Application of Brassica napus calcium channel gene BnCNGC3 in sclerotinia prevention and control

Abstract

The application provides application of a Brassica napus calcium ion channel gene BnCNGC3 in sclerotinia prevention and control, and is obtained by creating transgenic rapeseed to change the rapeseed material with disease resistance. The application constructs a BnCNGC3 overexpression transgenic rapeseed and a CRISPR gene knockout rapeseed, firstly discloses the positive regulation function of the gene on sclerotinia resistance, provides application of the BnCNGC3 gene in obtaining high-sclerotinia-resistant rapeseed material by creating overexpression rapeseed, and application of the BnCNGC3-CRISPR gene knockout rapeseed in obtaining sclerotinia-resistant rapeseed material with weakened sclerotinia resistance. The BnCNGC3 gene provided by the application is a new gene resource suitable for creating and breeding new sclerotinia-resistant rapeseed materials and new varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and relates to application of a Brassica napus calcium ion channel gene BnCNGC3 in sclerotinia disease prevention and control. BACKGROUND

[0002] 1. Plant gene function analysis technology

[0003] The function of a plant gene is usually determined and clarified by comparing and analyzing the phenotype or function of the gene under the condition of over-expression and normal expression. The over-expression of the gene includes two categories: over-expression higher than normal expression and over-expression lower than normal expression. The over-expression higher than normal expression is mainly over-expression, which is mainly achieved by connecting a strong promoter to drive the expression of the target gene. The over-expression lower than normal expression is mainly achieved by RNA interference (RNAi) and gene knock-out. The RNAi is achieved by constructing and transforming a hairpin structure containing a fragment of the same sequence in opposite directions and other non-expressed sequences, so as to reduce the expression of the target gene. The gene knock-out is achieved by inserting a long non-plant sequence or removing the target gene in the plant genome, so as to completely or nearly completely inhibit the expression of the target gene. By constructing over-expression plants and / or RNAi plants and gene knock-out mutants, and comparing and analyzing the differences between the phenotypes and traits of the plants and wild type / normal plants, the regulatory function of the target gene on the traits can be determined.

[0004] 2. Plant disease resistance genetic regulation technology

[0005] Plant disease resistance is the result of activating disease resistance signaling and activating a series of defense responses by plant receptors recognizing pathogen ligands. From the perspective of genetics, after the recognition of receptors and ligands, the genes involved in resistance regulation include early disease resistance signaling genes and late defense-related genes. Generally speaking, the effect of disease resistance regulation by early disease resistance signaling genes is better. Calcium is a second messenger. Calcium signaling pathway is a recognized key early signaling pathway. The key calcium signal driving plant disease resistance comes from the rapid and massive calcium influx from the cell membrane to the cytoplasm. The mechanism of triggering calcium influx is currently known to come from calcium 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 few public reports on this aspect. Currently, only one patent related to the application of Arabidopsis thaliana calcium channel gene AtCNGC3 and one Brassica napus calcium channel gene BnCNGC4 in the control of sclerotinia stem rot has been published. There are 56 members of CNGC in the Brassica napus variety Shuang 11, and the function and application of other member genes in the control of sclerotinia stem rot have not been published.

[0006] 3. Sclerotinia stem rot control technology

[0007] Plant sclerotinia stem rot is caused by Sclerotinia sclerotiorum infection. Sclerotinia sclerotiorum is a dead body nutrition type pathogenic fungus with a very wide host range, and is the main 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. Due to the ecological pollution, toxicity to humans and animals, and easy to produce drug resistance of some pesticides, it is very important to identify important sclerotinia stem rot resistance regulation genes, create and use disease-resistant varieties for green control of sclerotinia stem rot.

[0008] 4. Plant disease resistance breeding technology

[0009] Plant disease resistance breeding techniques are mainly divided into traditional disease resistance breeding and disease resistance breeding by genetic engineering and molecular modification design. Traditional disease resistance breeding is significantly limited in the use range of disease resistance resources due to natural genetic isolation, and can only use disease resistance resources with close genetic relationship, and needs multiple crosses and backcrosses, etc. Therefore, the breeding cycle is long, and a large amount of manpower and material resources are needed. The genetic engineering and molecular modification design breeding method is to introduce an exogenous disease resistance regulatory gene or a modified gene into a plant by Agrobacterium-mediated method, or to knock out or increase the expression of the target gene of the plant by gene editing technology, so that the plant obtains the original disease resistance or improves the disease resistance. Therefore, the genetic engineering and molecular modification design breeding method breaks the limitation of natural genetic isolation, widens the use range of disease resistance resources, and has the characteristics of relatively simple and convenient operation, short breeding cycle and no need for a large amount of manpower and material resources. In addition, a broad-spectrum disease resistance regulatory gene can be modified, or multiple genes with different disease resistance spectra can be introduced to create a variety with broad-spectrum disease resistance. Therefore, it has the advantages of being particularly suitable for breeding broad-spectrum and persistent disease resistance varieties. SUMMARY

[0010] The purpose of the present application is to provide an application of Brassica napus calcium ion channel gene BnCNGC3 in sclerotinia disease prevention and control. The Brassica napus calcium ion channel gene BnCNGC3 has a positive regulation function on disease resistance, and can be applied to the creation of Sclerotinia sclerotiorum crop germplasm.

[0011] The application is the application of the transgenic Brassica napus to obtain disease resistance changed Brassica napus material. It is the application of the transgenic Brassica napus overexpressing BnCNGC3 to obtain Brassica napus material with increased sclerotinia disease resistance, or the application of the Brassica napus with BnCNGC3 gene knockout to obtain Brassica napus material with reduced sclerotinia disease resistance.

[0012] The present application takes Brassica napus double 11 variety cDNA as a template, and obtains Brassica napus gene BnCNGC3 through PCR cloning, the nucleotide sequence of which is shown as SEQ ID No. 1, the open reading frame (ORF) of the gene is 2112 bp long, and the encoded protein is composed of 703 amino acids, the sequence of which is shown as 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 in the present application has a deletion of 3 consecutive bases and a difference of 4 bases from the ZS11C04G000660 nucleotide sequence of Brassica_napuscv.ZS11 PacBio V1.0 database of Brassica napus variety ZS11, and these base changes result in a deletion of 1 amino acid and a change of 3 amino acids. Specifically, T75, T76, C77 are deleted, resulting in the deletion of amino acid Ser26; G1589 is changed to C, resulting in the change of amino acid Ser530 to Thr; A1731 is changed to C, and the amino acid does not change; A2009 is changed to G, resulting in the change of amino acid Lys670 to Arg; G2012 is changed to A, resulting in the change of amino acid Ser671 to Asn.

[0013] Before the present application, there is no any public report on the function of the gene. The present application first constructs the super-expression transgenic Brassica napus and the CRISPR gene knockout Brassica napus of the gene, carries out disease resistance analysis, and clarifies the regulation effect and mechanism of the gene on the resistance of Brassica napus to sclerotinia disease. The analysis results of the inoculated leaf lesion area and pathogen biomass, stem lesion length and the like show that, compared with the wild type Brassica napus plants, the super-expression transgenic Brassica napus plants are significantly more resistant to Sclerotinia sclerotiorum, and the gene knockout plants are significantly more susceptible to Sclerotinia sclerotiorum, indicating that BnCNGC3 positively regulates the resistance of Brassica napus to Sclerotinia sclerotiorum.

[0014] Based on the BnCNGC3 gene function clarified in the present application, the application purpose of the present application is to provide the application of the Brassica napus BnCNGC3 gene in obtaining the Brassica napus material with changed disease resistance through creating transgenic Brassica napus, including (1) the application in obtaining the Brassica napus material with increased resistance to sclerotinia disease through creating the transgenic Brassica napus with super-expression of BnCNGC3 (see the description in embodiment 1 for details); and (2) the application in obtaining the Brassica napus material with reduced resistance to sclerotinia disease through creating the Brassica napus with BnCNGC3-CRISPR gene knockout (see the description in embodiment 2 for details).

[0015] The application of Brassica napus BnCNGC3 gene in obtaining the Brassica napus material with increased resistance to sclerotinia disease through creating the transgenic Brassica napus with super-expression of BnCNGC3 is specifically realized through the following steps:

[0016] (1) Construction and acquisition of BnCNGC3 gene overexpression structure: the open reading frame (ORF) of BnCNGC3 gene is cloned into a plant expression vector to be expressed under the drive of a strong promoter;

[0017] (2) Acquisition of Agrobacterium transformed with BnCNGC3 gene overexpression structure: the constructed BnCNGC3 gene overexpression structure is transformed into an Agrobacterium strain with strong invasiveness to Brassica napus by electroporation or the like;

[0018] (3) Creation and acquisition of transgenic Brassica napus overexpressing BnCNGC3: the BnCNGC3 gene overexpression structure is introduced into Brassica napus by Agrobacterium-mediated method to obtain Brassica napus transformed with the BnCNGC3 gene overexpression structure;

[0019] (4) Acquisition of homozygous line of transgenic Brassica napus overexpressing BnCNGC3: the segregation of the progeny of the transgenic plants is detected by taking antibiotic resistance and BnCNGC3 gene expression as detection indexes to obtain a homozygous line of transgenic Brassica napus overexpressing BnCNGC3 which is no longer segregated and can be stably inherited;

[0020] (5) Screening, identification and acquisition of homozygous line of transgenic Brassica napus overexpressing BnCNGC3 with increased resistance to sclerotinia: the homozygous line of transgenic Brassica napus overexpressing BnCNGC3 is taken as material to detect and analyze the resistance to sclerotinia, and Brassica napus transformed with the BnCNGC3 gene with increased disease resistance is obtained.

[0021] Application of Brassica napus BnCNGC3 gene in obtaining Brassica napus with reduced resistance to sclerotinia by creating BnCNGC3-CRISPR gene knockout Brassica napus. The following steps are taken to achieve the application:

[0022] (1) Construction and acquisition of BnCNGC3 gene CRISPR knockout structure: three 20-bp knockout targets are preset according to the BnCNGC3 genomic sequence, and the target sequences are cloned into a plant expression vector pBSbdcas9i to obtain the recombination structure pBSbdcas9i-BnCNGC3 through colony PCR identification and sequencing;

[0023] (2) Acquisition of Agrobacterium transformed with BnCNGC3 gene CRISPR knockout structure: the BnCNGC3 gene CRISPR knockout structure (pBSbdcas9i-BnCNGC3) is transformed into an Agrobacterium strain with strong invasiveness to Brassica napus by electroporation or the like;

[0024] (3) Creation and acquisition of Brassica napus transformed with BnCNGC3 gene CRISPR knockout structure: the BnCNGC3 gene CRISPR knockout structure is introduced into Brassica napus by Agrobacterium-mediated method to obtain Brassica napus transformed with the BnCNGC3 gene CRISPR knockout structure;

[0025] (4) Obtaining homozygous rapeseed lines with BnCNGC3 gene CRISPR knockout structure: Using antibiotic resistance screening, the phenotypic segregation of the offspring of transgenic plants was detected, and homozygous rapeseed lines with BnCNGC3 gene CRISPR knockout structure that no longer segregate in the offspring and can be stably inherited were obtained.

[0026] (5) Screening, identification and acquisition of homozygous rapeseed lines with reduced resistance to sclerotinia stem rot by transgenic BnCNGC3 gene CRISPR knockout structure: Using homozygous rapeseed lines with transgenic BnCNGC3 gene CRISPR knockout structure (pBSbdcas9i-BnCNGC3) as materials, the resistance to sclerotinia stem rot was detected and analyzed, and rapeseed with reduced resistance to sclerotinia stem rot by transgenic BnCNGC3 gene CRISPR knockout structure was obtained.

[0027] Advantages of the present invention: (1) The BnCNGC3 gene provided by the present invention is a high-quality anti-sclerotinia stem rot regulatory gene resource. The disease-resistant materials obtained by using this gene have the advantages of strong disease resistance. Sclerotinia stem rot resistance in rapeseed is a quantitative trait resistance, controlled by multiple genes. Generally speaking, the degree of regulation of this resistance by a single gene is low. Therefore, there is a shortage of rapeseed sclerotinia stem rot resistant materials worldwide, and there are no highly resistant materials. The BnCNGC3 gene is a calcium ion channel gene, an early disease resistance regulatory gene, which acts upstream of signal transduction and has a good resistance regulation effect, thus 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 control diseases in a green manner. Therefore, the BnCNGC3 gene is a new gene resource suitable for creating and breeding new rapeseed materials and varieties resistant to sclerotinia stem rot. (2) The cycle for obtaining disease-resistant materials is short. The main methods for obtaining disease-resistant plant materials and varieties are conventional traditional breeding methods and genetic engineering breeding methods using disease resistance regulatory genes. Traditional breeding methods have drawbacks such as the limited range of available disease-resistant resources due to natural genetic isolation, long breeding cycles, and the need for substantial human and material resources. In contrast, genetic engineering breeding methods offer advantages such as a wide range of available disease-resistant resources, relatively simple and convenient operation, short breeding cycles, no need for large amounts of human and material resources, and particular suitability for cultivating broad-spectrum, durable, and highly disease-resistant varieties. This invention utilizes the disease-resistance regulatory gene BnCNGC3 and employs genetic engineering methods to create and cultivate rapeseed materials with high resistance to Sclerotinia stem rot, characterized by short cycles and rapid breeding. Attached Figure Description

[0028] Figure 1This invention provides evidence for various transgenic rapeseed plants obtained in this invention, including gene expression detection in BnCNGC3 overexpressing plants and gene editing identification in CRISPR gene knockout plants. It shows the gene expression detection results in BnCNGC3 overexpressing plants and the gene editing form in gene knockout plants. The expression level of BnCNGC3 in overexpressing (OE) plants was detected using real-time quantitative PCR (qRT-PCR) (A), with rapeseed BnACTIN7 as an internal reference gene and ZS11 expression set to 1. The qRT-PCR analysis was performed in triplicate (biological replicates, each containing three technical replicates). The expression results were statistically analyzed using GraphPad Prism with Student's t-test. Data are expressed as mean ± standard deviation. Significance is indicated by * (**, P < 0.001). The results showed that the expression level of BnCNGC3 was significantly increased in both overexpressing lines #3 and #15, being 16.2-fold and 29.2-fold higher than the control, respectively (A). Two gene knockout mutant lines, #1 and #7, exhibited different insertion mutations at three common target sites of two BnCNGC3 homologs located on chromosomes C04 and A05. However, both lines produced a total of 2bp insertions at each of the three target sites of these two homologs, resulting in the inability to properly encode functional BnCNGC3. C04 and BnCNGC3 A05 Gene (B). This indicates that these plants are true BnCNGC3 gene overexpression plants and CRISPR gene knockout plants.

[0029] Figure 2 This study provides evidence for the anti-Sclerotinia sclerotiorum function of the BnCNGC3 gene, demonstrating that BnCNGC3 positively regulates rapeseed resistance to Sclerotinia sclerotiorum. The study shows that BnCNGC3 positively regulates rapeseed resistance to Sclerotinia sclerotiorum. Inoculation analysis of rapeseed BnCNGC3 transgenic plants with Sclerotinia sclerotiorum UF-1 was performed. The figures show the phenotype (A), lesion area, and mycelial biomass (B) of leaves after inoculation in overexpressing plants and knockout mutants, and the phenotype (C) and lesion length (D) of stems after inoculation. The inoculation experiment was repeated three times. Student's t-test was used to statistically analyze lesion area using GraphPad Prism. Data are expressed as mean ± standard deviation. Significance is indicated by different numbers of asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Leaf inoculation analysis showed that overexpression plants exhibited significantly greater disease resistance than the control (ZS11) plants, while knockout mutant plants were significantly more susceptible to the disease than the control (A). Quantitative analysis of lesion area showed that the lesion area of ​​the two overexpression lines was 142.3 mm². 2 and 144.0mm2 It was significantly lower than the control group's 234.2 mm. 2 Only 60.8% and 61.5% of the control (B) were observed; bacterial count also showed that the bacterial count in the lesions of the overexpressing plants was significantly lower than that of the control plants. The lesion area of ​​the two knockout mutant lines was 293.2 mm². 2 and 282.8mm 2 The result was significantly higher than the control group's 234.2 mm. 2 The bacterial counts of the knockout mutant plants were 1.3 times and 1.2 times that of the control (B). Bacterial count analysis also showed that the bacterial count in the lesions of the knockout mutant plants was significantly higher than that of the control plants. Stem inoculation analysis showed that the stem lesion lengths of the two overexpression lines were 17.8 mm and 15.58 mm, respectively, significantly lower than the control's 30.48 mm, representing only 58.4% and 51.1% of the control (D). In contrast, the stem lesion lengths of the two knockout mutant lines were 45.66 mm and 50.55 mm, respectively, significantly higher than the control's 30.48 mm, representing 1.5 times and 1.7 times that of the control (D). These results indicate that BnCNGC3 positively regulates the resistance of rapeseed to Sclerotinia sclerotiorum. Detailed Implementation

[0030] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0031] Example 1

[0032] This invention cloned a rapeseed gene, BnCNGC3, and for the first time elucidated the function of this gene in conferring resistance to Sclerotinia stem rot in rapeseed by constructing transgenic rapeseed with overexpression. Overexpression of the BnCNGC3 gene led to a significant increase in rapeseed resistance to Sclerotinia stem rot. Therefore, new rapeseed materials with enhanced resistance to Sclerotinia stem rot can be created and obtained by constructing homozygous lines of rapeseed with overexpression of the BnCNGC3 gene. These materials can be used for the creation and breeding of rapeseed varieties resistant to Sclerotinia stem rot, and for analyzing the function and mechanism of action of the BnCNGC3 gene. 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 stem rot include:

[0033] 1) Cloning and preservation of the BnCNGC3 gene in rapeseed

[0034] The rapeseed BnCNGC3 gene provided by this invention was cloned through the following steps. First, primers BnCNGC3-F (5'-atg gag atg atg aat ctc aaa-3') (sequence shown as SEQ ID No. 3) and BnCNGC3-R (5'-tta ggt ttc atc cat agg aaa-3') (sequence shown as SEQ ID No. 4) were designed based on the BnCNGC3 sequence in the rapeseed genome database. Total RNA was extracted from the leaves of the rapeseed variety Shuang 11 using TRIZOL reagent. BnCNGC3 cDNA was amplified using high-fidelity PFU-mediated RT-PCR. The PCR product was purified by gel extraction after 1% agarose gel electrophoresis, ligated into the pEASY-Blunt Cloning Vector, and transformed into *E. coli* DH5α by heat shock. The culture was then incubated overnight in LB medium, and plasmids were extracted. PCR was performed using primers BnCNGC3-F / BnCNGC3-R to verify the presence of the BnCNGC3 gene. Sequencing was then performed to confirm the cloning. The full-length cDNA sequence of the BnCNGC3 gene on chromosome C04 was successfully obtained. The nucleotide sequence of BnCNGC3 is shown in SEQ ID No. 1. The open reading frame (ORF) of this gene is 2112 bp long, encoding a protein of 703 amino acids, the sequence of which is shown in SEQ ID No. 2. The gene-encoded product contains a transmembrane domain, a cyclic nucleotide-binding domain, and a CaM-binding domain. BLAST analysis revealed that the cloned BnCNGC3 gene sequence showed 86% nucleotide sequence similarity and 84% amino acid sequence similarity compared to the Arabidopsis CNGC3. Compared to the ZS11C04G000660 nucleotide sequence of rapeseed variety ZS11 in the Brassica_napus cv.ZS11 PacBio V1.0 database, there were three consecutive base deletions and four base differences. These base changes resulted in the deletion of one amino acid and the alteration of three amino acids. Specifically, deletions of T75, T76, and C77 resulted in the deletion of amino acid Ser26; the change from G1589 to C resulted in the change from Ser530 to Thr; the change from A1731 to C did not change the amino acid; the change from A2009 to G resulted in the change from Lys670 to Arg; and the change from G2012 to A resulted in the change from Ser671 to Asn. Prior to this invention, the function of the BnCNGC3 gene had not been publicly reported.

[0035] E. coli carrying the pEASY-BnCNGC3 vector were transformed and stored at -80°C. The plasmid can be extracted at any time by activating the strain, and the BnCNGC3 gene can be subcloned into the target vector via PCR amplification for use in transgenic and other research.

[0036] 2) Construction and acquisition of the BnCNGC3 gene overexpression structure

[0037] Primers BnCNGC3-F2 (5'-agagaacacgggggactctag atg gag atg atg aat ctc aaa-3', the italicized portion being a sequence containing an Xba I restriction site and identical to that on the vector pCAMBIA1300) (sequence shown in SEQ ID No. 5) and BnCNGC3-R2 (5'-gtccttgtagtccatgtcgac ggt ttc atc cat agg aaa ctc-3', the italicized portion being a sequence containing a Sal I restriction site and identical to that on the linearized vector pCAMBIA1300) (sequence shown in SEQ ID No. 6) were designed based on the cloned BnCNGC3 sequence according to the present invention. Using the pEASY-BnCNGC3 plasmid obtained in Example 1(1) as a template, and BnCNGC3-F2 / BnCNGC3-R2 as the primer pair, the target gene was obtained by PCR amplification.

[0038] The overexpression vector pCAMBIA1300 plasmid was digested with Xba I and Sal I. The amplified target gene was then ligated into the digested pCAMBIA1300 vector via homologous recombination. The reagents used in this experiment were: A one-step PCR directional cloning kit. The ligation product is transformed into *E. coli*, screened using kanamycin plates, identified by PCR, and sequenced to obtain the overexpression structure pCAMBIA1300-BnCNGC3 of the BnCNGC3 gene. The overexpression vector pCAMBIA1300 drives the expression of the target gene using the CaMV 35S promoter and carries both FLAG and HA dual tags for easy molecular identification and gene function studies.

[0039] 3) Obtaining Agrobacterium tumefaciens transformed with the BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3

[0040] The BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3 was transformed into Agrobacterium strains with strong infectivity against rapeseed, such as EHA105, using methods such as electroporation. Transformants were screened on YEP medium containing kanamycin, and then identified by double digestion with XbaI and SalI and PCR to obtain Agrobacterium strains carrying the BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3. This was used for the next step of genetic transformation of rapeseed.

[0041] 4) Creation and acquisition of rapeseed with the transgenic BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3

[0042] The BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3 was introduced into rapeseed using Agrobacterium-mediated transformation to obtain the T0 generation of rapeseed transgenic with pCAMBIA1300-BnCNGC3. The specific operational steps are as follows:

[0043] (i) Seed washing and germination

[0044] 75% ethanol for 30-60 seconds, sterile water once, 1 min / time; 0.15% mercuric chloride for 10 min, sterile water rinse twice, 1 min / time; sterile water rinse for 30 min, inoculate onto sterile filter paper and air dry; inoculate seeds into culture flasks and incubate in the dark at 23℃ for 5-6 days.

[0045] (ii) Pre-culture

[0046] Cut the hypocotyls of germinating rapeseed seedlings into 0.4-0.6 cm segments, inoculate them into pre-culture medium, and culture at 23°C under light for 2-3 days.

[0047] (iii) Agrobacterium infection and co-culture

[0048] Agrobacterium was picked into the infection solution to prepare OD. 600 The explants were inoculated into a 0.2 μL Agrobacterium suspension and incubated for 10 min. After inoculation, the explants were inoculated onto sterile filter paper and air-dried, then inoculated onto co-culture medium and incubated in the dark at 23°C for 48-72 h.

[0049] (iv) Destermination (extended screening)

[0050] The co-cultured explants were inoculated onto sterile culture medium and cultured at 23°C under light for 6 days.

[0051] (v) Screening / Differentiation

[0052] The sterilized explants were inoculated onto selection / differentiation medium, with 30 explants per plate, and cultured at 23°C under light. The plates were changed every 15 days.

[0053] (vi) Rooting culture

[0054] The differentiated buds were inoculated into rooting medium and cultured under light at 23°C until roots were formed.

[0055] (vii) Detection

[0056] Genomic DNA was extracted from rapeseed using the CTAB method, and resistance genes were detected by PCR.

[0057] (viii) Soil cultivation

[0058] After the kanamycin-resistant seedlings have grown to full root system, the T0 generation sterile seedlings are cultured and hardened off at 27℃ for 2 days with the cover off. Then they are transplanted into the soil and placed in an incubator for routine management. Finally, T0 seeds are harvested.

[0059] 5) Screening, identification, and acquisition of homozygous rapeseed lines overexpressing the BnCNGC3 gene with the structure pCAMBIA1300-BnCNGC3.

[0060] Hygromycin resistance and BnCNGC3 gene expression were used as indicators to detect phenotypic segregation in the offspring of transgenic plants. Hygromycin resistance screening was conducted on rapeseed seeds on hygromycin-containing plates to observe whether they could grow into healthy seedlings normally on hygromycin-resistant plates. Gene expression was detected using methods such as real-time quantitative PCR. Homozygous rapeseed lines with the BnCNGC3 gene overexpression structure pCAMBIA1300-BnCNGC3, whose offspring no longer exhibited phenotypic segregation and could be stably inherited, were obtained.

[0061] This invention obtained two homozygous rapeseed lines, OE-line 3 and OE-line 15, both of which were able to grow normally into healthy seedlings on hygromycin resistance plates, and the expression level of the BnCNGC3 gene was significantly higher than that of the control plants, being 16.2 times and 29.2 times higher, respectively, than that of the control plants. Figure 1 A). This indicates that these plants are true BnCNGC3 gene overexpression plants.

[0062] 6) Disease resistance detection and analysis of homozygous rapeseed lines overexpressing the BnCNGC3 gene

[0063] Using the homozygous rapeseed line overexpressing the BnCNGC3 gene obtained in step 5) as material, we detected and analyzed its resistance to Sclerotinia sclerotiorum by inoculating it with Sclerotinia sclerotiorum, thereby clarifying the regulatory role of the BnCNGC3 gene in the resistance of rapeseed to Sclerotinia sclerotiorum, and laying the foundation for creating and obtaining rapeseed resistant to Sclerotinia sclerotiorum using this gene.

[0064] Activation culture of Sclerotinia sclerotiorum: Select plump and uncontaminated Sclerotinia sclerotiorum sclerotia, cut the sclerotia in half with a sterile blade that has been flammed with an alcohol lamp, place them cut side down on a PDA solid plate, and incubate at 23°C in the dark for 3 days. Use a 4mm diameter punch to take a piece of hyphae 3-5mm inward from the edge of the colony, and inoculate it onto a new PDA solid plate with the hyphae side down. Incubate at 23°C in the dark for about 36 hours before inoculation.

[0065] Inoculation with *Sclerotinia sclerotiorum*: Select rapeseed plants with uniform growth for inoculation. Use a 4mm diameter punch to collect mycelial blocks 3-5mm inward from the edge of the colony. Inoculate the mycelial blocks, mycelial side down, into the middle of fully developed leaves, two mycelial blocks symmetrically inoculated on each half of each leaf. Cover with a film to maintain moisture and incubate at 23℃. Take photos after an appropriate time (approximately 30 hours). Similarly, inoculate the mycelial blocks, mycelial side down, onto the stems of flowering plants, one mycelial block per stem. Cover with a film to maintain moisture and incubate at 23℃. Take photos after an appropriate time (approximately 30 hours). Analyze the lesion area using ImageJ software.

[0066] The inoculation experiment was repeated three times. Student's t-test was used for statistical analysis of lesion area. Results showed that BnCNGC3 overexpressing plants were significantly more resistant to the disease than the non-transgenic control plants (ZS11). Figure 2 (A, C) Quantitative analysis of lesion area showed that the leaf lesion area of ​​the ZS11 control plant was 234.2 mm². 2 The leaf lesion area of ​​the two overexpressing plant lines was 142.3 mm. 2 and 144.0mm 2 Only 60.8% and 61.5% of the control group (…) Figure 2 B). The stem lesion length of the ZS11 control plant was 30.48 mm, while the stem lesion lengths of the two overexpression lines were 17.8 mm and 15.58 mm, respectively, only 58.4% and 51.1% of the control. Figure 2 D).

[0067] These results indicate that BnCNGC3-overexpressing plants exhibited significantly higher resistance to Sclerotinia stem rot than non-transgenic control plants. Overexpression of the BnCNGC3 gene led to a significant increase in rapeseed resistance to Sclerotinia stem rot; therefore, BnCNGC3 plays a positive regulatory role in rapeseed resistance to Sclerotinia stem rot. This invention successfully created a new rapeseed material with high resistance to Sclerotinia stem rot by constructing BnCNGC3-OE rapeseed.

[0068] Example 2

[0069] Based on the positive regulatory function of the rapeseed gene BnCNGC3 on sclerotinia disease resistance as elucidated in this invention as described above, a technical system was established to create and obtain new rapeseed materials with weakened resistance to sclerotinia disease by constructing a CRISPR gene knockout of this gene and using genetic engineering technology. The main steps include:

[0070] (1) Construction and acquisition of the CRISPR knockout structure of the BnCNGC3 gene

[0071] Three suitable target sites were designed based on the BnCNGC3 genome sequence. Each gRNA unit contains 20 bp gRNA and a gRNA backbone. The three gRNA units were combined to form a complete knockout functional sequence. Using this as a template, amplification was performed using primers BnCNGC3-CR-F1 (5'-cagtggtctc aag tga aca aag cac cag tgg tct agt ggt ag-3', the italicized part is a sequence containing the Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (sequence shown in SEQ ID No. 7) and BnCNGC3-CR-R1 (5'-cagtggtctc aaa acg agg aat agg gag aat agaatg cac cag-3', the italicized part is a sequence containing the Bsa I (Eco31 I) restriction site and consistent with the linearized vector pBSbdcas9i) (sequence shown in SEQ ID No. 8). The vector pBSbdcas9i was digested with Bsa I (Eco31 I), and recombinant ligation was performed using T4 ligase. Subsequent transformation and screening on kanamycin agar plates were conducted, and positive single clones were picked. PCR identification was performed using the pBSbdcas9i identification primers Pbw2+(5'-gca acg ctc tgt cat cgt tac aat-3') (sequence shown in SEQ ID No. 9) and Pbw2-(5'-gcg att aag ttg ggt aac gcc agg g-3') (sequence shown in SEQ ID No. 10). Further sequencing identification yielded the CRISPR knockout structure pBSbdcas9i-BnCNGC3.

[0072] (2) Obtaining Agrobacterium tumefaciens transformed with the BnCNGC3 gene CRISPR knockout structure

[0073] The CRISPR knockout structure of the BnCNGC3 gene (pBSbdcas9i-BnCNGC3) was transformed into Agrobacterium strains with strong infectivity against rapeseed, such as EHA105, by methods such as electroporation. Transformants were screened on YEP medium containing kanamycin and then identified by PCR to obtain Agrobacterium carrying the CRISPR knockout structure of the BnCNGC3 gene, pBSbdcas9i-BnCNGC3.

[0074] (3) Creation and acquisition of rapeseed with BnCNGC3 gene CRISPR knockout structure

[0075] The CRISPR knockout structure pBSbdcas9i-BnCNGC3 of the BnCNGC3 gene was introduced into rapeseed using Agrobacterium-mediated transformation to obtain the T0 generation of rapeseed transformed with pBSbdcas9i-BnCNGC3. The specific operational steps are the same as described in step 4) of Example 1. The transformation seedlings were tested for gene editing using BnCNGC3-CR-F2 (5'-gga gat gat gaa tct caa aag aaa cac-3') (sequence shown in SEQ ID No. 11) and BnCNGC3-CR-R2 (5'-acg cat gaa tgc aac ctt tta tc-3') (sequence shown in SEQ ID No. 12). Sequence sequencing and analysis of the target site near the gene-edited seedlings were performed, ultimately confirming them as mutant plants with successful BnCNGC3 gene knockout.

[0076] (4) Obtaining homozygous rapeseed lines with BnCNGC3 gene CRISPR knockout structure

[0077] Antibiotic resistance screening was used to detect phenotypic segregation in the progeny of transgenic plants. Rapeseed seeds were screened on Basta-resistant plates to observe whether they could grow into healthy seedlings normally. Homozygous rapeseed lines transgenic with the BnCNGC3 gene CRISPR knockout structure pBSbdcas9i-BnCNGC3, whose progeny no longer segregated and could be stably inherited, were obtained. All of these homozygous rapeseed lines grew into healthy seedlings normally on Basta-resistant plates.

[0078] This invention obtained a homozygous BnCNGC3-CRISPR gene knockout rapeseed line that could grow into healthy seedlings normally on Basta resistance plates. Sequencing results showed that the two mutant lines #1 and #7 had different insertion mutations at three common target sites of two BnCNGC3 homologs located on chromosomes C04 and A05. However, both lines produced a total of 2bp insertions at the three target sites of these two homologs, resulting in the inability to properly encode functional BnCNGC3. C04 and BnCNGC3A05 Gene( Figure 1 B). This indicates that these plants are true BnCNGC3 gene CRISPR knockout plants.

[0079] (5) Screening, identification and acquisition of homozygous rapeseed lines with weakened resistance to sclerotinia stem rot via CRISPR knockout of the BnCNGC3 gene.

[0080] Using homozygous rapeseed lines transgenic with the CRISPR knockout structure of the BnCNGC3 gene (pBSbdcas9i-BnCNGC3) as materials, resistance to sclerotinia stem rot was tested and analyzed. The inoculation method and resistance evaluation were the same as described in step 6) of Example 1).

[0081] Analysis of the leaves of the BnCNGC3-CRISPR plants constructed in this invention using Sclerotinia sclerotiorum inoculation showed that the BnCNGC3-CRISPR gene knockout plants were significantly more susceptible to the disease than the non-transgenic plant (ZS11) control. Figure 2 A). Quantitative analysis of lesion area showed that the lesion area on the leaves of the ZS11 control plant was 234.2 mm². 2 The leaf lesion areas of the two knockout mutant lines were 293.2 mm², respectively. 2 and 282.8mm 2 The number of plants was significantly higher than that of the control plants. Figure 2 B). Bacterial counts also showed that the bacterial count in lesions of the knockout mutant plants was significantly higher than that of the control plants. Figure 2 B). Stem inoculation analysis also showed that gene knockout plants were significantly more susceptible to the disease than non-transgenic plants (ZS11) controls. Figure 2 C). Quantitative analysis of lesion length showed that the stem lesion length of the ZS11 control plant was 30.48 mm, while the stem lesion lengths of the two knockout mutant lines were 45.66 mm and 50.55 mm, respectively, significantly longer than those of the control plant. Figure 2 D). This indicates that the resistance of BnCNGC3-CRISPR plants to sclerotinia stem rot was significantly lower than that of non-transgenic control plants. Inhibition of BnCNGC3 gene expression led to a significant decrease in rapeseed resistance to sclerotinia stem rot. This invention successfully created a new rapeseed material with weakened resistance to sclerotinia stem rot by constructing BnCNGC3-CRISPR gene knockout rapeseed.

[0082] In summary, this invention combines Figures 1-2 The results revealed for the first time that the rapeseed calcium ion channel gene BnCNGC3 plays a positive regulatory role in rapeseed resistance to Sclerotinia stem rot, provided the application pathways and technologies of BnCNGC3 in the creation of Sclerotinia stem rot resistant crop germplasm, provided examples, and successfully obtained rapeseed with high resistance to Sclerotinia stem rot.

Claims

1. A type of rapeseed ( Brassica napus Calcium channel gene BnCNGC3 In the prevention and control of Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Its application in treating rapeseed sclerotinia stem rot caused by ), characterized in that, The gene BnCNGC3 The nucleotide sequence of the gene is shown as SEQ ID No. 1, and the protein sequence encoded by the gene is shown as SEQ ID No.

2.

2. Use according to claim 1, characterized in that, In the transgenic rape plants obtained by creating transgenic rape plants overexpressing BnCNGC3 the gene for enhanced resistance to sclerotinia blight.

3. Use according to claim 2, characterized in that, The use of Brassica napus material with increased resistance to Sclerotinia sclerotiorum is achieved by the following steps: (1) BnCNGC3 Construction and obtaining of gene overexpression constructs The BnCNGC3 Gene open reading frames are cloned into a plant expression vector for expression under the control of a strong promoter. (2) Transformation BnCNGC3 Agrobacterium containing a gene overexpression construct The constructed BnCNGC3 The gene overexpression structure is transformed into an Agrobacterium strain with strong invasion ability to rape by an electric shock method. (3) Overexpression BnCNGC3 Creation and acquisition of genetically modified rapeseed The gene overexpression structure is introduced into rape by means of agrobacterium mediation BnCNGC3 The gene overexpression structure is introduced into rape by means of agrobacterium mediation BnCNGC3 The gene overexpression structure is introduced into rape by means of agrobacterium mediation (4) Obtaining homozygous lines of transgenic rape overexpressing BnCNGC3 the gene respectively, with antibiotic resistance and BnCNGC3 Gene expression as detection index, detection of transgenic plant offspring trait segregation, get offspring trait no longer segregation, and can be stable inheritance of super expression BnCNGC3 of transgenic rape homozygous line; (5) Overexpression of sclerotinia stem rot resistance BnCNGC3 Screening, identification and acquisition of transgenic rapeseed homozygous lines Using transgenic rapeseed homozygous line overexpressing BnCNGC3 of the gene as material, the resistance to sclerotinia stem rot is detected and analyzed, and transgenic rapeseed with increased disease resistance is obtained. BnCNGC3 BnCNGC3 of the gene as material, the resistance to sclerotinia stem rot is detected and analyzed, and transgenic rapeseed with increased disease resistance is obtained.

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