Rapeseed gene BnLYK and its application in the prevention and control of Sclerotinia sclerotiorum

Abstract

The present invention belongs to the field of plant biotechnology. Specifically, the present invention provides a rapeseed gene BnLYK The present invention uses gene editing technology to knock out two homologous copies of rapeseed. BnLYK.A05 , BnLYK.C05 ,get BnLYK The double mutant rapeseed with gene mutation was cultivated and its disease resistance function was analyzed, which revealed for the first time that this gene has a positive regulatory function on rapeseed's resistance to bacterial sclerotinia. BnLYK The homozygous double mutant transgenic rapeseed plants of the gene are more susceptible to sclerotinia. The discovery of the invention has important significance and application value for the prevention and treatment of sclerotinia of rapeseed, and can be used in the breeding of new rapeseed varieties.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and specifically relates to Brassica napus BYZGR Genes and their application in the prevention and control of Sclerotinia sclerotiorum. Background Art

[0002] Before gene editing technology, natural, physical or chemical mutagenesis and random insertion of transgenic DNA were the main methods for generating gene mutations in cells. However, by their nature, none of these methods can achieve gene editing at a specific desired site. Traditional methods are at a disadvantage due to the high randomness of mutagenesis, low efficiency, and time-consuming, labor-intensive and high cost in most cases. Traditional gene editing tools mainly include zinc finger nucleases (ZFNs) and transcription activator effector nucleases (TALENs). It was not until 2020 that CRISPR technology gradually came into people's view. Compared with the first and second generation gene editing technologies, CRISPR technology is simple, highly operable, and its target recognition can identify the same site of different genes only through a short RNA sequence. These advantages have greatly promoted the development of this technology. After the successful modification of the Arabidopsis and rice genomes with the CRISPR system, the system has been widely used in gene editing of various crops such as rapeseed, wheat, cucumber and tomato.

[0003] Plant immune response is a series of defense responses triggered by plants after being subjected to exogenous stress, and these responses are regulated by genes. Plants use many immune receptors on the cell surface and inside cells to perceive various immunogenic signals related to pathogen infection and activate plant defense. Pattern recognition receptors (PRRs) on the cell surface are key factors for plants to perceive pathogen elicitors and can activate PTI, including two cell surface proteins: receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Therefore, using this type of gene to study the immune regulation function of plants against pathogens is conducive to creating excellent materials with good disease resistance and broad-spectrum resistance to different pathogens.

[0004] Sclerotinia is caused by infection by Sclerotinia sclerotiorum. Sclerotinia sclerotiorum is extremely harmful to plants and has more than 400 host species, including important economic crops such as tomatoes, beans, sunflowers and rapeseed. Sclerotinia sclerotiorum causes stem and leaf rot, reducing rapeseed oil production by about 10-20% each year. In severe cases, field yields drop by 80%, causing huge economic losses each year. At present, there is a lack of highly resistant varieties of sclerotinia sclerotiorum in rapeseed, and the prevention and control of sclerotinia sclerotiorum is mainly chemical control. However, chemical control faces problems such as ecological environmental pollution and chemical pesticide residues that endanger human health. Summary of the invention

[0005] Identifying key anti-sclerotinia sclerotinia regulatory genes and creating and rationally utilizing highly resistant varieties through breeding technology are of great significance for the prevention and control of rapeseed sclerotinia sclerotinia and green development. Based on this, the present invention discovered the Brassica napus type through long-term research and gene editing technology. Rapeseed resistance to Sclerotinia sclerotiorum gene BnLYK, BnLYK The gene consists of two homologous copies, namely BnLYK.A05 Genes and BnLYK.C05 gene; BnLYK.A05 The nucleotide sequence is shown in SEQ ID NO. 1, BnLYK.C05 The nucleotide sequence is shown in SEQ ID NO.2.

[0006] The present invention uses the Westar variety as the background and uses CRISPR / CAS9 technology to target and knock out BYZGR Two homologous copies of the gene ( BnLYK.A05 and BnLYK.C05 ), a homozygous double mutant line was obtained, which resulted in the BYZGR Loss of function, resulting in reduced resistance to Sclerotinia infection ， Sclerotinia disease is prone to occur.

[0007] Thus, the present invention provides a method for BYZGR Genes are used in the prevention and control of sclerotinia disease, that is, by upregulating the rape BYZGR The expression of the gene is used to control the sclerotinia disease of rapeseed, which is caused by infection with Sclerotinia sclerotiorum.

[0008] The present invention also provides a method for screening rapeseed with resistance to sclerotinia sclerotiorum by detecting BYZGR Gene, if it appears in rapeseed BYZGR If the homozygous double mutation of the gene leads to loss of function, the rapeseed will have poor resistance to sclerotinia. BYZGR If the expression of the gene is significantly higher than that of the wild type, then the variety has a strong ability to resist bacterial sclerotinia.

[0009] Based on the research of the present invention, the present invention also provides a Brassica oleracea Rapeseed BnLYK A method for preparing a homozygous mutant transgenic plant, the method comprising the following steps:

[0010] (1) Designed to simultaneously target BnLYK.A05 and BnLYK.C05 The universal sgRNA sequence is used as the target, and the sequence is cloned into the CRISPR / CAS9 vector; wherein the sgRNA sequence is shown in SEQ ID NO. 3, BnLYK.A05 The nucleotide sequence is shown in SEQ ID NO. 1, BnLYK.C05 The nucleotide sequence is shown in SEQ ID NO. 2;

[0011] (2) introducing the CRISPR / CAS9 vector containing sgRNA in step (1) into Agrobacterium with strong infectivity to rapeseed by electroporation;

[0012] (3) transferring the above-mentioned Agrobacterium vector into rapeseed by infecting the hypocotyl of rapeseed with Agrobacterium to obtain transgenic rapeseed plants;

[0013] (4) Testing the transgenic plants and screening and identifying the transgenic plants with homozygous genotypes.

[0014] The above method can be used for Brassica napus BYZGR Gene function research has important practical value for exploring plant immune and antibacterial mechanisms and signal pathways.

[0015] The present invention uses the Westar variety as the background and uses CRISPR / CAS9 technology to target and knock out BYZGR Two homologous copies of the gene ( BnLYK.A05 and BnLYK.C05 ), and obtained homozygous double-copy mutant strains. The specific mutation types are shown in the attached Figure 1 Furthermore, the present invention reveals that the mutation of this gene can significantly enhance the sensitivity of rapeseed to Sclerotinia sclerotiorum, and reveals the strong positive regulatory effect of this gene in rapeseed's resistance to Sclerotinia sclerotiorum, providing a new idea for the green prevention and control of rapeseed to Sclerotinia sclerotiorum in the future. Beneficial Effects

[0016] This invention is the first to discover rapeseed resistance to bacterial sclerotinia related genes BYZGR Genes have laid the foundation for the prevention and control of rapeseed sclerotinia and the breeding of disease-resistant rapeseed plants.

[0017] The invention provides Brassica napus BYZGR Preparation method of homozygous mutant transgenic plants, rapeseed BYZGR Research on gene function and plant immune and antibacterial mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The T2 generation of the transformed plant in the embodiment of the present invention BYZGR Two copies of homozygous mutation ( BnLYK.A05 and BnLYK.C05 ) in the sgRNA site nucleotide sequence and its targeting site, and the results showed that the sgRNA targeted the gene BnLYK.A05 This leads to a 5-base deletion, and the sgRNA targets the gene BnLYK.C05 This resulted in a 16-base deletion, meaning that the sgRNA targeted both BnLYK.A05 and BnLYK.C05. Gene.

[0019] Figure 2In the embodiments of the present invention, the wild type and BYZGR Results of inoculation of double-copy mutants with Sclerotinia sclerotiorum. Figure 2 A in the middle shows the phenotypic results after 24 hours of inoculation with Sclerotinia sclerotiorum. Figure 2 B shows the wild type and BYZGR Statistical quantitative analysis of the lesion area of ​​double-copy mutant plants. The results showed BYZGR The double-copy mutant plants showed a significantly more susceptible phenotype than the wild type 24 hours after inoculation, and the lesions of the mutants were significantly expanded. BYZGR The mutation significantly reduced the resistance of rapeseed to Sclerotinia sclerotiorum; DETAILED DESCRIPTION

[0020] Unless otherwise specified, the relevant reagents in the present invention are all conventional reagents, and the relevant operation steps are all conventional operations, which can be performed according to the operating instructions of the reagent supplier or the guidance of classic reference books such as "Molecular Cloning Experiment Guide". Embodiment 1:

[0021] 1.1 CRISPR / Cas9 vector construction

[0022] (1) Cloning of target sgRNA sequences

[0023] Obtained from the Brassica napus pan-genome Information Resource (BnPIR) BYZGR Two homologous genes ( BnLYK.A05 and BnLYK.C05 ) sequences, the sequence registration numbers are BnaA05T0293700WE and BnaC05G0347700WE, and the sequence design can simultaneously target BnLYK.A05 and BnLYK.C05 The universal sgRNA sequence was synthesized by the company and connected to the intermediate vector pCBC through PCR using PCBC as a template. The PCR product was then purified after gel electrophoresis and gel cutting. BnLYK.A05 The nucleotide sequence is shown in SEQ ID NO. 1, BnLYK.C05 The nucleotide sequence is shown in SEQ ID NO.2; the sgRNA sequence is shown in SEQ ID NO.3.

[0024] (2) Construction of CAS9 final vector

[0025] The intermediate vector is subjected to restriction digestion-enzyme ligation reaction to finally construct the target sgRNA sequence into the CRISPR / CAS9 vector. In this step, the endonuclease used is Bsa Ⅰ(Thermo Scientific™), and T4 ligase (ThermoScientific™) was used as the ligase. The enzyme digestion-enzyme ligation product was transformed into E. coli DH5α competent cells by heat shock method and activated in liquid LB medium at 37°C for 45 After 10 min, the cells were spread on plates with corresponding resistance and cultured overnight. A single colony was picked for verification the next day, and the positive colony was sent to the company for sequencing. Finally, a CRISPR / CAS9 vector carrying the correct sgRNA target sequence was obtained.

[0026] 1.2. Agrobacterium transformation of CRISPR / CAS9 vectors

[0027] The CRISPR / CAS9 vector carrying the sgRNA target sequence was transformed into the plant-infecting Agrobacterium strain GV3101 by electroporation, and then cultured and screened at 28°C on LB medium containing kanamycin and rifampicin antibiotics. Positive strains were subsequently identified by PCR, and finally positive single colonies were shaken and stored in a -80°C refrigerator with 15% glycerol.

[0028] 1.3. Creation of transgenic rapeseed plants by Agrobacterium-mediated hypocotyl genetic transformation

[0029] (1) Seed disinfection and germination

[0030] The seeds that had been exposed to the sun for two weeks were stirred in 70% ethanol for 2 minutes and then washed with sterile water for 2 minutes. Then they were stirred in 84 solution for 15 minutes and finally washed with sterile water for 5 times, each time for 5 minutes. The sterilized seeds were evenly arranged on M0 medium (MS: 2.2 g / L) and cultured for 5-6 days. All the above operations need to be carried out in a clean bench, and the experimental equipment used has been sterilized.

[0031] (2) Agrobacterium infection and co-cultivation

[0032] Take out the rapeseed seedlings that have been cultured for 5-6 days, cut the hypocotyls into 0.8-1.0 cm segments in DM buffer (ms: 4.4 g / L, sucrose: 30 g / L, acetosyringone: 100 umol / L), and then add Agrobacterium to OD=0.6 for infection for 10 min. Dry the infected hypocotyls on sterile filter paper for 30 min and then move them to M1 medium (ms: 4.4 g / L, sucrose: 30 g / L, 2,4-D: 1 mg / L, KT: 0.3 mg / L, acetosyringone: 100 umol / L) for two days. All the above operations need to be performed in a clean bench, and the experimental instruments and reagents used have been sterilized.

[0033] (3) Degerming culture

[0034] After two days of culture on M1, the seedlings were transferred to M2 medium (ms: 4.4 g / L, sucrose: 30 g / L, 2,4-D: 1 mg / L, KT: 0.3 mg / L, acetosyringone: 100 umol / L, STS: 30 mg / L, timentin: 300 mg / L) and cultured at 25°C for two weeks. All the above operations need to be carried out in a clean bench, and the experimental instruments and reagents used have been sterilized.

[0035] (4) Inducing differentiation and germination

[0036] The seedlings cultured in M2 for two weeks were transferred to M3 medium (MS: 4.4 g / L, glucose: 10 g / L, xylose: 0.25 g / L, MES: 0.6 g / L, ZT: 20 mg / L, IAA: 0.1 mg / L, Timentin: 300 mg / L) and subcultured every two weeks until green shoots differentiated.

[0037] (5) Rooting culture

[0038] The green shoots differentiated in step (4) were cultured on M4 rooting medium (MS: 4.4 g / L, sucrose: 10 g / L, TMT: 100 mg / L) until they took root.

[0039] (6) Obtaining transgenic plants through soil planting

[0040] The M4 T0 seedlings with fully developed root systems were transplanted into the soil until the seeds were finally harvested and sown for reproduction. Genomic DNA was extracted from leaves at the seedling stage, and those that were confirmed positive were sent to the company for sequencing, and finally a homozygous double-copy mutant rapeseed was obtained.

[0041] 1.4. BYZGR Disease resistance detection of double-copy homozygous mutants

[0042] (1) Activation of Sclerotinia sclerotiorum

[0043] Use an alcohol lamp to burn the sterilized inoculation loop of the laboratory-stored Sclerotinia sclerotiorum plate, place the inoculation loop with the bacterial surface facing down in the middle of the PDA solid culture medium, and culture it at 25°C for 3 days until the Sclerotinia sclerotiorum grows to the edge of the culture medium.

[0044] (2) Sclerotinia inoculation

[0045] Use a 5 mm diameter cork puncher to divide the Sclerotinia into small plates along the growing edge of the fungus, and then select the wild type and BYZGR The mutant rapeseed leaves were placed flat on the leaves to keep them moist. After 24 hours, photos were taken to record the disease situation. The area of ​​the lesions was then statistically analyzed. The experiment was repeated three times. BYZGRThe double-copy mutant plants showed a significantly more susceptible phenotype than the wild type 24 hours after inoculation, and the lesions in the mutants also expanded significantly. BYZGR The mutation significantly reduced rapeseed's resistance to Sclerotinia sclerotiorum.

[0046] In summary, the present invention uses CRISPR / CAS9 technology for the first time to obtain the rapeseed gene BYZGR The loss-of-function strain clarified the regulatory role of this gene on rapeseed sclerotinia disease.

[0047] The present invention utilizes CRISPR / Cas9 technology to target BYZGR Two homologous genes ( BnLYK.A05 and BnLYK.C05 ), and obtained a double-copy homozygous mutant through genetic transformation, and identified and analyzed the resistance phenotype of the mutant to Sclerotinia sclerotiorum. BYZGR The gene acts upstream of the plant immune response and can trigger the plant PTI immune response. It has the advantages of being broad-spectrum, strong, stable and persistent. Therefore, it can be used as a high-quality disease-resistant gene resource and provide guidance for the screening of highly resistant rapeseed varieties.

Claims

1. A method for screening rapeseed that is resistant to sclerotinia sclerotiorum, characterized in that: Detection of rapeseed BnLYK Gene, if rapeseed BnLYK If the gene has a homozygous double mutation resulting in functional loss, the rapeseed has poor resistance to bacterial sclerotinia; BnLYK The gene consists of two homologous copies, namely BnLYK.A05 Genes and BnLYK.C05 gene; BnLYK.A05 The nucleotide sequence is shown in SEQ ID NO. 1, BnLYK.C05 The nucleotide sequence is shown in SEQ ID NO.2.

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