Application of MEB2 gene in negatively regulating resistance of plants to Sclerotinia sclerotiorum

Abstract

The present invention relates to the field of molecular biotechnology, and particularly to the application of the MEB2 gene in negatively regulating the resistance of plants to Sclerotinia sclerotiorum. Through genome-wide association analysis, the present invention identifies the locus where the BnMEB2 gene is located as a key regulatory locus for Sclerotinia sclerotiorum resistance in the rapeseed natural population. At the same time, using the Arabidopsis meb2 mutant, overexpression, and the BnMEB2 susceptible haplotype, it is confirmed that the loss of MEB2 function can enhance the disease resistance of plants, while overexpression leads to enhanced susceptibility, establishing a dose-effect relationship between gene expression and disease-resistant phenotypes. The present invention discloses the alternative splicing transcript AtMEB2.2 of the Arabidopsis MEB2 gene, and overexpression experiments show that this transcript significantly enhances the susceptibility to Sclerotinia sclerotiorum. In addition, the present invention develops a haplotype typing method for 16 SNP loci in the BnMEB2 gene region of rapeseed, which has important application prospects in the rapid screening of disease-resistant phenotypes.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to the application of the MEB2 gene in negatively regulating the resistance of plants to Sclerotinia sclerotiorum. Background Art

[0002] Plant fungal diseases, as a major threat in agricultural production, have long severely restricted the yield and quality of crops. Among them, diseases caused by necrotrophic pathogenic fungi have become one of the key problems to be urgently solved in agricultural production due to their unique pathogenic mechanisms and wide host ranges. Sclerotinia sclerotiorum, as a typical representative of such pathogenic fungi, can infect a variety of important economic crops including crucifers and legumes, causing Sclerotinia disease and resulting in significant economic losses.

[0003] Taking rapeseed as an example, as the second largest oil crop in the world, its yield and quality are directly related to national edible oil security and the stable development of the agricultural economy. However, the frequent outbreak of Sclerotinia disease in rapeseed production has become the main bottleneck restricting the sustainable development of the rapeseed industry. This disease not only directly causes a yield loss of up to 10%-20% in rapeseed, but also significantly reduces the quality of rapeseed oil, including a decrease in oil content and changes in fatty acid composition, seriously threatening the long-term healthy development of the rapeseed industry. In the face of the severe challenge of Sclerotinia disease, traditional prevention and control measures such as chemical control can alleviate the disease pressure to a certain extent, but long-term use is likely to lead to problems such as environmental pollution, enhanced pathogen resistance, and excessive pesticide residues in agricultural products, which do not meet the requirements of sustainable development of modern agriculture. Therefore, cultivating disease-resistant varieties as a green, economic, and safe way for disease prevention and control has become the consensus and key research direction in the current agricultural research field.

[0004] However, the process of breeding for Sclerotinia disease resistance faces many challenges. On the one hand, since no plant materials with complete immunity to Sclerotinia disease have been found, it is difficult to quickly obtain ideal disease-resistant varieties by traditional breeding methods. On the other hand, existing research shows that the resistance of plants to Sclerotinia disease is controlled by multiple genes, showing quantitative genetic trait characteristics, that is, disease resistance is determined by the combined action of multiple major genes and minor genes. This genetic complexity increases the difficulty of locating and cloning disease-resistant genes, restricting the in-depth development of disease-resistant breeding work. Therefore, exploring new disease-resistant gene resources and their application potential in disease-resistant breeding is of great significance for breaking through the current technical bottleneck of Sclerotinia disease-resistant breeding and promoting the genetic improvement of disease resistance in crops such as rapeseed. Summary of the Invention

[0005] In view of this, the present invention proposes the application of the MEB2 (Membrane Protein of Endoplasmic ReticulumBody2) gene in negatively regulating the resistance of plants to Sclerotinia sclerotiorum.

[0006] The technical solution of the present invention is achieved as follows:

[0007] In a first aspect, the present invention provides an application of the MEB2 gene in negatively regulating plant resistance to sclerotinia sclerotiorum, wherein the MEB2 gene is the BnaC07G30170D gene or the AT5G24290 gene; and the plant is rapeseed or Arabidopsis thaliana;

[0008] The negative regulation is as follows (1) or (2):

[0009] (1) The MEB2 gene is functionally lost, and the plant's resistance to sclerotinia sclerotiorum is enhanced;

[0010] (2) The MEB2 gene is overexpressed, and the plant is susceptible to sclerotinia disease.

[0011] The nucleotide sequence of the MEB2 gene is one of SEQ ID NOs: 1-3.

[0012] In some specific embodiments, the MEB2 gene functional loss includes reducing or eliminating the expression of the MEB2 gene by using gene mutation, gene knockout, gene interference or gene silencing technology.

[0013] In a second aspect, the present invention provides the use of the transcript AtMEB2.1 or AtMEB2.2 in regulating resistance to sclerotinia sclerotinia in Arabidopsis thaliana. The locus of the transcript AtMEB2.1 in the TAIR database is AT5G24290.1; the cDNA sequence of the transcript AtMEB2.2 is shown in SEQ ID NO: 4. In some specific embodiments, overexpression of the transcript AtMEB2.1 or AtMEB2.2 increases plant susceptibility to sclerotinia sclerotinia.

[0014] In a third aspect, the present invention provides the application of the rapeseed BnMEB2 gene in the identification of rapeseed Sclerotinia sclerotiorum resistance. Referring to the rapeseed genome Darmor-bzh v4.1, at the physical positions 34748484, 34748494, 34748655, 34748690, 34748835, 34749302, 34749303, 34749330, 34749439, 34749451, 34749513, 34749655, 34750334, 34750736, 34751328, and 34751950 on chromosome C07 of the BnMEB2 gene, there are a total of 16 SNP loci; the Sclerotinia sclerotiorum resistance of rapeseed with the haplotype SEQ ID NO: 29 (GATATTGGCAAAAAGG) at the said SNP loci is superior to that of rapeseed with the haplotype SEQ ID NO: 30 (AGCTCCCATGTGGTCA) at the said SNP loci. The haplotype typing results of the said SNP loci and their corresponding rapeseed germplasm resources are specifically shown in the following table:

[0015]

[0016] In a fourth aspect, the present invention provides a method for identifying rapeseed Sclerotinia sclerotiorum resistance. Referring to the rapeseed genome Darmor-bzh v4.1, detect the genotypes of rapeseed at 16 SNP loci at the physical positions 34748484, 34748494, 34748655, 34748690, 34748835, 34749302, 34749303, 34749330, 34749439, 34749451, 34749513, 34749655, 34750334, 34750736, 34751328, and 34751950 on chromosome C07 of the BnMEB2 gene. The Sclerotinia sclerotiorum resistance of rapeseed with the haplotype SEQ ID NO: 29 at the said SNP loci is superior to that of rapeseed with the haplotype SEQ ID NO: 30 at the said SNP loci.

[0017] In some specific embodiments, the detection method includes sequencing, PCR amplification, specific probe hybridization method, specific primer extension method, or gene chip method.

[0018] In a fifth aspect, the present invention provides the application of the said identification method in rapeseed breeding for resistance to Sclerotinia sclerotiorum.

[0019] The beneficial effects of the present invention at least include the following:

[0020] The present invention accurately identified the locus where the MEB2 gene is located as the key locus for regulating Sclerotinia sclerotiorum resistance in a rapeseed natural population through genome-wide association study (GWAS). By constructing Arabidopsis meb2 mutants and overexpression lines and combining with transgenic function verification in rapeseed, it was confirmed that the loss of function of the MEB2 gene could significantly improve plant disease resistance, while overexpression led to enhanced susceptibility.

[0021] The present invention first disclosed the transcript AtMEB2.2, and its overexpression significantly enhanced the susceptibility of plants to Sclerotinia sclerotiorum, indicating that AtMEB2.2 is involved in the negative regulation pathway of disease resistance, providing a new theoretical basis for molecular breeding of disease resistance by targeting the regulation of specific splicing isoforms.

[0022] In view of the breeding requirements of rapeseed, the present invention developed a haplotype typing method based on 16 SNP loci in the BnMEB2 gene region, realizing molecular marker-assisted selection of disease resistance phenotypes, which has important application prospects in fields such as rapid screening of field disease resistance phenotypes and germplasm creation of rapeseed and other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 For the GWAS analysis (Genome-Wide Association Study) of Sclerotinia sclerotiorum resistance in rapeseed populations and the haplotype analysis map (Haplotype Map, HapMap): Figure 1 A is the Manhattan plot of GWAS analysis of rapeseed populations; Figure 1 B is the quality control plot of GWAS analysis of rapeseed populations; Figure 1 C is the linkage disequilibrium analysis map of the locus where the BnMEB2 gene is located; Figure 1 D is the haplotype typing map of single-base variations in the BnMEB2 gene region in the population;

[0025] Figure 2 For the sequence comparison analysis map of rapeseed MEB2 and related species: Figure 2 A is the phylogenetic tree constructed from homologous sequences of BnMEB2 in rapeseed and its ancestral species Brassica rapa and Brassica oleracea and the model plant Arabidopsis thaliana; Figure 2 B is the collinearity display map of homologous sequences of BnMEB2 in rapeseed and its ancestral species Brassica rapa and Brassica oleracea;

[0026] Figure 3Diagram of the sequences, expression, and molecular identification of genetic materials created for two alternative splice variants of AtMEB2, AtMEB2.1 and AtMEB2.2: Figure 3 A is a diagram of the gene structure analysis of AtMEB2.1 and AtMEB2.2; Figure 3 B is a diagram of the expression of AtMEB2.1 and AtMEB2.2 in roots, stems, leaves, flowers (flowers include small buds, middle buds, fully open flowers), and siliques; Figure 3 C is a diagram of the expression of AtMEB2.1 and AtMEB2.2 in transgenic lines overexpressing them in wild-type and meb2 mutant plants, respectively; where lanes: 1 is 35S:MEB2.1 / WT (a line overexpressing AtMEB2.1 in wild-type plants); 2 is 35S:MEB2.2 / WT (a line overexpressing AtMEB2.2 in wild-type plants); 3 is WT (wild-type); 4 is the meb2 mutant; 5 - 7 are 35S:MEB2.1 / meb2 (lines overexpressing AtMEB2.1 in meb2 mutant plants); 8 - 10 are 35S:MEB2.2 / meb2 (lines overexpressing AtMEB2.2 in meb2 mutant plants);

[0027] Figure 4 Diagram of the phenotypic identification of wild-type Arabidopsis thaliana, meb2 mutants, and transgenic lines overexpressing AtMEB2.1 and AtMEB2.2 after inoculation with Sclerotinia sclerotiorum: Figure 4 A and Figure 4C are diagrams of the phenotypes of Sclerotinia sclerotiorum lesions; Figure 4 B and Figure 4D are statistical graphs of the areas of Sclerotinia sclerotiorum lesions; (Note: Experimental time points, such as "60 hpi" indicate 60 hours after inoculation; the control group is untransformed wild-type: WT);

[0028] Figure 5 Diagram of the phenotypic identification of T1 transgenic lines overexpressing BnMEB2 in rapeseed after inoculation with Sclerotinia sclerotiorum: Figure 5 A is a diagram of the expression of BnMEB2 in BnMEB2 overexpressing lines; Figure 5 B is a statistical graph of the areas of Sclerotinia sclerotiorum lesions; Figure 5 C is a diagram of the phenotypes of Sclerotinia sclerotiorum lesions; (Note: Experimental time points, such as "24 hpi" indicate 24 hours after inoculation; the control groups are untransformed wild-type ZS11 and empty vector EV). Detailed implementation methods

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention. For those embodiments where specific conditions are not indicated, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. The experimental methods without indicating specific conditions in the embodiments are all carried out according to "Molecular Cloning: A Laboratory Manual" (Fourth Edition) or the instructions of relevant reagent kits.

[0030] Table 1 Sequence Information Table

[0031]

[0032] Example 1

[0033] 1. Genome-wide association study (GWAS) of resistance to Sclerotinia sclerotiorum in rapeseed

[0034] In this application, 128 natural rapeseed germplasm resources (Table 2) were used. When the rapeseed plants grew to the 4-5 leaf stage, the second and third leaves were cut, and an in vitro leaf inoculation experiment with Sclerotinia sclerotiorum was carried out in a greenhouse (22-24 °C, 16 h light / 8 h dark). The sclerotia of Sclerotinia sclerotiorum first germinated mycelia on a potato medium (potato dextrose agar purchased from BD, USA, product number 6117503, and the preparation method was referred to the instructions). The activated mycelial blocks (5 mm × 5 mm in diameter) were inoculated onto the leaves. The lesion area was measured 24 h after inoculation to obtain the phenotypic data of the population's resistance to Sclerotinia sclerotiorum (for the specific method, refer to the paper: Zhang et al., Syntenic QTL and genomic divergence for Sclerotinia resistance and flowering time in Brassica napus. Journal of Integrative Plant Biology. 2019, 61(1): 75-88).

[0035] Population resequencing was commissioned to BGI-Shenzhen, and 7.36 Gb of short sequences without adapters that could be aligned to the rapeseed reference genome (Darmor-bzh v4.1, http: / / www.genoscope.cns.fr / plants) were obtained using the Illumina HiSeqXTen platform, with the sequencing depth ranging from 6.02× to 17.03×. SNP identification for each material was performed using the Sentieon DNAseq pipeline. The initially obtained SNPs were first filtered using GATK's function variantFiltration (filtering parameters: QUAL < 30 || MQ < 50 || QD < 2), and then filtered for low-frequency bases and mismatch rate (MAF > 0.05 || missing rate < 0.1). Finally, 2,288,712 high-quality SNPs were obtained for GWAS analysis. GWAS analysis was completed using the mixed linear model of the EMMAX software.

[0036] The analysis results showed that there was a significant association locus on chrC07 (chromosome C07). Linkage disequilibrium analysis showed that this region was a highly linked module ( Figure 1 A-C). As shown in Table 3, a candidate gene BnMEB2 (BnaC07g30170D) was screened at this locus. Its haplotype typing (Table 4) showed that the single-base variations in this gene region were divided into two haplotypes, and there were extremely significant differences in Sclerotinia sclerotiorum resistance between the two haplotypes. The materials with haplotype II had significantly better Sclerotinia sclerotiorum resistance than those with haplotype I at the SNP locus. ( Figure 1 D).

[0037] Table 2. Information table of 128 rapeseed germplasm resources

[0038]

[0039] Table 3. A locus significantly associated with Sclerotinia sclerotiorum resistance in rapeseed identified by GWAS

[0040]

[0041] Table 4. Haplotype typing of the BnMEB2 gene in the rapeseed population

[0042]

[0043] Based on the GWAS analysis of 128 rapeseed germplasms (P = 1.35×10⁻¹ 5, Manhattan plot threshold -Log10(P) = 5), the BnMEB2 gene (BnaC07g30170D) was located as the major locus for Sclerotinia sclerotiorum resistance. Haplotype typing showed that the average lesion area of the materials carrying haplotype II (SEQ ID NO: 29) was 1.08 cm² at 24 h after inoculation, and the resistance was significantly improved compared with that of the materials with haplotype I (SEQ ID NO: 30) (t-test P < 0.01). The LD value (r²) in this region was greater than 0.73, indicating that 16 SNP loci (ChrC07: 34,748,484 - 34,751,950) were tightly linked markers.

[0044] 2. Comparative analysis of the BnMEB2 gene sequence with its homologous gene in Arabidopsis thaliana

[0045] The BnMEB2 gene sequence was obtained through genomic data resources, and the genetic relationship was analyzed by homologous search, sequence alignment, and phylogenetic tree construction. The results showed that BnMEB2 was highly homologous to the AtMEB2 gene in Arabidopsis thaliana ( Figure 2 A), and collinearity analysis confirmed that BnMEB2 had orthologous genes in Brassica rapa (Bra037799) and Brassica oleracea (Bo5g024370), and the genomic region where MEB2 was located was very conserved ( Figure 2 B). It indicated that the gene structures of BnMEB2 and AtMEB2 were highly conserved and their functions might be similar.

[0046] Example 2 Cloning and expression pattern analysis of two alternative splicing variants AtMEB2.1 and AtMEB2.2 of Arabidopsis thaliana MEB2

[0047] For ease of subsequent description, the two transcripts of AtMEB2 were named AtMEB2.1 (Locus: AT5G24290.1; https: / / www.arabidopsis.org / locus?key=134054#) and AtMEB2.2 respectively. AtMEB2.1 is the reported major transcript. AtMEB2.2 (nucleotide sequence as shown in SEQ ID NO: 4) is an alternative splicing variant first disclosed in this application. Compared with AtMEB2.1, there are two key differences: the 5' UTR (5' untranslated region) of AtMEB2.2 is 86 bp less than that of AtMEB2.1, and the second exon of AtMEB2.2 is 48 bp less than that of AtMEB2.1.

[0048] The method of this example is as follows:

[0049] (1)Primer design and template preparation: According to the AtMEB2 gene sequence information on the Arabidopsis TAIR website (http: / / www.arabidopsis.org / ), three pairs of primers were designed as shown in SEQ ID NO: 5 - 10. Young leaves of Arabidopsis thaliana Columbia ecotype were flash-frozen in liquid nitrogen and total RNA was extracted using the Trizol method (Invitrogen, catalog number 15596026). Reverse transcription was performed using Superscript TM reverse transcriptase (Promega, catalog number M1701) to obtain cDNA; this was used as the template.

[0050] (2)PCR amplification: PCR amplification was carried out using Ex Taq DNA high-fidelity polymerase. The amplification system was a 50 μl system including 4 μl of dNTP (10 mmol / L), 5 μl of 10× Taq buffer, 1 μl of 5’ primer (10 μmmol / L), 1 μl of 3’ primer (10 μmmol / L), 0.5 μl of Taq (5 U / μl), 1 μl of cDNA template, and 37.5 μl of ddH2O. The PCR amplification reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 58°C for 45 s, extension at 72°C for 2.5 min, and the above was repeated for 30 cycles; extension at 72°C for 10 min. The PCR products were identified by 1% (mass / volume ratio) agarose gel electrophoresis. The purification and recovery of the PCR products were carried out using the ordinary agarose gel DNA recovery kit (DP209, TIANGEN) from Tiangen Biotech. The experimental steps were referred to the kit instructions. The concentration of the gel-purified PCR products was measured using Nanodrop 2000, and then the PMD19-T Vector kit (D102A, Takara) from Takara Bio Inc. was used.

[0051] (3)Cloning construction and alternative splicing identification: The PCR products were ligated into the PMD19-T vector. The specific experimental steps and methods refer to the kit instructions. In a 10 μl reaction system, 4 μl of the purified PCR product, 1 μl of PMD19-T Vector, and 5 μl of ligase were used. After gently mixing with a pipette, the ligation was carried out overnight at 16 °C. The ligation products were transformed into Escherichia coli DH5α cells by heat shock method (after thawing the competent cells, add 10 μL of the recombinant plasmid and mix well, incubate on ice for 5 min; freeze in liquid nitrogen for 5 min; water bath in a 37 °C constant temperature water bath for 5 min). Then, add 800 μl of liquid LB medium (2.5 g of yeast extract, 5 g of peptone, 5 g of sodium chloride, and 0.5 L of sterile water, autoclaved at 120 °C for 20 min) and resuscitate for 1 h, and spread on an LB solid plate with ampicillin resistance (2.5 g of yeast extract, 5 g of peptone, 5 g of sodium chloride, 7 g of agar powder, and 0.5 L of sterile water, autoclaved at 120 °C for 20 min), and culture at 37 °C for 14 h. The next day, randomly pick monoclonal colonies with a sterilized toothpick, and perform colony PCR amplification using the M13 universal primer to obtain positive colonies (the PCR amplification conditions, procedures, and detections are the same as above).

[0052] The positive clones were inoculated into LB liquid medium containing ampicillin antibiotics and cultured overnight at 37 °C for plasmid extraction (the plasmid mini-prep kit from Tiangen Biotech, DP103), and then sent to Qingke Company for sequencing. The sequencing results analysis showed that alternative splicing occurred in the 5’ UTR and the second exon of AtMEB2, generating two alternative splice variants, AtMEB2.1 and AtMEB2.2. The 5’ UTR (5’ untranslated region) of AtMEB2.2 was 86 bp less than that of AtMEB2.1, and the second exon of AtMEB2.2 was 48 bp less than that of AtMEB2.1 ( Figure 3 A).

[0053] (4)Tissue expression pattern detection: To further detect the expression patterns of the two alternative splice variants, samples of different tissues of Arabidopsis thaliana (including leaves, stems, small flower buds, medium flower buds, fully open flowers, roots, and siliques) were collected, RNA was extracted and reverse transcribed into cDNA. After diluting this cDNA to the same concentration (20 ng / L), it was used as a template for semi-quantitative PCR amplification. The primers for AtMEB2 in semi-quantitative PCR are shown in SEQ ID NO: 11 and 12, and the internal reference gene is the Arabidopsis thaliana β-actin encoding (Actin) gene (Locus: AT1G13810; https: / / www.arabidopsis.org, primers are shown in SEQ ID NO: 13 and 14). The PCR amplification conditions, procedures, and detection methods are the same as above.

[0054] ResultIt was shown that the two alternative splice variants were expressed in multiple tissues (including leaves, stems, small flower buds, medium flower buds, fully open flowers, roots, and siliques), but the expression level of AtMEB2.1 was higher than that of AtMEB2.2 ( Figure 3 B).

[0055] Example 3 Construction of Overexpression and Complementation Genetic Materials of AtMEB2.1 and AtMEB2.2

[0056] To explore the function of AtMEB2 in resistance to Sclerotinia sclerotiorum, the present application constructed overexpression vectors of AtMEB2.1 and AtMEB2.2 and transferred them into wild-type Arabidopsis thaliana and meb2 mutants (GABI_059D03, T-DNA inserted into the second exon of AtMEB2, Figure 3 A), and the steps were as follows:

[0057] (1) Construction of overexpression vectors and transformation and verification of recombinant plasmids: Using primers (as shown in SEQ ID NO: 15-18), the complete coding region sequences of AtMEB2.1 and AtMEB2.2 (as shown in SEQ ID NO: 2-3) were amplified by PCR from the templates of recombinant plasmids PMD-19-AtMEB2.1 and PMD-19-AtMEB2.2. The PCR reaction system (50 μl) was: 2 μl cDNA, 2 μl each of forward / reverse primers (10 μM), 25 μl of 2×Phanta Max Master Mix, and 19 μl of ddH2O. After adding samples on ice, mix well. The PCR reaction conditions were: 95 o °C for 3 min; 95 o °C for 15 sec, 53 o °C for 15 sec, 72 o °C for 90 sec, 32 cycles; 72 oC 10 min. The target fragment and the expression vector PBI121 plasmid (Shenzhen Kangti Life Technology Co., Ltd.) were recovered by agarose gel electrophoresis and digested with enzymes respectively. The digestion system was as follows: 30 μl of target fragment / expression vector, 5 μl of 10×buffer, 2 μl each of Bam HI and Xba I (10 U / μl), and 11 μl of ddH2O. Incubate in a 37°C water bath for 3 - 5 h, and identify the digestion result by agarose gel electrophoresis. The digestion products were purified and recovered using the ordinary agarose gel DNA recovery kit (DP209) from Tiangen Company, and the experimental steps and operations were referred to the instruction manual. The ligation system of the digestion products was: 5 μl of target fragment, 1 μl of target vector fragment, 1 μl of 10×buffer, 1 μl of T4 DNA ligase (350 U / μl), and 1 μl of ddH2O. After gently mixing with a pipette, seal and ligate overnight at 16°C. The ligation products were transferred into Escherichia coli cells (the antibiotic is kanamycin resistance) by the same method as above. After identifying positive clones by colony PCR, shake the bacteria to extract the plasmid and send it to Qingke Company for sequencing. For the recombinant strain plasmid without base errors in sequencing, it was transferred into Agrobacterium tumefaciens EHA105 competent cells by the heat shock method. After the competent cells were frozen and thawed, 10 μL of the recombinant plasmid was added and mixed evenly, and incubated on ice for 5 min; frozen in liquid nitrogen for 5 min; incubated in a 37°C constant temperature water bath for 5 min; then added with fresh liquid LB medium without antibiotics and mixed evenly, activated at 28°C, 220 rpm / min for 2 h; finally spread on an LB solid plate containing kanamycin and rifampicin and cultured in the dark at 28°C for 2 days, and identify positive monoclonal clones using the above primers.

[0058] (2) Arabidopsis thaliana transformation and verification of transgenic results: Pick positive clones and culture them in an enlarged manner in an LB liquid medium containing kanamycin and rifampicin for the transformation of Arabidopsis thaliana. Use the Agrobacterium-mediated floral dip method to transform wild-type Arabidopsis thaliana (Columbia ecotype) and meb2 mutants, and construct overexpression materials of AtMEB2.1 and AtMEB2.2 in wild-type and complementary materials in meb2 mutants respectively. Extract RNA from the leaves of transgenic Arabidopsis thaliana overexpressing and complementing AtMEB2.1 and AtMEB2.2, as well as the leaves of wild-type and meb2 mutants, and reverse transcribe them into cDNA. Dilute their concentrations to the same concentration (20 ng / μl) as the template for semi-quantitative PCR to detect the expression of the target gene. The specific primers for AtMEB2.1 and AtMEB2.2 are shown in SEQ ID NO: 19 - 22.

[0059] Results: As Figure 3As shown in C, compared with the band intensities of AtMEB2.1 and AtMEB2.2 in the wild type in the third lane, the band intensities of the target genes of the AtMEB2.1 and AtMEB2.2 overexpression lines in the first and second lanes were significantly enhanced, i.e., overexpressed; compared with the almost undetectable expression bands of AtMEB2.1 and AtMEB2.2 in the meb2 mutant in the fourth lane, the bands of AtMEB2.1 in the transgenic lines in which AtMEB2.1 was complemented into meb2 in the fifth to seventh lanes were clearly visible, and the bands of AtMEB2.2 in the transgenic lines in which AtMEB2.2 was complemented into meb2 in the eighth to tenth lanes were equally bright, i.e., the complementation was successful.

[0060] Example 4 Identification of Sclerotinia sclerotiorum Resistance of AtMEB2 Transgenic Plants

[0061] The transgenic positive plants obtained above were cultured in a greenhouse until they were 4 weeks old for an in vitro inoculation experiment with Sclerotinia sclerotiorum. The steps were as follows:

[0062] 1) Activation of Sclerotinia sclerotiorum: First, the sclerotia of Sclerotinia sclerotiorum stored in the laboratory were disinfected (treated with 75% alcohol for 1 min, then with 0.1% mercuric chloride for 5 min, and then washed with sterile water 3 - 5 times). After that, the sclerotia of Sclerotinia sclerotiorum were cut with a sterilized knife, and the cross-section of the sclerotia was inoculated into the center of the PDA solid medium (the PDA solid medium was dissolved according to the potato dextrose agar instructions, autoclaved at 121 °C for 15 min). After sealing with a sealing film, it was cultured in an incubator at 24 °C for 72 h; in a sterilized ultra-clean workbench, 5 mm×5 mm mycelial blocks were punched along the mycelial edge with a sterile punch and inoculated into the center of the Minimal solid medium (ammonium sulfate 2 g, sodium hydroxide 1 g, DL-malic acid 3 g, magnesium sulfate heptahydrate 0.1 g, bacteriological agar powder 39 g, supplemented with sterile water to 1 L, adjusted the pH to 5.8 - 6.0, autoclaved at 115 °C for 15 min), and also cultured in an incubator at 24 °C for 48 h.

[0063] 2) Inoculation of Arabidopsis thaliana leaves with Sclerotinia sclerotiorum: When the mycelia grew to 2 / 3 of the medium, 2 mm×2 mm mycelial blocks were punched along the mycelial edge with a sterile punch for inoculation. The AtMEB2.1 and AtMEB2.2 overexpression and complementation transgenic lines, as well as Arabidopsis thaliana and the meb2 mutant, were sown in a growth room and cultured until they were 4 weeks old. When the plants grew to the state where the leaves were the largest and had not bolted, in vivo inoculation was carried out, that is, the prepared mycelial blocks were picked out with a paper clip, and the mycelial surface-containing side was inoculated onto the Arabidopsis thaliana leaves (3 - 5 mycelial blocks were inoculated on each plant). After inoculation, water was sprayed to keep the humidity (humidity > 80%), and the culture was sealed.

[0064] 3) Statistical analysis of lesion area: The length and width of the leaf lesions were measured with a ruler at 24 h, 36 h, 48 h, and 60 h after inoculation, respectively.

[0065] Results: As Figure 4 shown, the lesion area of Arabidopsis meb2 mutants inoculated with Sclerotinia sclerotiorum was significantly reduced compared with that of the wild type (P<0.001). The lesion areas of AtMEB2.1 and AtMEB2.2 overexpression lines (verified by RT-PCR to have significantly increased expression levels) were significantly increased compared with that of the wild type. Complementation experiments showed that the lesion area of meb2 mutants overexpressing AtMEB2.1 / AtMEB2.2 was restored to a level not significantly different from that of the wild type (P>0.05), proving that these two alternative splicing variants of AtMEB2 can, to a certain extent, compensate for the reduced resistance caused by the mutation in the meb2 mutant background.

[0066] Example 5

[0067] 1. Cloning of susceptible haplotype genes and vector construction

[0068] To clone the susceptible haplotype of BnMEB2, in this invention, the leaves of rapeseed Westar were taken to extract RNA and reverse-transcribed into cDNA as a template. Based on the sequence information of BnMEB2 in the C07 copy (BnaC07T0360300WE) in the published Westar genome (https: / / bnaomics.ocri-genomics.net / ), primers were designed (as shown in SEQ ID NO: 23 and 24), and the complete open reading frame of BnMEB2ws_C07 (BnaC07g30170D) (as shown in SEQ ID NO: 1) was amplified by PCR. After the amplified product was detected by agarose gel electrophoresis, gel-purified and recovered, and the concentration of the recovered fragment was determined using the same method as above, the recovered fragment was ligated to the linear vector of the plant expression vector PGTV II-3FLAG modified in the laboratory by homologous recombination (the homologous recombination kit was purchased from Novoprotein Co., Ltd., and the recombination system and method refer to the kit instruction manual). The ligation system (20 μl): 12 μl of the PGTVII-3FLAG linear vector, 1 μl of the purified product of BnMEB2ws_C07, 2 μl of Exnase II, and 5 μl of 5×CE buffer. Take 10 μl of the ligation product and transform the competent cells of Escherichia coli by the same method as described above. Add 800 μl of liquid LB medium and resuscitate for 1 h, then spread it on the LB solid plate with kanamycin resistance and culture at 37 °C for 14 h. Colony PCR verified the size of the target gene fragment in the recombinant vector (the primers used are as shown in SEQ ID NO: 23 and 24). The positive colonies were cultured on a large scale, and the recombinant plasmid was extracted and sent to Tsingke Biotechnology Co., Ltd. for sequencing. After sequencing analysis, the recombinant plasmid contained a complete open reading frame, indicating that the overexpression vector of BnMEB2ws_C07 was successfully constructed. The recombinant plasmid was extracted, transferred into Agrobacterium cells, single colonies were picked and identified by PCR for positive clones, the plasmid was extracted and sent for sequencing, and the Agrobacterium strain containing the positive plasmid was used for subsequent rapeseed genetic transformation using the same method as above.

[0069] 2. Rapeseed Genetic Transformation and Line Selection

[0070] Using the Agrobacterium-mediated hypocotyl transformation method, the overexpression vector of BnMEB2ws_C0 was transferred into the rapeseed variety Zhongshuang 11 (ZS11). After screening for hygromycin resistance (universal primers) and PCR verification with gene-specific primers (primers: SEQ ID NO: 23 and 24), 4 overexpression lines (#2, #7, #11, #13) of the T1 generation were obtained. Total RNA was extracted from the above-mentioned transgenic positive plants and the young tissues of ZS11 using the same method as described above, and reverse-transcribed into cDNA as a template (the template concentration was adjusted to the CT value after fluorescence quantitative PCR (qRT-PCR) of the rapeseed internal reference gene Actin (AF111812, primers as shown in SEQ ID NO: 25 and 26) being between 18 and 22). The expression of BnMEB2ws_C07 in each material was detected using primers SEQ ID NO: 27 and 28. PCR reaction system (20 μL): 1 μl cDNA, 1 μL each of forward / reverse primers (10 μM), 10 μL 2×SYBR Green Master Mix (Yeasen Biotech, product number 11184ES), and 7 μL ddH2O. Mix well after loading on ice. PCR reaction conditions were: 95°C for 5 min; 95°C for 15 sec, 58°C for 30 sec, 72°C for 15 sec, 40 cycles; 95°C for 15 s; 60°C for 60 s; 95°C for 15 s. The 2 -ΔΔCT -method was used to calculate the relative expression level of the target gene.

[0071] The results showed that the expression level of BnMEB2 in the transgenic lines was significantly higher than that in ZS11 and the empty vector (EV) ( Figure 5 A).

[0072] 3. Sclerotinia sclerotiorum detached leaf inoculation experiment

[0073] The 4 lines overexpressing BnMEB2ws_C07, the empty vector (PGTV II-3FLAG vector without the target fragment), and ZS11 were cultured in the growth chamber until the 4-5 leaf stage for Sclerotinia sclerotiorum detached leaf inoculation:

[0074] 1) The activation of Sclerotinia sclerotiorum was the same as the activation method of the bacteria inoculated on Arabidopsis thaliana described above, that is, sterilized cut sclerotia were placed on PDA solid medium. When the mycelium grew to 2 / 3 of the medium, a sterilized puncher was used to punch holes (5 mm×5 mm in diameter) at the edge of the mycelium, and the resulting mycelium blocks were used for rapeseed leaf inoculation;

[0075] 2) The second and third leaves of each plant of each material at the 4-5 leaf stage were laid flat in a box with two layers of absorbent paper (add water to make it in a moist state) at the bottom. 15-20 leaves were cut from each material;

[0076] 3) Use a paper clip to inoculate the prepared mycelium blocks with the mycelium facing down on both sides of the main vein of the rape leaf (avoiding the vein position). After inoculation, spray water to keep the humidity (80%) and then cover with a lid and seal for cultivation;

[0077] 4) Measure the lesion size 24 h after inoculation using the same method as described above. The results showed that: compared with the controls (ZS11 and empty vector), the lesion size of the BnMEB2 overexpression lines was significantly enlarged. Phenotypic observation showed that large areas of water-soaked lesions appeared on the transgenic leaves and the mycelium spread faster ( Figure 5 B and 5C).

[0078] Conclusion : Overexpression of BnMEB2 in rape significantly enhanced the susceptibility to Sclerotinia sclerotiorum and promoted the occurrence of Sclerotinia sclerotiorum in rape, which was consistent with its function of negatively regulating resistance in Arabidopsis. This result corroborated the effect of the susceptible haplotype associated with GWAS, and there was a certain correlation between the gene dosage effect of BnMEB2 and susceptibility (R² = 0.02).

[0079] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Transcript AtMEB2.2 Application in regulating Sclerotinia sclerotiorum resistance of Arabidopsis thaliana, characterized in that The transcript AtMEB2.2 has a cDNA sequence as shown in SEQ ID NO:

4.

2. The application according to claim 1, wherein By means of the said transcript AtMEB2.2 Overexpression promotes the susceptibility of plants to Sclerotinia sclerotiorum.

3. Rapeseed BnMEB2 Application of the gene in the identification of rapeseed resistance to Sclerotinia sclerotiorum, characterized in that Refer to the rapeseed genome Darmor- bzh v4.

1. At the physical positions of the BnaC07g30170D gene on chromosome C07, namely positions 34748484, 34748494, 34748655, 34748690, 34748835, 34749302, 34749303, 34749330, 34749439, 34749451, 34749513, 34749655, 34750334, 34750736, 34751328, and 34751950, there are a total of 16 SNP sites. The sclerotinia resistance of rapeseed with the haplotype of SEQ ID NO: 29 at the said SNP sites is superior to that of rapeseed with the haplotype of SEQ ID NO: 30 at the said SNP sites.

4. A method for identifying the resistance to Sclerotinia sclerotiorum in rapeseed, characterized in that, Reference rapeseed genome Darmor- bzh v4.1, detect rapeseed in BnMEB2 The genotypes of the 16 SNP sites at the physical positions 34748484, 34748494, 34748655, 34748690, 34748835, 34749302, 34749303, 34749330, 34749439, 34749451, 34749513, 34749655, 34750334, 34750736, 34751328 and 34751950 of the chromosome of gene C07, and the rapeseed whose haplotype at the SNP site is SEQ ID NO: 29, have better resistance to sclerotinia disease than the rapeseed whose haplotype at the SNP site is SEQ ID NO:

30.

5. The identification method according to claim 4, characterized in that, The detection method includes sequencing, PCR amplification, specific probe hybridization method, specific primer extension method or gene chip method.

6. Use of the identification method according to claim 4 or 5 in breeding Brassica napus for resistance to Sclerotinia sclerotiorum.

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