Gene BnaCLE20 related to sclerotiniose resistance of oilseed rape and application of gene BnaCLE20 in oilseed rape breeding

Abstract

The invention belongs to the field of rape breeding, and particularly discloses a sclerotinia rot resistance related gene BnaCLE20 of rape and application of the sclerotinia rot resistance related gene BnaCLE20 in rape breeding. The sequence of the BnaCLE20 gene is as shown in SEQ ID NO. 1, and the coded protein is as shown in SEQ ID NO. 2; the BnaCLE20 gene of the brassica napus is identified to be capable of regulating the sclerotinia rot resistance of the brassica napus for the first time, a molecular network for regulating the sclerotinia rot resistance of plants is enriched, and the BnaCLE20 gene plays an important role in researching a molecular mechanism formed by the disease resistance of the plants. The overexpression of the BnaCLE20 gene disclosed by the invention can reduce the content of active oxygen, malondialdehyde and oxalic acid in rape leaves infected by sclerotinia sclerotiorum; meanwhile, the activity of catalase in the rape leaves infected by sclerotinia sclerotiorum is enhanced; the sclerotiniose resistance of the oilseed rape is remarkably improved, and the method has important significance in guaranteeing the high and stable yield of the oilseed rape by improving the sclerotiniose resistance of the oilseed rape.

Description

Technical Field

[0001] The invention belongs to the field of rapeseed breeding, and particularly relates to a rapeseed anti-bacterial sclerotinia rot-related gene BnaCLE20 and its application in rapeseed breeding. Background Art

[0002] Rapeseed is the oilseed crop with the largest planting area in my country. Sclerotinia sclerotiorum, caused by infection with the fungus Sclerotinia sclerotiorum, is the "cancer" of rapeseed, resulting in a 10-20% yield loss in rapeseed each year and direct economic losses exceeding 7 billion yuan (Zhang Ka et al., 2018). Extreme weather conditions can cause a surge in the incidence of this disease.

[0003] Sclerotinia resistance in rapeseed is a quantitative trait. Whole-genome association analysis revealed that the gene BnaA05.RLK902, encoding an LRR receptor protein kinase, regulates Sclerotinia resistance in rapeseed through a jasmonic acid-mediated immune response (Zhao et al., 2023). The gene BnaA07.MKK9, encoding a mitogen-activated protein kinase kinase 9, regulates the accumulation of immune response-related substances such as ethylene, antifungal substances (phytoalexins and glucosinolates), and hydrogen peroxide, thereby regulating Sclerotinia resistance in rapeseed (Lin et al., 2024). Through multi-omics data mining, some genes regulating rapeseed sclerotinia resistance were identified using homologous cloning methods, such as BnF5H, which inhibits disease resistance by regulating lignin monomer composition (Cao et al., 2021); the GDSL lipase gene BnaC07.GLIP1, which induces phospholipid molecule production and maintains reactive oxygen homeostasis, positively regulating rapeseed sclerotinia resistance (Ding et al., 2024); and the zinc finger transcription factor BnaSTOP2s, which confers rapeseed sclerotinia resistance by regulating sulfur metabolism (Dai et al., 2025). However, there have been no reports on CLE-type small peptides in rapeseed regulating sclerotinia resistance.

[0004] The present invention found that after being infected by Sclerotinia sclerotiorum, the BnaCLE20 gene in the leaves of the disease-resistant rapeseed Zhongyou 821 and the susceptible rapeseed Westar had different response patterns, and further functional analysis was performed using transient overexpression in rapeseed. Summary of the Invention

[0005] The present invention aims to provide a rapeseed resistance to sclerotinia sclerotiorum-related gene BnaCLE20 for rapeseed breeding. The gene sequence is shown in SEQ ID NO.1, and the protein encoded by the gene is a small peptide protein shown in SEQ ID NO.2.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Screening and acquisition of the rapeseed sclerotinia resistance-related gene BnaCLE20:

[0008] Based on the transcriptome data of rapeseed in response to Sclerotinia infection published by Wu et al. (2017), Xu et al. (2021) and Walker (2022), 2640 core DEGs responding to Sclerotinia infection were obtained, of which 78 DEGs showed different response patterns in disease-resistant (Zhongyou 821) and susceptible (Westar) rapeseed. The location and function of the 78 DEGs were annotated, and it was found that the CLE-like gene BnaCLE20 was located in the uqA09.1 interval of the disease resistance-related QTL site located by Zhang et al. (2023). RT-qPCR analysis verified that after infection with Sclerotinia, the expression level of BnaCLE20 in disease-resistant rapeseed was significantly increased, and there was no significant expression change in susceptible rapeseed. Therefore, it was listed as a candidate gene regulating rapeseed resistance to Sclerotinia. The BnaCLE20 gene sequence is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2; the promoter sequence of BnaCLE20 in Zhongyou 821 is shown in SEQ ID NO.3, and the promoter sequence of BnaCLE20 in Westar is shown in SEQ ID NO.4.

[0009] The present invention also provides a primer pair for cloning the rapeseed sclerotinia resistance-related gene BnaCLE20, as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0010] The present invention also provides a method for constructing a BnaCLE20 overexpression strain, comprising the following steps:

[0011] 1) Ligate the BnaCLE20 gene into the pCAMBIA1300 vector;

[0012] 2) Transform the successfully constructed vector into Agrobacterium tumefaciens GV3101;

[0013] 3) Transiently transform the target rapeseed (e.g., Zhongshuang 11) using Agrobacterium-mediated transformation to obtain transgenic leaves overexpressing BnaCLE20.

[0014] The present invention also provides a positive identification primer for an overexpression rapeseed transgenic line, characterized in that the primer pair comprises: 1300F as shown in SEQ ID NO.7: '-CGCCAGGGTTTTCCCAGTCACGAC-3', 1300R as shown in SEQ ID NO.8: 5'-AGCGGATAACAATTTCACACAGGA-3';

[0015] The pCAMBIA1300-BnaCLE20 vector-specific primer pair 1300F is shown in SEQ ID NO. 7: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3' and 1300-CLE20R is shown in SEQ ID NO. 6: 5'-catggtaccggatccactagtTCGCTTGTTGTGCAAGGGA-3'.

[0016] The present invention also provides a method for positive identification of overexpressed rapeseed transgenic leaves, using the gDNA of transgenic plant leaves as a template and using the above two pairs of positive identification primers for overexpressed rapeseed transgenic leaves to positively identify the obtained overexpressed rapeseed transgenic strains, ultimately obtaining rapeseed plants that transiently overexpress the target gene.

[0017] The present invention also provides a primer pair for performing fluorescence quantitative analysis on the rapeseed sclerotinia resistance-related gene BnaCLE20, as shown in SEQ ID NO.9 and SEQ ID NO.10.

[0018] The present invention also provides a method for detecting the expression level of a gene BnaCLE20 related to resistance to sclerotinia sclerotiorum in rapeseed, comprising the following steps:

[0019] 1) Extracting total RNA from rapeseed leaves;

[0020] 2) reverse transcribing the RNA into cDNA;

[0021] 3) Fluorescence quantitative PCR analysis was performed using the primer pair shown as SEQ ID NO. 9 and SEQ ID NO. 10 to quantitatively determine the expression level of the gene BnaCLE20.

[0022] The present invention also provides an application of the rapeseed sclerotinia resistance-related gene BnaCLE20 in rapeseed breeding, including:

[0023] A. Reduce the content of malondialdehyde and oxalic acid in rapeseed leaves infected by Sclerotinia sclerotiorum;

[0024] B. Enhance the activity of catalase in rapeseed leaves infected by Sclerotinia sclerotiorum.

[0025] The above application is preferably to introduce a substance that increases the expression level of the rapeseed BnaCLE20 gene into the plant;

[0026] In the above application, the substance is a nucleic acid molecule containing the BnaCLE20 gene, or its expression cassette, recombinant vector, or recombinant microorganism; in the above application, the plant is Brassica napus.

[0027] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0028] Through homologous cloning, the present invention identifies for the first time the BnaCLE20 gene from Brassica napus as regulating Sclerotinia sclerotiorum resistance in rapeseed, enriching the molecular network regulating Sclerotinia sclerotiorum resistance in plants and playing an important role in studying the molecular mechanisms of plant disease resistance. While the gene sequence of CLE20 in rapeseed is known, its specific biological function is unclear. Through research, the present invention discovered that overexpression of the BnaCLE20 gene in rapeseed can reduce the levels of malondialdehyde and oxalic acid in rapeseed leaves infected with Sclerotinia sclerotiorum; at the same time, it enhances catalase activity in rapeseed leaves infected with Sclerotinia sclerotiorum, significantly enhancing Sclerotinia sclerotiorum resistance in rapeseed. This is of great significance for ensuring high and stable yields of rapeseed by improving Sclerotinia sclerotiorum resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solution of the present invention, the applicant will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] Figure 1 (AD) shows the effect of BnaCLE20 peptide on the growth, development and pathogenicity of Sclerotinia sclerotiorum; Figure 1-A The expression pattern of CLE20 peptide in Brassica napus; Figure 1-B This is a phylogenetic analysis of CLE20 small peptides in Brassica napus; Figure 1-C For protein sequence analysis of BnaCLE20; Figure 1-D The phenotype of Sclerotinia sclerotiorum treated with the BnaCLE20 core peptide; Figure 1-E Pathogenicity analysis of Sclerotinia sclerotiorum treated with BnaCLE20 core peptide; Figure 1-F Statistical data for the plaque area of ​​Sclerotinia sclerotiorum treated with BnaCLE20 core peptide;

[0031] Figure 2 (AG) shows the disease resistance analysis of the transiently overexpressed gene BnaCLE20 in rapeseed; Figure 2-A For the identification of DNA levels of transiently overexpressed rapeseed; Figure 2-B Analysis of the expression patterns of BnaCLE20 in overexpressed and control rapeseed leaves. CK: no infection; 48 hpi: 48 h after infection; Figure 2-C The phenotypes of BnaCLE20 overexpression (OE-BnaCLE20) and wild type after infection with Sclerotinia sclerotiorum; Figure 2-D is the statistical data of plaque area (unit: cm 2 ); Figure 2-E is the catalase (CAT) activity (unit) of transgenic and wild-type rapeseed leaves before and after infection; Figure 2-F is the malondialdehyde content (unit) in transgenic and wild-type rapeseed leaves before and after infection; Figure 2-G is the oxalic acid content (unit) in transgenic and wild-type rapeseed leaves before and after infection. DETAILED DESCRIPTION

[0032] The applicant will now provide a clear and complete description of the technical solutions in the embodiments of the present invention with reference to Figures 1 and 2, and will explain the present invention in detail. These embodiments are only intended to illustrate the present invention and are not intended to limit the scope of protection claimed in the present invention.

[0033] The technical solutions described in the present invention, unless otherwise specified, are all conventional techniques in the art; the reagents and materials described, unless otherwise specified, are all commercially available. The present invention refers to the annotated sequence of the rapeseed (Zhongshuang 11) genome ZS11.V0 (http: / / cbi.hzau.edu.cn / bnapus / index.php).

[0034] All nucleotide sequences in this specification are in the 5'-3' direction.

[0035] Example 1: Screening and obtaining of the gene BnaCLE20 related to rapeseed sclerotinia resistance:

[0036] Based on the transcriptome data of rapeseed in response to Sclerotinia infection published by Wu et al. (2017), Xu et al. (2021) and Walker (2022), 2640 core DEGs responding to Sclerotinia infection were obtained, of which 78 DEGs showed different response patterns in disease-resistant (Zhongyou 821) and susceptible (Westar) rapeseed. The location and function of the 78 DEGs were annotated, and it was found that the CLE-like gene BnaCLE20 was located in the uqA09.1 interval of the disease resistance-related QTL site located by Zhang et al. (2023). RT-qPCR analysis verified that after infection with Sclerotinia, the expression level of BnaCLE20 in disease-resistant rapeseed was significantly increased, and there was no significant expression change in susceptible rapeseed. Therefore, it was listed as a candidate gene regulating rapeseed resistance to Sclerotinia. The BnaCLE20 gene sequence is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2; the promoter sequence of BnaCLE20 in Zhongyou 821 is shown in SEQ ID NO.3, and the promoter sequence of BnaCLE20 in Westar is shown in SEQ ID NO.4.

[0037] The experimental steps for analyzing the effect of BnaCLE20 peptide on the growth, development and pathogenicity of Sclerotinia sclerotiorum are as follows:

[0038] S1: RNA was collected from Westar and Zhongyou 821 leaves 48 hours after Sclerotinia sclerotiorum infection (48 hpi), reverse transcribed into cDNA, and then subjected to fluorescence quantitative PCR analysis using quantitative primers;

[0039] S2: CLE20 protein sequences from other species of the Brassicaceae were obtained from NCBI using protein sequence alignment, and phylogenetic analysis was performed using MEGAX.

[0040] S3: CLE20 protein sequences from different species were aligned using CLUSTALW and visualized using ESPript;

[0041] S4: Based on the core peptide of CLE20, BnaCLE20 small peptide (SP) was synthesized by Qingke Biotechnology Co., Ltd. After filtration, 5 μM was added to PDA culture medium, and the growth of S. sclerotiorum hyphae was observed after 48 h of culture;

[0042] S5: Sclerotinia sclerotiorum grown on a medium containing 5 μM SP PDA was inoculated onto rapeseed leaves, and the plaque area was counted after 48 h.

[0043] like Figure 1-A As shown, the expression pattern of the BnaCLE20 peptide is as follows: after infection with Sclerotinia sclerotiorum, the expression of BnaCLE20 in the susceptible rapeseed Westar did not change significantly, but the expression level in the resistant rapeseed Zhongyou 821 increased significantly;

[0044] like Figure 1-B As shown in the figure, the phylogenetic analysis results of CLE20 small peptides in Brassicaceae are as follows: there are 4 homologous proteins of CLE20 in Brassica napus, and the highest homology is with Brassica rapa;

[0045] like Figure 1-C As shown, the results of protein sequence analysis of BnaCLE20 are as follows: the core of BnaCLE20 protein is too short and highly conserved;

[0046] like Figure 1-D As shown, the phenotypic results of Sclerotinia sclerotiorum treated with BnaCLE20 core peptide are as follows: 5 μM SP significantly reduced the hyphal density of S. sclerotiorum;

[0047] like Figure 1-E As shown, the results of the pathogenicity analysis of Sclerotinia sclerotiorum treated with the BnaCLE20 core peptide showed that the pathogenicity of Sclerotinia sclerotiorum grown on a medium containing 5 μM SPPDA was significantly reduced.

[0048] Example 2: Application of the gene BnaCLE20 related to rapeseed sclerotinia resistance in rapeseed breeding

[0049] As shown in FIG2 , this example used the gene BnaCLE20 related to rapeseed sclerotinia resistance in the breeding of rapeseed variety Double 11:

[0050] (1) Primers were designed to amplify the coding region sequence of BnaCLE20 in Zhongyou 821 (shown in SEQ ID NO. 1). The left primer sequence is shown in SEQ ID NO. 5:

[0051] The right primer sequence is shown in SEQ ID NO. 6: catggtaccggatccactagtTCGCTTGTTGTGCAAGGGA. The 5' ends of the left and right primers contain restriction sites for XbaI and SpeI, respectively. The PCR reaction program was 95°C for 3 minutes, 95°C for 15 seconds, 60°C for 2 minutes, 72°C for 35 cycles, and 72°C for 5 minutes. The PCR product was recovered using a DNA purification kit (OMEGA).

[0052] The CDS sequence of BnaCLE20 from Zhongyou 821 (as shown in SEQ ID NO. 3) was ligated with the pCAMBIA1300 vector carrying the 35S promoter and transformed into Escherichia coli DH5α. The positive cloning primer pair sequences were 1300F (SEQ ID NO. 7): CGCCAGGGTTTTCCCAGTCACGAC and 1300R (SEQ ID NO. 8): AGCGGATAACAATTTCACACAGGA; identification was performed by PCR.

[0053] (2) The identified recombinant plasmid vector was transformed into Agrobacterium tumefaciens GV3101, and the cells were shaken overnight and centrifuged. The supernatant was removed and an appropriate amount of infection solution (10 nM MES-KOH + 10 mM MgCl2 + 200 μM AS) was added for resuspending and the D600 value was adjusted to 0.8-1.0. After incubation at room temperature in the dark for 3 h, the cells were injected into the right side of the leaves of Zhongshuang No. 11 rapeseed (cultured in hydroponics for 4 weeks) with pCAMBIA1300 as a negative control. After 24 h of dark culture, the detached leaves were infected with Sclerotinia sclerotiorum in vitro. Pictures and samples were taken 48 h after infection.

[0054] (3) Samples were taken from rapeseed leaves injected with empty pCAMBIA1300 and BnaCLE20-pCAMBIA1300, and genomic DNA was extracted for transient overexpression identification. The primer pairs used were shown in SEQ ID NO.7: CGCCAGGGTTTTCCCAGTCACGAC and SEQ ID NO.6:

[0055] catggtaccggatccactagtTCGCTTGTTGTGCAAGGGA

[0056] The test results showed that the expected size electrophoresis bands were amplified in the rapeseed leaves injected with BnaCLE20-pCAMBIA1300, while the negative control did not, indicating that the target DNA fragments were already present in the genome of the transgenic rapeseed ( Figure 2-A ).

[0057] (4) The expression level of BnaCLE20 was analyzed by RT-qPCR. The quantitative primer pair sequences are shown in SEQ ID NO.9: GACGACGCTTCTCCTCCG and SEQ ID NO.10: TCCCGTCTTAACCTTCCGT. The results showed that the expression level of BnaCLE20 gene in rapeseed leaves injected with BnaCLE20-pCAMBIA1300 was significantly increased, indicating that the gene was successfully transiently overexpressed in rapeseed leaves, and the expression level of the gene was significantly increased after infection with Sclerotinia sclerotiorum ( Figure 2-B ).

[0058] (5) The detached leaves cultured in the dark for 24 hours were infected with Sclerotinia sclerotiorum. After 48 hours, the lesion area on both sides of the rapeseed leaves was counted. The results showed that the lesion area on the right side of the rapeseed leaves overexpressing BnaCLE20 was significantly reduced ( Figure 2-C and Figure 2-D ).

[0059] (6) The malondialdehyde (MDA) content detection kit (BC0025) produced by Solarbio (Beijing Solarbio Science & Technology Co., Ltd.) was used to determine the changes in the malondialdehyde content in rapeseed leaves before and after infection with S. sclerotiorum. The specific experimental steps were as follows:

[0060] S1: Weigh about 0.1 g of leaves (stored at -80°C), add 1 mL of extract solution and homogenize in an ice bath, centrifuge at 8000 rpm at 4°C for 10 min, collect the supernatant, and place on ice for testing.

[0061] S2: Add 20 mL of reagent 1 to 1 bottle of reagent 2, dissolve and mix well, and use it as the MDA detection working solution.

[0062] S3: Add 300 μL of the MDA working solution from step S2, 100 μL of sample, and 100 μL of Reagent 3 to each assay tube. Add 300 μL of MDA working solution, 100 μL of distilled water, and 100 μL of Reagent 3 to each blank tube. Mix thoroughly.

[0063] S4: The mixed solution in step S3 was kept in a water bath at 100° C. for 60 min, cooled in an ice bath, and centrifuged at 10,000 rpm for 10 min at room temperature.

[0064] S5: Preheat the microplate reader for 30 minutes and adjust the zero value with distilled water. Pipette 200 μL of the supernatant from step S4 into a 96-well plate and measure the absorbance of each sample at 532 nm and 600 nm.

[0065] S6: Calculate the MDA content using the formula. The calculation formula is as follows:

[0066] ΔA532=A532 测定 -A532 空白

[0067] ΔA600=A600 测定 -A600 空白

[0068] ΔA=ΔA532-ΔA600

[0069] MDA content (nmol / g mass) = [ΔA × V total (ε × d) × 10 9 ]-(W×Vsample÷Vextract)

[0070] =53.763×ΔA÷W

[0071] Where, Vreaction: total volume of the reaction system, 5×10 -4 L;

[0072] ε: MDA molar absorptivity, 1.55×10 5 L / mol / cm;

[0073] V sample: added sample volume, 0.1 mL;

[0074] d: 96-well plate optical diameter, 0.6 cm;

[0075] V extraction: volume of extract added, 1 mL;

[0076] W: sample mass, g;

[0077] 10 9 : Unit conversion factor, 1mol=10 9 nmol.

[0078] The results showed that the malondialdehyde content in rapeseed leaves overexpressing BnaCLE20 did not increase significantly after infection with Sclerotinia sclerotiorum, and was significantly lower than that in the negative control. Malondialdehyde is a marker product of lipid peroxidation. This result indicates that overexpression of BnaCLE20 enhances the oxidative stress capacity of rapeseed ( Figure 2-E ).

[0079] (7) The oxalic acid content in rapeseed leaves before and after S. sclerotiorum infection was determined using an oxalic acid assay kit (BC4365) from Solarbio (Beijing Solarbio Science & Technology Co., Ltd.). The specific experimental steps were as follows:

[0080] S1: Weigh approximately 0.1 g of leaves (stored at -80°C) and add 1 mL of distilled water. Homogenize thoroughly, then add approximately 3-5 mg of Reagent IV. Vortex to mix thoroughly, then decolorize in a 75°C waterbath for 30 minutes, shaking 2-3 times. After decolorization, centrifuge at 3000 rpm for 15 minutes at room temperature. Remove the supernatant and place on ice for analysis.

[0081] S2: Dilute 100 μmol / mL oxalic acid standard solution (provided in the kit) with distilled water to 18, 15, 12, 6, 3, 1.5, 0.75, and 0.375 μmol / mL standard solutions for later use.

[0082] S3: Add 20 μL of Reagent 1, 150 μL of Reagent 2, 10 μL of Reagent 3, and 20 μL of sample to each assay tube. Add 20 μL of Reagent 1, 150 μL of Reagent 2, 10 μL of Reagent 3, and 20 μL of distilled water to each blank tube. Add 20 μL of Reagent 1, 150 μL of Reagent 2, 10 μL of Reagent 3, and 20 μL of standard solution to each standard tube. Mix thoroughly and let stand at room temperature for 20 minutes.

[0083] S4: Preheat the microplate reader for at least 30 minutes, adjust the wavelength to 510 nm, and zero the sample with distilled water. Pipette 200 μL of the mixed solution from step S3 into a 96-well plate and measure the absorbance (A) at 510 nm. These values ​​are designated as A blank tube, A assay tube, and A standard tube, respectively. Calculate ΔA = A blank tube - A assay tube, and ΔA standard = A blank tube - A standard tube.

[0084] S5: Draw a standard curve: Use the concentration of each standard solution as the x-axis and its corresponding ΔA standard as the y-axis to draw a standard curve to obtain the standard equation y=kx+b. Substitute ΔA into the equation to obtain x (μmol / mL).

[0085] S6: Calculate the oxalic acid content using the formula. The calculation formula is as follows:

[0086] Oxalic acid content (mg / g mass) = x × V extraction × M × 10 -3 ÷W=0.09x÷W

[0087] Where, Vextraction: volume of distilled water added, 1 mL;

[0088] W: sample mass, g;

[0089] M: Oxalic acid molecular weight 90.04:

[0090] 10 -3 :Unit conversion factor, 1μg=10 -3 mg.

[0091] The results showed that after infection with S. sclerotiorum, the oxalic acid content in the negative control increased by 1.39 times, while the oxalic acid content in rapeseed leaves overexpressing BnaCLE20 was significantly lower than the negative control. S. sclerotiorum produces oxalic acid to enhance infection of rapeseed leaves. This result shows that BnaCLE20 can reduce the production of oxalic acid during infection to resist infection with S. sclerotiorum. Figure 2-F ).

[0092] (8) The catalase activity in rapeseed leaves before and after infection with S. sclerotiorum was determined using a catalase activity assay kit (catalase (CAT) activity assay kit, BC0205) from Solarbio (Beijing Solarbio Science & Technology Co., Ltd.). The specific experimental steps were as follows:

[0093] S1: Weigh approximately 0.1g of leaves (stored at -80°C), add 1mL of the extract, and homogenize on ice. Centrifuge at 8000g for 10 minutes at 4°C, remove the supernatant, and place on ice until assayed.

[0094] S2: Pipette 25 μL of reagent 2 into 5 mL of reagent 1, mix thoroughly, and place in a 25°C water bath for 10 minutes to serve as the detection working solution.

[0095] S3: Preheat the microplate reader for 30 minutes, adjust the wavelength to 240 nm, and zero the sample with distilled water. Add 10 μL of sample and 190 μL of the working solution from step S2 to a 96-well UV plate. Immediately mix and start measuring. Record the initial absorbance (A1) at 240 nm after 5 seconds and the absorbance (A2) after 1 minute and 5 seconds. Calculate ΔA = A1 - A2.

[0096] S4: Calculate catalase (CAT) activity using the formula. The calculation formula is as follows:

[0097] CAT (U / g mass) = [ΔA × V total ÷ (ε × d) × 10 6 ]÷(V sample ÷ V sample total × W)÷T×F

[0098] =764.5×ΔA÷W×F

[0099] Where, Vreaction: total volume of reaction system, 2×10 -4 L;

[0100] ε: H2O2 molar extinction coefficient, 43.6 L / mol / cm;

[0101] d: 96-well UV plate light path, 0.6 cm;

[0102] V sample: added sample volume, 0.01;

[0103] V total sample: volume of extract added, 1 mL;

[0104] T: reaction time, 1 min;

[0105] W: sample mass, g;

[0106] 10 6 : Unit conversion factor, 1mol=10 6 μmol;

[0107] F: Sample dilution factor.

[0108] The results showed that the CAT activity in rapeseed leaves overexpressing BnaCLE20 was significantly higher than that in the control, indicating that BnaCLE20 can enhance the activity of catalase and thus has a stronger ability to alleviate oxidative damage. Figure 2-G ).

[0109] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0110] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be understood by anyone familiar with the technology within the technical scope disclosed by the present invention should be included in the scope of the present invention.

[0111] The BnaCLE20 gene sequence is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2; the promoter sequence of BnaCLE20 in Zhongyou 821 is shown in SEQ ID NO.3, and the promoter sequence of BnaCLE20 in Westar is shown in SEQ ID NO.4.

[0112] SEQ ID NO.1

[0113] ATGAATCAGGGTCGTCTTCTTTGTCTCATTGGCTTTCTCTTCTTCCTTGTTGT

[0114] TTTATCTAAAGCTTCAAGAGTTCACGTTGAAAGACGACGCTTCTCCTCCGA

[0115] ACCTTTATCTAGCCAAGAAAGAGGGTTTATCCCTTCTAAACCTACCCTGCCG

[0116] GTCGCCAGTGCTGATGAAATCTTACCGGAGAAACGGAAGGTTAAGACGGG

[0117] ATCAAATCCCTTGCACAACAAGCGATGA

[0118] SEQ ID NO.2

[0119] MNQGRLLCLIGFLFFLVVLSKASRVHVERRRFSSEPLSSQERGFIPSKPTLPVAS

[0120] ADEILPEKRKVKTGSNPLHNKR

[0121] SEQ ID NO.3

[0122] TAAGCTGGAATATATGGTTTTAAGATATCTTTCCAATTTGTGTGAAAAGTACC

[0123] AATACTCATATAGTTTTCCATTTATTTGTGTGTGTGGTTTTGACCGCGAATTG

[0124] GATCTTGGTTGTTAGCAATATACTCGGAGCTCGCTACTTTGAACTGTGAAAC

[0125] ATATTTTTTGGTCGGAATGGCTGAACTTCCCAACAGATCATGTTTTTATTATC

[0126] AGTTTATCTCGTTCCGATTGAGCCTGTACCAGAGTTAATTGACAAAGCGTGT

[0127] TCCAACGGTCATTTTTTGCAACTGTTTATATGTCATCACTTACTGAGTCGGTT

[0128] TAGGTTTTGGATCAGGTTACTAATATCCCGGTTCAGGTTTGGATTGGATGGGCC

[0129] AGATTTCTTACACCCCCAAATCCACTAGTTGGTCCAGTGTCATATACTCACT

[0130] CATTCATGGTTGGCAACATTCAGTAATCATGAAATAATATTAAATCAACCGC

[0131] AACTATGAAAACAGACATTTAATTCACAATATAACTGACCATCACGATCAT

[0132] TTCAATATTTGACATGATGATGAGATGTTGTGAATTTTGAATTAAGCATAAGATAT

[0133] ACTAGGCTGTAAATATGCTATGCCACTAGACCAAGGTCGTTTTCCCAAGTAA

[0134] AAGCGACTTAAAATCGTAGATAGGTTATATTTTATAAGTGATAATCATGACTT

[0135] TGAATTAGTCTTTGAAGAATAGAAGGCAGTTTTTTTTTTCCGGTCTTATTT

[0136] CTCAAAAGGTCAATAGAAAAATTAAACTTTATAAGTCACTTTGTCTCTTTGT

[0137] ATTTGATATTCTTGATTATGATATTTACACAAAATCATGTCGCTATTCAAAGCT

[0138] TCATCATTCTGCATACGAACACTTTTCACCTTAAAGCTACATTATACTCACGC

[0139] GTAAGTAGCAAACTCTGATATGTTTAAAGAAAAGTTTTACGTTTCACATGTG

[0140] AAAATTAATAAGAAATCAATAAGGATCACTATATTTTTGGCACTTCCACATTT

[0141] ATGGCCACGTACTGGTGATATTTTTTTTCCTTTTGAGTTGAATTCCAAATCGT

[0142] TGCATCTATAATCAATACTCACTTTTAAAACCGGCCATTTTTAGGTGGGAAG

[0143] TGGGGTTCATTAGTTATAAAAAAAGTAAAAGACACGTTAACACTTAACATT

[0144] GCAATAATATAGTACGTCAGTTTGTAGTAAATGTTGTGTTACCATTACCATTT

[0145] AGACTTTGCTATGGTAGAATGCTTGTGAGATAAAAAAATAGTAAAATGAAT

[0146] GTTGTGGGAAAAAATAAAGAAAATTTAGTAGTTCGCATAAAATATTACTTAT

[0147] ACAAATTAATGTTGACAAAGAAGAAACTTTATGAAGTGGACCATGTATGTT

[0148] TACACACCTATTTCCCTAAATAAAAGGCAAAACATAAACGCTCTTAACAAC

[0149] ACATATGGAAGTTTGTGCATCACAGCTTCTTGTTTCTCATAATCATACCCTCT

[0150] [[ID=二十九]]CTATCTCTCTCATATAACAAGAAAAAAGTTAAAATSEQ ID NO.4(Promoter sequence of BnaCLE20 in Westar)

[0151] TAAGCTGGAATATATGGTTTTAAGATATCTTTCCAATTTGTGTGAAAAGTACC

[0152] AATACTTATATAGTTTTCCATTTATTTGTGTGTGTGGTTTTGACCGCGAATTG

[0153] Note: The Chinese number "二十九" in the translation should be adjusted according to the actual Arabic numeral in the original text. Here, it's just a placeholder for the correct numbering in the translation process.GATCTTGGTTGTTAGCAATATACTCGGAGCTCGCTACTTTGAACTGTGAAAC

[0154] ATATTTTTTGGTCGGAATGGCTGAACTTCCCAACAGATCATGTTTTTATTATC

[0155] AGTTTATCTCGTTCCGATTGAGCCTGTACCAGAGTTAATTGACAAAGCGTGT

[0156] TCCAACGGTCATTTTTTGCAACTGTTTATATGTCATCACTTACTGAGTCGGTT

[0157] TAGGTTTTGGATCAGGTTACTAATATCCCGGTTCAGGTTTGGATTGGATGGGCC

[0158] AGATTTCTTACACCCCCAAATCCACTAGATTGGTCCAGTGTCATATACTCAC

[0159] TCATTCAAGGTTGGCAATATTCAATAATCATGAAATAGTATTAAATCAACCGC

[0160] AACTATGAAAACAGACATTTAATTCACAATATAACTGACCATCACGATCAT

[0161] TTCAATATTTGACATGATGATGAGATGTTGTGAATTTTGAATTAAACATATAAGAT

[0162] ATACTAGGCTGTAAATATGCTATGCCACTAGACCAAGGTCGTTTTCACAGGT

[0163] AAAGCGACTTAAAATCGTAGATAGGTTATATTTTATAAGTGATAATCATGAC

[0164] TTTGAATTAGTCTTTGAAGAATAGAAGGCAGTTTTTTTTTTCCGGTCTTAT

[0165] TTCTCAAAAGGTCAATAGAAAAATTAAACTTTATAAGTCACTTTGTCTCTTT

[0166] GTATTTGATATTATATTCTTGATTATGATATTTACACAAAATCATGTCGCTATTA

[0167] AAAGCTTCATCATCTGCATACGAACACTTTTCACCTTAAAGCTACATTATA

[0168] CTCACGCGTAAGTAGCAAACTCTGATATGTTTAAAGAAAAGTTTTACGTTTC

[0169] ACATGTGAAAATTAAAAAAATCAAATAAGGATCACTATATTTTTGGCACTT

[0170] CCACATTTATGGCCACGTACTGGTGATATTTTTTTTCCTTTTGAGTTGAATTC

[0171] CAAATCGTTGCATCTATAATCAATACTCACTTTTAAAACCGGCCATTTTAGG

[0172] TAGGAAGTGGGGTTCATTAGTTATAAAAAAGTAAAAGACACGTTAACACT

[0173] TAACATTGCAATAATATAGTACGTCAGTTTGTAGTAAATGTTGTGTTACCATT

[0174] ACCATTTAGACTTTGCTATGGTAGAATGCTTGTGAGATTAAAAAAAAATATAGTA

[0175] AAAATGAATGTTGTGGGAAAAAAAAAAAAATTTAGTAGTTCGCATATAATA

[0176] TTACTTATACAAATTAATGTTGACAAAGAAAAACTTTATGAAGTGGACCAT

[0177] GTATGTTTACACACCTATTTCCCTAAATAAAGGCAAAACATAAACGCTCTT

[0178] AACAACACATATGGAAGTTTGTGCATCACAGCTTCTTGTTTCTCATAATCAT

[0179] ACCCTCTCTATCTCTCTCATATAACAAGAAAAAAGTTAAAATSEQ ID NO.5 Clone F

[0180] atacaccaaatcgactctagaATGAATCAGGGTCGTCTTCTTTG

[0181] SEQ ID NO.6 Clone R

[0182] catggtaccggatccactagtTCGCTTGTTGTGCAAGGGA

[0183] SEQ ID NO.7 1300F CGCCAGGGTTTTCCCAGTCACGAC

[0184] SEQ ID NO.8 1300R AGCGGATAACAATTTCACACAGGA

[0185] SEQ ID NO.9 Quantitative FGACGACGCTTCTCCTCCG

[0186] SEQ ID NO.10 Quantitative R TCCCGTCTTAACCTTCCGT。

Claims

1. A rapeseed resistance gene BnaCLE20 for rapeseed breeding, characterized in that: The gene BnaCLE20 comprises a nucleotide sequence selected from the group consisting of: A. The sequence is the nucleotide sequence shown in SEQ ID NO.1; B. The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

2. A protein encoded by the rapeseed anti-bacterial sclerotinia rot-related gene BnaCLE20 according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A primer pair for cloning the rapeseed anti-bacterial sclerotinia sclerotiorum-related gene BnaCLE20 according to claim 1, characterized in that: The nucleotide sequences of the primer pair are shown in SEQ ID NO.5 and SEQ ID NO.

6.

4. A method for constructing a BnaCLE20 overexpression strain, characterized in that: The method comprises the following steps: 1) Ligate the BnaCLE20 gene into the pCAMBIA1300 vector; 2) Transform the successfully constructed vector into Agrobacterium tumefaciens GV3101; 3) The target rapeseed is injected using a transient injection method to ultimately obtain transgenic rapeseed leaves transiently overexpressing BnaCLE20; preferably, the target rapeseed variety is Zhongshuang 11.

5. A positive identification primer for overexpression rapeseed transgenic strains, characterized in that: The primer pairs include a 1300F / R primer pair with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8; and a pCAMBIA1300-BnaCLE20 vector-specific primer pair 1300F and 1300-CLE20R with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.

6.

6. A method for positive identification of overexpressed rapeseed transgenic lines, characterized in that: Using rapeseed leaf DNA that transiently overexpresses the target gene as a template, the two pairs of primers described in claim 5 are used to perform positive identification on the obtained overexpression rapeseed transgenic lines, and finally obtain positive plants.

7. A method for identifying rapeseed leaves transiently overexpressing a target gene, characterized in that: Extract gDNA from rapeseed and clone it using the primer pair described in claim 5.

8. A primer pair for fluorescence quantitative analysis of the rapeseed sclerotinia resistance-related gene BnaCLE20, characterized in that: The primer pair is shown as SEQ ID NO.9 and SEQ ID NO.

10.

9. A method for detecting the expression level of the rapeseed anti-bacterial sclerotinia rot-related gene BnaCLE20 according to claim 1 in rapeseed, characterized in that: The method comprises the following steps: 1) Extracting total RNA from rapeseed leaves; 2) reverse transcribing the RNA into cDNA; 3) Performing fluorescence quantitative PCR analysis using the primer pair described in claim 8 to quantitatively determine the expression level of the gene BnaCLE20.

10. Application of rapeseed resistance gene BnaCLE20 in rapeseed breeding, characterized in that: The applications include: A. Reduce the contents of reactive oxygen species, malondialdehyde and oxalic acid in rapeseed leaves infected with Sclerotinia sclerotiorum; B. Enhance the activity of catalase in rapeseed leaves.

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