CN114752599B - Antipathogenic target gene fragment of Verticillium dahliae VdNRPS3 gene and its interference vector and application - Google Patents

Abstract

The invention discloses verticillium dahliaeVdNRPS3Gene antipathogen target gene fragment and its interference vector and application. The invention uses Verticillium dahliaeVdNRPS3As a target gene, the relation between the target gene and pathogenic bacteria pathogenicity is researched by a VIGS technology, a target gene fragment capable of obviously improving plant resistance is obtained by combining a transgene technology and an RNAi technology, and the nucleotide sequences of the target gene fragment are respectively shown as SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 5. The invention further provides a Gateway interference vector constructed by using the target gene fragment. Verticillium dahliae of the inventionVdNRPS3The gene anti-pathogenic bacteria target gene fragment and the Gateway interference vector constructed by adopting the target gene fragment can be applied to the aspects of improving the resistance of crops to diseases caused by verticillium dahliae, cultivating new varieties of transgenic plants resisting the verticillium dahliae and the like.

Description

Antipathogenic target gene fragment of Verticillium dahliae VdNRPS3 gene and its interference vector and application

technical field

The present invention relates to a target gene fragment of Verticillium dahliae against pathogenic bacteria and an RNA interference vector containing said target gene fragment, in particular to a target gene fragment of Verticillium dahliae VdNRPS3 gene against pathogenic bacteria and said RNA interference carrier in improving the performance of crops or vegetables The application to the disease resistance of Verticillium dahliae belongs to the field of the antipathogenic bacteria target gene fragment of Verticillium dahliae and the application field of disease resistance.

Background technique

Cotton Verticillium Wilt, known as "cotton cancer", reduces cotton production by an average of 10-35% in many countries, seriously harming cotton production and causing huge economic losses. The pathogenic bacteria is Verticillium dahliae, which is highly pathogenic and can infect throughout the cotton growth stage, resulting in wilting and yellowing of cotton leaves; In severe cases, the entire cotton leaves are scorched and broken, and eventually die. At the same time, it has a wide range of host plants, and can infect more than 600 kinds of plants, including annual herbs, perennial herbs and woody plants, among which there are many cruciferous, nightshade, Asteraceae and Rosaceae, etc. A variety of crops, seedlings and flowers with important economic value, and the range of infectable hosts is still expanding. Since Verticillium dahliae is a soil-borne plant pathogenic fungus, it is difficult to control and control, and there is no effective control agent in production at present.

Filamentous fungi can synthesize a series of low-molecular-weight polypeptide secondary metabolites with medicinal value through non-ribosomal pathways. These complex and diverse polypeptide compounds are collectively referred to as non-ribosomal peptides (non-ribosomal peptides, NRP). More than 80% of the non-ribosomal peptides found so far come from fungi and actinomycetes, followed by unicellular prokaryotes such as myxobacteria and cyanobacteria. Genome sequence analysis showed that archaea, metazoans and dinoflagellates also have the potential to synthesize nonribosomal peptides (Wang H, Fewer D P, Holm L, et al. Atlas of nonribosomalpeptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(5): 9259-9264.). They have various biological activities such as antibacterial, antiviral and anticancer, and have been widely used. The formation of NRP is mainly catalyzed by non-ribosomal peptide synthases (NRPS), the first NRPS identified was L-aminoadipoyl-L-cysteine-D-valine synthase, which catalyzes β- The first step in the biosynthesis of lactam antibiotics is the condensation of three amino acids into a tripeptide compound to form β-lactam antibiotics such as penicillin (Smith D J, Burnham M K R, Bull J H, et al. Beta-lactamantibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. Embo Journal, 1990, 9(3): 741-747). NRPS has a variety of physiological functions, which can be used as antibiotics (Antibiotics) to inhibit the competition of nutrients with itself; it can also be used as a toxin (Toxins), which plays an important role in the process of pathogenic bacteria invading the host and colonizing; and Some can be used as siderophores to participate in the growth of microorganisms; they can also be used as places to store nitrogenous substances or as signal factors to regulate the growth, reproduction and differentiation of microorganisms. Some NRPS also cooperate with other enzymes such as polyketide (Polyketide) or terpenoid (Terpenoid) synthetase to complete the synthesis of hybrid molecules. A typical NRPS consists of multiple modules (Module) arranged in a specific spatial order, each module consists of multiple functional domains (Domain) or catalytic units, structurally diverse non-ribosomal peptides in these modules and their Domain-ordered synthesis. The recent whole-genome sequencing data of Cochliobolus heterostrophus showed that there are 12 genes encoding NRPS in Cochliobolus heterostrophus. Among them, NPS6, as a typical virulence determinant in Cochliobolus heterostrophus, is not only involved in its pathogenicity but also Involved in H2O2 tolerance, is a virulence determinant of the corn pathogen (Cochliobolus miyabeanus), and is related to H2O2 tolerance. Deletions of genes homologous to NPS6 in the rice pathogen Cochliobolus miyabeanus, the wheat scab pathogen Fusarium graminearum and the Arabidopsis pathogen Alternaria brassicicola also cause their corresponding Decreased virulence and poor tolerance to H2O2 (Oide S, Moeder W, Krasnoff S , et al. NPS6, encoding a nonribosomal peptide synthetase involved in siderophore-mediated metabolism, is a conserved virulence determinant of plant pathogenic ascomycetes. Plant Cell , 2006, 18(10): 2836-2853.).

By analyzing the genome sequence of Verticillium dahliae, the inventors found that there are 6 NRPS genes in total, and analyzed the gene expression levels in different infection stages, and found that VdNRPS3 was significantly up-regulated in the early stage of infection, which may be related to Verticillium dahliae. The pathogenicity of bacteria is closely related. Therefore, screening the disease resistance target gene fragments from the VdNRPS3 gene and constructing the corresponding interference vector can be applied to improve the disease resistance of plants to Verticillium dahliae.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a disease resistance target gene fragment of Verticillium dahliae VdNRPS3 gene;

The second object of the present invention is to provide an RNA interference vector containing the disease resistance target gene fragment of the Verticillium dahliae VdNRPS3 gene;

The third purpose of the present invention is to apply the disease resistance target gene fragment of the Verticillium dahliae VdNRPS3 gene and the RNA interference vector containing the target gene fragment to plant disease resistance or to construct new disease-resistant transgenic plant varieties.

In order to achieve the above-mentioned purpose, the main technical scheme adopted by the present invention includes:

The present invention firstly discloses the VdNRPS3 target gene fragment of Verticillium dahliae which can improve plant resistance to pathogenic bacteria, and its nucleotide sequence is shown in SEQ ID No.2, SEQ ID No.3 or SEQ ID No.5 respectively; , the nucleotide sequence of the target gene fragment is shown in SEQ ID No.2.

The invention also discloses an RNA interference vector containing the VdNRPS3 target gene fragment of Verticillium dahliae and a host cell containing the RNA interference vector.

In addition, dsRNA transcribed from the target gene fragment shown in SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 5 is also included in the protection scope of the present invention.

The Verticillium dahliae VdNRPS3 target gene fragment of the present invention can be applied to improve the disease resistance of plants to diseases caused by Verticillium dahliae, including the following steps: (1) constructing a VdNRPS3 containing the Verticillium dahliae VdNRPS3 RNA interference vector of the target gene fragment; (2) transforming the constructed RNA interference vector into plants or plant cells; (3) screening to obtain a transgenic plant with improved disease resistance to Verticillium dahliae.

Preferably, a method for constructing the RNA interference vector comprises: by BP reaction, connecting the Verticillium dahliae VdNRPS3 target gene fragment into pDONR207 and then by LR reaction, constructing it into pK7GWIWG2(I) , 0 to obtain the Gateway interference vector.

The RNA interference vector of the present invention can be applied to improve the disease resistance of plants to diseases caused by Verticillium dahliae, comprising the following steps: (1) transforming the RNA interference vector into plants or plant cells; (2) ) screened to obtain transgenic plants with improved resistance to Verticillium dahliae.

The disease caused by Verticillium dahliae described in the present invention is preferably cotton Verticillium wilt.

The invention further discloses a method for cultivating a new variety of transgenic plants resistant to Verticillium dahliae, comprising the following steps: (1) constructing an RNA interference vector containing the VdNRPS3 target gene fragment of Verticillium dahliae; (2) Transform the constructed RNA interference vector into plants or plant cells; (3) screen to obtain new transgenic plant varieties with improved disease resistance to Verticillium dahliae.

The protocol for the transformation, and the protocol for introducing the nucleotides into the plant, may vary for the type of plant or plant cell being transformed. Suitable methods for introducing the nucleotides into plant cells include: microinjection, electroporation, Agrobacterium-mediated transformation, and direct gene transfer, among others.

The plants of the present invention are host plants of Verticillium dahliae, preferably crops or vegetables, including any one of tobacco, cotton, tomato, potato, melon, watermelon, cucumber or peanut.

The present invention adopts the host-induced gene silencing technology (HIGS), and uses the highly pathogenic Verticillium dahliae strain V991 as the experimental material to construct a plurality of non-ribosomal peptides targeting the target gene of Verticillium dahliae Tobacco rattle virus (TRV) interference plasmid for synthetase 3 (non-ribosomal peptide synthetase 3, NRPS3, VDAG_05314). Nicotina benthamiana was transformed by Agrobacterium injection and inoculated with Verticillium dahliae, and a Gateway interference vector was constructed with the target fragment that can significantly reduce the disease index of the plant to obtain a stable genetic transgenic plant. The fungal biomass and the transcription level of target genes were detected by disease index and molecular biological methods, and the best interference fragments were screened. The VdNRPS3 target gene fragment of Verticillium dahliae screened in the present invention and the RNA interference vector constructed by using the target gene fragment can be applied to improve the disease resistance of plants to diseases caused by Verticillium dahliae and cultivate the anti-dahliae wheel New varieties of transgenic plants of mycobacteria.

Detailed description of the overall technical solution of the present invention

According to the coding sequence information of Verticillium dahliae VdNRPS3 , the present invention designs primers and amplifies to obtain 5 different segments for the target gene. The five target genes are VdNRPS3-1 , VdNRPS3-2 , VdNRPS3-3 , VdNRPS3-4 and VdNRPS3-5 respectively, and their nucleotide sequences are SEQ ID No.1, SEQ ID No.2, SEQ ID No. 3. Shown in SEQ ID No.4 and SEQ ID No.5. The cloned target fragment was digested by Bam H I and Eco R I, and then constructed into the TRV2 vector to become the VIGS series RNAi vector. After verification by bacterial liquid amplification and DNA sequencing, it was found that the sequence was completely consistent with the target fragment sequence. Then, the positive material verified to be correct was transformed into Agrobacterium GV3101 for injection of N. benthamiana.

From the 7th day after injection, the young shoots of N. benthamiana began to appear albino. By the 10th day, the new leaves are all white, and this phenomenon of leaf whitening can last for 45 days. This indicated that on the 7th day after inoculation, the VIGS vector in N. benthamiana had already produced a large amount of dsRNA and played an interfering role. Therefore, the present invention chooses to carry out inoculation of 10 ⁶ cfu/mL spore suspension by dip-root method on the 7th day after the injection of VIGS series vectors. The disease index statistics of N. benthamiana were performed at 10 dpi (days post-inoculation), 11 dpi and 12 dpi after docking. The results showed that compared with the empty load, the disease index of N. benthamiana injected with fungal target gene fragments decreased; with the increase of days, the disease index gradually increased. In the three groups of tobacco VdNRPS3-2 , VdNRPS3-3 , and VdNRPS3-5 , the disease index has been kept at a low level, which preliminarily shows that the introduction of these three target fragments can reduce the disease index of plants.

The present invention obtains 3 target segments that can improve the resistance of plants to pathogenic bacteria by the method of VIGS screening, in order to further verify the relationship between VdNRPS3 and the pathogenicity of pathogenic bacteria, and the region that can significantly reduce the pathogenicity of pathogenic bacteria after interference A stable genetic transgenic N. benthamiana was obtained, and primers for the target gene were designed, with BP sites at both ends of the primers, and the target gene fragment was obtained by amplification. In the present invention, the three VdNRPS3 target fragments obtained by cloning are connected to the Gateway interference vector pK7GWIWG2(I),0 through the BP reaction and the LR reaction to form a plant transformation vector containing the fungal target gene.

In order to obtain dsRNA that can stably inherit the target gene VdNRPS3 of pathogenic bacteria, the constructed Gateway interference vector was transformed into Nicotiana benthamiana by Agrobacterium-mediated and tissue culture methods, and finally transgenic tobacco was obtained and the disease resistance of transgenic tobacco was analyzed. .

The obtained positive transgenic tobacco containing dsVdNRPS3-2 , 3, and 5 was inoculated with Verticillium dahliae. Disease index analysis was then performed from 10 dpi, 11 dpi and 12 dpi post-inoculation. It can be seen from the test results that the resistance of transgenic tobacco to pathogenic bacteria is significantly improved, and the disease index is reduced by about 40-85%. DNA was extracted from the roots of transgenic tobacco, and fungal biomass was analyzed by qRT-PCR. The fungal biomass of transgenic-positive tobacco was significantly reduced, about 20-40% of wild-type. Disease index statistics and fungal biomass analysis showed that transgenic-positive tobacco had stronger resistance to pathogens.

In order to further verify the relationship between the reduction of the plant disease index and the expression of the target gene, the present invention analyzes the expression of the target gene of the plant root pathogenic bacteria, and it can be observed that compared with the wild-type plant, the expression of the target gene in the transgenic plant has decreased by about approx. 50-80%. Meanwhile, the pictures show that the disease resistance of RNAi- VdNRPS3 transgenic tobacco is significantly better than that of wild-type tobacco. And according to the disease index, fungal amount and target gene expression detection of the three materials, it shows that the segment 2 of VdNRPS3 gene ( VdNRPS3-2 ) is used as the target fragment to design dsRNA, which can achieve the best interference effect, which can effectively reduce the pathogenic bacteria. pathogenicity.

Definitions of terms involved in the present invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences are also implicitly encompassed by a particular nucleic acid sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues.

The term "recombinant host cell" or "host cell" means a cell comprising the nucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell. Host cells can be prokaryotic or eukaryotic.

The term "RNA interference (RNAi)" means the phenomenon of inducing silencing of gene expression of homologous sequences in a cell by exogenous or endogenous double-stranded RNA.

Description of drawings

Figure 1 is a schematic diagram of VIGS vector.

Figure 2 shows the amplification results of different segments of VIGS.

Figure 3 shows the amplification verification of VIGS interference vector; M is the marker, N is the negative control, 1-4 (the first fragment), 5-8 (the second fragment), 9-12 (the third fragment), 13- 16 (fragment 4) and 17-20 (fragment 5) are the amplification results of bacterial liquid for constructing VIGS plasmid for VdNRPS3 gene.

Figure 4 shows the statistical results of the disease index.

Figure 5 shows the results of RNAi amplification; M is marker, M is marker, and 1, 2, and 3 are bands against VdNRPS3-2 , VdNRPS3-3 , and VdNRPS3-5 .

Figure 6 is a schematic diagram of the RNAi vector.

Figure 7 shows the PCR detection results of transgenic positive tobacco; M: marker, 1-4 (2nd fragment), 5-8 (3rd fragment), 9-12 (5th fragment) are transgenic positive tobacco, WT is wild type tobacco .

Figure 8 shows the results of the disease index analysis of transgenic tobacco.

Figure 9 shows the results of the analysis of fungal biomass in plants.

Figure 10 is the analysis result of fungal target gene expression in plants.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become clearer with the description. However, it should be understood that the described embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

Example 1 Screening of the target gene fragment of Verticillium dahliae VdNRPS3 against pathogenic bacteria, construction of RNA interference vector and verification of disease resistance of transgenic tobacco

⒈Materials and methods

⑴Material

Tobacco: Nicotiana benthamiana strain.

Cultivation conditions: planted in mixed nutrient soil sterilized by high temperature and high pressure (Fangjie nutrient soil: vermiculite = 1:1), temperature 23 ± 2 °C, relative humidity 75 ± 5 %, photoperiod L: D is 16 h : 8h.

⑵ strains and plasmids

Cotton Verticillium wilt pathogen: Verticillium dahliae V991, a highly pathogenic deciduous strain, donated by researcher Jian Guiliang, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

Viral vector: Tobacco rattle virus (TRV) binary vector (TRV1 and TRV2) was donated by Professor Liu Yule of Tsinghua University.

Agrobacterium: strains GV3101 and LBA4404, maintained by the inventors' laboratory.

Plant stable genetic vectors: pDONR207 and pK7GWIWG2(I),0 vectors are maintained in the laboratory of the inventors.

(3) Fungal culture and plant inoculation

Verticillium dahliae spores were cultured in liquid CM medium and cultured with shaking at 25°C for 5-7 days. Filter through 5 layers of gauze, collect spores by centrifugation, dilute with distilled water and observe under a microscope, and adjust the spore concentration to 10 ⁶ cfu/mL for later use.

When the leaves of N. benthamiana grow to 6-8 true leaves, select seedlings with the same growth vigor for inoculation. The seedlings were dug out from the roots with tweezers and the root soil was washed in distilled water. The roots of the seedlings were completely immersed in the diluted 10 ⁶ cfu/mL spore suspension or distilled water for 2 minutes, then the seedlings were moved back to the original plastic pot as soon as possible, and watered to make the soil moist, and the disease index was counted.

⑷ Plant disease index statistics

According to relevant literature (Wang HM, Lin ZX, Zhang XL, et al. Mapping and quantitative trait loci analysis of Verticillium wilt resistance genes incotton. Journal of Integrative Plant Biology , 2008, 50(2): 174-182.) and make appropriate Adjusted to formulate the disease level of N. benthamiana infection of Verticillium dahliae in this experiment (as shown in Table 1). The formula for calculating the disease index is as follows (Formula 1):

Table 1 Statistics of disease index

Formula 1 Disease Index = [Ʃ (number × level) / (total plant × highestlevel) ] ×100

⑸Construction of VIGS interference vector

In order to screen the target gene segment with the best interference effect, two pairs of specific primers were designed according to the coding sequence of non-ribosomal peptide synthetase 3 (NRPS3, VDAG_05314) of Verticillium dahliae. Both ends contain Eco R I and Bam H I restriction sites (as shown in Table 2), and the target fragments are amplified respectively. The PCR amplification products were then detected by 1% agarose gel electrophoresis, and fragments were recovered. The target fragment and the vector were subjected to enzyme digestion reaction respectively, and T4 ligase was used to construct them into the TRV2 vector. Finally, the verified positive plasmid was transformed into Agrobacterium GV3101 by enzyme digestion and sequencing analysis.

Table 2 Primer information of different segments of VdNRPS3

Note: Bold italics are the restriction sites.

⑹ VIGS transformation method

The Agrobacterium monoclones containing the positive plasmids were placed in LB liquid medium (25 µg/mL Rif and 50 µg/mL Kan), and incubated overnight at 28°C on a shaker. The next day, the bacterial solution (with a ratio of 2%) was added to the LB liquid medium for re-culturing, and the cells were collected by low-temperature centrifugation when the OD ₆₀₀ was 0.5 - 0.6. Discard the waste solution and resuspend the bacteria in injection matrix (10 mM MES, 10 mM MgCl ₂ , 100 µM acetosyringone) and adjust the OD ₆₀₀ to 0.8 - 1.0. Mix the two Agrobacterium strains (TRV1 and TRV2 + target gene fragment) at a ratio of 1:1 and let stand for 3-5 h at room temperature without shaking. Finally, the Agrobacterium mixture was injected into the youngest leaves using a syringe.

⑺Construction of stable genetic carrier

In order to obtain stably inherited N. benthamiana containing the target gene dsRNA, redesign primers (partial BP sites at both ends) for the DNA segment that can significantly improve plant resistance to pathogens, and then use attb primers for amplification ( As shown in Table 3), for the construction of stable genetic interference vector. Through BP reaction, the target sequence was ligated into pDONR207; then through LR reaction, it was constructed into pK7GWIWG2(I),0; finally, the constructed vector was transformed into Agrobacterium LBA4404.

Table 3 Information of stable genetic interference primers

(8) Transformation of Nicotiana benthamiana

The leaves of N. benthamiana planted on MS minimal medium were cut into 0.4 × 0.6 cm pieces (with the edges and main veins removed), and placed in the Agrobacterium LBA4404 bacterial solution containing positive plasmids with an OD ₆₀₀ of 0.1 - 0.2 Soak for 5 min, and dry the bacterial liquid on the surface of the plant material with sterile filter paper. The leaflets were then cultured on tobacco bud differentiation medium (MS + NAA 0.2 mg/L + 6-BA 2 mg/L) covered with a layer of filter paper, and cultured in the dark at 25°C for 3 days. Co-cultured tobacco explants were transferred to selection medium (MS + NAA 0.2 mg/L + 6-BA 2 mg/L + Kan 100 mg/L + Carb 500 mg/L) containing the corresponding antibiotics for culture, The photoperiod was 16 h light/8 h dark. After 2-3 weeks, when the resistant shoots grow to a height of 1-2 cm, use a sterile scalpel to cut off the small shoots and transfer them to rooting medium (MS + Kan 100 mg/L + Carb 500 mg/L) to induce rooting. Adventitous roots will form after 1 to 2 weeks. Then, the DNA of the transgenic plants was extracted, and PCR detection was performed (as shown in Table 4) to obtain transgenic positive plants.

Table 4 Detection primer information

⑼ Fungal biomass detection

To compare the biomass changes of the pathogens in transgenic N. benthamiana and wild-type N. benthamiana, we used qRT-PCR to measure the biomass of Verticillium dahliae in the roots of different plant genotypes. The total DNA from the roots of N. benthamiana was extracted 12 days after inoculation, and the relative quantitative determination was carried out with the internal transcription spacer ITS of Verticillium dahliae as the target fragment and the housekeeping gene actin of N. benthamiana as the housekeeping fragment (Table 5).

The qRT-PCR reaction was completed on ABI7500, and the results were analyzed by the 2- ^∆∆Ct method. The unimodality of the primers and the amplification efficiency meet the experimental requirements.

Table 5 Fluorescence quantitative primer information

⑽Expression analysis of target genes

In order to confirm that there is a certain relationship between the improvement of N. benthamiana resistance and the decline of target genes, we used qRT-PCR to further determine the transcription levels of target genes in N. benthamiana. Total RNA was extracted from N. benthamiana roots 12 days after inoculation, and reverse transcription analysis was performed. The primers were designed with the coding sequence of the VdNRPS3 gene in the pathogenic bacteria as the target fragment (Table 6), and the pathogenic bacteria actin was used as the housekeeping fragment for relative quantitative determination of the transcription level.

The qRT-PCR reaction was completed on ABI7500, and the results were analyzed by the 2- ^∆∆Ct method. The unimodality of the primers and the amplification efficiency meet the experimental requirements.

Table 6 Fluorescence quantitative primer information

⒉ Experimental results

(1) Construction of VdNRPS3 interference vector of Verticillium dahliae

In this experiment, the Tobacco rattle virus (TRV) vector provided by Mr. Liu Yule from Tsinghua University was used (Figure 1). The cDNA of TRV is located between the double 35S promoter and the nopaline synthase terminator (NOSt). TRV1 contains viral RNA-dependent RNA polymerase (RNA-dependent RNApolymerase, RdRp), mobile protein (Movement Protein, MP), 16 kDa cysteine-rich region and other elements. TRV2 contains other elements such as coat protein (CP) and multiple cloning site (MCS) of the virus. The introduction of multiple cloning sites facilitates the insertion of foreign genes.

Based on the coding sequence information of Verticillium dahliae VdNRPS3 , primers were designed and amplified to obtain 5 different segments for the target gene (Figure 2). The five target genes are VdNRPS3-1 , VdNRPS3-2 , VdNRPS3-3 , VdNRPS3-4 and VdNRPS3-5 , and their nucleotide sequences are SEQ ID No.1, SEQ ID No.2, SEQ ID No. 3. Shown in SEQ ID No.4 and SEQ ID No.5.

The cloned target fragment was digested by Bam H I and Eco R I, and then constructed into the TRV2 vector to become the VIGS series RNAi vector. After verification by bacterial liquid amplification and DNA sequencing, it was found that the sequence was consistent with the target fragment sequence (Figure 3). Then, the positive material that was verified correctly was transformed into Agrobacterium GV3101 for injection of N. benthamiana.

⑵Analysis of disease index of Tobacco Ben's

From the 7th day after injection, the young shoots of N. benthamiana began to appear albino. By the 10th day, the new leaves are all white, and this phenomenon of leaf whitening can last for 45 days. This indicated that on the 7th day after inoculation, the VIGS vector in N. benthamiana had already produced a large amount of dsRNA and played an interfering role. Therefore, we chose to carry out inoculation of 10 ⁶ cfu/mL spore suspension by dip-root method on the 7th day after the injection of VIGS series vectors. The disease index statistics of N. benthamiana were performed at 10 dpi, 11 dpi and 12 dpi after docking. The results showed (Fig. 4) that compared with the empty load, the disease index of N. benthamiana injected with fungal target gene fragments decreased; with the increase of days, the disease index gradually increased. In the three groups of tobacco VdNRPS3-2 , VdNRPS3-3 , and VdNRPS3-5 , the disease index has been kept at a low level, which preliminarily shows that the introduction of these three target fragments can reduce the disease index of plants.

(3) Obtaining of transgenic plants

Through VIGS screening, three target segments that can improve plant resistance to pathogens were obtained. In order to further verify the relationship between VdNRPS3 and the pathogenicity of pathogenic bacteria, as well as the segment that can significantly reduce the pathogenicity of pathogenic bacteria after interference, and obtain a stably inherited transgenic N. benthamiana, we designed primers for the target gene, with primers at both ends. With BP site, the target gene fragment was obtained by amplification. A bright target band can be found from the electrophoresis in Figure 5. After further DNA sequencing and comparison, it was found that the sequence was completely consistent with the target sequence.

Through BP reaction and LR reaction, the three VdNRPS3 target fragments obtained by cloning were ligated to the Gateway interference vector pK7GWIWG2(I),0 (Figure 6) to form a plant transformation vector containing fungal target genes.

In order to obtain dsRNA that can stably inherit the pathogenic target gene VdNRPS3 , the constructed Gateway interference vector was transformed into Nicotiana benthamiana by Agrobacterium-mediated and tissue culture methods, and finally transgenic tobacco was obtained (Figure 7).

(4) Disease resistance analysis of transgenic tobacco

The obtained positive transgenic tobacco containing dsVdNRPS3-2 , 3, and 5 was inoculated with Verticillium dahliae. Disease index analysis was then performed from 10 dpi, 11 dpi and 12 dpi post-inoculation. It can be seen from Figure 8 that the resistance of transgenic tobacco to pathogenic bacteria was significantly improved, and the disease index decreased by about 40-85%. DNA was extracted from the roots of transgenic tobacco, and fungal biomass was analyzed by qRT-PCR. Figure 9 shows that the fungal biomass of transgenic-positive tobacco was significantly reduced, about 20-40% of the wild type. Disease index statistics and fungal biomass analysis showed that transgenic-positive tobacco had stronger resistance to pathogens.

In order to further verify the relationship between the reduction of plant disease index and the expression of target genes, through the analysis of the expression of target genes of plant root pathogens, it can be observed that compared with wild-type plants, the expression of target genes in transgenic plants decreased by about 50- 80% (Figure 10). Meanwhile, the pictures show that the disease resistance of RNAi- VdNRPS3 transgenic tobacco is significantly better than that of wild-type tobacco. And according to the disease index, fungus amount and target gene expression detection of the three materials, it shows that the segment 2 of the VdNRPS3 gene ( VdNRPS3-2 ) is used as the target fragment to design dsRNA, which can achieve the best interference effect and can effectively reduce the pathogenic bacteria on plants. virulence.

sequence listing

<110> Institute of Biotechnology, Chinese Academy of Agricultural Sciences

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ctcgagcaag taaggaattg cctggaaaag atggtgcagc aaaacgacat tctccgatcg 240

ggtttcgttg agactggggg cagctttgtg cgtgtgattt tgaaatcact cccaaagtcc 300

cagatccaaa gcgtcaatca gtttacgttg tccgcgcaac agactgcgga tgatgactat 360

gagagtatct ggcttcgccc tttccaggcg caggttgcaa actcggcggc cggagctccc 420

ccacgccttc tcttgcgtat gcatcacgcc atttacgacg 460

<210> 3

<211> 471

<212> DNA

<213> Verticillium dahliae (2 Ambystoma laterale x Ambystomajeffersonianum)

<400> 3

atgctccagg ctgcgtggac tcggctgcta tctgcctatg ttggcgagca aacgcttgta 60

tttggagtcg tgctttcggg ccgcaccttg ccaatgacag atgatgcagt gttcccttgc 120

atcacgacag tcccggtcat ctctcacaac caagcgtcca acacggagct tctgtctcaa 180

atgatgcaat acagcagcca attgtcggcc caccagcatg cacctctttc taaaatacag 240

caatggctcg gccacccagg caccccccta ttcgacactc tgctcgtgta tcagaagact 300

gcaaacggac cgtcatcggc ccagaagctg ccgtggcgtc agatcagtga cgagggcaag 360

cttgactacg ctgtttccct ggagatcgag ccgtcaaacg acgggcatat cagtcttcat 420

ctatcgtctc atacaggtgt tcttccgcgg caacaggcgt caattgccct c 471

<210> 4

<211> 425

<212> DNA

<213> Verticillium dahliae (2 Ambystoma laterale x Ambystomajeffersonianum)

<400> 4

aatattgcga acggtcttct tacaagtcga cgatccacgg ctcgaacaaa cattttgcca 60

gattgctctg gctcaccact tgccctcgat ccaacacatt cagctccaag gcgaacaaga 120

aattgaggga gtctacacag cgcatcgcca aaatgcaagg aacgcacggg gtgcttccca 180

attgctccag ttgacgatcg cgcacgtcgg tgatcagagt tatgtcgttt tcagcatcgc 240

ccacgctctt tacgatggat ggtcattggc tctccttcat caagacgtcg attcggctta 300

ccacaatatg ctggagcaac gcccgtctcc acgaaatttg ttgcagagcc tgctgcagtc 360

accgactgac aaaagccaac gattctggtc ccaatatcta gctggtgccc ccccttcgct 420

tcttc 425

<210> 5

<211> 463

<212> DNA

<213> Verticillium dahliae (2 Ambystoma laterale x Ambystomajeffersonianum)

<400> 5

ctgccacgga taattaccga tgaagcacca tcccttcgct ccgttcgccg gctttacaac 60

gttgagagac tcagggaggc aagcaaagca ctgaacatca ccttgcagtc cgctgtcttg 120

tccttatggt ctggcacctt tggcaaatct ttctccccaa gagctgcgat gggggttgtg 180

gtttcagggc gcgttctcga catagaagac gtcgaggatg tgatgggccc ccttttcaac 240

accttgcctt tctacccaga cgtcgccacg aacaacacgc tacagaagct ggcgcagagg 300

tgccatgact tcaacgtgtc aaccctgcct tttcagcaca cggcgttacg aaacatccag 360

aaatggtgca gcggaggtca tcccattttc gacaatctct tcgtctacca gaatgacgtg 420

cctttggcca gcaacgccga gacattctgg acggtagaag aca 463

Claims (9)

1. A target gene fragment of the VdNRPS3 gene of Verticillium dahliae against pathogenic bacteria, characterized in that: its nucleotide sequence is shown in SEQ ID No.2. 2 . The dsRNA transcribed by the anti-pathogenic bacteria target gene fragment of the Verticillium dahliae VdNRPS3 gene according to claim 1 . 3 . The RNA interference vector comprising the anti-pathogenic bacteria target gene fragment of the Verticillium dahliae VdNRPS3 gene of claim 1 ; wherein the RNA interference vector is a Gateway interference vector. 4 . 4. A method for constructing the RNA interference vector of claim 3, characterized in that, comprising: inserting the anti-pathogenic bacteria target gene fragment of the Verticillium dahliae VdNRPS3 gene of claim 1 into the Gateway interference vector to obtain. 5. The application of the said Verticillium dahliae VdNRPS3 gene anti-pathogenic bacteria target gene fragment of claim 1 in improving plant resistance to diseases caused by Verticillium dahliae. The application according to claim 5, characterized in that it comprises the following steps: (1) constructing a Gateway interference vector containing the anti-pathogenic bacteria target gene fragment of the Verticillium dahliae VdNRPS3 gene of claim 1; (2) converting the The constructed Gateway interference vector is transformed into plants or plant cells; (3) screening to obtain transgenic plants with improved resistance to diseases caused by Verticillium dahliae. 7. The application of the RNA interference vector of claim 3 in improving the resistance of plants to diseases caused by Verticillium dahliae, comprising: (1) transforming the RNA interference vector into a plant or a plant cell; (2) ) screening to obtain transgenic plants with improved resistance to diseases caused by Verticillium dahliae. 8. A method for cultivating a new variety of transgenic plants resistant to diseases caused by Verticillium dahliae, comprising the following steps: (1) constructing an anti-pathogenic bacteria target containing the Verticillium dahliae VdNRPS3 gene of claim 1 RNA interference vector of gene fragment; (2) transforming the constructed RNA interference vector into plants or plant cells; (3) screening to obtain new transgenic plant varieties with improved resistance to diseases caused by Verticillium dahliae. 9. method according to claim 8, is characterized in that, described plant is the host plant of Verticillium dahliae, including but not limited to any in tobacco, cotton, tomato, potato, muskmelon, watermelon, cucumber or peanut A sort of.

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