Application of oilseed rape calcium channel gene BnCNGC3 in prevention and control of sclerotiniose

Abstract

The invention provides an application of a Brassica napus calcium channel gene BnCNGC3 in prevention and control of sclerotiniose, and particularly relates to an application of a Brassica napus calcium channel gene BnCNGC3 in obtaining a rape material with changed disease resistance by creating transgenic rape. According to the invention, overexpression transgenic rape of BnCNGC3 and CRISPR (clustered regularly interspaced short palindromic repeats) gene knockout rape are constructed, the positive regulation function of the gene on sclerotiniose resistance is disclosed for the first time, and application of the BnCNGC3 gene to acquisition of a high sclerotiniose resistance rape material by creating overexpression rape is provided; the invention also discloses an application of the BnCNGC3-CRISPR gene knockout rape to obtain the sclerotinia sclerotiorum rape material with weakened sclerotinia sclerotiorum resistance. The BnCNGC3 gene provided by the invention is a new gene resource suitable for creating and breeding new sclerotiniose-resistant rape materials and new varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an application of a rapeseed calcium ion channel gene BnCNGC3 in the prevention and control of sclerotinia disease. Background Art

[0002] 1. Plant gene function analysis technology Plant gene function is usually determined and clarified by comparing and analyzing the phenotype or function of abnormal gene expression and normal expression. Abnormal gene expression includes two categories: expression above normal level and expression below normal level. Expression above normal level is mainly over-expression, which is mainly achieved by connecting a strong promoter to drive the expression of the target gene. Expression below normal level is mainly achieved by RNA interference (RNAi), gene knockout and other methods. RNAi is achieved by constructing and transforming a hairpin structure containing the same sequence fragment with an intron or other non-expressed sequence inserted in the opposite direction, resulting in a reduction in the expression of the target gene. Gene knockout is achieved by inserting a long non-plant sequence into the target gene in the plant genome or removing the target gene, resulting in complete or nearly complete inhibition of the expression of the target gene. By constructing gene overexpression plants and / or RNAi plants, gene knockout mutants, and comparing and analyzing the differences in their phenotypes and traits with those of wild-type / normal plants, the regulatory function of the target gene on the trait can be clarified.

[0003] 2. Genetic regulation technology for plant disease resistance Plant disease resistance is the result of plant receptors recognizing pathogen ligands to activate disease resistance signal transduction and a series of defense responses. From a genetic point of view, after receptor-ligand recognition, genes involved in resistance regulation include early disease resistance signal transduction genes and late defense-related genes. Generally speaking, the effect of regulating disease resistance through early disease resistance signal transduction genes is better. Calcium is the second messenger. The calcium signal pathway is a recognized key early signal transduction pathway. The key calcium signal that drives plant disease resistance comes from the rapid and large amount of calcium influx from the outside of the cell membrane to the cytoplasm. It is currently known that the mechanism for stimulating calcium influx comes from calcium ion channels located in the plasma membrane, especially cyclic nucleotide-gated ion channels (CNGC). Using CNGC genes to create crop germplasm with enhanced disease resistance is expected to be effective, but there are currently few public reports in this regard. Currently, there is only one patent related to the application of Arabidopsis calcium ion channel gene AtCNGC3 and rapeseed calcium ion channel gene BnCNGC4 in the prevention and control of sclerotinia. There are 56 CNGC members in the rapeseed variety Double 11. Except for BnCNGC4, there are no public patents on the functions and applications of other member genes in the prevention and control of sclerotinia disease.

[0004] 3. Sclerotinia disease prevention and control technology Plant sclerotinia is caused by infection with Sclerotinia sclerotiorum. Sclerotinia sclerotiorum is a necrotrophic pathogenic fungus with a very wide host range. It is a major disease of oil crops and vegetable crops. It causes huge economic losses every year. Due to the lack of highly resistant varieties, chemical control is still an important means. Since some pesticides have problems such as ecological pollution, human and animal toxicity, and easy to make pathogens resistant, identifying important anti-sclerotinia regulatory genes and creating and using disease-resistant varieties are crucial for the green prevention and control of sclerotinia.

[0005] 4. Plant disease resistance breeding technology Plant disease resistance breeding technology is mainly divided into two categories: traditional disease resistance breeding and disease resistance breeding designed by genetic engineering and molecular modification. Traditional disease resistance breeding has a natural genetic isolation phenomenon, which significantly limits the scope of available disease resistance resources. Only disease resistance resources with close genetic relationships can be used, and multiple hybridization and backcrossing are required. Therefore, the breeding cycle is long and requires a lot of manpower and material resources. The genetic engineering and molecular modification design breeding method is to introduce exogenous disease resistance regulatory genes or modified genes into plants through Agrobacterium-mediated methods and other technologies, or to knock out or increase the expression of plant target genes through gene editing and other technologies, so that they can obtain disease resistance that they did not originally have or improve disease resistance. Therefore, the genetic engineering and molecular modification design breeding method breaks the limitations of natural genetic isolation, broadens the scope of available disease resistance resources, and has the characteristics of relatively simple and convenient operation, short cultivation cycle, and no need for a lot of manpower and material resources. In addition, varieties with broad-spectrum disease resistance can be created by modifying broad-spectrum disease resistance regulatory genes or introducing multiple genes with different disease resistance spectra. Therefore, it has the advantages of being particularly suitable for cultivating broad-spectrum and long-lasting disease-resistant varieties. Summary of the invention

[0006] The invention aims to provide an application of a rapeseed calcium ion channel gene BnCNGC3 in the prevention and control of sclerotinia disease. The rapeseed (Brassica napus) calcium ion channel gene BnCNGC3 has a positive regulatory function on disease resistance and can be applied to the creation of crop germplasm resistant to Sclerotinia sclerotiorum (Sclerotinia sclerotiorum).

[0007] The application is to obtain rapeseed materials with improved disease resistance by creating transgenic rapeseed, to obtain rapeseed materials with increased resistance to sclerotinia by creating transgenic rapeseed overexpressing BnCNGC3, or to obtain rapeseed materials with reduced resistance to sclerotinia by creating BnCNGC3 gene knockout rapeseed.

[0008] The present invention uses the cDNA of the double 11 variety in rapeseed (Brassica napus) as a template, and obtains the rapeseed gene BnCNGC3 by PCR cloning. The nucleotide sequence is shown in SEQ ID No.1. The open reading frame (ORF) of the gene is 2112bp long, and the encoded protein consists of 703 amino acids. The sequence is shown in SEQ ID No.2. The BnCNGC3 protein contains a transmembrane domain, a cyclic nucleotide binding domain and a CaM binding domain. The nucleotide sequence cloned by the present invention has a deletion of 3 consecutive bases and a difference of 4 bases from the ZS11C04G000660 nucleotide sequence of the rapeseed variety ZS11 in the Brassica_napuscv.ZS11 PacBio V1.0 database. The changes in these bases lead to the deletion of 1 amino acid and the change of 3 amino acids. Specifically, T75, T76, and C77 are missing, resulting in the loss of amino acid Ser26; G1589 becomes C, resulting in the change of amino acid Ser530 to Thr; A1731 becomes C, and the amino acid does not change; A2009 becomes G, resulting in the change of amino acid Lys670 to Arg; G2012 becomes A, resulting in the change of amino acid Ser671 to Asn.

[0009] Prior to the present invention, there was no public report on the function of this gene. The present invention constructed for the first time the overexpression transgenic rapeseed and CRISPR gene knockout rapeseed of this gene, carried out disease resistance analysis, and clarified the regulatory effect and mechanism of this gene on rapeseed's resistance to Sclerotinia sclerotiorum. The analysis results of the inoculated leaf lesion area, pathogen biomass, stem lesion length, etc. showed that compared with wild-type rapeseed plants, overexpression transgenic rapeseed plants were significantly more resistant to Sclerotinia sclerotiorum, while gene knockout plants were significantly more susceptible to Sclerotinia sclerotiorum, indicating that BnCNGC3 positively regulates rapeseed's resistance to Sclerotinia sclerotiorum.

[0010] Based on the function of the BnCNGC3 gene explained in the present invention, the application purpose of the present invention is to provide the application of the rapeseed BnCNGC3 gene in obtaining rapeseed materials with altered disease resistance by creating transgenic rapeseed, including (1) application in obtaining rapeseed materials with increased resistance to sclerotinia by creating transgenic rapeseed overexpressing BnCNGC3 (see the description in Example 1 for details); and (2) application in obtaining rapeseed materials with reduced resistance to sclerotinia by creating BnCNGC3-CRISPR gene knockout rapeseed (see the description in Example 2 for details).

[0011] The application of the rapeseed BnCNGC3 gene in obtaining a rapeseed material with enhanced resistance to sclerotinia sclerotiorum by creating a transgenic rapeseed overexpressing BnCNGC3 is specifically achieved by the following steps: (1) Construction and acquisition of BnCNGC3 gene overexpression structure: The BnCNGC3 gene open reading frame (ORF) was cloned into a plant expression vector so that it was expressed under the drive of a strong promoter; (2) Obtaining Agrobacterium for transformation of BnCNGC3 gene overexpression structure: The constructed BnCNGC3 gene overexpression structure is transformed into an Agrobacterium strain with strong infection ability to rapeseed by methods such as electric shock; (3) Creation and acquisition of transgenic rapeseed overexpressing BnCNGC3: The BnCNGC3 gene overexpression structure was introduced into rapeseed through Agrobacterium-mediated method to obtain the transgenic rapeseed overexpressing BnCNGC3 gene; (4) Obtaining homozygous lines of transgenic rapeseed overexpressing BnCNGC3: Using antibiotic resistance and BnCNGC3 gene expression as detection indicators, the segregation of traits in the offspring of transgenic plants was detected, and homozygous lines of transgenic rapeseed overexpressing BnCNGC3 whose offspring traits no longer segregated and could be stably inherited were obtained; (5) Screening, identification and acquisition of homozygous transgenic rapeseed lines overexpressing BnCNGC3 with enhanced resistance to sclerotinia sclerotinia: Using homozygous transgenic rapeseed lines overexpressing BnCNGC3 as materials, the resistance to sclerotinia sclerotinia was detected and analyzed, and transgenic rapeseed with the BnCNGC3 gene with enhanced disease resistance was obtained.

[0012] The application of the rapeseed BnCNGC3 gene in obtaining rapeseed materials with reduced resistance to sclerotinia sclerotiorum by creating BnCNGC3-CRISPR gene knockout rapeseed is achieved by the following steps: (1) Construction and acquisition of the CRISPR knockout structure of the BnCNGC3 gene: Three 20bp knockout targets were preset according to the BnCNGC3 genome sequence, and the target sequences were cloned into the plant expression vector pBSbdcas9i. The recombinant structure pBSbdcas9i-BnCNGC3 was obtained through colony PCR identification and sequencing; (2) Obtaining Agrobacterium transformed with the CRISPR knockout structure of the BnCNGC3 gene: The CRISPR knockout structure of the BnCNGC3 gene (pBSbdcas9i-BnCNGC3) was transformed into an Agrobacterium strain with strong infection ability to rapeseed by electroporation or other methods; (3) Creation and acquisition of rapeseed with CRISPR knockout structure of BnCNGC3 gene: The CRISPR knockout structure of BnCNGC3 gene was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed with CRISPR knockout structure of BnCNGC3 gene; (4) Obtaining homozygous lines of rapeseed with the CRISPR knockout structure of the BnCNGC3 gene: Screening with antibiotic resistance, detecting the trait segregation of the offspring of transgenic plants, and obtaining homozygous lines of rapeseed with the CRISPR knockout structure of the BnCNGC3 gene, in which the offspring traits no longer segregate and can be stably inherited; (5) Screening, identification and acquisition of homozygous rapeseed lines with CRISPR knockout structure of BnCNGC3 gene and reduced resistance to sclerotinia rot: The homozygous rapeseed lines with CRISPR knockout structure of BnCNGC3 gene (pBSbdcas9i-BnCNGC3) were used as materials to detect and analyze the resistance to sclerotinia rot, and obtain CRISPR knockout rapeseed with reduced resistance to sclerotinia rot.

[0013] Advantages of the present invention: (1) The BnCNGC3 gene provided by the present invention is a high-quality anti-bacterial sclerotinia disease regulatory gene resource, and the disease-resistant material obtained using the gene has the advantages of strong disease resistance. Rapeseed sclerotinia disease resistance is a quantitative trait resistance controlled by multiple genes. Generally speaking, the degree of regulation of the resistance by a single gene is low. Therefore, rapeseed sclerotinia disease resistant materials are very scarce worldwide, and there is no highly resistant material. The BnCNGC3 gene is a calcium ion channel gene and an early disease resistance regulatory gene. Its function is located upstream of signal transduction and has a good resistance regulation effect. Therefore, it is a high-quality disease resistance regulatory gene resource. Using CNGC to create disease-resistant varieties and germplasm is an economical, effective and safe way to greenly prevent and control diseases. Therefore, the BnCNGC3 gene is a new gene resource suitable for creating and breeding new materials and new varieties of rapeseed resistant to sclerotinia disease. (2) The period for obtaining disease-resistant materials is short. The methods for obtaining disease-resistant plant materials and varieties mainly include conventional traditional breeding methods and genetic engineering breeding methods using disease resistance regulatory genes. Traditional breeding methods have the disadvantages of limited range of available disease-resistant resources by natural genetic isolation, long breeding cycle, and requiring a lot of manpower and material resources. Genetic engineering breeding methods have the advantages of wide range of available disease-resistant resources, relatively simple and convenient operation, short breeding cycle, no need for a lot of manpower and material resources, and are particularly suitable for breeding broad-spectrum, durable, and highly disease-resistant varieties. The present invention utilizes the disease-resistant regulatory gene BnCNGC3 and adopts genetic engineering methods to create and cultivate rapeseed materials with high resistance to bacterial sclerotinia, which has the characteristics of short cycle and rapid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Provide evidence of various transgenic rapeseed plants obtained by the present invention, which is the gene expression detection of BnCNGC3 overexpression plants and the gene editing identification of CRISPR gene knockout plants, showing the gene expression detection results in BnCNGC3 overexpression plants and the editing form of the gene in gene knockout plants. Real-time fluorescence quantitative PCR was used to detect the expression level of the gene in BnCNGC3 gene overexpression (OE) plants (A), with rapeseed BnACTIN7 as the internal reference gene, and the expression level of ZS11 was set to 1. qRT-PCR analysis was performed three times biologically, each containing three technical replicates, and the expression results were statistically analyzed by Student's t-test using GraphPad Prism, and the data were expressed as mean ± standard deviation. The significance of the difference is indicated by * (**, P<0.001). The results showed that the expression level of BnCNGC3 in the two overexpression lines #3 and #15 was significantly increased, 16.2 times and 29.2 times that of the control, respectively (A). The two mutant strains #1 and #7 with successful gene knockout had different insertion mutation forms at the three common target sites of the two BnCNGC3 homologous genes located on chromosomes C04 and A05, but both produced a total of 2 bp insertions at the three target sites of the two homologous genes, resulting in the inability to encode functional BnCNGC3 normally. C04 and BnCNGC3 A05 Gene (B). These plants are true BnCNGC3 gene overexpression plants and CRISPR gene knockout plants.

[0015] Figure 2 Provide evidence for the anti-sclerotinia function of the BnCNGC3 gene, and BnCNGC3 positively regulates rapeseed's resistance to Sclerotinia. Show that BnCNGC3 positively regulates rapeseed's resistance to Sclerotinia. Rapeseed BnCNGC3 transgenic plants were inoculated with Sclerotinia UF-1. The figure shows the phenotype (A), lesion area and hyphal biomass quantitative statistics (B) of the leaves of overexpression plants and knockout mutant plants, as well as the phenotype (C) and lesion length (D) analysis results after stem inoculation. The inoculation experiment was repeated three times. GraphPad Prism was used to perform Student's t-test statistical analysis on the lesion area, and the data are expressed as mean ± standard deviation. The significance of the difference is indicated by different numbers of * (*, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001). Leaf inoculation analysis showed that the overexpression plants showed a significantly more disease-resistant phenotype than the control (ZS11) plants, while the knockout mutant plants were significantly more susceptible to the disease than the control (A). Quantitative analysis of lesion area showed that the lesion areas of the two strains of overexpression plants were 142.3 mm 2 and 144.0mm2 , significantly lower than the control group of 234.2 mm 2 , only 60.8% and 61.5% of the control (B); bacterial count determination also showed that the bacterial count in the lesions of the overexpression plants was significantly lower than that of the control plants. The lesion areas of the two knockout mutant lines were 293.2 mm 2 and 282.8mm 2 , significantly higher than the control group of 234.2 mm 2 , which were 1.3 and 1.2 times of the control (B); bacterial load determination also showed that the amount of bacteria contained in the lesions of the knockout mutant plants was significantly higher than that of the control plants. The results of stem inoculation analysis showed that the length of the stem lesions of the two strains of overexpression plants were 17.8 mm and 15.58 mm, respectively, which were significantly lower than the 30.48 mm of the control, and only 58.4% and 51.1% of the control (D); while the length of the stem lesions of the two knockout mutant strains were 45.66 mm and 50.55 mm, respectively, which were significantly higher than the 30.48 mm of the control, and were 1.5 and 1.7 times of the control (D). These results indicate that BnCNGC3 positively regulates rapeseed's resistance to Sclerotinia sclerotiorum. DETAILED DESCRIPTION

[0016] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0017] Example 1 The present invention cloned a rapeseed gene BnCNGC3, and for the first time, by constructing an overexpressed transgenic rapeseed, clarified the function of the gene in conferring rapeseed resistance to sclerotinia. Overexpression of the BnCNGC3 gene leads to a significant increase in rapeseed resistance to sclerotinia. Therefore, a new rapeseed material with enhanced resistance to sclerotinia can be created by constructing a homozygous line of overexpressed rapeseed of the BnCNGC3 gene, which can be used for the creation and breeding of sclerotinia-resistant rapeseed varieties, analysis of the function and mechanism of action of the BnCNGC3 gene, and the like. The main steps of cloning the BnCNGC3 gene, analyzing its function and mechanism, and creating and obtaining new rapeseed materials with enhanced resistance to sclerotinia include:

[0018] 1) Cloning and conservation of the rapeseed BnCNGC3 gene The rapeseed BnCNGC3 gene provided by the present invention is cloned by the following steps: First, primers BnCNGC3-F (5'-atg gag atg atg aat ctc aaa-3') (sequence as shown in SEQ ID No.3) and BnCNGC3-R (5'-tta ggt ttc atc cat agg aaa-3') (sequence as shown in SEQ ID No.4) are designed according to the BnCNGC3 sequence in the rapeseed genome database. TRIZOL reagent was used to extract total RNA from the leaves of rapeseed variety Zhong Shuang 11, and the high-fidelity enzyme pfu-mediated RT-PCR method was used to amplify and obtain BnCNGC3 cDNA. The PCR product was recovered by 1% agarose gel electrophoresis and then purified by cutting. The pEASY-Blunt Cloning Vector vector was connected and transformed into Escherichia coli DH5α by heat shock. The bacteria were cultured overnight in LB medium, and the plasmid was extracted. The PCR method using BnCNGC3-F / BnCNGC3-R as the primer pair was used to test whether the extracted plasmid contained the BnCNGC3 gene. Finally, the company was sent for sequencing verification, thereby successfully cloning and obtaining the full-length cDNA sequence of the BnCNGC3 gene on the C04 chromosome. The nucleotide sequence of BnCNGC3 is shown in SEQ ID No.1, the open reading frame (ORF) of the gene is 2112bp long, and the encoded protein consists of 703 amino acids, and its sequence is shown in SEQ ID No.2. The gene encoding product contains a transmembrane domain, a cyclic nucleotide binding domain, and a CaM binding domain. After BLAST analysis, it was found that the BnCNGC3 gene sequence cloned in the present invention had a nucleotide sequence identity of 86% and an amino acid sequence identity of 84% compared with the Arabidopsis CNGC3; compared with the ZS11C04G000660 nucleotide sequence of the rapeseed variety ZS11 in the Brassica_napus cv.ZS11 PacBio V1.0 database, there were 3 consecutive base deletions and 4 base differences, and the changes in these bases led to the deletion of 1 amino acid and the change of 3 amino acids. Specifically, T75, T76, and C77 were deleted, resulting in the deletion of amino acid Ser26; G1589 changed to C, resulting in the amino acid Ser530 changed to Thr; A1731 changed to C, and the amino acid did not change; A2009 changed to G, resulting in the amino acid Lys670 changed to Arg; G2012 changed to A, resulting in the amino acid Ser671 changed to Asn. Before the present invention, there was no public report on the function of the BnCNGC3 gene.

[0019] E. coli carrying the pEASY-BnCNGC3 vector were transformed and stored in a -80°C refrigerator. The strain can be activated at any time, the plasmid can be extracted, and the BnCNGC3 gene can be subcloned into the target vector by PCR amplification for transgenic research.

[0020] 2) Construction and acquisition of BnCNGC3 gene overexpression structure According to the BnCNGC3 sequence cloned in the present invention, primers BnCNGC3-F2 (5'-agagaacacgggggactctag atg gag atg atg aat ctc aaa-3', the italic part is a sequence containing an Xba I restriction site and consistent with the sequence on the vector pCAMBIA1300) (the sequence is shown in SEQ ID No.5), and BnCNGC3-R2 (5'-gtccttgtagtccatgtcgac ggt ttc atc cat agg aaa ctc-3', the italic part is a sequence containing a Sal I restriction site and consistent with the linearized vector pCAMBIA1300) (the sequence is shown in SEQ ID No.6). The pEASY-BnCNGC3 plasmid obtained in 1) of Example 1 was used as a template, and BnCNGC3-F2 / BnCNGC3-R2 was used as a primer pair to obtain the target gene by PCR amplification.

[0021] The overexpression vector pCAMBIA1300 plasmid was double-digested with Xba I and Sal I, and the amplified target gene was homologously recombined with the pCAMBIA1300 vector after digestion. The reagents used in this experiment were: PCR one-step directional cloning kit. The ligation product was transformed into E. coli, screened on kanamycin plates, identified by PCR and sequenced to obtain the BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3. The overexpression vector pCAMBIA1300 drives the expression of the target gene with the CaMV 35S promoter and carries the FLAG and HA double tags, which is convenient for molecular identification and gene function research.

[0022] 3) Obtaining Agrobacterium transformed with the BnCNGC3 gene overexpression construct pCAMBIA1300-BnCNGC3 The BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3 was transformed into an Agrobacterium strain with strong infectivity to rapeseed, such as EHA105, by electroporation and other methods, and the transformants were screened on YEP medium containing kanamycin, and then double-digested with XbaI and Sal I and identified by PCR to obtain Agrobacterium carrying the BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3, which was used for the genetic transformation of rapeseed in the next step.

[0023] 4) Creation and acquisition of the BnCNGC3 gene overexpression construct pCAMBIA1300-BnCNGC3 rapeseed The BnCNGC3 gene overexpression construct pCAMBIA1300-BnCNGC3 was introduced into rapeseed by Agrobacterium-mediated method to obtain the rapeseed T0 generation transformed with pCAMBIA1300-BnCNGC3. The specific operation steps are as follows:

[0024] (i) Seed cleaning and germination 75% ethanol for 30-60s, sterile water once, 1min / time; 0.15% mercuric chloride for 10min, wash twice with sterile water, 1min / time; wash with sterile water for 30min, inoculate on sterile filter paper and dry; inoculate the seeds in culture bottles and culture in the dark at 23℃ for 5-6d.

[0025] (ii) Pre-culture The germinated hypocotyls of rapeseed seedlings were cut into 0.4-0.6 cm segments, inoculated into pre-culture medium, and cultured at 23°C for 2-3 days.

[0026] (iii) Agrobacterium infection and co-cultivation Pick Agrobacterium in the infection solution and prepare OD 600 = 0.2 Agrobacterium resuspension, inoculate the explants in the Agrobacterium suspension for 10 minutes, inoculate the infected explants on sterile filter paper to dry, inoculate on co-culture medium, and culture at 23°C in the dark for 48-72 hours.

[0027] (iv) Degerming (extended screening) The co-cultivated explants were inoculated on sterile culture medium and cultured at 23°C under light for 6 days.

[0028] (v) Screening / differentiation The sterilized explants were inoculated on screening / differentiation medium, 30 explants per dish, cultured at 23°C under light, and the plate was changed every 15 days.

[0029] (vi) Rooting culture The differentiated buds were inoculated into rooting medium and cultured at 23°C under light until roots formed.

[0030] (vii) Detection The CTAB method was used to extract rapeseed genomic DNA, and PCR was used to detect resistance genes.

[0031] (viii) Soil Planting After the kanamycin-resistant seedlings have grown a complete root system, the T0 generation sterile seedlings are incubated with the lid open at 27°C for 2 days, then transplanted into the soil, placed in an incubator for routine management, and finally harvested to obtain T0 seeds.

[0032] 5) Screening, identification and acquisition of homozygous rapeseed lines transformed with the BnCNGC3 gene overexpression construct pCAMBIA1300-BnCNGC3 Hygromycin resistance and BnCNGC3 gene expression were used as detection indicators to detect the segregation of traits in the offspring of transgenic plants. Hygromycin resistance screening was carried out on rapeseed seeds on plates containing hygromycin to observe whether they could grow normally into healthy seedlings on hygromycin resistance plates. Gene expression was detected by real-time fluorescence quantitative PCR and other methods. Obtained rapeseed homozygous lines of the BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3, whose offspring traits no longer separated and could be stably inherited.

[0033] The present invention obtained two rapeseed homozygous lines, OE-line 3 and OE-line 15, which can all grow normally into healthy seedlings on hygromycin resistant plates, and the expression levels of BnCNGC3 gene are significantly higher than those of the control plants, which are 16.2 times and 29.2 times ( Figure 1 A). These results indicate that these plants are true BnCNGC3 gene overexpressing plants.

[0034] 6) Disease resistance analysis of homozygous rapeseed lines overexpressing BnCNGC3 gene The homozygous rapeseed line overexpressing the BnCNGC3 gene obtained in 5) was used as material, and its resistance to rapeseed sclerotinia sclerotiorum was detected and analyzed by inoculating it with Sclerotinia sclerotiorum, thereby clarifying the regulatory role of the BnCNGC3 gene on rapeseed sclerotinia sclerotiorum resistance, laying the foundation for using this gene to create and obtain rapeseed resistant to sclerotinia sclerotiorum.

[0035] Activation culture of Sclerotinia sclerotiorum: Select full and uncontaminated sclerotia of Sclerotinia sclerotiorum, cut the sclerotia into two halves with a sterile blade burned by an alcohol lamp, place them with the cut surface facing down on a PDA solid plate, and culture them at 23℃ in the dark for 3 days. Use a 4mm diameter puncher to punch out mycelium blocks 3 to 5mm inward from the edge of the colony, and inoculate them with the mycelium side facing down on a new PDA solid plate. Culture them at 23℃ in the dark for about 36 hours before inoculation.

[0036] Inoculation of Sclerotinia: Select rapeseed plants with consistent growth for inoculation. Use a 4mm diameter puncher to punch out hyphae blocks 3-5mm inward from the edge of the colony, and inoculate the hyphae face down into the middle of the fully developed leaves. Two hyphae blocks are symmetrically inoculated on the left and right halves of each leaf, covered with film to keep moisture, and placed in a 23℃ greenhouse for cultivation. After an appropriate time (about 30 hours), take photos and record. Similarly, the hyphae are inoculated face down onto the stems of plants in the flowering period, and one hyphae block is inoculated on each stem. Cover with film to keep moisture, and place in a 23℃ greenhouse for cultivation. After an appropriate time (about 30 hours), take photos and record, and the area of ​​the lesions is analyzed using ImageJ software.

[0037] The inoculation experiment was repeated three times. The lesion area was statistically analyzed by Student's t-test. The results showed that the BnCNGC3 overexpressing plants were significantly more resistant to the disease than the non-transgenic control plants (ZS11). Figure 2 A, C). Quantitative analysis of the lesion area showed that the leaf lesion area of ​​the ZS11 control plant was 234.2 mm 2 The leaf lesion areas of the two strains of overexpressing plants were 142.3 mm 2 and 144.0mm 2 , only 60.8% and 61.5% of the control group ( Figure 2 B). The length of the stem lesions of the ZS11 control plants was 30.48 mm, while the length of the stem lesions of the two strains of the overexpressing plants were 17.8 mm and 15.58 mm, respectively, which were only 58.4% and 51.1% of the control ( Figure 2 D).

[0038] These results show that the resistance of BnCNGC3 overexpressing plants to sclerotinia is significantly higher than that of non-transgenic control plants. Overexpression of the BnCNGC3 gene leads to a significant increase in the resistance of rapeseed to sclerotinia, so BnCNGC3 plays a positive regulatory role in the resistance of rapeseed to sclerotinia. The present invention successfully creates a new rapeseed material with high resistance to sclerotinia by constructing BnCNGC3-OE rapeseed.

[0039] Example 2 According to the positive regulatory function of the rapeseed gene BnCNGC3 on sclerotinia resistance explained in the present invention, a technical system for constructing CRISPR gene knockout rapeseed of the gene and using genetic engineering technology to create and obtain new rapeseed materials with reduced resistance to sclerotinia resistance has been established. The main steps include:

[0040] (1) Construction and acquisition of CRISPR knockout construct of BnCNGC3 gene According to the BnCNGC3 genome sequence, three suitable targets were designed, each gRNA unit contained 20 bp gRNA and a gRNA backbone, and the three gRNA units were combined as a complete knockout functional sequence. Using it as a template, primers BnCNGC3-CR-F1 (5'-cagtggtctc aag tga aca aag cac cag tgg tct agt ggt ag-3', the italic part is a sequence containing a Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (the sequence is shown in SEQ ID No.7) and BnCNGC3-CR-R1 (5'-cagtggtctc aaa acg agg aat agg gag aat agaatg cac cag-3', the italic part is a sequence containing a Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (the sequence is shown in SEQ ID No.8) were used for amplification. The vector pBSbdcas9i was digested with Bsa I (Eco31 I), and recombined with T4 ligase, followed by transformation and kanamycin medium plate screening. Positive single clones were picked and PCR identified using pBSbdcas9i identification primers Pbw2+ (5'-gca acg ctc tgt cat cgt tac aat-3') (sequence as shown in SEQ ID No.9) and Pbw2- (5'-gcg att aag ttg ggt aac gcc agg g-3') (sequence as shown in SEQ ID No.10). Further sequencing and identification was performed to obtain the CRISPR knockout structure pBSbdcas9i-BnCNGC3.

[0041] (2) Obtaining Agrobacterium transformed with the CRISPR knockout construct of the BnCNGC3 gene The BnCNGC3 gene CRISPR knockout structure (pBSbdcas9i-BnCNGC3) was transformed into an Agrobacterium strain with strong infection ability to rapeseed, such as EHA105, by electroporation and other methods. The transformants were screened on YEP medium containing kanamycin and then identified by PCR to obtain Agrobacterium carrying the BnCNGC3 gene CRISPR knockout structure pBSbdcas9i-BnCNGC3.

[0042] (3) Creation and acquisition of BnCNGC3 gene CRISPR knockout rapeseed The BnCNGC3 gene CRISPR knockout structure pBSbdcas9i-BnCNGC3 was introduced into rapeseed by Agrobacterium-mediated method to obtain the rapeseed T0 generation transformed with pBSbdcas9i-BnCNGC3. The specific operation steps are the same as those described in 4) of Example 1. BnCNGC3-CR-F2 (5'-gga gat gat gaa tct caa aag aaa cac-3') (sequence as shown in SEQ ID No.11) and BnCNGC3-CR-R2 (5'-acg cat gaa tgc aac ctt tta tc-3') (sequence as shown in SEQ ID No.12) were used to detect whether the transformed seedlings were gene-edited seedlings. The gene-edited seedlings were sequenced and analyzed near the target site, and finally determined to be mutant plants with successful knockout of the BnCNGC3 gene.

[0043] (4) Obtaining homozygous rapeseed lines with CRISPR knockout of BnCNGC3 gene The segregation of traits of the offspring of transgenic plants was detected by antibiotic resistance screening. The test was carried out on rapeseed seeds on plates containing Basta resistance to observe whether they can grow normally into healthy seedlings on Basta resistance plates. The homozygous rapeseed lines with the BnCNGC3 gene CRISPR knockout structure pBSbdcas9i-BnCNGC3, whose offspring traits no longer separated and could be stably inherited, were obtained. All these homozygous rapeseed lines can grow normally into healthy seedlings on Basta resistance plates.

[0044] The present invention obtains a homozygous BnCNGC3-CRISPR gene knockout rapeseed line that can grow normally into healthy seedlings on Basta resistance plates. Sequencing results show that the two mutant strains #1 and #7 have different insertion mutation forms at the three common target sites of the two BnCNGC3 homologous genes located on chromosomes C04 and A05, but both have a total of 2bp insertions at the three target sites of the two homologous genes, resulting in the inability to normally encode functional BnCNGC3. C04 and BnCNGC3 A05 Gene( Figure 1 B). These results indicate that these plants are true BnCNGC3 gene CRISPR knockout plants.

[0045] (5) Screening, identification and acquisition of homozygous rapeseed lines with reduced resistance to sclerotinia sclerotinia by CRISPR knockout of the BnCNGC3 gene The homozygous rapeseed line transformed with the BnCNGC3 gene CRISPR knockout structure (pBSbdcas9i-BnCNGC3) was used as the material to detect and analyze the resistance to sclerotinia. The inoculation method and disease resistance evaluation were the same as those described in 6) of Example 1).

[0046] The results of the Sclerotinia inoculation analysis of the leaves of the BnCNGC3-CRISPR plants constructed by the present invention showed that the BnCNGC3-CRISPR gene knockout plants were significantly more susceptible to the disease than the non-transgenic plants (ZS11) control ( Figure 2 A). Quantitative analysis of the lesion area showed that the lesion area of ​​the leaves of the ZS11 control plant was 234.2 mm 2 The lesion areas of the leaves of the two knockout mutant lines were 293.2 mm 2 and 282.8mm 2 , which was significantly higher than that of the control plants ( Figure 2 B). The bacterial count also showed that the bacterial count in the lesions of the knockout mutant plants was significantly higher than that in the control plants ( Figure 2 B). The results of stem inoculation analysis also showed that the knockout plants were significantly more susceptible to disease than the non-transgenic plants (ZS11) ( Figure 2 C). Quantitative analysis of lesion length showed that the length of the stem lesions of the ZS11 control plants was 30.48 mm, while the length of the stem lesions of the two knockout mutant lines were 45.66 mm and 50.55 mm, respectively, which were significantly longer than those of the control plants ( Figure 2 D). It shows that the resistance of BnCNGC3-CRISPR plants to Sclerotinia sclerotiorum is significantly lower than that of non-transgenic control plants. The inhibition of BnCNGC3 gene expression leads to a significant reduction in the resistance of rapeseed to Sclerotinia sclerotiorum. The present invention successfully creates and obtains a new rapeseed material with reduced resistance to Sclerotinia sclerotiorum by constructing BnCNGC3-CRISPR gene knockout rapeseed.

[0047] In summary, the present invention combines Figure 1-Figure 2 The results revealed for the first time that the rapeseed calcium channel gene BnCNGC3 plays a positive regulatory role in rapeseed's resistance to Sclerotinia sclerotinia, provided the application approach and technology of BnCNGC3 in creating Sclerotinia sclerotinia-resistant crop germplasm, provided an example, and successfully obtained highly resistant Sclerotinia sclerotinia-resistant rapeseed.

Claims

1. An application of a rapeseed (Brassica napus) calcium ion channel gene BnCNGC3 in the prevention and control of sclerotinia disease, characterized in that: The nucleotide sequence of the gene BnCNGC3 is shown as SEQ ID No.1, and the protein sequence encoded by it is shown as SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The application is to obtain rapeseed materials with altered disease resistance by creating transgenic rapeseed (Brassica napus).

3. The use according to claim 2, characterized in that: Application in obtaining rapeseed materials with enhanced resistance to sclerotinia sclerotinia by creating transgenic rapeseed overexpressing BnCNGC3.

4. The use according to claim 2, characterized in that: Application in obtaining rapeseed materials with weakened resistance to sclerotinia disease by creating BnCNGC3-CRISPR gene knockout rapeseed.

5. The use according to claim 3, characterized in that: The application of rapeseed materials with enhanced resistance to sclerotinia sclerotinia is achieved by the following steps: (1) Construction and acquisition of BnCNGC3 gene overexpression structure The open reading frame of the BnCNGC3 gene was cloned into a plant expression vector so that it was expressed under the drive of a strong promoter; (2) Obtaining Agrobacterium transformed with BnCNGC3 gene overexpression structure The constructed BnCNGC3 gene overexpression structure was transformed into an Agrobacterium strain with strong infection ability to rapeseed by electroporation; (3) Creation and acquisition of transgenic rapeseed overexpressing BnCNGC3 The BnCNGC3 gene overexpression structure was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed transformed with the BnCNGC3 gene overexpression structure; (4) Obtaining homozygous transgenic rapeseed lines overexpressing BnCNGC3 The antibiotic resistance and BnCNGC3 gene expression were used as detection indicators to detect the segregation of the traits of the offspring of the transgenic plants, and the homozygous transgenic rapeseed lines with overexpression of BnCNGC3 that could be stably inherited and whose offspring traits no longer separated were obtained; (5) Screening, identification and acquisition of homozygous transgenic rapeseed lines overexpressing BnCNGC3 with enhanced resistance to sclerotinia sclerotinia Using the homozygous transgenic rapeseed overexpressing BnCNGC3 as material, the resistance to sclerotinia sclerotinia was detected and analyzed, and transgenic rapeseed with BnCNGC3 gene with enhanced disease resistance was obtained.

6. The use according to claim 4, characterized in that: The application of the rape material with reduced resistance to sclerotinia sclerotinia is achieved by the following steps: (1) Construction and acquisition of CRISPR knockout construct of BnCNGC3 gene The three specific sequence fragments of the BnCNGC3 gene were cloned into the CRISPR vector pBSbdcas9i to obtain the CRISPR knockout structure pBSbdcas9i-BnCNGC3; (2) Obtaining Agrobacterium transformed with the CRISPR knockout construct of the BnCNGC3 gene The BnCNGC3 gene CRISPR knockout structure (pBSbdcas9i-BnCNGC3) was transformed into an Agrobacterium strain with strong infection ability to rapeseed by electroporation; (3) Creation and acquisition of BnCNGC3 gene CRISPR knockout rapeseed The CRISPR knockout structure of the BnCNGC3 gene was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed transformed with the CRISPR knockout structure of the BnCNGC3 gene; (4) Obtaining homozygous rapeseed lines with CRISPR knockout of BnCNGC3 gene The segregation of traits in the offspring of transgenic plants was detected by antibiotic resistance screening, and homozygous rapeseed lines with the CRISPR knockout structure of the BnCNGC3 gene were obtained, in which the offspring traits no longer separated and were stably inherited; (5) Screening, identification and acquisition of homozygous rapeseed lines with CRISPR knockout of the BnCNGC3 gene with reduced resistance to sclerotinia sclerotinia The rapeseed homozygous line transformed with the BnCNGC3 gene CRISPR knockout structure (pBSbdcas9i-BnCNGC3) was used as the material to detect and analyze the resistance to sclerotinia sclerotinia, and to obtain the BnCNGC3 gene CRISPR knockout rapeseed homozygous line with weakened resistance to sclerotinia sclerotinia.

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