Application of BnGAE1 gene in regulating resistance to sclerotinia sclerotiorum in brassica napus

Abstract

The application discloses application of BnGAE1 gene in regulation of resistance of Brassica napus to sclerotinia stem rot, and belongs to the technical field of plant genetic engineering. A gene BnGAE1 capable of enhancing the resistance of Brassica napus to sclerotinia stem rot is cloned from Brassica napus, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. It is found through experiment verification that the resistance of BnGAE1 (KO) strain to sclerotinia sclerotiorum is significantly enhanced, and the incidence rate is reduced by 25.46% after inoculation; physiological and molecular detection shows that the expression amount of defense-related genes (such as PR1 and PDF1.2) in the mutant strain is up-regulated, the SA (salicylic acid) and JA / ET (jasmonic acid / ethylene) signal pathways are activated, meanwhile, excessive ROS (reactive oxygen species) is effectively removed, and the oxidation-reduction balance is maintained. The application provides a new gene target and an efficient molecular breeding technology path for improvement of the resistance of Brassica napus to sclerotinia stem rot, and has important application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the BnGAE1 gene in regulating resistance to sclerotinia stem rot in Brassica napus. Background Technology

[0002] Rapeseed, belonging to the genus Brassica in the family Cruclferae, provides my country with a large amount of high-quality edible oil and protein feed, making it one of my country's most important oilseed crops. In recent years, with the development of the economy and tourism industry, rapeseed has gradually become an emerging ornamental plant. However, problems in production, such as lodging, pests, and diseases, severely limit the yield and ornamental value of rapeseed. In particular, Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, seriously affects the quality and yield of rapeseed, causing a yield reduction of 10% to 20%, and as high as 70% in severe years. Therefore, fully utilizing genetic engineering technology to discover resistance gene resources and applying them to the screening of rapeseed resistant germplasm resources is of great significance for improving the resistance of rapeseed to Sclerotinia stem rot.

[0003] *Sclerotinia sclerotiorum*, belonging to the genus *Sclerotinia* in the order Myxomycetes of the phylum Ascomycota, has a wide ecological distribution, parasitizing over 400 plant species belonging to 75 families and 278 genera, particularly causing serious damage to the growth and development of legumes, solanaceae, and cruciferous plants. Studies have shown that one of the key steps by which *Sclerotinia sclerotiorum* overcomes host defenses is the degradation of the cell wall. The plant cell wall is a flexible, thin layer (0.1–1.0 μm) composed of numerous complex polysaccharides and a small amount of structural proteins. Its main components include cellulose, hemicellulose, pectins, lignins, and small amounts of cell wall proteins. These components provide cells with support, protection, strength, flexibility, plasticity, and environmental adaptation. One of its most important functions is providing support and protection for cell morphology, considered the plant's first line of defense, capable of resisting infection by most pathogens.

[0004] Pectin, a crucial component of plant primary cell walls, is a complex polysaccharide composed of glucose and galactose units that link together to form linear or branched structures. It plays a vital role in increasing cell strength and maintaining cell shape within plant cell walls, while also possessing adhesive properties. Widely distributed in the primary cell walls of terrestrial plants, it plays a significant regulatory role in the synthesis and deposition of secondary cell walls. During cell growth, pectin is essential for cell wall remodeling and normal intercellular adhesion. It also plays a crucial role in plant structural resistance, pathogen defense, and plant development (morphogenesis and organogenesis), making it an important factor in plant immunity. Furthermore, pectin metabolism involves various enzymes, including pectin synthases and lyases, and studies have shown that it may be involved in plant-pathogenic microorganism interactions. Pectin is composed of over 60% poly(homocaluronic acid), a complex polymerized from galacturonic acid, which is an essential precursor for pectin synthesis. UDP-D-glucuronate 4-epimerase (GAE, EC: 5.1.3.6) is a key enzyme catalyzing the conversion of UDP-glucuronic acid (UDP-α-D-glucuronic acid, UDP-GlcA) to UDP-galacturonic acid (UDP-GalA), belonging to the short-chain dehydrogenase / reductase family (SDR). Therefore, plants contain a large amount of glucuronate isomerase.

[0005] Studies have shown that the Arabidopsis AtGAEs family contains six members, belonging to the SDR type II membrane proteins. All members of this family contain NAD(P)-related proteins. + Combined with the relevant conserved motif GxxGxxG. RT-PCR confirmed that all members of the AtGAE family are expressed in Arabidopsis pollen, suggesting that AtGAEs may provide essential precursors for pectin synthesis. Transforming tobacco with the antisense gene expression vector of Arabidopsis AtUGAE4 resulted in restricted tobacco growth and elongated leaves, indicating that the antisense gene interfered with the normal expression of GAE family genes, reducing pectin content. Pang et al. demonstrated that exogenous addition of UDP-galacturonic acid significantly promoted fiber elongation; GhGAE1 and GhGAE3 were highly expressed during the elongation phase of cotton fibers, potentially leading to increased pectin content and ultimately rapid fiber elongation. Bethke et al. found that the gae1 gae6 double mutant exhibited reduced cell wall pectin content, increased leaf brittleness, and decreased resistance to *Pseudomonas syringae*. The GhGAE08 gene in *Gossypium barbadense* affects secondary cell wall thickening and cotton fiber elongation. Studies by Ahmed et al. have found that downregulation of the NbGAE6 gene expression in tobacco affects the formation of "GalA" during pectin synthesis.

[0006] In summary, although cell wall-mediated broad-spectrum plant resistance has attracted widespread attention, the specific mechanisms are still not thoroughly studied. GAE family genes participate in plant growth, development, and abiotic stress by mediating pectin synthesis, but their functions in biotic stress are rarely reported, particularly the role of the BnGAE1 gene in rapeseed sclerotinia stem rot resistance, for which there are currently no relevant literature or patent reports. Therefore, exploring the molecular mechanisms of BnGAEs-mediated sclerotinia stem rot resistance through gene editing technology can provide a new theoretical basis and germplasm resources for genetic improvement and breeding of disease-resistant varieties, and has important reference value for the safe production of rapeseed. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the BnGAE1 gene in regulating resistance to Sclerotinia stem rot in rapeseed, thereby solving the problems existing in the prior art. This invention provides a gene for regulating pectin synthesis-related glucuronide isomerase BnGAE1, which regulates resistance to Sclerotinia stem rot in rapeseed. A knockout vector for this gene is constructed, and its knockout expression is achieved in rapeseed 'J9712' using Agrobacterium-mediated genetic transformation. This invention utilizes the BnGAE1 knockout gene to improve resistance to Sclerotinia stem rot in rapeseed, and employs transgenic genetic modification to enhance rapeseed's resistance to Sclerotinia stem rot, thus providing new genetic resources for breeding rapeseed resistant to Sclerotinia stem rot.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] In a first aspect, the present invention provides the application of knocking out the BnGAE1 gene in improving the resistance of Brassica napus to Sclerotinia stem rot, wherein the nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.1.

[0010] Secondly, the present invention also provides the application of the protein encoded by the BnGAE1 gene in improving the resistance of Brassica napus to Sclerotinia stem rot, the amino acid sequence of which is shown in SEQ ID NO.2.

[0011] Thirdly, the present invention also provides the application of the recombinant expression vector containing the BnGAE1 gene in improving the resistance of Brassica napus to Sclerotinia stem rot.

[0012] Preferably, the recombinant expression vector is transformed into microbial culture for expression, and then transformed into the target plant to obtain the BnGAE1 gene knockout mutant; the transformation method is Agrobacterium-mediated hypocotyl infection; the nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.1.

[0013] Fourthly, the present invention also provides the application of host bacteria containing the recombinant expression vector in improving resistance to sclerotinia stem rot in rapeseed.

[0014] Fifthly, the present invention also provides the application of the BnGAE1 gene in improving the resistance of Brassica napus to sclerotinia stem rot, wherein the nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.1.

[0015] In a sixth aspect, the present invention also provides the application of the BnGAE1 gene in molecular breeding for resistance to sclerotinia stem rot in Brassica napus, wherein the nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.1.

[0016] In a seventh aspect, the present invention also provides a method for improving the resistance of Brassica napus to Sclerotinia stem rot, the method comprising transferring a gene-directed editing recombinant expression vector into Brassica napus to obtain a BnGAE1 gene knockout mutant, the nucleotide sequence of the BnGAE1 gene being shown in SEQ ID NO.1.

[0017] The more detailed technical solution is as follows:

[0018] This invention uses pCBC-DT1DT2 (vector resistance is kanamycin) as the intermediate gene editing vector. Figure 2 pKSE401 (vector resistance is kanamycin) is the final gene editing vector. Figure 2 The applicant constructed a CRISPR / Cas9-BnGAE1 gene knockout vector for this gene. The applicant then transformed the vector into *Brassica napus* 'J9712' using Agrobacterium-mediated hypocotyl infection until regenerated differentiated seedlings were obtained. Figure 3Genomic DNA was extracted from leaves of wild-type Brassica napus (control) and transgenic rapeseed lines using the CTAB method. PCR amplification and identification were performed using primers U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3' (SEQ ID NO. 9) and U629-IDR: 5'-AGCCCTCTTCTTTCGATCCATCAAC-3' (SEQ ID NO. 10) to screen for CRISPR / Cas9 knockout lines (KO). Positive lines were then sequenced. Finally, positive lines KO-1, KO-2, KO-3, KO-4, KO-5, KO-6, and KO-7 were obtained. Figure 4 The above-mentioned transgenic materials were self-pollinated to obtain higher generations, which were then used as materials for subsequent experiments.

[0019] The wild-type control WT-'J9712' and the knockout line BnGAE1(KO) of Brassica napus were identified by inoculation with mycelial blocks from detached leaves at the seedling stage. Disease incidence was generally most pronounced after inoculation. The sclerotinia disease index was calculated 48 hours (48 hpi) after inoculation, following the method described by Wu J, Zhao Q, Yang Q, et al. Comparative transcriptomic analysis uncovers the complex genetic network for resistance to Sclerotinia sclerotiorumin Brassica napus[J]. Scientific Reports, 2016, 6:19007. The disease index results showed that BnGAE1(KO) plants exhibited significantly higher resistance than WT, with a significantly smaller lesion area. Figure 6 This indicates that the BnGAE1 gene negatively mediates rapeseed leaf resistance to sclerotinia stem rot. Leaf tissues from consistent locations around lesions in WT and BnGAE1(KO) lines were collected, RNA was extracted and reverse transcribed into cDNA, and expression patterns of ROS-related genes, jasmonic acid signaling pathway genes, and resistance-related genes were analyzed. The results showed that in the BnGAE1(KO) line, genes such as BnNPR1, BnPR2, and BnMRKY33 in the SA signaling pathway were strongly induced mainly in the early stage of inoculation (6 hpi), while genes such as BnEIN2, BnJAZ1, and BnPDF1.2 in the JA / ET signaling pathway were strongly induced mainly in the middle and late stages of Sclerotinia stem rot infection. ROS scavenging-related genes BnCAT1 and BnGPX7 were strongly induced in the later stages. Figure 7The results indicate that knocking out BnGAE1-mediated rapeseed disease resistance can activate the SA signaling pathway in the early stage of pathogen infection, but mainly relies on the JA / ET signaling pathway in the later stage. It can also clear excess ROS in the plant to achieve redox balance. These findings are of great significance for breeding disease-resistant rapeseed varieties.

[0020] The present invention discloses the following technical effects:

[0021] 1. Although multiple pectin synthase genes GAE have been cloned in species such as Arabidopsis thaliana and researchers have verified their functions, there are few reports on them in rapeseed so far. The BnGAE1 gene cloned in this invention enriches the research on this type of gene in rapeseed.

[0022] 2. The BnGAE1 gene cloned in this invention provides new genetic resources for disease resistance breeding of other crops and has guiding and reference value for improving the resistance of other crops to sclerotinia stem rot.

[0023] 3. The gene knockout vector CRISPR / Cas9-BnGAE1 constructed in this invention, after being transformed into rapeseed, yielded BnGAE1-deficient expression lines. Studies on the functional acquisition of BnGAE1 revealed that BnGAE1 is a negative regulator of rapeseed resistance to Sclerotinia stem rot, and knocking out BnGAE1 enhances the plant's disease resistance. Through genetic transformation, deficient expression of this gene resulted in new rapeseed lines resistant to Sclerotinia stem rot. BnGAE1 can serve as a potential marker gene for rapeseed resistance.

[0024] 4. This invention, through a series of studies on the resistance of BnGAE1-Cas9 knockout lines to Sclerotinia sclerotinia, fully demonstrates that BnGAE1 participates in and plays an important role in the plant's resistance response to Sclerotinia sclerotinia. Regulating the deficient expression of the BnGAE1 gene can regulate the accumulation of resistance factors in plants, thereby enhancing plant resistance to Sclerotinia sclerotinia. This is of great significance for elucidating the biological function of the BnGAE1 gene.

[0025] 5. The discovery of BnGAE1, a rapeseed sclerotinia disease resistance gene, has important theoretical guiding significance for the discovery and identification of crop sclerotinia disease resistance genes and for in-depth exploration of the molecular mechanism of action of sclerotinia disease resistance genes.

[0026] 6. The selection and breeding of rapeseed materials resistant to Sclerotinia stem rot has always been a focus for rapeseed breeders. The development and utilization of BnGAE1 will help solve the problem of Sclerotinia stem rot resistance in rapeseed production. Since Sclerotinia stem rot currently poses a significant threat to rapeseed yield and quality, the selection and breeding of disease-resistant varieties is particularly important. Knockout of the BnGAE1 gene will help cultivate highly resistant rapeseed varieties and provides a theoretical and practical basis for the application of BnGAE1 in Sclerotinia stem rot resistance in major oilseed crops (rapeseed). It also provides new breeding materials for further breeding rapeseed varieties with Sclerotinia stem rot resistance. Furthermore, it has significant practical guiding value in crop breeding practices, variety improvement, and variety promotion related to Sclerotinia stem rot resistance. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1 The amino acid sequence alignment results of the rapeseed BnGAE1 gene and the known Arabidopsis thaliana AtGAE1 gene are shown; where Bn (Brassica napus L.) is rapeseed and At (Arabidopsis thaliana) is Arabidopsis thaliana.

[0029] Figure 2 The vector maps used to construct CRISPR / Cas9 gene knockout vectors, where A is the pCBC-DT1T2 vector map and B is the pKSE401 vector map;

[0030] Figure 3 This is a schematic diagram of the Agrobacterium-mediated genetic transformation system of Brassica napus hypocotyls. A shows the germination of Brassica napus 'J9712' seeds sterilized with 0.1% mercuric chloride on MS medium; B shows the etiolated hypocotyls that have grown after approximately 5 days of dark incubation at 24°C; C shows the etiolated hypocotyls cut into approximately 0.8 cm segments under aseptic conditions and placed in Agrobacterium GV3101 containing the transformation vector for infection; D shows the explants from infected hypocotyls transferred to M1 medium and dark-cultured for 48 hours before being placed in M2 medium for 1... Selective culture was conducted for 15-20 days under a 6-hour light / 8-hour dark cycle; E represents the selected explants placed in M3 medium and cultured for differentiation for more than 15 days under a 16-hour light / 8-hour dark cycle; F represents the budding status of explants on M3 medium; G represents the new shoots from explants placed in fresh M3 medium and cultured for differentiation for more than 15 days under a 16-hour light / 8-hour dark cycle until they differentiate into seedlings; H represents the seedlings rooting on M4 medium; I represents the seedlings being transplanted into soil and ready for harvesting T1 generation seeds.

[0031] Figure 4Identification of positive seedlings of rapeseed BnGAE1-Cas9 transgenic knockout line; M: DL 1500 DNAmaker; 1-7 are KO-1, KO-2, KO-3, KO-4, KO-5, KO-6 and KO-7 transgenic lines respectively;

[0032] Figure 5 Gene editing status of the BnGAE1-Cas9 transgenic knockout line;

[0033] Figure 6 To identify the resistance of BnGAE1-Cas9 transgenic knockout lines to sclerotinia rot; where A is the disease phenotype of WT and BnGAE1(KO) seedling leaves 48 h after in vitro inoculation with the fungus; B is the size of lesions on WT and BnGAE1(KO) leaves 48 h after infection with Sclerotinia rotundifolia.

[0034] Figure 7 The expression levels of disease resistance-related genes were analyzed in the leaves of wild-type 'J9712' and transgenic BnGAE1(KO) seedlings after in vitro inoculation with Sclerotinia sclerotiorum at 0h, 6h, 12h, 24h, 36h, 48h and 60h. Among them, A represents the expression level of key ROS-related genes; B represents the expression level of key genes related to the jasmonic acid signaling pathway and disease resistance. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] This invention specifically relates to the BnGAE1 gene, a monosaccharide donor—glucuronide isomerase—required for regulating pectin synthesis in rapeseed to resist Sclerotinia stem rot, and its application. A gene, BnGAE1 (BnaC07g42640D), mediating cell wall resistance and thus resisting Sclerotinia stem rot in rapeseed, was screened. This gene showed 97.44% amino acid sequence homology and 86.16% nucleic acid sequence homology with the AtGAE1 (AT4G30440) gene in the model plant Arabidopsis thaliana. Using Agrobacterium-mediated transformation, BnGAE1-CRISPR / Cas9 knockout mutant lines were obtained and inoculated with Sclerotinia stem rot to rapidly identify its function in rapeseed resistance to Sclerotinia stem rot.

[0037] SEQ ID NO.1 is the nucleotide sequence of the rapeseed BnGAE1 gene cloned in this invention, and SEQ ID NO.2 is the protein sequence encoded by the BnGAE1 gene;

[0038] SEQ ID NO.1:

[0039]

[0040] SEQ ID NO.2:

[0041] MPSIEDELMFPSTPGKFKIDRSNRQLNRCFASTSTMFLWALFLIALTASYLSFQSFVDSGSRYLTASWGGIQWEKQVRTSAQIHRSGGISVLVTGATGFVGSHVSLA LRKRGDGVVGLDNFNNYYDPSLKRARMSLLSSRGIFVEGDLNDGKLLSKLFDVVAFTHVMHLAAQAGVRYALENPQSYVHSNIAGLVNLLETCKAANPQPAVVWASS SSVYGLNEKVPFSESDRTDQPASLYAATKKAGEEITHTYNHIYGLAITGLRFFTVYGPWGRPDMAYFSFTRNILQGKPITIYRGKNRVDLARDFTFIDDIVKGCLGSL DSSGKSTGSGGKKRGSAPYRIFNLGNTSPVTVPILVDILEKHLKVKAKRNFVEMPGNGDVPFTHANISSARREFGYKPTTDLETGLKKFVRWYLSYYGYNTKAKLVQ;

[0042] The following embodiments define the present invention and describe how the present invention obtained BnGAE1-Cas9 transgenic knockout rapeseed plants and the method for verifying the function of the BnGAE1 gene. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the present invention, and various changes and modifications can be made to the present invention to suit different uses and conditions without departing from the spirit and scope thereof.

[0043] Example 1: Obtaining BnGAE1-Cas9 knockout transgenic rapeseed plants

[0044] (1) Construction of CRISPR / Cas9 gene knockout vector

[0045] Based on the BnGAE1 gene sequence of Brassica napus obtained from the BnPIR database, and using analysis software from the CRISPR-Plant website (http: / / www.genome.arizona.edu / crispr / index.html) combined with protein sequence function prediction results, two candidate gene sgRNAs with the highest scores and strongest specificity were designed: gae1-gRNA1: 5'-TCACTCGCGCTTAGAAAACGCGG-3' (SEQ ID NO.3) and gae1-gRNA2: 5'-GCACCTCGCGGCTCAGGCCGGGG-3' (SEQ ID NO.4). These were ligated to the PKSE401 vector with a 35S Cas9 promoter via BsaI digestion and T4 ligase. Figure 2 (B). Amplification primers were designed and synthesized based on the gRNA sequence: BnGAE1-DT1-BsF: 5'-ATATATGGTCTCGATTGCACTCGCGCTTAGAAAACGGTT-3' (SEQ ID NO.5), BnGAE1-DT1-F0: 5'-TGCACTCGCGCTTAGAAAACGTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.6), BnGAE1-DT2-R0: 5'-AACCGGCCTGAGCCGCGAGGTGCAATCTCTTAGTCG ACTCTAC-3' (SEQ ID NO.7), BnGAE1-DT2-BsR: 5'-ATTATTGGTCTCGAAACCGGCCT GAGCCGCGAGGTGCAA-3' (SEQ ID NO.8). The intermediate vector pCBC-DT1T2 was used as a template. Figure 2 (A) Amplification was performed using four primers, and the product was preserved by enzyme digestion and ligation. The PCR product from the previous step was then ligated with the PKSE401 vector using BsaI restriction enzyme and T4 ligase at 37℃ for 12 hours. The product was then heat-shocked and transformed into *E. coli* DH5α. Single colonies were selected for positive detection and sequencing. Positive strains and plasmids were preserved, and the positive plasmid was transformed into *Agrobacterium* competent cells GV3101. Single colonies were picked and cultured in LB broth with appropriate antibiotic resistance at 28℃ with shaking for 36-48 hours. After PCR detection, positive strains were stored at -80℃ with an appropriate amount of glycerol for later use.

[0046] (2) Genetic transformation of rapeseed

[0047] The recombinant plasmid (i.e., the knockout vector) was screened and differentiated into seedlings using Agrobacterium-mediated hypocotyl infection. The specific steps are as follows: Figure 3 ):

[0048] a. Sowing: Wash the seeds of Brassica napus 'J9712' with 75% alcohol for 1 min, then wash them with 0.1% mercuric chloride solution for 5 min, and finally wash them 5 times with sterile water. Place the seeds into MO solid medium using sterilized tweezers and incubate the inoculated seeds in the dark at 24℃ for 5 days. The MO solid medium is prepared as follows: MS 4.404 g / L (1962 formula), adjust the pH to 5.8-5.9, add 7 g / L agar, and sterilize using conventional autoclaving.

[0049] b. Activation of Agrobacterium: Three days after inoculation, Agrobacterium strain GV3101 stored at -80℃ was streaked onto LB solid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL), and incubated at 28℃ for 16 h. Single colonies were picked and placed in 5 mL of LB liquid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL), and cultured at 28℃ with shaking at 200 rpm for 20-24 h to allow Agrobacterium to grow to the logarithmic phase; 500 μL of the cultured bacterial solution was then transferred to 50 mL of LB liquid medium (containing the three antibiotics) for expansion culture for about 12 h.

[0050] c. Preparation of Infected Bacterial Fluid: Pour the activated Agrobacterium tumefaciens solution into two 50 mL sterile centrifuge tubes, centrifuge at 3500 rpm for 15 min, discard the supernatant, place on ice, wash the bacterial solution with 1 mL of DM liquid medium, centrifuge, discard the supernatant, add 2-3 mL of DM liquid medium to resuspend the bacterial cells, and adjust the OD of the infected bacterial solution to 0.5%. 600 The value is approximately 0.6. The preparation method of the DM liquid culture medium is as follows: MS 4.404 g / L and sucrose 30 g / L according to the 1962 formula, adjust the pH of the culture medium to 5.8-5.9, sterilize at 121℃ for 20 min, and add 2,4-D 1 mg / L, AS 100 mmol / L and KT 0.3 mg / L to the aseptic operating table after sterilization.

[0051] d. Infecting explants: Place the infecting bacterial solution from step c on ice. At the same time, use a scalpel to cut the hypocotyls of the dark-cultured Brassica napus seedlings from step a (each hypocotyl segment should be 0.8-1.0 cm in length). Use sterile forceps to transfer the cut hypocotyls into a Petri dish containing the infecting bacterial solution. Infect for 15-30 minutes depending on the concentration of the bacterial solution (shake once every 3 minutes).

[0052] e. Co-culture: Transfer the infected explants to petri dishes lined with filter paper (which should be sterilized beforehand), blot dry any visible infection solution on the surface of the explants, and then transfer them to M1 solid medium. Incubate in the dark at 24°C for 40-48 hours. The M1 solid medium is prepared as follows: MS 4.404 g / L, mannitol 18 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, KT 0.3 mg / L (1962 formula), adjust the pH of the medium to 5.8-5.9, add 7 g / L agar, sterilize, and then add 100 mmol / L LAS.

[0053] f. Screening: The co-cultured explants were transferred to M2 solid medium and screened for 20 days under the following conditions: 24℃, 16h light / 8h dark. The M2 solid medium was prepared as follows: MS 4.404g / L (1962 formula), mannitol 18g / L, sucrose 30g / L, 2,4-D 2mg / L, KT 0.3mg / L; the pH of the medium was adjusted to 5.8-5.9, and 7g / L agar was added.

[0054] g. Differentiation Culture: The screened explants were transferred to M3 solid medium to begin differentiation culture. Subculture was performed every 15-20 days until shoots differentiated (culture conditions were the same as the screening conditions in step f). The M3 solid medium was prepared as follows: MS 4.404 g / L (1962 formula), glucose 10 g / L, xylose 0.25 g / L, yeast extract (MES) 0.6 g / L, pH adjusted to 5.8-5.9, agar 7 g / L added, sterilized, and then zeatin (ZT) 2 mg / L, IAA 0.1 mg / L, TMT 250 mg / L, and Kan 25 mg / L were added.

[0055] h. Rooting Culture: Once a clear growth point can be observed on the differentiated seedling, carefully cut the seedling along the junction of the callus and the bud using a scalpel (be extremely careful not to damage the growth point during this process). Then, transfer the seedling to M4 solid medium for rooting. The M4 solid medium is prepared as follows: MS 2.202g (1962 formula), sucrose 10g / L, IBA 0.5mg / L, adjust the pH of the medium to 5.8-5.9, add 7g / L agar, sterilize, and then add TMT 250mg / L and Kan 25mg / L.

[0056] i. Transplanting: When the plant has developed a certain number of roots and a relatively well-developed root system in the rooting medium, the regenerated plant is transferred to soil until the T1 generation seeds are harvested. Before transplanting, it is usually necessary to open the sealing film of the culture bottle to allow the plant to gradually adapt to the external environment.

[0057] (3) Identification of transgenic plants

[0058] a. Extraction of genomic DNA from rapeseed leaves

[0059] Fresh leaves of T0 generation transgenic rapeseed were used for DNA extraction using the conventional CTAB method. The specific steps were as follows: Young rapeseed leaves (1-2 cm in length) were ground into a fine paste in a clean mortar and transferred to a 1.5 mL centrifuge tube. 700 μL of 2×CTAB solution was added. The mixture was incubated at 70℃ for 30 min, gently shaken once every 10 min. After cooling to room temperature, 700 μL of a Tris-saturated phenol:chloroform:isoamyl alcohol mixture (25:24:1 v / v) was added, and the mixture was repeatedly inverted and gently shaken approximately 40 times. The mixture was centrifuged at 3100 rpm for 15 min at room temperature. Approximately 500 μL of the supernatant was collected and 500 μL of a chloroform-isoamyl alcohol mixture (24:1 v / v). After mixing, the mixture was centrifuged at 3100 rpm for 15 min at room temperature. Discard the supernatant, add 1 mL of frozen -20℃ anhydrous ethanol, incubate at -20℃ for 30 min, then centrifuge at 12000 rpm for 10 min at room temperature. Wash with 75% ethanol and repeatedly pipette the precipitate for 3 min to remove salt. Discard the ethanol, air dry, and dissolve each sample in 30-50 μL of ddH2O. Analyze the extracted genomic DNA from Brassica napus using a Nanodrop micro-nucleic acid analyzer.

[0060] b. Detection of positive transgenic plants

[0061] The genomic DNA obtained from the above-mentioned rapeseed leaves was detected using the following primers:

[0062] U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3' (SEQ ID NO.9);

[0063] U629-IDR: 5'-AGCCCTCTTTCTTTCGATCCATCAAC-3' (SEQ ID NO. 10);

[0064] The control group consisted of non-GMO wild-type plants (J9712).

[0065] The test results show that ( Figure 4 The seven transgenic plants, KO-1, KO-2, KO-3, KO-4, KO-5, KO-6, and KO-7, were all able to amplify the expected size electrophoretic band (572 bp), while the wild-type control did not have an electrophoretic band. This indicates that the transgenic rapeseed genome contains DNA fragments of exogenous knockout genes. However, the bands were not uniform in brightness, which may indicate that the expression level of the knockout gene differs among different positive seedlings, and the knockout efficiency may be different.

[0066] Example 2: Validation of the BnGAE1-Cas9 gene knockout mutant

[0067] Primers were designed before and after the BnGAE1 gene target site, with the primer sequences as follows:

[0068] Bngae1-F: 5'-CTCTTCCTCATCGCCCTCA-3' (SEQ ID NO. 11);

[0069] Bngae1-R: 5'-TCCGATTCCGAGAAAGGC-3' (SEQ ID NO. 12);

[0070] Using genomic DNA from BnGAE1-Cas9 knockout mutant rapeseed plants as a template, PCR amplification was performed. The PCR products were sequenced and compared to analyze the gene editing type (large deletion lines with obvious phenotypes were selected for high-generation propagation). The editing status was analyzed using the DSDecode website (http: / / www.ygliulab.club / dsdecode / ). The results are as follows: Figure 5 As shown, in the open reading frame coding region of BnGAE1, large fragment deletions of 679bp, 180bp, 180bp, and 328bp appeared in the double strands of KO-1, KO-2, KO-4, and KO-5, respectively, while no editing occurred in other strains.

[0071] Example 3: Evaluation of resistance to Sclerotinia stem rot in BnGAE1-Cas9 knockout transgenic rapeseed

[0072] (1) Immunological response of BnGAE1 knockout strains to Sclerotinia sclerotiorum.

[0073] The second-to-last leaves of rapeseed seedlings (at the four-leaf stage) were inoculated with *Sclerotinia sclerotiorum*, the pathogen of sclerotinia rot (preserved and activated in the laboratory of the College of Life Sciences, Huazhong Agricultural University). The second-to-last leaves of wild-type plants and positive lines from Example 1 were taken, cut, and placed in transparent square boxes. Leaves from each transgenic plant were placed side-by-side with non-transgenic controls in one box. Several layers of filter paper and damp gauze were placed under the leaves. Freshly prepared... Mycelial blocks, with the mycelial side down, were inoculated into the middle of the leaf, slightly off-center from the midrib, with two blocks inoculated per leaf. The leaves were covered to maintain humidity and then incubated in the dark at 22°C. Leaf observation was conducted continuously from 0 h to 72 h after inoculation. It was found that the growth area of ​​*Sclerotinia sclerotiorum* lesions in the BnGAE1-Cas9 transgenic line was significantly smaller than that in the wild type, with the most significant difference in resistance observed 48 h after inoculation. Figure 6 Compared to the wild type, transgenic rapeseed (Type A) showed a 25.46% reduction in the area of ​​sclerotinia lesions on leaves. Figure 6(B). Therefore, knocking out the BnGAE1 gene can effectively enhance the resistance of rapeseed leaves to sclerotinia stem rot.

[0074] (2) RNA extraction from rapeseed leaves after inoculation

[0075] use The Super Total RNA Extraction Kit (Promega, USA) was used to extract total RNA from leaves treated with *Sclerotinia sclerotiorum* for 48 hours in step (1). The RNA was then extracted using... The IIQ RT SuperMix for qRNA (+gDNAwiper) kit (Vazyme, China) reverse transcribes RNA into cDNA.

[0076] (3) qPCR detection of defense-related gene expression

[0077] qPCR use The Green Realtime PCR Master Mix-Plus kit contains the following primers for disease resistance-related genes:

[0078] The qPCR primers for the BnRbohD gene are:

[0079] Q-BnRbohD-F: 5'-TTAGGAACAAGACCAAGCTAGG-3' (SEQ ID NO. 13);

[0080] Q-BnRbohD-R: 5'-CACAACCATCACAATACCAGTC-3' (SEQ ID NO. 14);

[0081] The qPCR primers for the BnCAT1 gene are:

[0082] Q-BnCAT1-F: 5'-TACAGACACGAAACAGCA-3' (SEQ ID NO. 15);

[0083] Q-BnCAT1-R: 5'-GACAGAAACTAGCAAGCC-3' (SEQ ID NO. 16);

[0084] The qPCR primers for the BnGPX7 gene are:

[0085] Q-BnGPX7-F: 5'-ACATAGTATCGACCACGTGTTT-3' (SEQ ID NO. 17);

[0086] Q-BnGPX7-R: 5'-TGTCCGTATGTAGAACACAAGT-3' (SEQ ID NO. 18);

[0087] The qPCR primers for the BnEIN2 gene are:

[0088] Q-BnEIN2-F: 5'-GTTGGTGAGGAAGTCAGGAATA-3' (SEQ ID NO. 19);

[0089] Q-BnEIN2-R: 5'-CTTTTCTCTTGCAGCTACCTTG-3' (SEQ ID NO. 20);

[0090] The qPCR primers for the BnJAZ1 gene are:

[0091] Q-BnJAZ1-F: 5'-TACGGCGGGCAAGTGATT-3' (SEQ ID NO. 21);

[0092] Q-BnJAZ1-R: 5'-TTGGTTCGGGGTAGGAGC-3' (SEQ ID NO. 22);

[0093] The qPCR primers for the BnPDF1.2 gene are:

[0094] Q-BnPDF1.2-F: 5'-CATCACCCTTCTCTTCGCTGC-3' (SEQ ID NO23);

[0095] Q-BnPDF1.2-R: 5'-ATGTCCCACTTGACCTCTCGC-3' (SEQ ID NO.24);

[0096] The qPCR primers for the BnNPR1 gene are:

[0097] Q-BnNPR1-F: 5'-ATCTCCGAAAAGCCAGGGA-3' (SEQ ID NO. 25);

[0098] Q-BnNPR1-R: 5'-TACCATTTACCGCATCGCCC-3' (SEQ ID NO. 26);

[0099] The qPCR primers for the BnPR2 gene are:

[0100] Q-BnPR2-F: 5'-ATCTCCGAAAAGGCCAGGGA-3' (SEQ ID NO. 27);

[0101] Q-BnPR2-R: 5'-TACCATTTACCGCATCGCCC-3' (SEQ ID NO. 28);

[0102] The qPCR primers for the BnWRKY33 gene are:

[0103] Q-BnWRKY33-F: 5'-GTACTTTCCCAAACTGTCCAAC-3' (SEQ ID NO. 29);

[0104] Q-BnWRKY33-R: 5'-CGAAGAAGACGACGATCTTCTA-3' (SEQ ID NO. 30);

[0105] The qPCR primers for the BnActin7 gene are:

[0106] Q-BnActin7-F: 5'-TCTTCCTCACGCTATCCCTCG-3' (SEQ ID NO. 31);

[0107] Q-BnActin7-R: 5'-AGCCGTTCCAGCTCTTGC-3' (SEQ ID NO. 32);

[0108] Primers specific to the coding region of this gene were designed using Primer Premier 5.0 software, and primer specificity was verified by Primer-BLAST (NCBI) in the Brassica Gene Database (BRAD) and the Rapeseed EST database.

[0109] The qPCR reaction system consisted of 15 μL containing: 7 μL SYBR Mix, 0.5 μL each of forward and reverse primers (10 μmol / L), 2 μL template, and 5 μL DEPC-treated sterile water. Amplification conditions were: 95℃ for 30 s; then 95℃ for 5 s, 60℃ for 30 s, 72℃ for 27 s, for 40 cycles; fluorescence detection was performed at the annealed ends at 72℃ after each cycle.

[0110] After the reaction, the temperature was first heated to 95℃, then lowered to 72℃, and then slowly increased to 95℃. Changes in fluorescence signal were recorded to obtain the melting curve of the amplified product. Three biological replicates were performed for each experiment, and at least three technical replicates were performed for each biological replicate. Primer pairs Q-BnRbohD-F / R, Q-BnCAT1-F / R, Q-BnGPX7-F / R, Q-BnEIN2-F / R, Q-BnJAZ1-F / R, Q-BnPDF1.2-F / R, Q-BnNPR1-F / R, Q-BnPR2-F / R, and Q-BnWRKY33-F / R were used to target BnRbohD (BnaC02g38300D) and BnCAT1 (BnaA07g11370D), respectively. The genes BnGPX7 (BnaA03g51760D), BnEIN2 (BnaA10g26670D), BnJAZ1 (BnaA06g13250D), BnPDF1.2 (BnaC02g23620D), BnNPR1 (BnaA01g15290D), BnPR2 (BnaC04g24330D), and BnWRKY33 (BnaC04g06800D) were specifically amplified by PCR. Simultaneously, the BnActin7 (BnaC09g46850D) gene from rapeseed was specifically amplified using primer pair Q-BnActin7-F / R as an internal control for relative quantification. The expression changes of these disease resistance-related genes in rapeseed leaves were detected after 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 60 h of Sclerotinia sclerotiorum induction.

[0111] The results showed that the expression of BnRbohD, a gene related to superoxide anion synthesis, was significantly lower in the BnGAE1 knockout lines than in the wild type, but overall showed an increasing trend. Meanwhile, the expression of ROS scavenging-related genes BnCAT1 and BnGPX7 was strongly induced in the later stages. Figure 7 In the JA / ET signaling pathway, genes such as BnEIN2, BnJAZ1, and BnPDF1.2 are strongly induced mainly in the mid-to-late stages of inoculation, while genes such as BnNPR1, BnPR2, and BnMRKY33 in the SA signaling pathway are intensely induced mainly in the early stage of Sclerotinia sclerotiorum infection (6 hpi) in BnGAE1 knockout plants. Figure 7 (B). The above results indicate that BnGAE1-mediated resistance in rapeseed can activate the SA signaling pathway in the early stage of pathogen infection, but mainly relies on the JA / ET signaling pathway in the later stage. It can also clear excess ROS in the plant to achieve redox balance.

[0112] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of BnGAE1 gene knockout in improving resistance to Sclerotinia stem rot in Brassica napus, characterized in that, The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

2. The application of inhibiting the function of the protein encoded by the BnGAE1 gene as described in claim 1 in improving resistance to Sclerotinia stem rot in Brassica napus, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Application of the BnGAE1 gene knockout vector in improving resistance to Sclerotinia stem rot in Brassica napus, characterized by: The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, The BnGAE1 gene knockout vector was transformed into microbial culture for expression, and then transformed into target plants to obtain the BnGAE1 gene knockout mutant; the transformation method was Agrobacterium-mediated hypocotyl infection; the nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

5. The application of the host bacteria containing the BnGAE1 gene knockout vector as described in claim 3 in improving the resistance of Brassica napus to Sclerotinia stem rot.

6. The application of BnGAE1 gene knockout in improving resistance to Sclerotinia stem rot in Brassica napus, characterized by: The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

7. The application of BnGAE1 gene knockout in molecular breeding for resistance to Sclerotinia stem rot in Brassica napus, characterized by: The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

8. A method for improving resistance to sclerotinia stem rot in rapeseed, characterized in that, The method includes transferring a gene-directed editing knockout vector into rapeseed to obtain a BnGAE1 gene knockout mutant, the nucleotide sequence of which is shown in SEQ ID NO.1.

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