Application of rape gene BnDTX19 in prevention and control of sclerotiniose

Abstract

The invention provides an application of a Brassica napus gene BnDTX19 in prevention and control of sclerotiniose, which is an application in obtaining a rape material with changed disease resistance by creating transgenic rape. According to the invention, overexpression of BnDTX19 and RNAi transgenic oilseed rape are constructed, a positive regulation function of the gene on sclerotiniose resistance is disclosed for the first time, and application of the BnDTX19 gene to acquisition of a high sclerotiniose resistance oilseed rape material by creating overexpression oilseed rape and application of the BnDTX19 gene to acquisition of a sclerotiniose oilseed rape material with weakened sclerotiniose resistance by creating BnDTX19-RNAi oilseed rape are provided. The BnDTX19 gene coding product provided by the invention has the function of transferring the key pathogenic factor oxalic acid of sclerotinia sclerotiorum so as to promote disease resistance, and 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 rapeseed gene BnDTX19 in the prevention and control of sclerotinia disease. Background Art

[0002] 1. MATE transporters and regulation of plant disease resistance Transporters play an important role in plant immunity and disease resistance. Transporters are located in the cell membrane and are responsible for transporting various molecules (such as ions, metabolites, signaling molecules, etc.) in and out of the membrane, or between different organelles inside the cell. The plant MATE family is an important member of transporters, mainly involved in the transport of secondary metabolites and exogenous substances, the dynamic balance of iron, and the transport of organic molecules such as citric acid in response to aluminum. There are very few reports on the regulatory role of transporters in disease resistance.

[0003] The inventors have previously found that a member of the rapeseed MATE family, BnDTX19, can directly bind to and transport oxalic acid, an important pathogenic factor of Sclerotinia sclerotiorum, and thus plays an important regulatory role in Sclerotinia sclerotiorum resistance. The present invention uses a genetic method to clarify the regulatory role of BnDTX19 on Sclerotinia sclerotiorum resistance, and uses the gene to create disease-resistant germplasm.

[0004] 2. Plant Sclerotinia disease prevention and control technology Plant sclerotinia disease is caused by infection by Sclerotinia sclerotiorum. Sclerotinia sclerotiorum is a necrotrophic pathogenic fungus with a very wide host range. It is a major disease of crops such as oilseeds and vegetables. 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, it is crucial to identify important anti-sclerotinia disease regulatory genes and create and use disease-resistant varieties for green prevention and control of sclerotinia disease.

[0005] 3. 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 gene BnDTX19 in the prevention and control of sclerotinia sclerotiorum, which is an application of a rapeseed (Brassica napus) transport protein gene BnDTX19 in the prevention and control of sclerotinia sclerotiorum. The rapeseed gene BnDTX19 has a positive regulatory function on sclerotinia sclerotiorum resistance and can be used to improve or weaken the creation of crop germplasm resistant to 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 BnDTX19, or to obtain rapeseed materials with reduced resistance to sclerotinia by creating BnDTX19-RNAi transgenic 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 BnDTX19 by PCR cloning, the nucleotide sequence of which is shown in SEQ ID No.1, the open reading frame (ORF) of the gene is 1428bp long, and the encoded protein consists of 475 amino acids, and its sequence is shown in SEQ ID No.2. The BnDTX19 protein contains a transmembrane domain and two MatE domains. The nucleotide sequence cloned by the present invention is consistent with the BnaC01G0357100ZS nucleotide sequence and protein sequence of the rapeseed variety ZS11 in the database of the Brassica napus multi-omics database (information resource), a website created by Huazhong Agricultural University.

[0009] Prior to the present invention, there was no public report on the function of the BnDTX19 gene. The present invention, for the first time, clarified the regulatory effect of the gene on rapeseed resistance to Sclerotinia sclerotiorum by constructing overexpression and RNAi transgenic rapeseed and its disease resistance analysis. The results of inoculation analysis showed that compared with wild-type rapeseed plants, overexpression transgenic rapeseed plants were significantly more resistant to Sclerotinia sclerotiorum, while RNAi transgenic plants were significantly more susceptible to Sclerotinia sclerotiorum, indicating that BnDTX19 positively regulates rapeseed resistance to Sclerotinia sclerotiorum.

[0010] Based on the function of the BnDTX19 gene explained in the present invention, the application purpose of the present invention is to provide the application of the rapeseed BnDTX19 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 BnDTX19 (Example 1); and (2) application in obtaining rapeseed materials with reduced resistance to sclerotinia by creating BnDTX19-RNAi transgenic rapeseed (Example 2).

[0011] The application of the rapeseed BnDTX19 gene in obtaining rapeseed materials with enhanced resistance to sclerotinia sclerotiorum by creating over-expression transgenic rapeseed is achieved by the following steps: (1) Construction and acquisition of BnDTX19 gene overexpression structure: The BnDTX19 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 BnDTX19 gene overexpression structure: The constructed BnDTX19 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 BnDTX19: The BnDTX19 gene overexpression structure was introduced into rapeseed by Agrobacterium-mediated method to obtain the transgenic rapeseed overexpressing BnDTX19 gene; (4) Obtaining homozygous transgenic rapeseed lines overexpressing BnDTX19: Using antibiotic resistance and BnDTX19 gene expression as detection indicators, the trait segregation of the offspring of transgenic plants was detected, and homozygous transgenic rapeseed lines overexpressing BnDTX19 whose offspring traits no longer segregated and could be stably inherited were obtained; (5) Screening, identification and acquisition of homozygous transgenic rapeseed lines overexpressing BnDTX19 with enhanced resistance to sclerotinia sclerotinia: Using homozygous transgenic rapeseed lines overexpressing BnDTX19 as materials, the resistance to sclerotinia sclerotinia was detected and analyzed, and transgenic rapeseed with the BnDTX19 gene with enhanced disease resistance was obtained.

[0012] The application of the rapeseed BnDTX19 gene in obtaining rapeseed materials with reduced resistance to sclerotinia sclerotiorum by creating RNAi transgenic rapeseed is achieved by the following steps: (1) Construction and acquisition of RNAi structure of BnDTX19 gene: A specific sequence fragment of BnDTX19 gene was cloned into RNAi vector pBWA(V)BS-ccdB-RNAi to obtain RNAi structure pBWA(V)BS-RNAi-BnDTX19; (2) Obtaining Agrobacterium transformed with BnDTX19 gene RNAi structure: The BnDTX19 gene RNAi structure (pBWA(V)BS-RNAi-BnDTX19) was transformed into an Agrobacterium strain with strong infection ability to rapeseed by electroporation or other methods; (3) Creation and acquisition of rapeseed transgenic with BnDTX19 gene RNAi structure: The BnDTX19 gene RNAi structure was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed transgenic with BnDTX19 gene RNAi structure; (4) Obtaining homozygous lines of rapeseed with BnDTX19 gene RNAi structure: Screening with antibiotic resistance to detect the segregation of traits of the offspring of transgenic plants, and obtaining homozygous lines of rapeseed with BnDTX19 gene RNAi structure whose offspring traits no longer segregate and can be stably inherited; (5) Screening, identification and acquisition of homozygous rapeseed lines carrying the BnDTX19 gene RNAi structure with reduced resistance to sclerotinia sclerotinia: The homozygous rapeseed lines carrying the BnDTX19 gene RNAi structure (pBWA(V)BS-RNAi-BnDTX19) were used as materials to detect and analyze resistance to sclerotinia sclerotinia, and to obtain BnDTX19 gene RNAi rapeseed lines with reduced resistance to sclerotinia sclerotinia.

[0013] Advantages of the present invention: (1) The BnDTX19 gene provided by the present invention is a high-quality anti-sclerotinia disease regulatory gene resource, and its disease resistance mechanism is strongly targeted to Sclerotinia sclerotiorum. The disease-resistant material obtained by 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 this 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 BnDTX19 gene encodes a transporter protein that can transport oxalic acid, a key pathogenic factor of Sclerotinia sclerotiorum, thereby enhancing the plant's resistance to sclerotinia disease. Using BnDTX19 to create disease-resistant germplasm is an economical, effective and safe way to greenly prevent and control sclerotinia disease. Therefore, the BnDTX19 gene is a brand-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-resistant 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 BnDTX19 and adopts genetic engineering methods to create and cultivate highly resistant sclerotinia rapeseed materials, which have the characteristics of short cycle and rapid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The evidence of the transgenic rapeseed plants obtained in this paper is the gene expression detection of BnDTX19 overexpression plants and RNAi plants, showing the results of BnDTX19 gene expression detection in transgenic rapeseed plants. Real-time fluorescence quantitative PCR was used to detect the expression level of BnDTX19 gene in OE plants overexpressing BnDTX19 gene (A) and RNAi plants silencing BnDTX19 gene (B). Rapeseed BnACTIN7 was selected as the internal reference gene, and the relative expression level of wild-type ZS11 was set as 1. qRT-PCR analysis was performed three times, each containing three technical replicates. GraphPad Prism was used to perform Student's t-test statistical analysis on the expression results, and the data were expressed as mean ± standard deviation. The significance of the difference is indicated by different numbers of * (**, P<0.01; ***, P<0.001, ****, P<0.0001). The results showed that the expression levels of BnDTX19 in the three overexpression lines, line 2, line 10, and line 13, were significantly increased, 5.6 times, 8.3 times, and 3 times that of the control, respectively (A), while the expression level of BnDTX19 in the RNAi plants was significantly reduced, only 17.5% of the control plants (B), indicating that these plants are true BnDTX19 gene overexpression plants and RNAi plants.

[0015] Figure 2 The results provide evidence for the anti-bacterial sclerotinia function of the BnDTX19 gene, indicating that BnDTX19 positively regulates rapeseed's resistance to Sclerotinia sclerotiorum. The rapeseed BnDTX19 transgenic plants were inoculated with the Sclerotinia sclerotiorum UF-1 strain. The figure shows the phenotype and lesion area statistical analysis results of the overexpression plants (A) and RNAi plants (B) after inoculation. The inoculation experiment was repeated three times. The Student's t-test was used to analyze the lesion area using GraphPad Prism, and the data are expressed as mean ± standard deviation. The significant difference is indicated by different numbers of * (****, P<0.0001). The results showed that the overexpression plants showed a significantly more disease-resistant phenotype than the control (ZS11) plants (A), while the RNAi plants were significantly more susceptible than the control (B). The quantitative analysis of the lesion area showed that the lesion areas of the three strains of the overexpression plants were 144.1 mm 2 、165.3mm 2 and 155.2mm 2 , significantly lower than the control of 213mm 2 The lesion area of ​​the RNAi strain was 177.8 mm 2 , significantly higher than the control group of 123.7 mm 2These results suggest that BnDTX19 positively regulates rapeseed resistance to S. 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 BnDTX19, and for the first time, clarified the function of the gene in conferring resistance to sclerotinia sclerotinia by constructing an overexpressed transgenic rapeseed. Overexpression of the BnDTX19 gene leads to a significant increase in rapeseed's resistance to sclerotinia sclerotinia. Therefore, a new rapeseed material with enhanced resistance to sclerotinia sclerotinia can be created by constructing a homozygous line of overexpressed rapeseed of the BnDTX19 gene, which can be used for the creation and breeding of sclerotinia sclerotinia resistant rapeseed varieties, analysis of the function and mechanism of action of the BnDTX19 gene, and the creation and acquisition of new rapeseed materials with enhanced resistance to sclerotinia sclerotinia. The main steps of cloning the BnDTX19 gene, analyzing its function and mechanism, and creating and obtaining new rapeseed materials with enhanced resistance to sclerotinia sclerotinia sclerotinia include:

[0018] 1) Cloning and conservation of the rapeseed BnDTX19 gene The rapeseed BnDTX19 gene provided by the present invention is cloned by the following steps: First, primers BnDTX19-F (5'-atggctgatcgcatcaacacgg-3') (sequence as shown in SEQ ID No. 3) and BnDTX19-R (5'-gaccgacgcatttatctttgtc-3') (sequence as shown in SEQ ID No. 4) are designed according to the BnDTX19 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 BnDTX19 cDNA. The PCR product was recovered by 1% agarose gel electrophoresis and then purified by gel cutting. The pEASY-BluntCloning 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 with BnDTX19-F / BnDTX19-R as the primer pair was used to test whether the extracted plasmid contained the BnDTX19 gene. Finally, it was sent to the company for sequencing verification, thereby successfully cloning and obtaining the full-length cDNA sequence of the BnDTX19 gene. The nucleotide sequence of BnDTX19 is shown in SEQ ID No.1, the open reading frame (ORF) of the gene is 1428bp long, and the encoded protein consists of 475 amino acids, and its sequence is shown in SEQ ID No.2. The gene encoding product contains a transmembrane domain and two MatE domains. BLAST analysis revealed that the BnDTX19 gene sequence cloned in the present invention is consistent with the nucleotide sequence and protein sequence of BnaC01G0357100ZS of rapeseed variety ZS11 in the database of BnIR, Brassica napus multi-omics database (information resource) created by Huazhong Agricultural University. Prior to the present invention, there was no public report on the function of this gene.

[0019] E. coli carrying the pEASY-BnDTX19 vector were transformed and stored in a -80°C refrigerator. The BnDTX19 gene can be subcloned into the target vector at any time by activating the strain, extracting the plasmid, and amplifying by PCR for transgenic research.

[0020] 2) Construction and acquisition of BnDTX19 gene overexpression structure According to the BnDTX19 sequence cloned according to the present invention, primers BnDTX19-F2 (5'-agagaacacgggggactttgcaac atggctgatcgcatcaacacg-3', the italic part is the sequence consistent with the vector pBWA(V)BS-OE) (the sequence is shown in SEQ ID No.5), and BnDTX19-R2 (5'-gtactgaagacagagctagttaca tcagaccgacgcatttatctttgtcc-3', the italic part is the sequence consistent with the vector pBWA(V)BS-OE) (the sequence is shown in SEQ ID No.6) were designed. The pEASY-BnDTX19 plasmid obtained in 1) of Example 1 was used as a template, and BnDTX19-F2 / BnDTX19-R2 was used as a primer pair to obtain the target gene by PCR amplification.

[0021] The overexpression vector pBWA(V)BS-OE plasmid was digested with Eco 31I (Bsa I), and the amplified target gene was connected to the pBWA(V)BS-OE vector after digestion. The ligase used in this experiment was T4_ligase. The ligation product was transformed into E. coli, screened on kanamycin plates, identified by PCR, and sequenced to obtain the BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19. The overexpression vector pBWA(V)BS-OE drives the expression of the target gene with the CaMV 35S promoter and does not carry any tags.

[0022] 3) Obtaining Agrobacterium Transformed with BnDTX19 Gene Overexpression Structure pBWA(V)BS-OE-BnDTX19 The BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19 was transformed into an Agrobacterium strain with strong infectivity to rapeseed, such as GV3101, by electroporation and other methods, and transformants were selected on LB medium containing kanamycin and rifampicin, and then identified by PCR to obtain Agrobacterium carrying the BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19, which was used for the next step of rapeseed genetic transformation.

[0023] 4) Creation and acquisition of the BnDTX19 gene overexpression construct pBWA(V)BS-OE-BnDTX19 in rapeseed The BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19 was introduced into rapeseed by Agrobacterium-mediated method to obtain the rapeseed T0 generation transformed with pBWA(V)BS-OE-BnDTX19. 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 glufosinate-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 BnDTX19 gene overexpression construct pBWA(V)BS-OE-BnDTX19 The glufosinate resistance and BnDTX19 gene expression were used as detection indicators to detect the segregation of traits in the offspring of transgenic plants. The glufosinate resistance screening was carried out on rapeseed seeds on plates containing glufosinate to observe whether they could grow normally into healthy seedlings on the glufosinate resistance plates. Gene expression was detected by real-time fluorescence quantitative PCR and other methods. The homozygous rapeseed lines of the BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19 were obtained, and the offspring traits were no longer separated and could be stably inherited.

[0033] The present invention obtains three rapeseed homozygous lines, OE-line 2, OE-line 10 and OE-line 13, all of which can grow normally into healthy seedlings on glufosinate-resistant plates, and the expression levels of the BnDTX19 gene are significantly higher than those of the control plants, which are 5.6 times, 8.3 times and 3 times that of the control, respectively (( Figure 1 A). These plants are true BnDTX19 gene overexpressing plants.

[0034] 6) Disease resistance analysis of homozygous rapeseed lines overexpressing BnDTX19 gene The homozygous rapeseed line overexpressing the BnDTX19 gene obtained in 5) was used as material, and its resistance to rapeseed sclerotinia sclerotiorum was detected and analyzed by inoculation with Sclerotinia sclerotiorum, thereby clarifying the regulatory effect of the BnDTX19 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 them 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 3-5mm 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 sclerotiorum: Select rape 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 them with the hyphae facing down in the middle of fully developed leaves. Inoculate two hyphae blocks symmetrically on the left and right halves of each leaf, cover with film to keep moisture, and place in a 23℃ greenhouse for cultivation. After an appropriate period of time (about 24 hours), take photos and record them, and analyze the area of ​​lesions 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 BnDTX19 overexpressing plants were significantly more resistant to the disease than the non-transgenic control plants (ZS11). Figure 2A). Quantitative analysis of the lesion area showed that the lesion areas of the three strains of overexpression plants were 144.1 mm 2 、165.3mm 2 and 155.2mm 2 , significantly lower than the control of 213mm 2 ( Figure 2 B).

[0038] These results show that the resistance of BnDTX19 overexpressing plants to sclerotinia is significantly higher than that of non-transgenic control plants. Overexpression of the BnDTX19 gene leads to a significant increase in the resistance of rapeseed to sclerotinia, so BnDTX19 plays a positive regulatory role in the resistance of rapeseed to sclerotinia. The present invention successfully created a new rapeseed material with high resistance to sclerotinia by constructing BnDTX19-OE rapeseed.

[0039] Example 2 Based on the positive regulatory function of the rapeseed gene BnDTX19 on sclerotinia resistance explained in the present invention, a technical system for creating and obtaining new rapeseed materials with reduced sclerotinia resistance by constructing RNAi transgenic rapeseed of the gene and using genetic engineering technology has been established. The main steps include:

[0040] (1) Construction and acquisition of BnDTX19 gene RNAi structure According to the BnDTX19 sequence cloned according to the present invention, primers BnDTX19-F3 (5'-cagtGGTCTCacaacatctcatatgttctggtctacatcgctgg-3') (sequence as shown in SEQ ID No.7), and BnDTX19-R3 (5'-cgatGGTCTCacagggctaatggactccgtgttgacacatatag-3') (sequence as shown in SEQ ID No.8); BnDTX19-F4 (5'-cgatGGTCTCacctgcaggtctagttttctccttcattttc-3') (sequence as shown in SEQ ID No.9), and BnDTX19-R4 (5'-cgatGGTCTCagcccgggctctgtaactatcatc-3') (sequence as shown in SEQ ID No.10); BnDTX19-F5 (5'-cagtGGTCTCagggcgctaatggactccgtgttgacacatatag-3') (the sequence is shown in SEQ ID No.11), and BnDTX19-R5 (5'-cagtGGTCTCatacaatctcatatgttctggtctacatcgctgg-3') (the sequence is shown in SEQ ID No.12), wherein GGTCTC is the recognition site of Eco 31I (Bsa I). Using the pEASY-BnDTX19 plasmid obtained in 1) of Example 1 as a template, BnDTX19-F3 / BnDTX19-R3 as a primer pair, PCR amplification was performed to obtain the BnDTX19 forward fragment (fragment 1), BnDTX19-F4 / BnDTX19-R4 as a primer pair, PCR amplification was performed to obtain the BnDTX19Loop (fragment 2), BnDTX19-F5 / BnDTX19-R5 as a primer pair, PCR amplification was performed to obtain the BnDTX19 reverse fragment (fragment 3) and recovered respectively, and fragments 1, 2, 3 and Eco 31I (Bsa I) The linearized vector pBWA(V)BS-ccdB-RNAi was ligated with T4-ligase, transformed, screened on kanamycin culture medium, digested, PCR and sequenced to obtain the pBWA(V)BS-RNAi-BnDTX19 vector containing the forward, LooP and reverse BnDTX19 fragments. Thus, the RNAi structure pBWA(V)BS-RNAi-BnDTX19 was obtained.

[0041] (2) Obtaining Agrobacterium Transformed with BnDTX19 Gene RNAi Structure The BnDTX19 gene RNAi structure (pBWA(V)BS-RNAi-BnDTX19) was transformed into an Agrobacterium strain with strong infection ability to rapeseed, such as GV3101, by electroporation and other methods. Transformants were selected on LB medium containing kanamycin and rifampicin, and then identified by PCR to obtain Agrobacterium carrying the BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19.

[0042] (3) Creation and acquisition of BnDTX19 gene RNAi-transgenic rapeseed The BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19 was introduced into rapeseed by Agrobacterium-mediated method to obtain the rapeseed T0 generation transformed with pBWA(V)BS-RNAi-BnDTX19. The specific operation steps are the same as those described in 4) of Example 1.

[0043] (4) Obtaining homozygous lines of rapeseed transgenic for BnDTX19 gene RNAi structure The antibiotic resistance and BnDTX19 gene expression were used as detection indicators to detect the segregation of traits of the offspring of transgenic plants. The test was carried out on rapeseed seeds on plates containing glufosinate resistance to observe whether they can grow normally into healthy seedlings on the glufosinate resistance plates. The homozygous rapeseed lines with the BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19, whose offspring traits no longer separated and could be stably inherited, were obtained. All of these rapeseed homozygous lines were able to grow normally into healthy seedlings on glufosinate resistance plates, and the expression level of the BnDTX19 gene was significantly lower than that of the wild-type Zhong Shuang 11 control, with only 17.5% ( Figure 1 B).

[0044] The present invention obtains a BnDTX19-RNAi rapeseed homozygous line, which can grow normally into healthy seedlings on a glufosinate-resistant plate, and the expression level of the BnDTX19 gene is significantly lower than that of the wild-type Zhong Shuang 11 control, with only 17.5% ( Figure 1 A). These plants are true BnDTX19 gene RNAi plants.

[0045] (5) Screening, identification and acquisition of homozygous rapeseed lines with reduced resistance to sclerotinia sclerotinia The homozygous rapeseed line transformed with the BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19 was used as 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 BnDTX19-RNAi plants constructed by the present invention showed that the BnDTX19-RNAi plants were significantly more susceptible to the disease than the non-transgenic plants (ZS11) control ( Figure 2 B). Quantitative analysis of the lesion area showed that the lesion area of ​​the RNAi strain was 177.8 mm 2 , significantly higher than the control group of 123.7 mm 2 ( Figure 2 B). It shows that the resistance of BnDTX19-RNAi plants to sclerotinia is significantly lower than that of non-transgenic control plants. The inhibition of BnDTX19 gene expression leads to a significant reduction in the resistance of rapeseed to sclerotinia. The present invention successfully creates and obtains a new rapeseed material with reduced resistance to sclerotinia by constructing BnDTX19-RNAi rapeseed.

[0047] In summary, the present invention combines Figure 1-Figure 2 The results revealed for the first time the positive regulatory effect of rapeseed transporter gene BnDTX19 on rapeseed resistance to Sclerotinia sclerotinia, provided the application approach, application technology and implementation example of BnDTX19 in the creation of Sclerotinia sclerotinia-resistant crop germplasm, and successfully obtained highly resistant Sclerotinia sclerotinia-resistant rapeseed.

Claims

1. Application of a Brassica napus gene BnDTX19 in the prevention and control of sclerotinia sclerotiorum, characterized in that: The nucleotide sequence of the gene BnDTX19 is shown in SEQ ID No. 1, and the protein sequences encoded by the gene are shown in 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 BnDTX19.

4. The use according to claim 2, characterized in that: Application in obtaining rapeseed materials with reduced resistance to sclerotinia disease by creating BnDTX19-RNAi transgenic 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 BnDTX19 gene overexpression structure The open reading frame of the BnDTX19 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 BnDTX19 gene overexpression structure The constructed BnDTX19 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 BnDTX19 The BnDTX19 gene overexpression structure was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed transformed with the BnDTX19 gene overexpression structure; (4) Obtaining homozygous transgenic rapeseed lines overexpressing BnDTX19 The antibiotic resistance and BnDTX19 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 BnDTX19 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 BnDTX19 with enhanced resistance to Sclerotinia sclerotiorum The homozygous transgenic rapeseed overexpressing BnDTX19 was used as material to detect and analyze the resistance to Sclerotinia sclerotiorum, and to obtain transgenic rapeseed with BnDTX19 gene with enhanced disease resistance.

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 BnDTX19 gene RNAi structure A specific sequence fragment of the BnDTX19 gene was cloned into the RNAi vector pBWA(V)BS-ccdB-RNAi to obtain the RNAi structure pBWA(V)BS-RNAi-BnDTX19; (2) Obtaining Agrobacterium Transformed with BnDTX19 Gene RNAi Structure The BnDTX19 gene RNAi structure (pBWA(V)BS-RNAi-BnDTX19) was transformed into an Agrobacterium strain with strong infection ability to rapeseed by electroporation; (3) Creation and acquisition of BnDTX19 gene RNAi-transgenic rapeseed The BnDTX19 gene RNAi structure was introduced into rapeseed by Agrobacterium-mediated method to obtain rapeseed transformed with the BnDTX19 gene RNAi structure; (4) Obtaining homozygous lines of rapeseed transgenic for BnDTX19 gene RNAi structure The characteristics of the offspring of transgenic plants were detected by antibiotic resistance screening, and homozygous rapeseed lines with BnDTX19 gene RNAi structure were obtained, in which the offspring characteristics no longer separated and could be stably inherited; (5) Screening, identification and acquisition of homozygous rapeseed lines with reduced resistance to sclerotinia sclerotinia The rapeseed homozygous lines transformed with the BnDTX19 gene RNAi structure (pBWA(V)BS-RNAi-BnDTX19) were used as materials to detect and analyze the resistance to sclerotinia sclerotinia, and to obtain the BnDTX19 gene RNAi rapeseed homozygous lines with weakened resistance to sclerotinia sclerotinia.

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