Application of BnGAE1 gene in regulation and control of sclerotinia rot resistance of brassica napus

Abstract

The invention discloses application of a BnGAE1 gene in regulation and control of sclerotinia rot resistance of brassica napus, and belongs to the technical field of plant genetic engineering. The gene BnGAE1 capable of enhancing the sclerotinia rot resistance of the brassica napus is separated and cloned from the brassica napus, and the nucleotide sequence of the gene is as shown in SEQ ID NO. 1. Experimental verification shows that the resistance of the BnGAE1 (KO) strain to sclerotinia sclerotiorum is remarkably enhanced, and the incidence rate is reduced by 25.46% after inoculation; physiological and molecular detection shows that the expression quantity of defense related genes (such as PR1 and PDF1.2) in a mutant strain is up-regulated, SA (salicylic acid) and JA / ET (jasmonic acid / ethylene) signal channels are activated, and meanwhile, excessive ROS (reactive oxygen species) is effectively removed, so that the redox balance is maintained. The invention provides a new gene target and a high-efficiency molecular breeding technical path for improvement of sclerotinia rot resistance of colza, and has an important application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to application of a BnGAE1 gene in regulating resistance to sclerotinia sclerotiorum of Brassica napus. Background Art

[0002] Rapeseed, a Brassica plant in the Cruciferae family, provides 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 industries, rapeseed has gradually developed into an emerging ornamental plant. However, production problems such as lodging, insect pests, and diseases have severely limited its yield and ornamental value. In particular, Sclerotinia stem rot, caused by the fungus Sclerotinia sclerotiorum, severely impacts rapeseed quality and yield, resulting in a 10% to 20% yield reduction, and up to 70% in severely affected years. Therefore, fully utilizing genetic engineering technologies to identify resistance genes and apply them to the screening of rapeseed germplasm for resistance is of great significance for improving rapeseed's resistance to Sclerotinia stem rot.

[0003] Sclerotinia sclerotiorum belongs to the genus Sclerotinia of the order Mollicutes in the phylum Ascomycota. It has a wide ecological distribution and can parasitize over 400 plant species across 75 families and 278 genera. It particularly harms the growth and development of plants in the legume, solanaceae, and cruciferous families. Studies have shown that one of the key steps for Sclerotinia sclerotiorum to overcome host defenses is cell wall degradation. The plant cell wall is a thin, flexible layer (0.1–1.0 μm) composed of numerous complex polysaccharides and a small amount of structural proteins. Its main components include cellulose, hemicelluloses, pectins, lignins, and a small amount of wall proteins. These components provide support, protection, strength, flexibility, plasticity, and environmental adaptation to the cell. One of the most important functions is to support and protect cell morphology. This is considered the plant's first line of defense, protecting it from most pathogens.

[0004] Pectin, as an important component of plant primary cell wall, is a complex polysaccharide composed of glucose and galactose units, which are connected to form linear or branched structures, playing a role in increasing cell strength and maintaining cell shape in plant cell wall, and also having adhesion, widely existing in the primary cell wall of terrestrial plants, and having an important regulatory role in the synthesis and deposition of secondary wall. In the process of cell growth, pectin is essential for remodeling cell wall and normal cell adhesion, and plays an important role in plant structural resistance, resistance to pathogens and plant development (morphogenesis and organogenesis), and is an important factor of plant immunity. Moreover, the metabolism of pectin involves a variety of enzymes, including pectin synthesis enzymes and lytic enzymes, and studies have shown that it may be involved in the interaction between plants and pathogenic microorganisms. More than 60% of pectin is polyhomogalacturonide, and homogalacturonan is a complex of galacturonan polymerization, and galacturonic acid is a necessary precursor for synthesizing pectin; and UDP-D-glucuronate 4-epimerase (GAE, EC: 5.1.3.6) is a key enzyme for catalyzing the conversion of UDP-α-D-glucuronic acid (UDP-GlcA) to UDP-α-D-galacturonic acid (UDP-GalA), belonging to the short-chain dehydrogenases / reductases family (SDR). Therefore, there are a large number of glucuronate isomerases in plants.

[0005] Studies have shown that the Arabidopsis AtGAEs family contains 6 members, which belong to the type II membrane protein of SDR. All members of the family contain the conserved motif GxxGxxG associated with NAD(P) + binding. et al. used RT-PCR to confirm that all members of the AtGAE family are expressed in Arabidopsis pollen, speculating that AtGAEs may provide precursors necessary for pectin synthesis. Transformation of tobacco with an antisense gene expression vector for Arabidopsis AtUGAE4 resulted in restricted growth and slender leaves, suggesting that the antisense gene interfered with the normal expression of GAE family genes and reduced 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 an increase in pectin content and ultimately rapid fiber elongation. Bethke et al. found that gae1 gae6 double mutants exhibited reduced cell wall pectin content, increased leaf brittleness, and decreased resistance to Pseudomonas syringae. The GhGAE08 gene in Gossypium barbadense affects secondary wall thickening and fiber elongation. Ahmed et al. found that downregulation of tobacco NbGAE6 gene expression would affect the formation of "GalA" during pectin synthesis.

[0006] In summary, while cell wall-mediated broad-spectrum plant resistance has attracted widespread attention, the specific mechanisms remain understudied. While GAE family genes participate in plant growth and development and abiotic stresses by mediating pectin synthesis, their roles in biotic stresses are rarely reported. In particular, the role of the BnGAE1 gene in Sclerotinia sclerotiorum resistance in rapeseed is not yet documented in the literature or patents. Therefore, exploring the molecular mechanisms of BnGAEs-mediated Sclerotinia sclerotiorum resistance through gene editing could provide a new theoretical basis and germplasm resources for genetic improvement and breeding of disease-resistant varieties, and would be of great significance for the safe production of rapeseed. Summary of the Invention

[0007] The purpose of the present invention is to provide an application of the BnGAE1 gene in regulating the resistance of Brassica napus to sclerotinia rot, so as to solve the problems existing in the above-mentioned prior art. A pectin synthesis-related glucuronide isomerase BnGAE1 gene for regulating the resistance of Brassica napus to sclerotinia rot is provided, a knockout vector of the gene is constructed, and the knockout expression is carried out in Brassica napus 'J9712' through an Agrobacterium-mediated genetic transformation method. A method for improving the resistance of Brassica napus to sclerotinia rot by using the BnGAE1 knockout gene is provided, and rapeseed is genetically improved by using transgenics to improve the resistance of rapeseed to sclerotinia rot fungi, thereby providing new genetic resources for breeding rapeseed resistant to sclerotinia rot.

[0008] To achieve the above object, the present invention provides the following solutions:

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

[0010] In a second aspect, the present invention further provides the use of a protein encoded by the BnGAE1 gene in improving resistance to sclerotinia sclerotiorum in Brassica napus. The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0011] In a third aspect, the present invention further provides the use of a recombinant expression vector containing the BnGAE1 gene in improving the resistance of Brassica napus to sclerotinia sclerotiorum.

[0012] Preferably, the recombinant expression vector is transformed into a microorganism for culture and expression, and then transformed into a target plant to obtain a 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] In a fourth aspect, the present invention further provides the use of a host bacteria containing the recombinant expression vector in improving the resistance of Brassica napus to sclerotinia sclerotiorum.

[0014] In a fifth aspect, the present invention further provides an application of the BnGAE1 gene in improving the resistance of Brassica napus to bacterial sclerotinia. The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.1.

[0015] In a sixth aspect, the present invention further provides an application of the BnGAE1 gene in molecular breeding of Brassica napus for resistance to Sclerotinia sclerotiorum. The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.1.

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

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

[0018] The present invention uses pCBC-DT1DT2 (vector resistance is kanamycin) as the gene editing intermediate vector ( Figure 2 A), pKSE401 (vector resistance is kanamycin) is the final gene editing vector ( Figure 2 B), a CRISPR / Cas9-BnGAE1 gene knockout vector was constructed, and the applicant transformed the vector into Brassica napus 'J9712' by infecting the hypocotyls with Agrobacterium until regenerated differentiated seedlings were obtained ( Figure 3). The genomic DNA of the leaves of wild-type Brassica napus as a control and transgenic rapeseed lines was extracted by CTAB method, and PCR amplification was performed using primers U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3' (SEQ ID NO.9) and U629-IDR: 5'-AGCCCTCTTCTTTCGATCCATCAAC-3' (SEQ ID NO.10). The CRISPR / Cas9 knockout lines (Knockout, KO) of the gene were screened, and the positive lines were then sequenced and analyzed. Finally, a total of KO-1, KO-2, KO-3, KO-4, KO-5, KO-6 and KO-7 positive lines were obtained ( Figure 4 The above transgenic materials were self-fertilized to obtain high-generation seeds as subsequent experimental materials.

[0019] The wild-type control WT ('J9712') and the knockout strain BnGAE1 (KO) of Brassica napus were inoculated with mycelial blocks from detached leaves at the seedling stage. Disease was generally most pronounced after inoculation, and the sclerotinia disease index was calculated 48 hours after inoculation (48 hpi), when disease was most pronounced. The specific method was based on the Brassica napus sclerotinia disease resistance identification method (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 statistical results showed that BnGAE1 (KO) plants were significantly more resistant than WT plants, and the area of ​​lesion expansion was significantly reduced ( Figure 6 ), indicating that the BnGAE1 gene negatively mediates the resistance of rapeseed leaves to Sclerotinia sclerotiorum. Leaf tissues from the same area around the lesions of WT and BnGAE1 (KO) strains were taken, RNA was extracted and reverse transcribed into cDNA, and the 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) strain, genes such as BnNPR1, BnPR2 and BnMRKY33 on the SA signaling pathway were mainly induced strongly at the early stage of inoculation (6hpi), while genes such as BnEIN2, BnJAZ1 and BnPDF1.2 on the JA / ET signaling pathway were mainly induced strongly in the middle and late stages of Sclerotinia sclerotiorum infection. ROS scavenging-related genes BnCAT1 and BnGPX7 were strongly induced in the late stage ( Figure 7), indicating that BnGAE1-mediated rapeseed disease resistance can activate the SA signaling pathway in the early stage of pathogen infection, but mainly depends on the JA / ET signaling pathway in the later stage, and can clear excess ROS in the plant body to achieve redox balance in the plant body. The above results are of great significance for breeding rapeseed disease-resistant lines.

[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 relevant functions, there have been few reports in rapeseed so far. The BnGAE1 gene cloned in this paper enriches the research on this type of gene in rapeseed.

[0022] 2. The BnGAE1 gene cloned in the present invention provides a new genetic resource for disease resistance breeding of other crops, and has a guiding and reference role in improving the resistance of other crops to Sclerotinia sclerotiorum.

[0023] 3. The gene knockout vector CRISPR / Cas9-BnGAE1 constructed in this invention was used to transform rapeseed into a BnGAE1 underexpressing strain. Research into BnGAE1's gain of function revealed that BnGAE1 is a negative regulator of rapeseed's resistance to Sclerotinia sclerotiorum. Knockout of BnGAE1 can improve plant resistance to the disease. New rapeseed lines resistant to Sclerotinia sclerotiorum were obtained through genetic transformation and underexpression of this gene. BnGAE1 can serve as a potential marker gene for resistance in rapeseed.

[0024] 4. Through a series of studies on the resistance of BnGAE1-Cas9 knockout strains to Sclerotinia sclerotiorum, this paper fully demonstrates that BnGAE1 is involved in plant resistance to Sclerotinia sclerotiorum and plays a key role. By regulating the expression of the BnGAE1 gene, the accumulation of resistance factors in plants can be modulated, improving plant resistance to Sclerotinia sclerotiorum. This is of great significance for clarifying the biological function of the BnGAE1 gene.

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

[0026] 6. Breeding rapeseed materials resistant to Sclerotinia sclerotiorum has always been valued by rapeseed breeders. The development and utilization of BnGAE1 will help solve the problem of rapeseed resistance to Sclerotinia sclerotiorum in production. Since Sclerotinia sclerotiorum currently poses a significant threat to rapeseed yield and quality, breeding disease-resistant varieties is particularly important. Knockout of the BnGAE1 gene will help cultivate highly resistant rapeseed varieties and provide a theoretical and practical basis for the application of BnGAE1 in the resistance to Sclerotinia sclerotiorum in major oilseed crops (rape). It also provides new breeding materials for the further development of rapeseed varieties with Sclerotinia sclerotiorum resistance and has important practical guidance value in crop breeding practice, variety improvement, and variety promotion for Sclerotinia sclerotiorum resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

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

[0029] Figure 2 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 Schematic diagram of the Agrobacterium-mediated genetic transformation system for Brassica napus hypocotyls; A shows Brassica napus 'J9712' seeds disinfected with 0.1% mercuric chloride and germinated on MS medium; B shows etiolated hypocotyls grown after dark culture at 24°C for about 5 days; C shows etiolated hypocotyls cut into 0.8 cm segments under sterile conditions and infected with Agrobacterium GV3101 containing the transformation vector; D shows the infected hypocotyls transferred to M1 medium, dark cultured for 48 hours, and then placed on M2 medium for 1 minute. 6h light / 8h dark cycle selection culture for 15-20 days; E: explants after selection culture were placed in M3 medium and cultured for differentiation for more than 15 days under 16h light / 8h dark cycle; F: budding of explants on M3 medium; G: new shoots on explants were placed in fresh M3 medium and cultured for differentiation for more than 15 days under 16h light / 8h dark cycle until they differentiated into seedlings; H: seedlings were placed in M4 medium and rooted; I: seedlings were transplanted to soil and harvested for T1 generation seeds;

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

[0032] Figure 5 The gene editing status of the BnGAE1-Cas9 transgenic knockout strain;

[0033] Figure 6 Identification of the resistance of BnGAE1-Cas9 transgenic knockout lines to Sclerotinia sclerotiorum. A shows the disease phenotype of WT and BnGAE1(KO) seedling leaves 48 hours after inoculation; B shows the lesion size of WT and BnGAE1(KO) leaves 48 hours after infection with Sclerotinia sclerotiorum.

[0034] Figure 7 This figure shows the expression level analysis of disease resistance-related genes in the seedling leaves of wild-type 'J9712' and transgenic BnGAE1 (KO) plants at 0h, 6h, 12h, 24h, 36h, 48h and 60h after inoculation with Sclerotinia sclerotiorum; among them, A is the expression level of key genes related to ROS; B is the expression level of key genes related to jasmonic acid signaling pathway and disease resistance. DETAILED DESCRIPTION

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

[0036] The present invention specifically relates to the glucuronide isomerase BnGAE1 gene, a monosaccharide donor required for pectin synthesis and regulation of resistance to sclerotinia rot in Brassica napus, and its applications. A gene, BnGAE1 (BnaC07g42640D), was identified that mediates cell wall resistance and, consequently, resistance to sclerotinia rot in Brassica napus. This gene shares 97.44% amino acid sequence homology and 86.16% nucleic acid sequence homology with the model plant AtGAE1 (AT4G30440) in Arabidopsis thaliana. A BnGAE1-CRISPR / Cas9 knockout mutant strain was generated using Agrobacterium-mediated transformation and inoculated with Sclerotinia sclerotiorum to rapidly identify its function in sclerotinia rot resistance in Brassica napus.

[0037] SEQ ID NO.1 is the nucleotide sequence of the rapeseed BnGAE1 gene cloned in the present 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 examples define the present invention and describe how the present invention generates BnGAE1-Cas9 transgenic rapeseed knockout plants and methods for verifying BnGAE1 gene function. Based on the following description and these examples, those skilled in the art will be able to ascertain the essential features of the present invention and, without departing from the spirit and scope of the invention, may make various changes and modifications to adapt the invention to various uses and conditions.

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

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

[0045] Based on the BnGAE1 gene sequence of Brassica napus obtained from the BnPIR database, two sgRNAs with the highest scores and strongest specificity were designed using the analysis software of the CRISPR-Plant website (http: / / www.genome.arizona.edu / crispr / index.html) and the results of protein sequence function prediction: gae1-gRNA1: 5'-TCACTCGCGCTTAGAAAACGCGG-3' (SEQ ID NO. 3) and gae1-gRNA2: 5'-GCACCTCGCGGCTCAGGCCGGGG-3' (SEQ ID NO. 4). They were then ligated with the PKSE401 vector containing the Cas9 promoter and a 35S promoter by Bsa I digestion and T4 ligase ligation ( Figure 2 According to the gRNA sequence, synthetic amplification primers were designed: 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 In Figure A), four primers were mixed for amplification, and the product was saved for enzyme ligation. The PCR product from the previous step was ligated with the PKSE401 vector using Bsa I endonuclease and T4 ligase at 37°C for 12 hours. Heat-shock the reaction product to transform Escherichia coli DH5α, select a single colony for positive detection and sequencing, preserve the positive strain and plasmid, and transform the positive plasmid into competent Agrobacterium GV3101. Single colonies were selected and cultured in LB liquid medium with the corresponding resistance, shaking at 28°C for 36-48 hours. After PCR testing, positive strains were stored at -80°C with an appropriate amount of glycerol.

[0046] (2) Genetic transformation of rapeseed

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

[0048] a. Sowing: Wash Brassica napus 'J9712' seeds with 75% alcohol for 1 minute, then with 0.1% mercuric chloride solution for 5 minutes, and finally with sterile water five times. Place the seeds in M0 solid medium using sterilized tweezers and incubate the inoculated seeds in the dark at 24°C for 5 days. The M0 solid medium is prepared as follows: 4.404 g / L of MS (prepared in 1962), adjust the pH to 5.8-5.9, add 7 g / L of agar, and sterilize by conventional high-pressure steam sterilization.

[0049] b. Activation of Agrobacterium: Three days after sowing, streak Agrobacterium strain GV3101 stored at -80°C onto LB solid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL) and incubate at 28°C for 16 hours. Pick a single colony and incubate in 5 mL of LB liquid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL). Incubate at 28°C with shaking at 200 rpm for 20-24 hours until the Agrobacterium reaches the logarithmic phase. Take 500 μL of the culture and incubate in 50 mL of LB liquid medium (containing the three antibiotics) for approximately 12 hours.

[0050] c. Preparation of infection solution: Pour the activated Agrobacterium solution into two 50mL sterile centrifuge tubes, centrifuge at 3500rpm for 15min, remove the supernatant, place on ice, wash the solution with 1mL of DM liquid medium, centrifuge, remove the supernatant, add 2-3mL of DM liquid medium to resuspend the bacteria, and adjust the OD value of the infection solution to 0. 600 The DM liquid culture medium is prepared as follows: 4.404 g / L MS and 30 g / L sucrose of the 1962 formula are added, the pH of the culture medium is adjusted to 5.8-5.9, and the medium is sterilized at 121°C for 20 minutes. After sterilization, 1 mg / L 2,4-D, 100 mmol / L AS, and 0.3 mg / L KT are added in a sterile operating table to obtain the product.

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

[0052] e. Co-cultivation: Transfer the infected explants to a plate lined with filter paper (previously sterilized), remove any visible infection fluid from the explant surface, and then transfer to M1 solid culture medium and incubate in the dark at 24°C for 40-48 hours. The M1 solid culture medium is prepared as follows: 4.404 g / L MS (presumably a 1962 formulation), 18 g / L mannitol, 30 g / L sucrose, 2 mg / L 2,4-D, and 0.3 mg / L KT. Adjust the pH of the culture medium to 5.8-5.9, add 7 g / L agar, and after sterilization, add 100 mmol / L LAS.

[0053] f. Screening: Transfer the co-cultivated explants to M2 solid medium and culture for 20 days at 24°C with 16 hours of light and 8 hours of dark. The M2 solid medium is prepared as follows: 4.404 g / L MS (presumably a 1962 formulation), 18 g / L mannitol, 30 g / L sucrose, 2 mg / L 2,4-D, and 0.3 mg / L KT. Adjust the medium pH to 5.8-5.9 and add 7 g / L agar.

[0054] g. Differentiation culture: The screened explants were transferred to M3 solid culture medium and differentiation culture was initiated, with subculturing every 15-20 days until buds emerged (culture conditions were consistent with the screening conditions in step f). The M3 solid culture medium was prepared as follows: 4.404 g / L MS (prepared in 1962), 10 g / L glucose, 0.25 g / L xylose, and 0.6 g / L yeast extract (MES). The pH of the culture medium was adjusted to 5.8-5.9, and 7 g / L agar was added. After sterilization, 2 mg / L zeatin (ZT), 0.1 mg / L IAA, 250 mg / L TMT (timentin), and 25 mg / L Kan were added.

[0055] h. Rooting Culture: After a distinct growth point is visible on the seedlings that have differentiated and sprouted, carefully excise the seedlings using a scalpel at the junction of the callus and bud (taking care not to damage the growth point during this operation). The seedlings are then transferred to M4 solid medium for rooting. The M4 solid medium is prepared as follows: 2.202 g of MS (prepared in 1962), 10 g / L sucrose, and 0.5 mg / L IBA. The pH of the medium is adjusted to 5.8-5.9, and 7 g / L agar is added. After sterilization, 250 mg / L TMT and 25 mg / L Kan are added.

[0056] i. Transplanting: When the plant has grown a sufficient number of roots in the rooting medium and a well-developed root system, transfer the regenerated plant to soil for harvesting T1 seeds. Before transplanting, it is usually necessary to remove the sealing film from the culture bottle to allow the plant to gradually acclimate to the external environment.

[0057] (3) Identification of transgenic plants

[0058] a. Extraction of genomic DNA from Brassica napus leaves

[0059] DNA was extracted from fresh leaves of T0 transgenic rapeseed using the conventional CTAB method. The specific steps are as follows: Take young, tender leaves of Brassica napus (Brassica napus) 1-2 cm in length, place them in a clean mortar and pestle, grind them into a fine pulp, transfer them to a 1.5 mL centrifuge tube, and add 700 μL of 2× CTAB solution. Incubate at 70°C for 30 minutes, gently shaking every 10 minutes. Cool to room temperature, then add 700 μL of Tris-saturated phenol:chloroform:isoamyl alcohol (volume ratio:25:24:1), invert repeatedly to mix, and gently shake approximately 40 times. Centrifuge at 3100 rpm for 15 minutes at room temperature. Aspirate approximately 500 μL of the supernatant and add 500 μL of a 24:1 chloroform-isoamyl alcohol mixture. Shake thoroughly, then centrifuge at 3100 rpm for 15 minutes at room temperature. Discard the supernatant, add 1 mL of ice-cold -20°C anhydrous ethanol, and incubate at -20°C for 30 minutes. Centrifuge at 12,000 rpm for 10 minutes at room temperature. Wash the pellet with 75% ethanol and repeatedly pipette for 3 minutes to remove salts. Discard the alcohol, air-dry, and dissolve each sample in 30-50 μL of ddH₂O. Analyze the concentration of the extracted Brassica napus genomic DNA using a Nanodrop micro-nucleic acid analyzer.

[0060] b. Detection of positive transgenic plants

[0061] The genomic DNA of Brassica napus leaves obtained above 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 was a non-transgenic wild-type plant (J9712).

[0065] The test results showed that ( Figure 4 ), the seven transformed plants, KO-1, KO-2, KO-3, KO-4, KO-5, KO-6 and KO-7, were able to amplify the expected size of the electrophoresis band (572bp), while the wild-type control had no electrophoresis band, indicating that the transgenic rapeseed genome contained the DNA fragment of the exogenous knockout gene, but the brightness of the band was inconsistent, which may indicate that the expression levels of the knockout gene in different positive seedlings were different and the knockout efficiency may be different.

[0066] Example 2: Verification of BnGAE1-Cas9 gene knockout mutants

[0067] Primers were designed at both ends of the BnGAE1 gene target site. The primer sequences were:

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

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

[0070] Using the genomic DNA of the BnGAE1-Cas9 knockout mutant rapeseed plant as a template, PCR amplification was performed, and the PCR products were sequenced and compared to analyze the gene editing type (large fragment deletion lines with obvious phenotypes were selected for high-generation breeding). 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 coding region of the BnGAE1 open reading frame, large deletions of 679 bp, 180 bp, 180 bp, and 328 bp appeared in the double-stranded regions of KO-1, KO-2, KO-4, and KO-5, respectively, while no editing occurred in the other strains.

[0071] Example 3 Evaluation of resistance of BnGAE1-Cas9 knockout transgenic rapeseed to sclerotinia rot

[0072] (1) Immune response of BnGAE1 knockout strain to Sclerotinia sclerotiorum

[0073] The second leaf of rapeseed seedlings (at the four-leaf, one-heart stage) was inoculated in vitro with the pathogen of sclerotinia sclerotiorum (Sclerotinia sclerotiorum was preserved in the laboratory of the College of Life Sciences of Huazhong Agricultural University and activated in the laboratory). The second leaf of the wild type and the positive strain in Example 1 was cut and placed in a transparent square box. Each transgenic plant leaf was placed side by side with the non-transgenic control in a box. Several layers of filter paper and wet gauze were placed under the leaves. Freshly prepared Inoculate the mycelial block, with the mycelium facing downward, in the middle of the leaf, slightly away from the main vein. Two mycelial blocks are inoculated per leaf. Cover with a lid to maintain moisture, and then incubate in the dark at 22°C. Observe the leaves continuously from 0 to 72 hours after inoculation and find that the growth area of ​​sclerotinia lesions in the BnGAE1-Cas9 transgenic line is significantly smaller than that in the wild type. The most significant difference in resistance is observed 48 hours after inoculation ( Figure 6 Middle A), compared with the wild type, the area of ​​sclerotinia lesions on transgenic rapeseed leaves was reduced by 25.46% ( Figure 6(B) Therefore, knocking out the BnGAE1 gene can effectively enhance the resistance of rapeseed leaves to Sclerotinia sclerotiorum.

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

[0075] use Super total RNA extraction kit (Promega, USA) was used to extract total RNA from leaves 48 hours after Sclerotinia sclerotiorum treatment in step (1). RNA was reverse transcribed into cDNA using IIQ RT SuperMix for qRNA (+gDNAwiper) Kit (Vazyme, China).

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

[0077] qPCR use Green Realtime PCR Master Mix-Plus-Kit, disease resistance-related gene primers are as follows:

[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] Primer Premier 5.0 software was used to design specific primers for the coding region of the gene, and Primer-BLAST (NCBI) was used to perform primer specificity comparison analysis in the Brassica gene database (BRAD) and the rapeseed EST database to verify the specificity of the primers.

[0109] The qPCR reaction system (15 μL) contained 7 μL of SYBR Mix, 0.5 μL of each forward and reverse primer (10 μmol / L), 2 μL of template, and 5 μL of DEPC-treated sterile water. Amplification conditions were: 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 27 seconds. Fluorescence detection was performed after annealing at 72°C between cycles.

[0110] After the reaction, the reaction mixture was first heated to 95°C, then lowered to 72°C, and then slowly raised to 95°C. The changes in the fluorescence signal were recorded to obtain the melting curve of the amplified product. Each set of experiments was repeated three times, and each biological replicate was repeated at least three times. 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 amplify BnRbohD (BnaC02g38300D), BnCAT1 (BnaA07g11370D), and Specific PCR amplification of the BnGPX7 (BnaA03g51760D), BnEIN2 (BnaA10g26670D), BnJAZ1 (BnaA06g13250D), BnPDF1.2 (BnaC02g23620D), BnNPR1 (BnaA01g15290D), BnPR2 (BnaC04g24330D), and BnWRKY33 (BnaC04g06800D) genes was performed. The BnActin7 (BnaC09g46850D) gene was also amplified using the primer pair Q-BnActin7-F / R, serving as an internal control for relative quantitative analysis. The expression of these disease resistance-related genes in rapeseed leaves was examined at 0, 6, 12, 24, 36, 48, and 60 hours after S. sclerotiorum induction.

[0111] The results showed that the expression of superoxide anion synthesis-related gene BnRbohD in the BnGAE1 knockout strain was significantly lower than that in the wild type, but showed an overall upward trend, while the ROS scavenging-related genes BnCAT1 and BnGPX7 were strongly induced in the later stage ( Figure 7 The genes BnEIN2, BnJAZ1, and BnPDF1.2 in the JA / ET signaling pathway were strongly induced in the middle and late stages of inoculation, while the genes BnNPR1, BnPR2, and BnMRKY33 in the SA signaling pathway were strongly induced in the early stages of S. sclerotiorum infection (6 hpi) in BnGAE1 knockout plants ( Figure 7 These results indicate that BnGAE1-mediated disease resistance in rapeseed can activate the SA signaling pathway in the early stages of pathogen infection, but is primarily dependent on the JA / ET signaling pathway in the later stages. Furthermore, BnGAE1 can scavenge excess ROS in the plant to maintain redox balance.

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

Claims

1. Application of knocking out the BnGAE1 gene in improving resistance to sclerotinia sclerotiorum in Brassica napus, characterized in that: The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

2. Use of the protein encoded by the BnGAE1 gene according to claim 1 in improving resistance to sclerotinia sclerotiorum in Brassica napus, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Use of a recombinant expression vector containing the BnGAE1 gene according to claim 1 in improving the resistance of Brassica napus to sclerotinia sclerotiorum.

4. The use according to claim 3, characterized in that The recombinant expression vector is transformed into microorganisms for culture and expression, and then transformed into target plants to obtain BnGAE1 gene knockout mutants; the transformation method is Agrobacterium-mediated hypocotyl infection; the nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

5. Use of a host bacterium containing the recombinant expression vector according to claim 3 in improving the resistance of Brassica napus to sclerotinia sclerotiorum.

6. Application of the BnGAE1 gene in improving resistance to sclerotinia sclerotiorum in Brassica napus, characterized in that: The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

7. Application of the BnGAE1 gene in molecular breeding of Brassica napus for resistance to Sclerotinia sclerotiorum, characterized in that: The nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.

1.

8. A method for improving the resistance of Brassica napus to Sclerotinia sclerotiorum, characterized in that: The method comprises the steps of introducing a gene-directed editing recombinant expression vector into Brassica napus to obtain a BnGAE1 gene knockout mutant, wherein the nucleotide sequence of the BnGAE1 gene is shown in SEQ ID NO.1.

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