mutation is caused by frame shift mutation after 365 amino acid sites. One strand of the C01 copy lacks 12 bases, L is replaced with R at amino acid 357, and the four amino acids-V-Q-P-G are subsequently lost. One strand of copy a09 lacks 3 bases and V is deleted at amino acid 356. The copy C09 lacks 2 bases on one strand, resulting in a frame shift mutation after amino acid 355. The sequence analysis results show that mutation on the DNA sequence causes the amino acid sequence of each copy of the mlo6-212 single strain to be changed, and the functional change of the BnMOO 6 gene can be caused.

(3)T ₁ Analysis of substitution mutations and observation of powdery mildew resistance

10T of mlo6-212 single plants are arbitrarily selected ₁ DNA was extracted from the generation of individuals, and sequencing of a part of the individuals showed that these T ₁ Mutation types of six BnMOO 6 gene copies in the generation plant are all the same as T ₀ The generation is consistent, the mutation of the previous generation is inherited, no new mutation type is found, and the mutation generated by gene editing can be inherited to the next generation effectively.

The observation of the disease condition of powdery mildew of offspring and wild powdery mildew of gene coding mutation planted between a transgenic base and basement growth shows that mutation of BnMOO 6 gene can obviously improve the resistance of rape to powdery mildew. All wild plant stems in the base are coated with a layer of powdery mildew, and the powdery mildew is very serious. But T of mlo6-212 ₁ No powdery mildew was visible on the plants of the generation and no trace of powdery mildew was observed (FIG. 5A). Plants grown in the room plant growth room also had the same phenotype (fig. 5B). The strain mlo6-212 is used in Hanchuan transgenic base to make statistical investigation on powdery mildew disease condition of field. Because of more overcast and rainy days in the spring rape flowering and pod development period in 2021, powdery mildew of field rape is serious, powdery mildew is infected on almost all stems of wild plants, the incidence rate is close to 100%, and the incidence rate of mutant materials of BnMOO 6 genes is 50-61% and is extremely lower than that of wild materials.

(4) Callose analysis of BnMOO 6 Gene mutant Material

T for mlo6-212 mutant material ₁ The observation of the generation callose microscope showed that callose accumulation occurred spontaneously in the presence of BnMLO6 mutant material in the absence of powdery mildew infection (see FIG. 6). Mutant material is single compared to wild typeThe number of callose spots observed in each field increased from 7.33 to 35.29, with an increase of 486.3%. The increase in callose may be associated with the presence of powdery mildew resistance caused by mutation of the BnMOO 6 gene.

(5) Sclerotinia rot resistance identification of BnMOO 6 gene mutant material

Selecting T with mutation in all six BnMOO 6 homologous copies ₁ T of individual strain mlo6-212 ₂ And (3) planting seeds of the generation in a growth room, taking leaves after the five-leaf period, and inoculating sclerotinia sclerotiorum, wherein the wild type is used as a control. The inoculation identification results show (see figures 7a-7 c) that there is a significant difference between leaf plaque area of the Bnmlo6 mutant material and wild type. 24 hours after inoculation (FIG. 7 a), the Bnmlo6 mutant material had an average plaque area of from 1.37cm relative to the wild type ² Reduced to 1.10cm ² Reduced by 19.5% (p=0.012), reaching significant levels; 36 hours after inoculation (FIG. 7 b), the average plaque area of Bnmlo6 mutant material was from 3.95cm relative to wild type ² Reduced to 3.45cm ² A reduction of 12.5% (p=0.045) was achieved, also to a significant level.

(6) Mutant material disease resistance related gene expression analysis

Identification of ERF1, ERF2, PR4, ORA059 in wild type and mlo6-212 mutant T by RT-PCR ₂ And (3) gene expression change after inoculation. The result shows that the expression quantity of two copies of ERF1 gene in the mutant material is higher than that of the wild type after 24 hours of non-inoculation and inoculation, and the expression quantity of the two copies tends to be consistent after 36 hours of inoculation. When the three copies of the ERF2 gene are not inoculated, the expression quantity of the mutant and the wild type is not greatly different; however, after 24h and 36h of inoculation, the expression level of 3 copies in the mutant is obviously higher than that of the wild type. When PR4 gene is not inoculated, the expression quantity in the mutant and the wild type is very low; after 24h and 36h inoculation, 4 copies of PR4 gene were hardly induced in wild type, and the expression level was not detected, but in mutants, the expression level of 4 copies of PR4 gene was significantly improved. When ORA059 gene is not inoculated, the expression level is low in the mutant and the wild type, and after inoculation, the expression level of two copies of ORA059 gene in the mutant and the wild type is improved, but the expression level is improved in the mutantSignificant (see fig. 8).

In FIG. 8, each row represents the variation in expression level of one homologous copy of the relevant gene, and three copies of ERF2, each from top to bottom, are: bnaA09G0212800ZS, bnaC02G0416900ZS, bnaC09G0247600ZS; two copies of ERF1 are: bnaA01G0293300ZS, bnaC01G0362300ZS; four copies of PR4 are: bnaA03G0296000ZS, bnaA03G0296200ZS, bnaC03G0354600ZS, bnaC03G0354700ZS; two copies of OAR059 are: bnaA10G0042700ZS, bnaC05G0044000ZS; samples from left to right were in order: the wild type strain is not inoculated, the mutant strain is not inoculated, the wild type strain is inoculated for 24 hours, the mutant strain is inoculated for 24 hours, the wild type strain is inoculated for 36 hours, and the mutant strain is inoculated for 36 hours.

Because of the existence of gene redundancy in polyploid plants, multiple copies often need to be edited simultaneously for gene knockout. Brassica napus is used as an heterotetraploid crop, in most cases carrying more than two homologous copies of a gene. The CRISPR/Cas9 system is an effective tool for multiplex genome editing of brassica napus. Two targets are selected by using the conserved sequence of BnMOO 6 homologous genes in the genome of Darmor, and the conserved exon sequences of 6 homologous copies of the rape BnMOO 6 genes are respectively targeted. The mutation detection result shows that the sgRNA1 designed according to the first target point does not detect editing at the target site, and the BnMOO 6-sgRNA2 designed according to the second target point realizes editing mutation of the target site. The editing efficiency of the BnMOO 6-sgRNA2 target point is higher and reaches 29.9 percent. Six copies of the edited plant were mutated at the same time, accounting for 7.32% (3/41) of the edited plant, and 2.19% of the positive plants. From the 3 individual plants with 6 gene copies edited at the same time, the individual plant mlo6-212 with ideal mutation is selected for subsequent function identification.

After multiple gene copies of the rape BnMOO 6 gene are mutated simultaneously, T ₁ And T ₂ The generation lines show strong powdery mildew resistance under indoor plant growth room and field conditions. Spontaneous accumulation of callose occurs in the rape Bnmlo6 editing material, and the quantity of callose is markedly increased over the wild type. Callose, in addition to acting as a physical barrier against entry of fungal pathogens at an early stage, may form a diffusion barrier against pathogenic bacteriaPlays an important role in the mechanical defense of plants in early disease resistance. It can be seen that powdery mildew resistance generated after mutation of the rape BnMOO 6 gene is related to the increase of accumulation of callose in cells.

The study of the relation between MLO gene and sclerotinia resistance has not been reported yet. After the sclerotinia sclerotiorum is inoculated, the expression quantity of 6 homologous copies of the rape BnMOO 6 gene is detected to be increased to different degrees, and the sclerotinia sclerotiorum is possibly involved in the defense reaction. The sclerotinia inoculation result shows that the average plaque area of the mlo6-212 mutant is significantly smaller than that of the wild type after 24h and 36h inoculation. The result shows that the BnMOL6 gene can negatively regulate the early-stage defense response of rape to sclerotinia.

The plant hormone mediated signaling pathways SA and JA/ET are involved in the defense of plants against different nutritional pathogens, and the mutation of the Arabidopsis MLO gene affects the expression of the JA/ET signaling pathway gene. The increased expression level of the marker genes ERF1, ERF2, PR4 and ORA59 of the JA/ET pathway in Bnmlo6 mutant after sclerotinia inoculation shows that the expression of the JA/ET signal pathway gene is promoted after MLO gene knockout, thereby possibly mediating the defense of the signal pathway and improving the resistance to sclerotinia at early stage of infectious diseases.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the design of the present invention.

Sequence listing

<110> institute of oil crop and oil crop at national academy of agricultural sciences

WUHAN ZHONGYOU DADI HOPE SEED Co.,Ltd.

<120> an MLO gene mutant, and preparation method and application thereof

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<213> Artificial sequence ()

<400> 19

ttcggttttt ttttttttta catgaacc 28

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 20

gaaagatgtt agtttgatgt tttctctctg 30

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 21

ggtcttaacg gcatagcaaa acgat 25

<210> 22

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 22

cgtggaatta ttttgtttgc ttaccgt 27

<210> 23

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 23

ttcttaccgc attgcaaaat gattctt 27

<210> 24

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 24

tgtgggatac tactttgctt gcttacc 27

<210> 25

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 25

ctcagaatgt ttctgcatcc gatatct 27

<210> 26

<211> 31

<212> DNA

<213> Artificial sequence ()

<400> 26

cttttactta cccaactgaa attctgatga c 31

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 27

gaattgctct gcttgcttac cgttg 25

<210> 28

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 28

caacaatccc agaactaata tatttgctct 30

<210> 29

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 29

aagcagagct tatgctgat 19

<210> 30

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 30

cttgttcagc ccggtatc 18

<210> 31

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 31

atcaatcctt cctctcagtt agc 23

<210> 32

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 32

tcttgatccg taacaatact ctg 23

<210> 33

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 33

caatcattcc tctcagctgt tagc 24

<210> 34

<211> 24

<212> DNA

<213> Artificial sequence ()

<400> 34

cttgattcgt aacaacactt tgtt 24

<210> 35

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 35

cgcgtttgcg gctaaaaca 19

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 36

tggtttcctt tccacgttga g 21

<210> 37

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 37

cagacttagc atagccatca tat 23

<210> 38

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 38

gctctctcca gagccgctt 19

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 39

agccgctcaa tccgctaata 20

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 40

taccgcaaga atcacgacca 20

<210> 41

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 41

cagacttagc ataaccctcg ta 22

<210> 42

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 42

ctctctccgg aaccgcttg 19

<210> 43

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 43

tcgtccaaca actactctct cc 22

<210> 44

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 44

gtacgacttc tctggctttc ttg 23

<210> 45

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 45

ggaatctttc tcctcctcgt ctt 23

<210> 46

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 46

tgacttctct ggttttctcg atg 23

<210> 47

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 47

agatttagaa ccgtcgtcga ag 22

<210> 48

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 48

tatttctcct ccgtttcggt gt 22

<210> 49

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 49

gagttgtatc accgaatttg tag 23

<210> 50

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 50

acgaagacga tgaagacgaa gta 23

<210> 51

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 51

tagataagcc cccggctaat c 21

<210> 52

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 52

tccgtttcgg cgtcccgtta 20

<210> 53

<211> 1776

<212> DNA

<213> rape ()

<400> 53

atggctgata aggtagatga aagaacgcta cagcagactt ccacgtgggc ggtcgccgtg 60

gtttgcttct tcttgcttct catttcgatt gtcattgaga aactgattca caaattagga 120

acctgtttta aaaggaagaa taaaaaagct ctgtatgaag ctcttgaaaa ggtgaaagca 180

gagcttatgc tgatgggatt catatcacta ctgctaacaa ttggacaaaa ctatatatca 240

caaatttgca tctccgagag catcgcagca tcaatgcgcc cttgcagtaa atcagaagag 300

ttgcaagagt atccacctaa gaacaaagat actggaaatg acgaaggaga tgaagaaacc 360

tctggtcgaa agcttctcga attagtcgaa actttcattc ctcggaggag tttggctacc 420

aaaggctatg acaaatgtgc agagaaggga aaagtggctt ttgtatcgtc gtacggtatg 480

catcagctgc atatattcat ttttgttttg gccgtttgtc atgtgatcta ctgcattgtt 540

acttatgctt tgggaaagat caagatgaga aggtggaaga ggtgggaaga ggagacaaag 600

acaatcgaat atcagtattc acatgatcct gagaggttta ggtttgcaag ggatacatct 660

ttcgggcgta gacatctgaa cttctggagc aagtcaacta ttactctttg gattgcatgt 720

ttcttcagac agttctttgg atctgttacc aaagttgatt acttaacact gagacatggt 780

ttcatcatgg cccatttggc tcctgggagt gacgcgaggt tcgatttccg gaagtacatt 840

caaagatcat tagaggaaga cttcaaaacc atcgtcgaaa tcagtcctgt gatctggttt 900

gtggctgtgc tattcctcct taccaccaca cacggattga attcttaccg ctggcaacca 960

ttcattccct tagttgtgat acttatagtt ggaacgaaac ttcaaatgat aataacaaaa 1020

ctaggacttc taatccaaga gaaaggagag atagtgaaag gtatgccgct tgttcagccc 1080

ggtgatcatc tcttctggtt tggtcgtcca cgattcattc tcttcctcgt tcacttggtc 1140

cttttcacga atgcgtttca actagcgttc tttgcgtgga gtacgtatga gtttgggatt 1200

aagaactgtt tccacaaaag tactgtagat gtggtcatca gaatttcaat tggacttatt 1260

gtacagtttc tttgcagcta cgttactctt cctctttacg ctcttgttac tcagatgggt 1320

tcgaagatga aaccgacagt gttcaacgag agagtagcag tagctttaaa gagttggcat 1380

cacactgcca agaagcagat caaacatgga agaacctcag agtcaacgac gcctttctct 1440

agccgtccag ctacacctac gcacggttct tctccgatac atctccttcg caacgtccac 1500

aaacgaagca gaagcgccga tgagagcttc gctaactcgc tgtctccgag gagaaactct 1560

gacttcgata cgtgggatgt tgagtcgcaa caagaacctt cttcttcttc cataaagtat 1620

cattctaggt ttagggaaga agactcagag aagaagaagg cttcttcttc ttcttcttct 1680

tcttcagctg tggagcttcc tcctggacct ggaataataa gaactcagca tgagattagt 1740

actattagct taagagattt ttcatttaag cgatga 1776

<210> 54

<211> 1755

<212> DNA

<213> rape ()

<400> 54

atggctgata aggttgatga aagaacgcta cagcagactt ccacgtgggc ggtcgccgtg 60

gtttgcttct tcttgcttct aatttcaatt gtcattgaga aactgattca caaattagga 120

acctgtttta aaaggaagaa taaaaaagct ctgtatgaag ctcttgaaaa ggtcaaagca 180

gagcttatgc tgatgggatt catatcactg ctgctaacaa ttggacaaaa ctatatatca 240

caaatttgca tctccgagag catcgcagca tcaatgcgcc cttgcagtaa atccgaagag 300

ttgaaagagt atccacctaa gaacaaagat acgggaaatg acgaaggaga tgaagaaaac 360

tctggtcgaa agcttctcga actagtcgaa tctttcattc ctcggaggag tttggctacc 420

aaaggctatg acaaatgtgc agagaaggga aaagtggctt ttgtatcgtc ctacggtatg 480

catcagctgc atatattcat ctttgttttg gccgtttgtc atgtgatcta ctgtattgtt 540

acttatgctt tgggaaagat caagatgaga aggtggaaga ggtgggaaga ggagacaaag 600

acaatcgaat atcagtattc acatgatcct gagaggttta ggtttgcgag ggatacatct 660

ttcgggcgta gacatctgaa cttctggagc aaatcgacta ttacgctttg gattgcatgt 720

ttcttcagac agttctttgg gtctgttacc agagttgatt acttaacatt gagacatggt 780

ttcatcatgg cacatttggc tcctgggagt gacgcgaggt tcgatttccg gaagtacatt 840

caaagatcat tagaggaaga cttcaaaacc atcgtcgaaa tcagccctgt gatctggttt 900

atcgctgtgc tattcctcct cacaaccaca cacggattga attcttacct ctggcaacca 960

ttcattccct tagttgtgat acttatagtt ggaacaaaac ttcaagtgat aataacaaaa 1020

ctaggacttc taatccaaga gaaaggagag atagtgaaag gcatgccgct tgttcagccc 1080

ggtgatcatc ttttctggtt cggtcgtcca cgtttcattc tcttcctcgt tcacttagtc 1140

cttttcacga atgcgtttca actagcgttc tttgcgtgga gtacgtatga gtttgggatt 1200

aagaactgtt tccacaaaag tactgtagat gtggtcatca gaatttcaat tggacttatt 1260

gtacagtttc tttgcagcta cgttactctt cctctttacg ctcttgttac tcagatgggt 1320

tcgaagatga aaccgacagt gttcaacgag agagtagcag tagctttaaa gagttggcat 1380

cacacagcta agaagcagat caaacatgga agaacttcag agtcaacgac gcctttctct 1440

agccgtccag ctacacctac gcacgattct tctccgattc atctcctccg caacgtccac 1500

aaacgaagca gaagcgccga tgaaagcttc gccaactcgc tgtctccgag gagaaactct 1560

gacttcgata cgtgggatgt tgagtctcaa caagaacctt cttcttcttc gataaagtat 1620

cattctaggt ttagggaaga agactcagag aagaagaagc cttctgctgt ggagcttcct 1680

gctggacctg gaataataag aactcagcat gagattagta ctattagctt aagggatttt 1740

tcatttaagc gatga 1755

<210> 55

<211> 1671

<212> DNA

<213> rape ()

<400> 55

atggctgata aggtggctga taaggtggct gaaaaggtgg ctgaaagaac gctacagcag 60

acttccacgt gggcggtcgc cgttgtttgc ttcttcttgc ttctaatttc aattgtcatt 120

gagaaactga tccacaaatt aggaaactgg tttaaaagga agaacaaaaa agcactgtat 180

gaagctcttg aaaaagtgaa agcagagctt atgctgatgg gattcatatc actactgcta 240

acaattggac aaaactatat ctcacaaatt tgcgtctccg agagcgtcgc agcatcaatg 300

cgcccttgca gtagatccga agaagagaag aagtatccca atacaaaaaa agataccggg 360

aaggacctag gagatgaaga aaactctggt cgaaagcttc tcgagttagt cgaatctttc 420

attcctcgaa ggagtttggc taccaaaggc tatgacaagt gtgcagaaaa gggacaagtg 480

gcttttgtat cgtcctacgg gatgcatcag cttcatatat tcatcttcgt tcttgctgtt 540

tgtcatgtga tctactgcat tcttacttat gctttgggaa agaccaagat gagaagatgg 600

aagaggtggg aagaggagac aaagacaatt gaatatcagt attcacacga tcctgagagg 660

tttaggtttg cgagggatac atctttcggg cgtagacatc tgaacttctg gagcaaatcg 720

actattacac tgtggattgc atgtttcttt agacagtttt ttggatctgt aaccaaagtt 780

gattacttaa cgctgagaca tggtttcatt atggcccatt tggctccagg gagcgatgcg 840

aggttcgatt tccgaaagta cattcagaga tcattagagg aagatttcaa aacaatcgtt 900

gaaatcagtc ctgtgatatg gtttgtagcc gtgctattcc tcctgaccaa cacaaacgga 960

ttgaattctt acgtctggca accattcatt cccttagtag tgatacttat agttggaaca 1020

aaacttcaag tgataataac aaaactagga cttctaatcc aagagaaagg ggacatagtg 1080

aaaggcatgc cgcttgttca gcccggtgat catctcttct ggttcggtcg tccacgtttc 1140

attctcttcc tcgttcactt agtccttttc acgaatgcgt ttcaactagc tttctttgcc 1200

tggactacgt atgagttcaa gctcaagaac tgtttccaca agaacacagt agatgtggtc 1260

atcagaattt cagttggagt tattgtacag gttctttgca gctacgttac tcttcctcta 1320

tacgctctag ttactcagat gggttccaag atgaaaccta cagtgttcaa cgagagagta 1380

gcagtagctt taaagagttg gcaccacaca gctaagaagc aaatgaaaca tggaagaacc 1440

tcagagtcga ctacgccttt ctctagtcgt ccagctacac caacacacgg ttcttctccg 1500

atccatctcc tccacaatgt ccacaaacga agcagaagcg ctgatgaaag tttcgctaac 1560

tcaatgtctc cgaggagaaa ctctgacttc gatacgtggg atgttgagtc tcaacaagaa 1620

ccttcttctt cttccgtgaa gtatcattct aggtttaggg aagaaatata a 1671

<210> 56

<211> 1416

<212> DNA

<213> rape ()

<400> 56

atggcagata aggtggatga aagaacgctt cagcagactt ccacgtgggc ggtcgccgtt 60

gtttgcttct tcttgcttct aatttcgatt gtcattgaga aactgatcca caaattagga 120

aattggttta aaaggaagaa caaaaaggct ctgtatgaag ctcttgaaaa agtgaaagca 180

gagcttatgc tgatgggatt catatcacta ctgctaacaa ttggacaaaa ctatatctca 240

caaatttgcg tctccgagag cattgcagca tcaatgcgcc cttgcagtag atccgaagaa 300

gagaagaagt atcccaatac gaaaaaagat accgggaagg acgtaggaga tgaagaaaac 360

tctggtcgaa agcttctcga gttagtcgat tctttcattc ctcgaaggag tttggctacc 420

aaaggctatg acaagtgtgc agagaaggga caagtggctt ttgtatcgtc ctacgggatg 480

catcagctgc atatattcat cttcgttctt gctgtttgtc atgtgatcta ctgcattcta 540

acttatgctt tgggaaagac caagatgaga agatggaaga ggtgggaaga ggagacaaag 600

acaattgaat atcagtattc acacgatcct gagaggttta ggtttgcgag ggatacatct 660

ttcgggcgta gacatctgaa cttctggagc aaatcgacta ttacactgtg gattgcatgt 720

ttcttcagac agttttttgg atctgtaacc aaagttgatt acttaacact gagacatggt 780

ttcatcatgg cccatttggc tccagggagt gatgcgaggt tcgatttccg aaagtacatt 840

caaagatcat tagaggaaga tttcaaaact atcgttgaaa tcagtcctgt gatatggttt 900

gtagccgtgc tattcctcct gaccaacaca aacggattga attcttacgt ctggcaacca 960

ttcattccct tagttgtgat acttatagtt ggaacaaaac ttcaagtgat aataacaaaa 1020

ctaggacttc taatccaaga gaaaggggac atagtgaaag gcatgccgct tgttcagccc 1080

ggtgatcatc tcttctggtt cggtcgtcca cgtttcattc tcttcctcgt tcacttagtc 1140

cttttcacga atgcgtttca attagctttc tttgcctgga ctacgtatga attcaagctc 1200

aagaactgct tccacaagaa cactgtagat gtggtcatca gaatttcagt tggagttatt 1260

gtacaggttc tttgcagcta cgttactctt cctctttacg ctctagttac tcagatgggt 1320

tcgaagatga aacctacagt gttcaacgag agagtagcag tagctttaaa gagttggcac 1380

cacacagcta agaagcagat gaaaaaccga acctaa 1416

<210> 57

<211> 1749

<212> DNA

<213> rape ()

<400> 57

atggcggata aggtggacga aaaaacgcta cagcagactt ccacctgggc ggtcgctgtg 60

gtttgcttct tcttgcttct attttcgatt gtcattgaga aactgattca caaacttgga 120

acctggttta agattaagaa caaaaaagct ctgtacgaag ctctagaaaa agtgaaagca 180

gagcttatgc tgatgggatt catatcacta ttgctaacaa ttggacaaaa ctatatatcg 240

caaatttgcg tctccgagag catcgcagca tcaatgcacc cttgcagtga atctgaagag 300

gcgaaaaagt atccacctaa gaaaaaagat accaaaaatg ctgaagaaaa ctctggtcga 360

aagcttcatg agttagtcca atcttacatt cctcgccgga gtttggctac caaaggttat 420

gacaagtgtg cagagaaggg aaaagtcgct tttgtatcgt cttatgggat gcatcagctg 480

catatattta tcttcgttct cgcggtttgt catgtgatct actgcgttgt tacttatgct 540

ctaggcaaga ccaagatgag aaggtggaag agatgggaag aggagaccaa gacaattgaa 600

tatcagtatt ctcacgcaga tcctgagagg tttaggtttg cgagggatac atctttcggg 660

cgtagacatt tgaatttctg gagcaaatca actattacac tgtggattgt atgtttcttc 720

cgacagttct ttggatctgt tactagagtt gattacttaa cactaagaca tggtttcatc 780

atggcccatt tggctccagg gagtgatggg aggttcgatt tccggaagta cattcagaga 840

tcattagaag aagacttcaa aaccatcgtc gaaatcagtc ctgtgatttg gtttgtcgcc 900

gtgctatttc tcctgaccaa cacaaacgga ttgcattctt tcctctggca accattcatt 960

ccattagttg tgattcttat agttgggaca aaacttcaag tgataataac aaaacttgga 1020

cttttaatcc aagagaaagg agacatagtg aagggcatgc cgcttgttca gcccggtgat 1080

cacctcttct ggttcggtcg cccacgtttt attctcttcc tcattcactt agtccttttc 1140

acgaatgcgt ttcaactagc tttttttgcc tggacaacgt atgagttcca gctcgagaac 1200

tgttaccaca aaaccattgt agatgtggtc atcagaattt cagttggagt tattgtacaa 1260

attctttgca gctacgttac tcttcctctt tatgctctag tgactcaggc atgggcaaaa 1320

aacccaatat ccaagatccg aacagaaccc aaccgaaaac ctgactttgt acccaatccg 1380

agatgcaaga aatatctgaa tggatcctat atggtatatc gggtaatgga agtattatta 1440

atcaaatcga acgtataccc aaaaatcccg agaacagaca aaaatccaaa aaacagattt 1500

aacgattcaa cttatagtac ggcgaacacg ctgtctccaa gaaactctga cttcgattca 1560

tggcgtcctg agcctcagca agaaccgtct tcttcttctg caaatcatca ttctaggttc 1620

agagaagaag attcagaaaa gatgaagcac tcttcttcat ctttggagct tcctcctgga 1680

cctggacaaa tacgaactca ccagcatgag attagtataa gcttaaggga tttttccttt 1740

aagggatga 1749

<210> 58

<211> 1341

<212> DNA

<213> rape ()

<400> 58

atggcggata aggtggacga aaaaacgcta cagcagactt ccacctgggc ggtcgctgtg 60

gtttgcttct tcttgcttct attttcgatt gtcattgaga aactgattca caaacttgga 120

acctggttta agattaagaa caaaaaagct ctgtacgaag ctctagaaaa agtgaaagca 180

gagcttatgc tgatgggatt catatcacta ttgctaacaa ttggacaaaa ctatatatcg 240

caaatttgcg tctccgagag catcgcagca tcaatgcacc cttgcagtga atctgaagag 300

gcgaaaaagt atccacctaa gaaaaaagat accaaaaatg atgaagaaaa ctctggtcga 360

aagcttcttg agttagtcca atcttacatt cctcgccgga gtttggctac caaaggttat 420

gacaagtgtg cagagaaggg aaaggttgct tttgtatcgt cttatgggat gcatcagctg 480

catatattca tcttcgttct cgcggtttgt catgtgatct actgcattgt tacttatgct 540

ctaggcaaga ccaagatgag aaggtggaag aggtgggaag aggagaccaa gacaattgaa 600

tatcagtatt ctcacgcaga tcctgagagg tttaggtttg cgagggatac atctttcggg 660

cgtagacatt tgaatttctg gagcaaatca actattacac tgtggattgt atgtttcttc 720

cgacagttct ttggatctgt tactagagtt gattacttaa cactaagaca tggtttcatc 780

atggcccatt tggctccagg gagtgatggg aggttcgatt tccggaagta cattcagaga 840

tcattagagg aagacttcaa aaccatcgtc gaaatcagtc ctgtgatttg gtttgtcgcc 900

gtgctatttc tcctgaccaa cacaaacgga ttgcattctt tcctctggca accattcatt 960

ccattagttg tgattcttat agttgggaca aaacttcaag tgataataac aaaacttgga 1020

cttctaatcc aagagaaagg agacatagtg aagggcatgc cgcttgttca gcccggtgat 1080

cacctctttt ggttcggtcg cccacgtttt attctcttcc tcattcactt agtccttttc 1140

acgaatgcgt ttcaactagc tttttttgcc tggacaacgt atgagttcca gctcgagaac 1200

tgttaccaca aaaccattgt agatgtggtc atcagaattt cagttggagt tattgtacaa 1260

attctttgca gctacgttac tcttcctctt tatgctctag tgactcaggt aagttccaca 1320

cacatataca cacatacata g 1341

Claims

1. A mutant MLO6 gene for simultaneously improving sclerotinia resistance and powdery mildew resistance of rape, characterized in that the wild type MLO6 gene comprises 6 homologous copies, the names of which are respectively BnaA03g60240D (MLO 6-a 03), bnacng 04240D (MLO 6-C03), bnaA01g22680D (MLO 6-a 01), bnaC01g29530D (MLO 6-C01), bnaA09g13700D (MLO 6-a 09) and bnacng 31840D (MLO 6-C09) in the genome of rape darmor, the corresponding nucleotide sequences of which are respectively shown in SEQ ID No.53-58, the mutant MLO6-a03 copies are homozygous mutant compared with the wild type MLO6 gene, 15 bases are deleted from 1069 to 1083 of both strands of the MLO6-a03 copies, and the deleted bases are CTTGTTCAGCCCGGT; MLO6-C03 copies are heterozygous mutations, 13 bases are deleted from positions 1069 to 1081 of one strand of the MLO6-C03 copies, 2 bases are deleted from positions 1072 and 1073 of the other strand of the CTTGTTCAGCCCG, MLO-C03 copies, and the deleted bases are GT; MLO6-A01 copies are heterozygous mutations, positions 1096 and 1097 of one strand of the MLO6-A01 copies are deleted for 2 bases TT, and position 1096 of the other strand of the MLO6-A01 copies is inserted with a single base G; the MLO6-C01 copy lacks 12 bases from 1070 to 1081 of the site of one strand, the deleted base is TTGTTCAGCCCG, the other strand is identical to the wild type; sites 1066 to 1068 of one strand of the MLO6-A09 copy are deleted by 3 bases, the deleted base is GTT, and the other strand is identical to the wild type; sites 1065 and 1066 of one strand of the MLO6-C09 copy were deleted for 2 bases, the deleted base being TG, and the other strand being identical to the wild-type.

2. A polypeptide encoded by the mutant MLO6 gene of claim 1.

3. A construct comprising the mutant MLO6 gene of claim 1.

4. Use of a mutant MLO6 gene according to claim 1 for the simultaneous improvement of sclerotinia resistance and powdery mildew resistance in canola.