Application of Brassica napus gene BnDTX19 in sclerotinia prevention and control

Abstract

The application provides application of Brassica napus gene BnDTX19 in sclerotinia prevention and control, and is obtained by creating transgenic rapeseed to change the disease resistance of rapeseed material. The application constructs the super-expression and RNAi transgenic rapeseed of BnDTX19, firstly discloses the positive regulation function of the gene on sclerotinia resistance, provides the application of the BnDTX19 gene in obtaining high-sclerotinia-resistant rapeseed material by creating super-expression rapeseed, and the application of the BnDTX19-RNAi rapeseed in obtaining sclerotinia-resistant rapeseed material with weakened sclerotinia resistance. The BnDTX19 gene product provided by the application has the function of transporting the key pathogenic factor oxalic acid of sclerotinia to promote disease resistance, and is a new gene resource suitable for creating and breeding new sclerotinia-resistant rapeseed materials and new varieties.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to application of a Brassica napus gene BnDTX19 in sclerotinia disease prevention and control. BACKGROUND

[0002] 1. MATE transporter and plant disease resistance regulation

[0003] 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, signal molecules, etc.) in and out of the membrane, or between different organelles in the cell. The plant MATE family is an important member of transporters, mainly involved in the transport of secondary metabolites and exogenous substances, iron homeostasis, and the transport of organic molecules such as citric acid in response to aluminum. There are few reports on the regulation of disease resistance.

[0004] The present inventors have found in previous studies that a Brassica napus MATE family member BnDTX19 can directly bind and transport oxalic acid, an important pathogenic factor of Sclerotinia sclerotiorum, and thus plays an important regulatory role in sclerotinia resistance. The present application illustrates the regulatory role of BnDTX19 in sclerotinia resistance through genetic methods, and uses the gene to create disease-resistant germplasm.

[0005] 2. Plant sclerotinia disease prevention and control technology

[0006] Plant sclerotinia disease is caused by infection of Sclerotinia sclerotiorum. Sclerotinia sclerotiorum is a dead body nutrition type pathogenic fungus with a wide host range, and is a major disease of oil 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 development of drug resistance of some pesticides, it is important to identify important sclerotinia disease resistance regulation genes, create and use disease-resistant varieties for green prevention and control of sclerotinia disease.

[0007] 3. Plant disease resistance breeding technology

[0008] Plant disease-resistant breeding techniques are mainly divided into traditional disease-resistant breeding and disease-resistant breeding by genetic engineering and molecular modification. Traditional disease-resistant breeding is significantly limited in the use of disease-resistant resources due to natural genetic isolation, and only disease-resistant resources with close genetic relationship can be used. In addition, it requires multiple crosses and backcrosses, and thus has a long breeding cycle and requires a large amount of manpower and material resources. The genetic engineering and molecular modification breeding method is to introduce an exogenous disease-resistant regulatory gene or a modified gene into a plant by Agrobacterium-mediated method, or to knock out or increase the expression of a target gene in a plant by gene editing technology, so that the plant obtains or improves the disease resistance. Therefore, the genetic engineering and molecular modification breeding method breaks the limitation of natural genetic isolation, widens the range of available disease-resistant 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-resistant 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 is particularly suitable for breeding varieties with broad-spectrum and durable disease resistance. SUMMARY

[0009] The application of the oilseed rape gene BnDTX19 in sclerotinia disease prevention and control is provided, which is the application of the Brassica napus transporter gene BnDTX19 in sclerotinia disease prevention and control. The oilseed rape gene BnDTX19 has a positive regulation function on sclerotinia disease resistance, and can be applied to the creation of crop germplasm with increased or decreased resistance to Sclerotinia sclerotiorum.

[0010] The application is the application of the transgenic oilseed rape to obtain oilseed rape materials with changed disease resistance. It is the application of transgenic oilseed rape overexpressing BnDTX19 to obtain oilseed rape materials with increased sclerotinia disease resistance, or the application of BnDTX19-RNAi transgenic oilseed rape to obtain oilseed rape materials with reduced sclerotinia disease resistance.

[0011] The application takes Brassica napus double 11 variety cDNA as a template, and obtains Brassica napus gene BnDTX19 through PCR cloning, the nucleotide sequence of which is shown as SEQ ID No. 1, the open reading frame (ORF) of the gene is 1428 bp long, and the encoded protein is composed of 475 amino acids, and the sequence is shown as SEQ ID No. 2. The BnDTX19 protein contains a transmembrane domain and two MatE domains. The nucleotide sequence cloned in the application is consistent with the BnaC01G0357100ZS nucleotide sequence of Brassica napus variety ZS11 in the database of the website BnIR, Brassica napus multi-omics database (information resource) created by Huazhong Agricultural University.

[0012] Before the application, there is no any public report on the function of the BnDTX19 gene. The application first clarifies the regulation effect of the gene on the resistance of Brassica napus to Sclerotinia sclerotiorum by constructing super-expression and RNAi transgenic Brassica napus of the gene and analyzing the disease resistance of the transgenic plants. The inoculation analysis result shows that the super-expression transgenic Brassica napus plants are significantly more resistant to Sclerotinia sclerotiorum than the wild-type Brassica napus plants, and the RNAi transgenic plants are significantly more susceptible to Sclerotinia sclerotiorum, which indicates that BnDTX19 positively regulates the resistance of Brassica napus to Sclerotinia sclerotiorum.

[0013] Based on the function of the BnDTX19 gene clarified in the application, the application aims to provide the application of the Brassica napus BnDTX19 gene in obtaining the Brassica napus material with changed disease resistance by creating transgenic Brassica napus, including (1) the application in obtaining the Brassica napus material with increased resistance to Sclerotinia sclerotiorum by creating the transgenic Brassica napus with super-expressed BnDTX19 (Example 1); and (2) the application in obtaining the Brassica napus material with reduced resistance to Sclerotinia sclerotiorum by creating the transgenic Brassica napus with BnDTX19-RNAi (Example 2).

[0014] The application of the Brassica napus BnDTX19 gene in obtaining the Brassica napus material with increased resistance to Sclerotinia sclerotiorum by creating the transgenic Brassica napus with super-expression is realized through the following steps:

[0015] (1) Construction and acquisition of BnDTX19 gene super-expression structure: the open reading frame (ORF) of the BnDTX19 gene is cloned into a plant expression vector to be expressed under the drive of a strong promoter;

[0016] (2) Acquisition of Agrobacterium transformed with the BnDTX19 gene super-expression structure: the constructed BnDTX19 gene super-expression structure is transformed into an Agrobacterium strain with strong invasiveness to Brassica napus through methods such as electroporation;

[0017] (3) Transgenic rape over-expressing BnDTX19 is created and obtained: BnDTX19 gene over-expression structure is introduced into rape by Agrobacterium-mediated method, and transgenic rape over-expressing BnDTX19 is obtained;

[0018] (4) Transgenic rape over-expressing BnDTX19 homozygous line is obtained: antibiotic resistance and BnDTX19 gene expression are taken as detection indexes respectively, trait segregation of transgenic plant offspring is detected, and transgenic rape over-expressing BnDTX19 homozygous line which is not segregated in offspring traits and can be stably inherited is obtained;

[0019] (5) Transgenic rape over-expressing BnDTX19 homozygous line with increased resistance to sclerotinia is screened, identified and obtained: transgenic rape over-expressing BnDTX19 homozygous line is taken as material, resistance to sclerotinia is detected and analyzed, and transgenic rape over-expressing BnDTX19 with increased resistance to sclerotinia is obtained.

[0020] The application of rape BnDTX19 gene in obtaining rape material with reduced resistance to sclerotinia by creating RNAi transgenic rape is realized by the following steps:

[0021] (1) BnDTX19 gene RNAi structure is constructed and obtained: a specific sequence fragment of BnDTX19 gene is cloned into RNAi vector pBWA(V)BS-ccdB-RNAi, and RNAi structure pBWA(V)BS-RNAi-BnDTX19 is obtained;

[0022] (2) Agrobacterium transformed with BnDTX19 gene RNAi structure is obtained: BnDTX19 gene RNAi structure (pBWA(V)BS-RNAi-BnDTX19) is transformed into Agrobacterium strain with strong invasion ability to rape by electric shock or other methods;

[0023] (3) Transgenic rape over-expressing BnDTX19 is created and obtained: BnDTX19 gene RNAi structure is introduced into rape by Agrobacterium-mediated method, and transgenic rape over-expressing BnDTX19 is obtained;

[0024] (4) Transgenic rape over-expressing BnDTX19 homozygous line is obtained: antibiotic resistance is screened, trait segregation of transgenic plant offspring is detected, and transgenic rape over-expressing BnDTX19 homozygous line which is not segregated in offspring traits and can be stably inherited is obtained;

[0025] (5) Screening, identifying and obtaining of homozygous line of BnDTX19 gene RNAi structure rapeseed with weakened resistance to sclerotinia: using homozygous line of BnDTX19 gene RNAi structure rapeseed (pBWA(V)BS-RNAi-BnDTX19) as material, detecting and analyzing the resistance to sclerotinia, and obtaining BnDTX19 gene RNAi rapeseed with weakened resistance to sclerotinia.

[0026] Advantages of the present application: (1) the BnDTX19 gene provided by the present application is a high-quality resistance to sclerotinia regulating gene resource, and the disease resistance mechanism is strongly targeted to sclerotinia sclerotiorum, and the disease-resistant material obtained by using the gene has the advantages of strong disease resistance. The resistance of rapeseed to sclerotinia is quantitative resistance, which is controlled by multiple genes. Generally speaking, the degree of regulation of a single gene on the resistance is low. Therefore, the rapeseed material with resistance to sclerotinia is very scarce worldwide, and there is no high-resistance material. BnDTX19 gene encodes a transporter protein that can transport the key pathogenic factor oxalic acid of sclerotinia sclerotiorum, thereby enhancing the resistance of plants to sclerotinia. Using BnDTX19 to create disease-resistant germplasm is an economical, effective and safe way for green prevention and control of sclerotinia. Therefore, BnDTX19 gene is a new gene resource suitable for creating and breeding new materials and new varieties of rapeseed resistant to sclerotinia. (2) The period of obtaining disease-resistant material is short. The methods of obtaining disease-resistant plant material and variety mainly include conventional traditional breeding method and genetic engineering breeding method using disease-resistant regulating gene. The traditional breeding method has the disadvantages of limited range of available disease-resistant resources due to natural genetic isolation, long breeding period, and the need for a large amount of labor and material. The genetic engineering breeding method has the advantages of wide range of available disease-resistant resources, relatively simple and convenient operation, short breeding period, no need for a large amount of labor and material, and is particularly suitable for breeding broad-spectrum, durable and highly disease-resistant varieties. The present application uses disease-resistant regulating gene BnDTX19 and adopts genetic engineering method to create and breed rapeseed material with high resistance to sclerotinia, which has the characteristics of short period and rapid breeding. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1The evidence of the obtained transgenic Brassica napus plants is provided for the gene expression detection of BnDTX19 overexpression plants and RNAi plants, and the BnDTX19 gene expression detection results in the transgenic Brassica napus plants are shown. The BnDTX19 gene expression levels in the OE plants overexpressing BnDTX19 gene (A) and the RNAi plants silencing BnDTX19 gene (B) are detected by real-time fluorescent quantitative PCR. Brassica napus BnACTIN7 is selected as the internal reference gene, and the relative expression of the wild type ZS11 is set as 1. The qRT-PCR analysis is performed for three biological repeats, each of which includes three technical repeats, the expression results are statistically analyzed by Student's t-test test using GraphPad Prism, and the data are represented by mean ± standard deviation. The significant differences are represented by different number of asterisks (**, P < 0.01; ***, P < 0.001, ****, P < 0.0001). The results show that the expression levels of BnDTX19 in three overexpression lines line 2, line 10 and line 13 are significantly improved, which are 5.6 times, 8.3 times and 3 times of the control (A) respectively, and the expression level of BnDTX19 in the RNAi plants is significantly reduced, which is only 17.5% of the control plants (B). It is shown that these plants are true BnDTX19 gene overexpression plants and RNAi plants.

[0028] Figure 2 The evidence of the antibacterial sclerotinia function of BnDTX19 gene is provided for the positive regulation of BnDTX19 on the resistance of Brassica napus to Sclerotinia sclerotiorum, and the positive regulation of BnDTX19 on the resistance of Brassica napus to Sclerotinia sclerotiorum is shown. The Brassica napus BnDTX19 transgenic plants are inoculated with Sclerotinia sclerotiorum strain UF-1, and the phenotypes and lesion area statistical analysis results of the inoculated overexpression plants (A) and RNAi plants (B) are shown. The inoculation experiment is repeated three times. The lesion area is statistically analyzed by Student's t-test test using GraphPad Prism, and the data are represented by mean ± standard deviation. The significant differences are represented by different number of asterisks (****, P < 0.0001). The results show that the overexpression plants exhibit a significantly more resistant phenotype than the control (ZS11) plants (A), and the RNAi plants are significantly more susceptible than the control (B). The quantitative analysis results of the lesion area show that the lesion areas of the three lines of overexpression plants are 144.1 mm 2 , 165.3 mm 2 and 155.2 mm 2 , which are significantly lower than 213 mm 2 of the control; and the lesion area of the RNAi line is 177.8 mm 2 , which is significantly higher than 123.7 mm 2These results show that BnDTX19 positively regulates the resistance of Brassica napus to S. sclerotiorum. DETAILED DESCRIPTION

[0029] The present application is further illustrated in conjunction with the accompanying drawings and examples.

[0030] Example 1

[0031] The present application clones a Brassica napus gene BnDTX19, and for the first time clarifies the function of the gene conferring resistance to Sclerotinia sclerotiorum by constructing overexpression transgenic Brassica napus. Overexpression of BnDTX19 gene leads to significantly increased resistance of Brassica napus to Sclerotinia sclerotiorum, and therefore, a new Brassica napus material with enhanced resistance to Sclerotinia sclerotiorum can be created by constructing a homozygous line of Brassica napus overexpressing BnDTX19 gene, which can be used for creating and breeding Sclerotinia sclerotiorum-resistant Brassica napus varieties, analyzing the function and mechanism of BnDTX19 gene, and the like. The main steps of cloning, function and mechanism analysis of BnDTX19 gene, and creating and obtaining a new Brassica napus material with increased resistance to Sclerotinia sclerotiorum include:

[0032] 1) Cloning and preservation of Brassica napus BnDTX19 gene

[0033] The BnDTX19 gene of the oilseed rape provided in the present application is cloned through the following steps. First, primers BnDTX19-F (5'-atggctgatcgcatcaacacgg-3') (the sequence is shown as SEQ ID No. 3) and BnDTX19-R (5'-gaccgacgcatttatctttgtc-3') (the sequence is shown as SEQ ID No. 4) are designed according to the BnDTX19 sequence in the oilseed rape genome database. The total RNA of the leaves of the oilseed rape Zhuan 11 is extracted by using the TRIZOL reagent, and the BnDTX19 cDNA is obtained by using the high-fidelity enzyme pfu-mediated RT-PCR method. The PCR product is purified by cutting the gel after 1% agarose gel electrophoresis, and is connected to the pEASY-Blunt Cloning Vector carrier. The E. coli DH5a is transformed by heat shock, and is cultured in the LB medium overnight. Whether the extracted plasmid contains the BnDTX19 gene is tested by using the BnDTX19-F / BnDTX19-R primer pair for the PCR method, and finally, the company is sent for sequencing verification, so that the full-length sequence of the BnDTX19 gene cDNA is successfully cloned and obtained. The BnDTX19 nucleotide sequence is shown as SEQ ID No. 1, the open reading frame (ORF) of the gene is 1428 bp long, and the encoded protein is composed of 475 amino acids, and the sequence is shown as SEQ ID No. 2. The gene product contains a transmembrane domain and two MatE domains. It is found through BLAST analysis that the BnDTX19 gene sequence cloned in the present application is consistent with the BnaC01G0357100ZS nucleotide sequence and protein sequence of the oilseed rape variety ZS11 in the database of the BnIR, Brassica napus multi-omics database (information resource) created by Huazhong Agricultural University. Before the present application, there is no report on the function of the gene.

[0034] The E. coli carrying the pEASY-BnDTX19 vector is transformed, and is stored in a refrigerator at -80 DEG C. At any time, the strain can be activated, the plasmid can be extracted, the BnDTX19 gene can be subcloned into the target vector by PCR amplification, and is used for transgenic research.

[0035] 2) Construction and acquisition of the BnDTX19 gene overexpression structure

[0036] The BnDTX19 sequence cloned according to the application is used to design primers BnDTX19-F2 (5'-agagaacacgggggactttgcaac atggctgatcgcatcaacacg-3', the italicized part is the sequence consistent with the vector pBWA(V)BS-OE) (the sequence is shown as SEQ ID No. 5) and BnDTX19-R2 (5'-gtactgaagacagagctagttaca tcagaccgacgcatttatctttgtcc-3', the italicized part is the sequence consistent with the vector pBWA(V)BS-OE) (the sequence is shown as SEQ ID No. 6). The pEASY-BnDTX19 plasmid obtained in 1) of Example 1 is used as a template, and BnDTX19-F2 / BnDTX19-R2 is used as a primer pair to obtain the target gene by PCR amplification.

[0037] The pBWA(V)BS-OE plasmid is digested with Eco 31I (Bsa I) enzyme, and the amplified target gene is ligated with the digested pBWA(V)BS-OE vector. The ligase used in the experiment is T4_ligase. The ligation product is subjected to steps such as E. coli transformation, kanamycin plate screening, PCR identification, and sequencing identification 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 tag.

[0038] 3) Obtaining Agrobacterium transformed with the BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19

[0039] The BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19 is transformed into Agrobacterium strains with strong invasiveness to Brassica napus, such as GV3101, by methods such as electroporation. The transformants are screened on LB medium containing kanamycin and rifampicin, and then identified by PCR detection to obtain Agrobacterium carrying the BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19. The Agrobacterium is used for the genetic transformation of Brassica napus in the next step.

[0040] 4) Creation and obtaining of Brassica napus transformed with the BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19

[0041] The BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19 is introduced into Brassica napus by Agrobacterium-mediated method to obtain Brassica napus T0 generation transformed with pBWA(V)BS-OE-BnDTX19. The specific operation steps are as follows:

[0042] (i) Seed washing and germination

[0043] 75% ethanol 30-60 s, sterile water 1 time, 1 min / time; 0.15% mercury 10 min, sterile water washing 2 times, 1 min / time; sterile water washing 30 min, inoculating on sterile filter paper and drying; inoculating the seeds on culture bottles, 23°C dark culture for 5-6 d.

[0044] (ii) Pre-culture

[0045] Cutting the hypocotyls of the germinated rape seedlings into 0.4-0.6 cm segments, inoculating on pre-culture medium, 23°C light culture for 2-3 d.

[0046] (iii) Agrobacterium infection and co-culture

[0047] Picking Agrobacterium in the infection liquid, preparing Agrobacterium resuspension liquid with OD 600 =0.2, inoculating explants in Agrobacterium suspension liquid for infection for 10 min. Inoculating the infected explants on sterile filter paper and drying, inoculating on co-culture medium, 23°C dark culture for 48-72 h.

[0048] (iv) Debacterization (delayed screening)

[0049] Inoculating the co-cultured explants on debacterization medium, 23°C light culture for 6 d.

[0050] (v) Screening / differentiation

[0051] Inoculating the debacterized explants on screening / differentiation medium, 30 explants per dish, 23°C light culture, changing the plate every 15 d.

[0052] (vi) Rooting culture

[0053] Inoculating the differentiated shoots on rooting culture medium, 23°C light culture until rooting.

[0054] (vii) Detection

[0055] Extracting rape genomic DNA by CTAB method, and performing PCR detection of resistance genes.

[0056] (viii) Soil planting

[0057] After the glufosinate-ammonium resistant seedlings grow to the complete root system, inoculating the T0 sterile seedlings in a culture box at 27°C for 2 d, then transplanting into soil, placing in a culture box for routine management, and finally obtaining T0 seeds.

[0058] 5) Screening and obtaining of homozygous lines of BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19 in Brassica napus

[0059] The trait segregation of the transgenic plant offspring is detected by glufosinate resistance and BnDTX19 gene expression, respectively. The glufosinate resistance screening is performed on a plate containing glufosinate for Brassica napus seeds, and whether it can grow into healthy seedlings on the glufosinate-resistant plate is observed. The gene expression is detected by real-time fluorescent quantitative PCR and other methods. The Brassica napus homozygous lines of BnDTX19 gene overexpression structure pBWA(V)BS-OE-BnDTX19 that are not segregated in offspring traits and can be stably inherited are obtained.

[0060] The present application obtains three Brassica napus homozygous lines, OE-line 2, OE-line 10 and OE-line 13, which can all grow into healthy seedlings on the glufosinate-resistant plate, and the BnDTX19 gene expression levels are all significantly higher than those of the control plants, being 5.6 times, 8.3 times and 3 times of the control, respectively. Figure 1 A) which indicates that these plants are true BnDTX19 gene overexpression plants.

[0061] 6) Detection and analysis of disease resistance of BnDTX19 gene overexpression Brassica napus homozygous lines

[0062] The BnDTX19 gene overexpression Brassica napus homozygous lines obtained in 5) are used as materials, Sclerotinia sclerotiorum is inoculated, the resistance to Sclerotinia sclerotiorum of the Brassica napus is detected and analyzed, so as to clarify the regulation of BnDTX19 gene on the resistance to Sclerotinia sclerotiorum of Brassica napus, and lay a foundation for creating and obtaining Sclerotinia sclerotiorum-resistant Brassica napus by using the gene.

[0063] Activation culture of Sclerotinia sclerotiorum: full and uncontaminated Sclerotinia sclerotiorum sclerotia are selected, a sterile knife blade burned by an alcohol lamp is used to cut the sclerotia into two halves, the cut surfaces are placed on PDA solid plates, and 23℃ dark culture is performed for 3d; a puncher with a diameter of 4mm is used to punch a mycelium block of 3-5mm inward from the edge of the colony, the mycelium side is inoculated onto a new PDA solid plate, and 23℃ dark culture is performed for about 36h, and then inoculation can be performed.

[0064] Inoculation of Sclerotinia sclerotiorum: Brassica napus plants with consistent growth are selected for inoculation. A puncher with a diameter of 4mm is used to punch a mycelium block of 3-5mm inward from the edge of the colony, the mycelium side is inoculated onto the middle position of a fully developed leaf, two mycelium blocks are inoculated on the left and right halves of each leaf symmetrically, a film is covered for moisturizing, and the culture is placed in a 23℃ greenhouse; after a proper time (about 24h), photographing is performed for recording, and the lesion area is analyzed by using ImageJ software.

[0065] The inoculation experiment was repeated three times. The Student's t-test was used for statistical analysis of the lesion area. The results showed that the BnDTX19 overexpression plants were significantly more resistant to disease than the non-transgenic control plants (ZS11) Figure 2 (A). The quantitative analysis results of the lesion area showed that the lesion areas of the three lines of overexpression plants were 144.1 mm 2 , 165.3 mm 2 and 155.2 mm 2 , respectively, which were significantly lower than the control of 213 mm 2 ( Figure 2 B).

[0066] These results showed that the resistance of the BnDTX19 overexpression plants to sclerotinia was significantly higher than that of the non-transgenic control plants. The overexpression of the BnDTX19 gene led to a significant increase in the resistance of the rape to sclerotinia, and therefore, BnDTX19 positively regulated the resistance of the rape to sclerotinia. The present application successfully created a new material of rape with high resistance to sclerotinia by constructing BnDTX19-OE rape.

[0067] Example 2

[0068] According to the positive regulation function of the rape gene BnDTX19 to sclerotinia resistance elucidated by the present application, a technical system for creating and obtaining a new material of rape with weakened resistance to sclerotinia by constructing RNAi transgenic rape of the gene and using genetic engineering technology was established. The main steps include:

[0069] (1) Construction and acquisition of BnDTX19 gene RNAi structure

[0070] According to the BnDTX19 sequence cloned in the application, 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'-cgatGGTCTCacctgcaggtctagtttttctccttcattttc-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') (sequence as shown in SEQ ID No. 11), and BnDTX19-R5 (5'-cagtGGTCTCatacaatctcatatgttctggtctacatcgctgg-3') (sequence as shown in SEQ ID No. 12) were designed, wherein GGTCTC is the recognition site of Eco 31 I (Bsa I). The pEASY-BnDTX19 plasmid obtained in 1) of Example 1 was used as a template, BnDTX19-F3 / BnDTX19-R3 was used as a primer pair, and a BnDTX19 forward fragment (fragment 1) was obtained by PCR amplification; BnDTX19-F4 / BnDTX19-R4 was used as a primer pair, and a BnDTX19 Loop (fragment 2) was obtained by PCR amplification; BnDTX19-F5 / BnDTX19-R5 was used as a primer pair, and a BnDTX19 reverse fragment (fragment 3) was obtained by PCR amplification, and fragments 1, 2, and 3 were recovered, respectively. The fragments 1, 2, 3, and Eco 31 I (Bsa I) linearized vector pBWA(V)BS-ccdB-RNAi were connected with T4_ligase, transformed, screened on kanamycin medium, subjected to enzyme digestion, PCR, and sequencing identification, and the like 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.

[0071] (2) Obtaining Agrobacterium transformed with the BnDTX19 gene RNAi structure

[0072] The BnDTX19 gene RNAi structure (pBWA(V)BS-RNAi-BnDTX19) is transformed into an agrobacterium strain with strong infection ability to rape by methods such as electric shock, and the transformants are screened on LB medium containing kanamycin and rifampicin, and then identified by PCR to obtain the agrobacterium carrying the BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19.

[0073] (3) Creation and obtaining of BnDTX19 gene RNAi structure rape

[0074] The BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19 is introduced into rape by agrobacterium-mediated method to obtain T0 generation of rape transformed with pBWA(V)BS-RNAi-BnDTX19. The specific operation steps are as described in 4) of Example 1.

[0075] (4) Obtaining of homozygous line of BnDTX19 gene RNAi structure rape

[0076] The trait segregation of the transgenic plant offspring is detected by taking antibiotic resistance and BnDTX19 gene expression as detection indexes. The detection is carried out on a plate containing glufosinate resistance, and it is observed whether the rape seeds can grow into healthy seedlings on the glufosinate resistance plate. The homozygous line of BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19 rape which is not segregated in offspring traits and can be stably inherited is obtained. These homozygous lines of rape can all grow into healthy seedlings on the glufosinate resistance plate, and the expression level of the BnDTX19 gene is significantly lower than that of the wild type Zhongshuang 11 control, only 17.5% of the control (B). Figure 1

[0077] The BnDTX19-RNAi rape homozygous line is obtained in the application, which can grow into healthy seedlings on the glufosinate resistance plate, and the expression level of the BnDTX19 gene is significantly lower than that of the wild type Zhongshuang 11 control, only 17.5% of the control (A). It is shown that these plants are true BnDTX19 gene RNAi plants. Figure 1

[0078] (5) Screening, identification and obtaining of BnDTX19 gene RNAi structure rape homozygous line with weakened resistance to sclerotinia disease

[0079] The homozygous line of BnDTX19 gene RNAi structure pBWA(V)BS-RNAi-BnDTX19 rape is taken as material to detect and analyze the resistance to sclerotinia disease. The inoculation method and disease resistance evaluation are as described in 6) of Example 1. ​​

[0080] Analysis of the BnDTX19-RNAi plants constructed in this invention using Sclerotinia sclerotiorum inoculation showed that the BnDTX19-RNAi plants were significantly more susceptible to the disease than the non-transgenic plant (ZS11) control. Figure 2 B). Quantitative analysis of lesion area showed that the lesion area of ​​the RNAi strain was 177.8 mm². 2 The result was significantly higher than the control group's 123.7 mm. 2 ( Figure 2 B). This indicates that the resistance of BnDTX19-RNAi plants to sclerotinia stem rot was significantly lower than that of non-transgenic control plants. Inhibition of BnDTX19 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 BnDTX19-RNAi rapeseed.

[0081] In summary, this invention combines Figures 1-2 The results revealed for the first time the positive regulatory role of the rapeseed transporter gene BnDTX19 in the resistance of rapeseed to Sclerotinia stem rot, provided the application pathways, application technologies and examples of BnDTX19 in the creation of germplasm for Sclerotinia stem rot resistant crops, and successfully obtained rapeseed with high resistance to Sclerotinia stem rot.

Claims

1. A type of rapeseed ( Brassica napus )Gene BnDTX19 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 BnDTX19 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 BnDTX19 the gene for enhanced resistance to sclerotinia blight.

3. Use according to claim 2, characterized in that, The use in Brassica napus material of increased resistance to Sclerotinia sclerotiorum is achieved by the following steps: (1) BnDTX19 Construction and obtaining of gene overexpression constructs The BnDTX19 Gene open reading frames are cloned into a plant expression vector for expression under the control of a strong promoter. (2) Transformation BnDTX19 Agrobacterium containing a gene overexpression construct The constructed BnDTX19 The gene overexpression structure is transformed into an Agrobacterium strain with strong invasion ability to rape by an electric shock method. (3) Overexpression BnDTX19 Creation and acquisition of genetically modified rapeseed The gene overexpression structure is introduced into rape by means of agrobacterium mediation BnDTX19 The gene overexpression structure is introduced into rape by means of agrobacterium mediation BnDTX19 The gene overexpression structure is introduced into rape by means of agrobacterium mediation (4) Obtaining homozygous lines of transgenic rape overexpressing BnDTX19 the gene respectively, with antibiotic resistance and BnDTX19 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 BnDTX19 of transgenic rape homozygous line; (5) Overexpression of increased resistance to sclerotinia stem rot BnDTX19 Screening, identification and acquisition of transgenic rapeseed homozygous lines Using transgenic rapeseed homozygous line overexpressing BnDTX19 of the gene as material, the resistance to sclerotinia stem rot is detected and analyzed, and transgenic rapeseed with increased disease resistance is obtained. BnDTX19 BnDTX19 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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