Use of a pectin synthase gene BnGATL10

Abstract

The application discloses application of a pectin synthase gene BnGATL10 and belongs to the field of plant genetic engineering.The nucleotide sequence of the pectin synthase gene BnGATL10 is shown as SEQ ID NO.3, and the protein sequence encoded by the pectin synthase gene BnGATL10 is shown as SEQ ID NO.4.Through construction of a recombinant vector containing the gene, the recombinant vector is transformed into rape by means of agrobacterium infection, and a transgenic plant is obtained, the expression amount of BnGATL10 is increased in Brassica napus through genetic engineering technology, the transgenic rape leaf can reduce the sclerotinia patch area by 47.39%, the plant shows more resistance to sclerotinia, and the application has important application prospects for enhancing the sclerotinia resistance of Brassica napus.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and in particular to the application of a pectin synthase gene, BnGATL10. Background Technology

[0002] Rapeseed belongs to the genus Brassica in the family Cruclferae. It is one of the most widely cultivated oil crops in the world, providing 15% of the world's edible vegetable oil. In my country, rapeseed oil accounts for half of the domestic edible vegetable oil each year (Li Jiana, Chen Li, Liang Ying. Research and Reflection on Yellow-Seed Rapeseed of the Brassica Variety. China Agricultural Science and Technology Press, 2004, 29-39).

[0003] In recent years, with the development of the economy and tourism industry, rapeseed has not only become an important oil crop, but has also gradually developed into an emerging ornamental plant. However, problems such as lodging, pests, and diseases in production have seriously limited the yield and ornamental value of rapeseed. In particular, sclerotinia stemrot, caused by Sclerotinia sclerotiorum, has seriously affected the quality and yield of rapeseed. When sclerotinia stemrot occurs severely, it can lead to a yield reduction of up to 80%. At present, chemical control methods are not only costly, but also easily cause environmental pollution. Therefore, selecting and cultivating disease-resistant varieties has become the most economical and effective way to control sclerotinia stemrot in rapeseed. However, at present, there are no varieties that are immune to sclerotinia stemrot (Wang Hanzhong et al. Study on the breeding of double-low sclerotinia stemrot-resistant rapeseed variety Shuang 9 and the change law of important defensive enzyme activity. Chinese Agricultural Science, 2004, 37(1):23-28). Therefore, making full use of genetic engineering technology to discover resistance gene resources and apply them to the screening of rapeseed resistance germplasm resources is of great significance for improving rapeseed resistance to sclerotinia stem rot.

[0004] *Sclerotinia sclerotiorum* belongs to the genus *Sclerotinia* in the order Mycota of the phylum Ascomycota. It has a wide ecological distribution and can parasitize more than 400 plant species, causing particularly serious damage to the growth and development of legumes, solanaceous plants, and cruciferous plants. Plant diseases caused by *Sclerotinia sclerotiorum* are called sclerotium rot, also known as white rot, stem rot, and soft rot. Previously, researchers generally believed that *Sclerotinia sclerotiorum* was a typical necrotrophic fungus, but increasing evidence suggests that it also has a biotrophic stage. In the early stages of infection, the hyphae first spread in the apoplastic space; at this time, the sclerotia have not penetrated the cell wall, and the plant cell remains alive. In this process, *Sclerotinia sclerotiorum* secretes oxalic acid and other pathogenic factors to interfere with the host's signal transduction, avoid being recognized by the host, and inhibit the host's defense response. Subsequently, *Sclerotinia sclerotiorum* induces host cell death and secretes a large number of cell wall degrading enzymes through oxalic acid and other pathogenic factors. PTI, or pathogen-associated molecular pattern triggered immune process, refers to the process by which PRRs on the surface of plant cells recognize pathogen-associated molecular patterns (PAMPs, i.e., conserved molecular structures on the surface of pathogens) and stimulate the host's defense response. Thus, it infects the host and causes host cell death. The pathogenic factors produced by the pathogen mainly include: (1) various hydrolytic enzymes that degrade plant cell walls and cuticles, such as pectinase, cellulase, lignin-degrading enzymes, and glycosidases, which can soften and degrade the host cell wall, making it easier for hyphae to invade. The degraded host tissue can also serve as a carbon source for the growth of the pathogen; (2) toxins that damage the host cell membrane and affect the normal metabolism of cells.

[0005] Studies have shown that one of the key steps in *Sclerotinia sclerotiorum* overcoming host defenses is the degradation of its cell wall. The cell wall, a natural mechanical barrier unique to plant cells, surrounds the protoplasmic membrane and can be divided into three parts: the middle lamella (ML), the primary cell wall (PCW), and the secondary cell wall (SCW). It is mainly composed of polysaccharides such as cellulose, hemicellulose, and pectin, as well as polymers such as lignin, interconnected with xyloglucan and embedded in the pectin hydrated matrix, providing structural support and protection against pathogens. The middle lamella is the first cell wall to form during cell division, mainly composed of pectin and proteins. The primary cell wall forms before cell growth ceases, possessing a rigid framework of cellulose microfibrils, with pectin, hemicellulose, and glycosylated proteins embedded within, exhibiting considerable plasticity. The secondary cell wall forms after cell growth ceases, is rigid, and enhances the mechanical strength of the cell wall; it is composed of cellulose, hemicellulose, and lignin. Cell walls physically resist pests and diseases, and further physiologically perform defense and disease resistance functions. When pathogens infect plants, oligosaccharides in the primary cell wall can induce the formation of antitoxins, causing localized allergic death and inhibiting the continued spread of pathogens. Many studies have also shown that cell wall polysaccharide components are a decisive factor in limiting disease development. For most necrophorous pathogens, the first line of defense they must overcome to successfully infect plant cells is the cell wall. Cell wall-mediated resistance is an important component of the plant immune response system. Research has found that in the interaction between plants and pathogens at the cell wall level, the cell wall can act as an active defense system, dynamically responding to changes. When the cell wall suffers minor damage or its structural components change, the plant cell wall itself can produce damage-associated molecular patterns (DAMPs), which act as an "alarm" signal to activate a DAMPs-triggered immunity (DTI), known as cell wall-mediated resistance. Although cell wall-mediated broad-spectrum plant resistance has attracted widespread attention, the specific mechanisms have not been studied in depth, especially the diseases caused by Sclerotinia sclerotiorum.

[0006] Pectin, a crucial component of primary plant cell walls, is a complex polysaccharide containing α-D-galacturonic acid. During cell growth, it plays a vital role in cell wall remodeling, cell wall structural integrity and strength, and normal intercellular adhesion. The complex structure and preservation properties of pectin indicate its important role in structural resistance (intercellular adhesion and cell wall structure), defense against pathogens (biological, necrotic, phytophagous, endosymbiotic, and insect-related pathogens), the physiological and biochemical properties of the final product, and plant development (morphogenesis and organogenesis), making it a key factor in plant immunity. Pectin metabolism involves various enzymes, including pectin synthases and lyases. Studies have shown that pectin-related enzymes may be involved in plant-pathogenic microorganism interactions. Enzymes involved in the synthesis and degradation of cell wall pectin polysaccharides are collectively called carbohydrate-active enzymes (CAZYs), which are divided into glycosyltransferases (GTs), glycosyl hydrolases (GHs), polysaccharide lyases (PLs), and carbohydrae esterases (CEs). The largest class is glycosyltransferases (GTs), which are present in all organisms and participate in the biosynthesis of oligosaccharides, polysaccharides, and sugar copolymers (Cantarel et al. 2009). In addition, GTs catalyze the glycosylation of a wide variety of molecules, participating in biological processes such as development, signal transduction, and defense. Studies have shown that several members of the GT8 family are galacturonyltransferases involved in the biosynthesis of pectin and xylan. Yin et al. found that proteins in the GT8 gene family contain a Glyco-transf-8 domain (approximately 256 amino acids), which can catalyze the transfer of glycosyl groups to lipooligosaccharides, proteins, inositol, oligosaccharides, or polysaccharide acceptors, thereby participating in the synthesis of pectin polysaccharides in the cell walls of higher plants. Among these, galacturonyltransferases (GAUTs) and galacturonyltransferase-like enzymes (GATLs) are classified as unique plant cell wall-related class I enzymes.GAUTs are the largest family in GT8 and are α-1,4-galacturonotransferases (1,4-linked-D-galactronosyltransferase, GalAT). It is generally believed that the genes in plant GATL subclasses are also mainly related to the biosynthesis of pectin and xylan. They can transfer galacturonic acid to pectin polysaccharides to synthesize pectin polysaccharides in plant cell walls—homogalacturon polysaccharides—which play an important role in plant growth and development, cell wall structure and function.

[0007] Sterling et al. identified 15 GAUT family members in Arabidopsis thaliana, divided into three branches: GAUT-A (AtGAUT1–AtGAUT7), GAUT-B (AtGAUT8–AtGAUT11), and GAUT-C (AtGAUT12–AtGAUT15). The Golgi localization of AtGATL proteins in Arabidopsis thaliana is consistent with their roles in the biosynthesis of non-cellulose polysaccharides. The amino acid sequences of AtGATLs contain characteristic domains of GTs, indicating that these proteins may be involved in the biosynthesis of cell wall polysaccharides. Galacuronosyltransferases (GAUTs) include two subclasses: GAUT and GATL (GAUT-Like). Current research suggests that these genes are mainly involved in the biosynthesis of pectin and xylan. For example, the protein encoded by GAUT1 in Arabidopsis thaliana is a galacturonoglycosyltransferase involved in pectin synthesis. Mohnen et al.'s research also showed that GAUT1 is a galacturonosyltransferase involved in pectin synthesis. GAUT1 is the first gene confirmed to be involved in pectin synthesis, catalyzing the transfer of GalA to HG and exhibiting characteristics of a type II membrane protein localized to the Golgi apparatus.

[0008] GAUT1 protein anchors to the Golgi apparatus and requires binding with GAUT7 to form the GAUT1:GAUT7 isomerase complex. The GAUT1:GAUT7 complex is soluble in vitro and catalyzes the elongation of HG products. Mutations in At GAUT12 significantly reduce xylan content in plants. Furthermore, GAUT8 mutations exhibit a dwarfing phenotype, reduced cell adhesion, and the GAUT8 gene has been shown to affect the biosynthesis of HG and xylan.

[0009] GAUT12 is highly expressed in xylem vessels and interfascicular fiber cells. GAUT12 mutants exhibit xylem vessel collapse and significant reductions in xylan and HG in vascular tissues.

[0010] Caffall et al. systematically analyzed the somatic cell wall glycosyl residue composition of homozygous T-DNA insertion mutants of 13 out of 15 Arabidopsis GAUT genes. The results showed that the gaut6, 8, 9, 10, 11, 12, 13, and 14 mutants significantly altered the cell wall glycosyl residue composition compared with the wild type, indicating that the mutations in these GAUT genes affect the biosynthesis of pectin and xylan.

[0011] Tomato plants with silenced GAUT4 exhibited altered pectin composition in their fruits, with reduced starch accumulation and pectin quantity. Besides its role in pectin biosynthesis, GAUT4 also interfered with carbon metabolism and allocation. GAUT13 and GAUT14, located in the Golgi apparatus, participate in plant developmental processes, such as promoting the synthesis of pectin and xylan in pollen tube walls and vegetative cell walls. Zheng et al. detected the expression level and pectin content of the cotton GhGATL15 gene. The results showed that as the expression level of the GhGATL15 gene decreased, the pectin content was significantly downregulated, indicating that the GhGATL15 gene is related to pectin production in cotton.

[0012] Based on the conservation of amino acid motifs, the GATL (GAUT-Like) gene family has been identified as a gene family closely related to the GAUT family. Currently, GATL family genes have been studied in model plants such as Arabidopsis thaliana and rice.

[0013] Currently, the functions of GATL family genes in the resistance of Brassica napus to Sclerotinia stem rot are unclear. Therefore, exploring the molecular mechanisms by which BnGATLs regulate Sclerotinia stem rot resistance can provide a new theoretical basis and germplasm resources for genetic improvement and breeding of disease-resistant varieties. However, there are currently no reports in the literature or patent literature regarding the role of the BnGATL10 gene in Sclerotinia stem rot resistance in rapeseed. Summary of the Invention

[0014] The purpose of this invention is to provide an application of the pectin synthase gene BnGATL10 to solve the problems existing in the prior art. This invention found that after treatment with sclerotinia stem rot to induce stress in Brassica napus, the expression level of BnGATL10 significantly increased, leading to a significant enhancement of resistance to sclerotinia stem rot in transgenic Brassica napus plants overexpressing BnGATL10. This result has significant application prospects in enhancing resistance to sclerotinia stem rot and increasing rapeseed oil yield.

[0015] To achieve the above objectives, the present invention provides the following solution:

[0016] This invention provides a method for improving the resistance of rapeseed to sclerotinia stem rot, comprising the process of introducing the pectin synthase gene BnGATL10 into rapeseed to cause it to be overexpressed; the nucleotide sequence of the pectin synthase gene BnGATL10 is shown in SEQ ID NO.3.

[0017] Furthermore, it includes the following steps:

[0018] (1) Construct a recombinant expression vector including the pectin synthase gene;

[0019] (2) Transform the recombinant expression vector obtained in step (1) into Agrobacterium;

[0020] (3) Rapeseed is transformed by Agrobacterium infection to obtain a transgenic line overexpressing the pectin synthase gene BnGATL10; the transgenic line has a higher resistance to sclerotinia stem rot than other lines.

[0021] Further, in step (3), the Agrobacterium infection method is to infect the hypocotyl of rape with Agrobacterium.

[0022] Furthermore, the rapeseed is Brassica napus.

[0023] The present invention also provides the application of the pectin synthase gene BnGATL10 in improving the resistance of rapeseed to sclerotinia stem rot, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0024] Furthermore, the rapeseed is Brassica napus.

[0025] This invention also provides the application of the pectin synthase gene BnGATL10 in the screening of rapeseed germplasm resources resistant to sclerotinia stem rot, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0026] Furthermore, the rapeseed is Brassica napus.

[0027] This invention also provides the application of the pectin synthase gene BnGATL10 in the construction of transgenic rapeseed plants resistant to sclerotinia stem rot.

[0028] Furthermore, the rapeseed is Brassica napus.

[0029] The present invention discloses the following technical effects:

[0030] This invention isolated and cloned the gene BnGATL10, a member of the GATL family (GT8 family), from Westerland rapeseed (Brassica napus), which can regulate resistance to Sclerotinia sclerotinia disease in Brassica napus. Functional verification and preliminary transformation applications of this gene were then conducted. Increasing the expression level of BnGATL10 in Brassica napus through genetic engineering activated the jasmonic acid / ethylene resistance signaling pathway, increased reactive oxygen species accumulation and the expression of resistance-related genes, resulting in a 47.39% reduction in Sclerotinia sclerotinia disease lesion area on transgenic rapeseed leaves. The plants exhibited greater resistance to Sclerotinia sclerotinia disease, demonstrating significant potential for enhancing the resistance of Brassica napus to this disease. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The amino acid sequence alignment results of the sequenced rapeseed BnGATL10 gene with the known Arabidopsis thaliana AtGATL10 gene are shown.

[0033] Figure 2 A schematic diagram illustrating the construction process of the plant transformation vector pGWB418(4xMyc)-BnGATL10-NOS: A represents the rapeseed overexpression vector map, B represents the fusion cloning method, and C represents the gene structure of BnGATL10(BnaC09g01660D). LB represents the left boundary of the T-DNA; 35S represents the cauliflower virus 35S promoter; NOS represents the terminator; and RB represents the right boundary of the T-DNA.

[0034] Figure 3 This is a schematic diagram of the Agrobacterium-mediated genetic transformation system for hypocotyls of Brassica napus. A shows the germination of Brassica napus Westar seeds sterilized with 0.1% mercuric chloride on 1 / 2 MS medium; B shows etiolated hypocotyls grown after approximately 5 days of dark incubation at 24°C; C shows 0.8 cm segments of etiolated hypocotyls aseptically inoculated with Agrobacterium GV3101 containing the transformation vector; D shows the hypocotyls after transfer to M1 medium and dark incubation for 48 hours. Explants were placed in M2 medium and cultured for 20 days under a 16-hour light / 8-hour dark cycle. E represents explants that, after selection, were placed in M3 medium and cultured for at least 15 days under a 16-hour light / 8-hour dark cycle until budding. These explants were then placed in fresh M3 medium and cultured for at least 15 days under a 16-hour light / 8-hour dark cycle until seedlings differentiated. F represents seedlings that rooted on M4 medium. G represents seedlings that were transplanted into soil and ready for harvesting T1 generation seeds.

[0035] Figure 4This study aimed to identify and analyze the expression levels of positive seedlings of BnGATL10 overexpressing transgenic lines in rapeseed. A represents the identification of positive seedlings of BnGATL10 overexpressing transgenic lines, with PCR amplification of the Kan gene fragment on the vector, targeting a size of 289 bp. B represents the detection of BnGATL10 expression levels in transgenic overexpressing lines and non-transgenic wild-type lines by qPCR. WT represents the wild-type control. OE-29, OE-36, OE-37, OE-40, OE-45, OE-48, and OE-51 are all independent overexpressing transgenic lines.

[0036] Figure 5 To identify the resistance of BnGATL10 overexpressing transgenic lines to sclerotinia stem rot; where A represents the disease phenotype of WT and BnGATL10(OE) seedlings 36 hours after in vitro inoculation with the sclerotinia stem rot; B corresponds to... Figure 5 The size of lesions on leaves of WT and BnGATL10(OE) in the A group after 36 hours of infection with Sclerotinia sclerotiorum; 36 hpi, 36 hours after inoculation; WT+S. sclerotiorum and BnGATL10(OE)+S. sclerotiorum represent wild-type and BnGATL10 overexpression lines inoculated with Sclerotinia sclerotiorum, respectively; the results showed that the mycelial expansion area of ​​the leaves of the BnGATL10(OE) line was significantly smaller than that of the control, indicating that the plant's resistance to Sclerotinia sclerotiorum was enhanced after BnGATL10 overexpression;

[0037] Figure 6 Analysis of the expression levels of disease resistance-related genes in the leaves of wild-type (WT) and transgenic BnGATL10 (OE) seedlings 36 hours after in vitro inoculation with Sclerotinia sclerotiorum; where A represents the expression level of disease resistance-related genes in the leaves of wild-type (WT) and transgenic BnGATL10 (OE) seedlings. Figure 5 Using the material as a template, the expression levels of key ROS-related genes were detected; B was used to detect the expression levels of key genes related to the jasmonic acid signaling pathway and disease resistance. The results showed that after BnGATL10 was overexpressed, all of the above key genes were activated, indicating that BnGATL10 can enhance the innate immune signal by accumulating more reactive oxygen species in plants and regulate the resistance of Brassica napus to Sclerotinia sclerotiorum through the jasmonic acid signaling pathway. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] The following embodiments define the present invention and describe the methods for isolating and cloning cDNA fragments containing the complete coding region of the BnGATL10 gene, and for verifying the function of the BnGATL10 gene. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the present invention.

[0044] Example 1: Isolation and Cloning of the BnGATL10 Gene

[0045] (1) Isolation and cloning of the BnGATL10 gene

[0046] Using the Westar variety of Brassica napus (provided by the Rapeseed Research Laboratory of Huazhong Agricultural University) as the experimental material, the following methods were employed: Total RNA was extracted from leaves of the Wester cultivar of Brassica napus using the Super Total RNA Extraction Kit (Promega, USA). After extraction, RNA was treated with DNase I (Promega kit). RNA integrity was assessed by 1.2% (w / v) agarose gel (EtBr) electrophoresis (5V / cm). Nucleic acid concentrations were determined using an IMPLEN NanoPhotometer-N50 series ultra-micro UV spectrophotometer (Germany). RNA with a 260 / 280 ratio between 1.9 and 2.1, a 260 / 230 ratio greater than 2.0, and a concentration greater than 500 ng / μL was used for further analysis. cDNA synthesis was performed using… The cDNA was analyzed using the II Q RT SuperMix for qRNA (+gDNA wiper) kit (purchased from Vazyme, China). 1 μg total RNA was used as a template and mixed with 4 μL of 4×gDNAwiper Mix and DEPC-water to a total volume of 16 μL. The mixture was incubated at 42°C for 2 min, then quenched on ice for 2-3 min. Next, 4 μL of 5×Hiscript II qRT Super Mix II was added and mixed to a total volume of 20 μL. The mixture was then incubated at 50°C for 15 min, followed by 85°C for 5 sec. Each cDNA sample was diluted to 200 μL and stored at -20°C for later use.

[0047] The target band was amplified using in-fusion cloning primers BnGATL10-418F (5'-GACTTGAACGGTAGCGCTGTTCTCTCTGATGATGTCTGGCTC-3', SEQ ID NO.1) and BnGATL10-418R (5'-TCGGGGAAATTCGAGCTCTCCTCAATGTATTTCCGTTTTATATAC-3', SEQ ID NO.2). Amplification was performed using TransTaq HiFi DNA Polymerase (Beijing TransGen Biotech Co., Ltd.). The PCR reaction conditions were: 94℃ pre-denaturation for 3 min; 31 cycles of 94℃ for 30 sec, 57℃ for 30 sec, and 72℃ for 1 min 20 sec; extension at 72℃ for 5 min. The PCR product was then cloned into the pGWB418 vector. Recovery of the target fragment was performed using the UNIQ-10 column-based DNA gel extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). The ligation system for the target fragment and the pGWAB418 linearized vector was as follows: 4.5 μL of the target fragment, 0.5 μL of the pGWB418 vector (Wuhan Protech Biotechnology Co., Ltd.), and 5 μL of Solution I (Takara Bio Engineering (Dalian) Co., Ltd.). Ligation was carried out overnight at 16°C. The ligation product was transformed into DH5α competent cells by heat excitation. The bacterial culture was plated on LB agar plates containing 100 mg / L Kan antibiotic. After approximately 10-12 hours of growth, single colonies were selected for PCR using primers BnGATL10-418F (5'-TCGGGGAAATTCGAGCTC-3', SEQ ID NO. 35) and BnGATL10-418R (5'-TCGGGGAAATTCGAGCTCTCCTCAATGTATTTCCGTTTTATATAC-3', SEQ ID NO. 2). Positive colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0048] (2) BnGATL10 gene sequence analysis

[0049] Sequencing revealed that the amplified nucleotide sequence of the BnGATL10 gene was 1403 bp long, containing a 1062 bp open reading frame (ORF) (SEQ ID NO. 3). Amino acid sequence alignment using ClustalX (Thompson JD, Gibson TJ, Plewniak F, et al. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 1997, 25: 4876-82) showed high amino acid homology (86.85%) with Arabidopsis thaliana GATL10, consistent with the results obtained from NCBI alignment. Figure 1 The amino acid sequence encoded by the BnGATL10 gene is shown in SEQ ID NO.4.

[0050] SEQ ID NO.3:

[0051]

[0052] SEQ ID NO.4:

[0053] MMSGSRLIIFAIISTTVFTVGSIRLLPEDASSDFMEAPAYINGPECSVLPQNRLLLACDPSAVHIAMTLDPAYLRGTVSAVHSILKHTSCPQNIFFHFIASGSSHVKTLSSVFPSLSFKVYTFDETLVKDLISSSIRQALDSPLNYARSYLSEILSSCVHRVIYLDSDVIVVDDIKK LWKTSLTGSRTIGAPEYCHANFTKYFSDSFWSDRNLSRVFDSKSPCYFNTGVMVIDLERWREGDYTRKIENWMRIQKEERIYELGSLPPFILVFSGEIEAIDHQWNQHGLGGDNVVSSCRSLHPGPVSLIHWSGKGKPWVRLDDGKPCPVDYLWAPYDLHKSQRQYLQYNQELEIL.

[0054] Example 2: Obtaining transgenic rapeseed plants overexpressing BnGATL10

[0055] (1) Construction of plant overexpression vectors

[0056] Afel and Sacl restriction sites were added to both ends of the cloning primers SEQ ID NO.1-2, respectively. The pGWB418 vector was double-digested with AfeI and SacI. The digestion system was as follows: 15 μL pGWB418 (1700 ng / μL), 5 μL rCutsmartt Buffer, 1 μL AfeI (10000 U / L), 1 μL SacI (20000 U / L), and sterile water was added to a final volume of 50 μL. The digestion was carried out overnight at 37°C. After 1% agarose gel electrophoresis, the single linearized vector fragment was recovered.

[0057] The PCR product obtained from the above amplification was ligated into the pGWB418(4xMyc) vector using an in-fusion enzyme. Positive clones were screened and sequenced for identification to obtain the required gene ORF (1062 bp), thus obtaining the Brassica napus BnGATL10 transgenic overexpression vector pGWB418(4xMyc)-BnGATL10-NOS( Figure 2 BnGATL10 contains a kanamycin-resistant gene sequence in its T-DNA region, and the promoter for overexpression of BnGATL10 is the D35s promoter. Figure 2 ).

[0058] (2) Genetic transformation of rapeseed

[0059] The recombinant plasmid (i.e., the overexpression vector) pGWB418(4xMyc)-BnGATL10-NOS was transformed into Agrobacterium GV3101 using the conventional hypocotyl infection method. The cells were then screened and differentiated into seedlings. The specific steps are as follows: Figure 3 ):

[0060] a. Sowing: Wash the Wester seeds of Brassica napus with 75% alcohol for 1 min, then wash the seeds with 0.1% mercuric chloride solution for 5 min, and finally wash the seeds 5 times with sterile water; use sterilized tweezers to put the seeds into MO solid medium (MS (inorganic salts and trace elements) 4.404 g / L of the 1962 formula, adjust the pH to 5.8, add 7 g / L of agar, and sterilize according to the conventional high-pressure steam sterilization method), and incubate the inoculated seeds in the dark at 24℃ for 5 days.

[0061] b. Activation of Agrobacterium: Three days after inoculation, Agrobacterium strain GV3101 stored at -80℃ was streaked onto LB solid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL), and incubated at 28℃ for 16 h. Single colonies were picked and placed in 5 mL of LB liquid medium containing rifampicin (50 mg / mL), gentamicin (50 mg / mL), and kanamycin (50 mg / mL), and cultured at 28℃ with shaking at 200 rpm for 24 h to allow Agrobacterium to reach the logarithmic growth phase. 500 μL of the cultured bacterial solution was transferred to 50 mL of LB liquid medium (containing the three antibiotics) for expansion culture for 12 h.

[0062] c. Preparation of Infected Bacterial Fluid: Pour the activated Agrobacterium tumefaciens solution into two 50 mL sterile centrifuge tubes, centrifuge at 3500 rpm for 15 min, discard the supernatant, place on ice, and wash the bacterial fluid with 1 mL of DM liquid medium (MS (inorganic salts and trace elements) 4.404 g / L, sucrose 30 g / L, pH adjusted to 5.8, sterilized at 121℃ for 20 min, and after sterilization, add 2,4-D 1 mg / L, AS 100 mmol / L, KT 0.3 mg / L in the aseptic operating room), centrifuge, discard the supernatant, and resuspend the bacterial cells in 3 mL of DM liquid medium to adjust the OD of the infected bacterial fluid. 600 It is 0.6.

[0063] d. Infecting explants: Place the infecting bacterial solution from step c on ice. At the same time, use a scalpel to cut the hypocotyls of the dark-cultured Brassica napus seedlings from step a (each hypocotyl segment is 1.0 cm long). Use sterile forceps to transfer the cut hypocotyls into a Petri dish containing the infecting bacterial solution and infect for 30 minutes depending on the bacterial solution concentration (shake once every 3 minutes).

[0064] e. Co-culture: Transfer the infected explants to petri dishes containing filter paper (which should be sterilized in advance), blot dry any visible infection solution on the surface of the explants, and then transfer them to M1 solid medium (MS (inorganic salts and trace elements) 4.404 g / L, mannitol 18 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, KT 0.3 mg / L, adjust the pH of the medium to 5.8, add 7 g / L agar, and after sterilization, add 100 mmol / L AS), and incubate in the dark at 24°C for 48 h.

[0065] f. Screening: The co-cultured explants were transferred to M2 solid medium (MS (inorganic salts and trace elements) 4.404 g / L, mannitol 18 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, KT 0.3 mg / L, pH adjusted to 5.8, and agar 7 g / L) for 20 days of screening culture under the following conditions: 24℃, 16 h light / 8 h dark culture.

[0066] g. Differentiation culture: The selected explants were transferred to M3 solid medium (MS (inorganic salts and trace elements) 4.404 g / L, glucose 10 g / L, xylose 0.25 g / L, yeast extract (MES) 0.6 g / L, pH adjusted to 5.8, agar 7 g / L added, sterilized and then zeatin (ZT) 2 mg / L, IAA 0.1 mg / L, Timentin (TMT) 250 mg / L, Kan 25 mg / L) were added to start the differentiation culture. Subculture was performed every 15 days until shoots differentiated (culture conditions were the same as the screening conditions in step f).

[0067] h. Rooting Culture: Once a clear growth point can be found on the seedling that has differentiated buds, carefully cut the seedling along the junction of the callus and the bud with a scalpel (be careful not to damage the growth point during the operation). Then transfer the seedling to M4 solid medium (MS (inorganic salts and trace elements) 2.202g, sucrose 10g / L, IBA 0.5mg / L, adjust the pH of the medium to 5.8, add 7g / L agar, sterilize and then add 250mg / L TMT and 25mg / L Kan) to root.

[0068] (3) Identification of transgenic plants

[0069] a. Extraction of genomic DNA from rapeseed leaves

[0070] DNA was extracted using the conventional CTAB method. The specific steps were as follows: Young rapeseed leaves (1-2 cm in length) were placed in a pre-cooled mortar and ground with liquid nitrogen three times until a fine paste was formed. The paste was then transferred to a 1.5 mL centrifuge tube, and 700 μL of 2×CTAB solution was added. The mixture was incubated at 70°C for 30 min, with gentle shaking every 6 min, followed by another 30 min incubation at 70°C with gentle shaking every 10 min. After cooling to room temperature, 700 μL of a 25:24:1 Tris-saturated phenol:chloroform:isoamyl alcohol solution was added, and the mixture was repeatedly inverted and gently shaken 40 times. The mixture was centrifuged at 3100 rpm for 15 min at room temperature. Approximately 500 μL of the supernatant was collected, and an equal volume of a 24:1 chloroform:isoamyl alcohol solution was added. The mixture was then centrifuged at 3100 rpm for 15 min at room temperature. Discard the supernatant, add 1 mL of frozen -20℃ anhydrous ethanol, incubate at -20℃ for 30 min, then centrifuge at 12000 rpm for 10 min at room temperature. Wash with 75% ethanol and repeatedly pipette the precipitate for 3 min to remove salt. Discard the ethanol, air dry, and dissolve each sample in 30 μL of ddH2O. Analyze the extracted genomic DNA from Brassica napus using a Nanodrop micro-nucleic acid analyzer.

[0071] b. Detection of positive transgenic plants

[0072] Young leaves of the transformed plants were collected, and DNA was extracted using the CTAB method. Positive seedlings were identified by PCR amplification of the Kan gene in the vector. The primers used were:

[0073] Bn-Kan-F: 5'-ACTGGGCACAACAGACAATCG-3' (SEQ ID NO.5) and Bn-Kan-R: 5'-GCATCAGCCATGATGGATACTTT-3' (SEQ ID NO.6), with the control being a non-transgenic wild-type plant (WT).

[0074] The test results show that ( Figure 4 In the A section, seven transgenic plants, namely OE-29, OE-36, OE-37, OE-40, OE-45, OE-48, and OE-51, were able to amplify electrophoretic bands of the expected size (289 bp), while the wild-type control did not have any electrophoretic bands, indicating that the transgenic rapeseed genome already contains exogenous gene DNA fragments.

[0075] (4) qPCR identification of transgenic rapeseed overexpressing BnGATL10

[0076] a. Extraction of genomic RNA from rapeseed leaves

[0077] use The Super Total RNA Extraction Kit (purchased from Promega, USA) was used to extract total RNA from the leaves of WT and BnGATL10 overexpression positive lines identified in step (3). The total RNA was then extracted using... The II Q RT SuperMix for qRNA (+gDNAwiper) kit (Vazyme, China) reverse transcribes RNA into cDNA.

[0078] b. Real-time quantitative PCR

[0079] To determine whether BnGATL10 was overexpressed in rapeseed, real-time quantitative PCR (qPCR) was used to analyze the transgenic plants identified in step (3). qPCR used... The Green Realtime PCR MasterMix-Plus kit (Takara Bio Engineering (Dalian) Co., Ltd.) uses the rapeseed housekeeper gene BnActin7 (Brassicanapus actin-7) as an internal control gene, and its primers were synthesized by Nanjing Genscript Biotech Co., Ltd. The qPCR primers for BnActin7 (Gene ID: 106418315) are as follows:

[0080] BnActin7-F1: 5'-TCTTCCTCACGCTATCCCTCG-3' (SEQ ID NO.7) and BnActin7-R1: 5'-AGCCGTCTCCAGCTCTTGC-3' (SEQ ID NO.8)

[0081] The qPCR primers for the BnGATL10 gene are:

[0082] Q-BnGATL10-F: 5'-TGTTACTATGCTGACGACCCTAG-3' (SEQ ID NO.9) and Q-BnGATL10-R: 5'-TGCTACAGCGTTACAGCCTACT-3' (SEQ ID NO.10) (These primer pairs are qPCR-specific primers designed based on the nucleotide sequences obtained from sequencing in Example 1).

[0083] PCR program: 95℃ pre-denaturation for 30 seconds, followed by 40 cycles (95℃ for 10 seconds, 60℃ for 10 seconds, 72℃ for 26 seconds). The transgenic plants identified in step (3) were analyzed, and the overexpressing plants were numbered with OE (abbreviation for overexpression) added before their names.

[0084] like Figure 4As shown in Figure B, the BnGATL10 expression levels in lines OE-29, OE-36, OE-37, OE-40, OE-45, OE-48, and OE-51 were significantly higher than the non-transgenic wild-type (WT) control. The expression levels in lines OE-45 and OE-51 were 3 and 4 times higher than the wild-type lines, respectively; the expression levels in lines OE-36, OE-40, and OE-45 were more than 5 times higher; and the expression levels in lines OE-37 and OE-29 were approximately 5 and 6 times higher than the wild-type, respectively. This indicates that these lines are independent BnGATL10 overexpressing transgenic lines.

[0085] Example 3: Evaluation of resistance to Sclerotinia stem rot in BnGATL10 overexpressing transgenic rapeseed.

[0086] (1) Immunological response of BnGATL10 overexpression lines to Sclerotinia sclerotiorum.

[0087] The second-to-last leaves of rapeseed seedlings (four-leaf stage) were inoculated with *Sclerotinia sclerotiorum* (preserved and activated in the laboratory of the College of Life Sciences, Huazhong Agricultural University) as the pathogen of sclerotinia rot. The second-to-last leaves of wild-type plants and positive lines with significantly elevated expression levels as shown in Example 2 were taken. The leaves were cut and placed in transparent square boxes. Each transgenic plant leaf was placed side-by-side with a non-transgenic control in one box. Several layers of filter paper and damp gauze were placed under the leaves. Freshly prepared... Mycelial blocks, with the mycelial side down, were inoculated into the middle of the leaf, slightly off-center from the midrib, with two blocks inoculated per leaf. The leaves were covered to maintain humidity and then incubated in the dark at 22°C. Leaf observation was conducted continuously from 0 h to 72 h after inoculation. It was found that the growth area of ​​*Sclerotinia sclerotiorum* lesions in the BnGATL10 overexpressing transgenic lines was significantly smaller than that in the non-transgenic lines, and the area of ​​damaged tissue was also smaller. The most significant difference in resistance was observed at 36 h after inoculation. Figure 5 (A) Compared to the wild type, the area of ​​sclerotinia rot lesions on transgenic rapeseed leaves was reduced by 47.39% (A). Figure 5 (B in the text). Therefore, the BnGATL10 gene can effectively enhance the resistance of rapeseed leaves to sclerotinia stem rot.

[0088] (2) RNA extraction from rapeseed leaves after inoculation

[0089] use The Super Total RNA Extraction Kit (Promega, USA) was used to extract total RNA from leaves treated with *Sclerotinia sclerotiorum* for 36 hours in step (1). The RNA was then extracted using... The II Q RT SuperMix for qRNA (+gDNAwiper) kit (Vazyme, China) reverse transcribes RNA into cDNA.

[0090] (3) qPCR detection of disease resistance-related gene expression

[0091] qPCR use The Green Realtime PCR Master Mix-Plus kit contains the following primers for disease resistance-related genes:

[0092] The qPCR primers for the BnCAT1 gene are:

[0093] Q-BnCAT1-F:5'-TACAGACACGAAACAGCA-3' (SEQ ID NO.11) and

[0094] Q-BnCAT1-R:5'-GACAGAAACTAGCAAGCC-3' (SEQ ID NO.12);

[0095] The qPCR primers for the BnRbohD gene are:

[0096] Q-BnRbohD-F: 5'-TTAGGAACAAGACCAAGCTAGG-3' (SEQ ID NO. 13) and

[0097] Q-BnRbohD-R: 5'-CACAACCATCACAATACCAGTC-3' (SEQ ID NO. 14);

[0098] The qPCR primers for the BnPER21 gene are:

[0099] Q-BnPER21-F 5'-TTGCCCAAGTCCAAACCC-3' (SEQ ID NO. 15) and

[0100] Q-BnPER21-R 5'-GACCAGGAGCCCTCTATG-3' (SEQ ID NO. 16);

[0101] The qPCR primers for the BnPDF1.2 gene are:

[0102] Q-BnPDF1.2-F:5'-CATCACCCTTCTCTTCGCTGC-3'(SEQ ID NO.17) and

[0103] Q-BnPDF1.2-R:5'-ATGTCCCACTTGACCTCTCGC-3'(SEQ ID NO.18);

[0104] The qPCR primers for the BnPDF1.3 gene are:

[0105] Q-BnPDF1.3-F:5'-TCATGGCTAAGGCTGCTACC-3'(SEQ ID NO.19) and

[0106] Q-BnPDF1.3-R:5'-ACTCCTGACCATGTCCCACT-3'(SEQ ID NO.20);

[0107] The qPCR primers for the BnWRKY33 gene are:

[0108] Q-BnWRKY33-F:5'-GTACTTTCCCAAACTGTCCAAC-3'(SEQ ID NO.21) and

[0109] Q-BnWRKY33-R:5'-CGAAGAAGACGACGATCTTCTA-3'(SEQ ID NO.22)

[0110] The qPCR primers for the BnPR1 gene are:

[0111] Q-BnPR1-F:5'-ATCTCCGAAAAGGCCAGGGA-3'(SEQ ID NO.23) and

[0112] Q-BnPR1-R:5'-TACCATTTACCGCATCGCCC-3' (SEQ ID NO. 24);

[0113] The qPCR primers for the BnPR2 gene are:

[0114] Q-BnPR2-F:5'-ATCTCCGAAAAGCCAGGGA-3' (SEQ ID NO.25) and

[0115] Q-BnPR2-R:5'-TACCATTTACCGCATCGCCC-3' (SEQ ID NO.26);

[0116] The qPCR primers for the BnNPR1 gene are:

[0117] Q-BnNPR1-F:5'-ATCTCCGAAAAGCCAGGGA-3' (SEQ ID NO.27) and

[0118] Q-BnNPR1-R:5'-TACCATTTACCGCATCGCCC-3' (SEQ ID NO. 28);

[0119] The qPCR primers for the BnGPX7 gene are:

[0120] Q-BnGPX7-F:5'-ACATAGTATCGACCACGTGTTT-3' (SEQ ID NO.29) and

[0121] Q-BnGPX7-R:5'-GTCCGTATGTAGAACACAAGT-3' (SEQ ID NO.30);

[0122] The qPCR primers for the BnCSD1 gene are:

[0123] Q-BnCSD1-F:5'-TCGATCATTGACATGTTTGCTC-3' (SEQ ID NO.31) and

[0124] Q-BnCSD1-R:5'-AGACACAACCGGATATGAGATC-3' (SEQ ID NO.32);

[0125] The qPCR primers for the BnEXPA16 gene are:

[0126] Q-BnCSD1-F:5'-TCCTCCATAAATCACCCACAAA-3' (SEQ ID NO.33) and

[0127] Q-BnCSD1-R:5'-AAGAGAAAGAGAGGGAACATGG-3' (SEQ ID NO.34);

[0128] Specific primers were designed for the coding region of the gene using Primer Premier 5.0 software, and primer specificity was verified by comparing and analyzing the primers in the Brassica Genus Database (BRAD) and the Rapeseed EST database using Primer-BLAST (NCBI).

[0129] The qPCR reaction system consisted of 15 μL containing 7 μL SYBR Mix, 0.5 μL each of forward and reverse primers (10 μmol / L), 2 μL template, and 5 μL DEPC-treated sterile water. Amplification conditions were: 95℃ for 30 sec; then 95℃ for 5 s, 60℃ for 30 s, and 72℃ for 27 s, for 40 cycles; fluorescence detection was performed at the annealed ends at 72℃ after each cycle. After the reaction, the temperature was first raised to 95℃, then lowered to 72℃, and then slowly raised back to 95℃. Changes in fluorescence signal were recorded to obtain the melting curve of the amplified products. Each experiment was performed in triplicate, and each biological replicate was performed in at least three technical replicates. Primer pairs Q-BnCAT1-F / R, Q-BnRbohD-F / R, Q-BnPER21-F / R, Q-BnPDF1.2-F / R, Q-BnPR1-F / R, BnPR2-F / R, and Q-BnNPR1-F / R were used to target BnCAT1 (BnaA07g11370D), BnRbohD (BnaC02g38300D), and BnBnPER21 (BnaC03g20530D), respectively. Specific PCR amplification was performed on the genes BnPDF1.2 (BnaC02g23620D), BnPR1 (BnaC08g03660D), BnPR2 (BnaC04g24330D), BnNPR1 (BnaA01g15290D), BnGPX7 (BnaA03g51760D), BnCSD1 (BnaA06g05150D), and BnEXPA16 (BnaC08g26400D). Simultaneously, the BnActin7 gene from rapeseed was specifically amplified using primer pair BnActin7-F1 / R1 as an internal control for relative quantification. The expression changes of these disease resistance-related genes in rapeseed leaves 36 h after Sclerotinia sclerotiorum induction were detected. Figure 6 ).

[0130] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the resistance of rapeseed to sclerotinia stem rot, characterized in that, Including the pectin synthase gene BnGATL10 The process of introducing the pectin synthase gene into rapeseed and overexpressing it; BnGATL10 The nucleotide sequence is shown in SEQ ID NO.3; the rapeseed is Brassica napus.

2. The method according to claim 1, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector including the pectin synthase gene; (2) Transform the recombinant expression vector obtained in step (1) into Agrobacterium; (3) Rapeseed was transformed by Agrobacterium infection to obtain the pectin synthase gene. BnGATL10 The transgenic lines overexpressed; the transgenic lines showed higher resistance to sclerotinia stem rot than other lines.

3. The method according to claim 2, characterized in that, In step (3), the Agrobacterium infection method is to infect the hypocotyl of rape with Agrobacterium.

4. Overexpression of pectin synthase gene BnGATL10 Its application in improving the resistance of rapeseed to sclerotinia stem rot is characterized by... The pectin synthase gene BnGATL10 The nucleotide sequence is shown in SEQ ID NO.3; the rapeseed is Brassica napus.

5. Overexpression of pectin synthase gene BnGATL10 Its application in constructing transgenic rapeseed plants resistant to Sclerotinia stem rot is characterized by... The pectin synthase gene BnGATL10 The nucleotide sequence is shown in SEQ ID NO.3; the rapeseed is Brassica napus.

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