CN114736915A - Verticillium dahliae VdNRPS2 gene antipathogen target gene fragment and interference vector and application thereof - Google Patents

Abstract

The invention discloses Verticillium dahliaeVdNRPS2A gene antipathogen target gene fragment, an interference vector and application thereof. The present invention provides Verticillium dahliaeVdNRPS2The nucleotide sequences of the target gene segments of the gene antipathogenic bacteria are respectively shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5. The invention further provides a Gateway interference vector constructed by adopting the target gene fragment. The verticillium dahliae provided by the inventionVdNRPS2The gene antipathogen target gene fragment and the Gateway interference vector constructed by adopting the target gene fragment can be applied to improving the resistance of crops to diseases caused by verticillium dahliae and cultivating new varieties of transgenic plants resisting the verticillium dahliae, and the likeAnd (5) kneading.

Description

Verticillium dahliae VdNRPS2 gene antipathogen target gene fragment and interference vector and application thereof

Technical Field

The invention relates to a verticillium dahliae antipathogen bacterium target gene segment and an RNA interference vector containing the target gene segment, in particular to verticillium dahliaeVdNRPS2A gene antipathogenic bacterium target gene fragment, an RNA interference vector constructed by adopting the antipathogenic bacterium target gene fragment and application of the RNA interference vector in improving the disease resistance of crops or vegetables belong to the field of the verticillium dahliae antipathogenic bacterium target gene fragment and the application of the disease resistance.

Background

Cotton Verticillium Wilt (Cotton Verticillium wild) is called "Cotton cancer", which causes an average Cotton yield reduction of 10-35% in many countries, seriously harms Cotton production and causes huge economic loss. The pathogenic bacteria is verticillium dahliae (A.Merr.) (Verticillium dahliae) The cotton has strong pathogenicity, and can be infected in the whole cotton growth stage, so that the cotton has the phenomena of leaf wilting, yellowing and the like; when the disease is serious, the whole cotton leaf is scorched and broken, and finally dies. Meanwhile, the host plant range of the plant is wider, the plant types capable of being infected can be more than six hundred, including annual herbaceous plants, perennial herbaceous plants and woody plants, wherein various crops, nursery stocks, flowers and the like with important economic values of cruciferae, solanaceae, compositae, rosaceae and the like are not lacked, and the range of the host capable of being infected is continuously expandedIs large. As the verticillium dahliae is a soil-borne plant pathogenic fungus and is difficult to control, no control agent with good effect exists in the prior production.

Filamentous fungi can synthesize a series of low-molecular-weight polypeptide secondary metabolites with medicinal value through a non-ribosomal pathway, and the complex and various polypeptide compounds are collectively called non-ribosomal peptides (NRP). More than 80% of the non-ribosomal peptides found to date are derived from fungi and actinomycetes, followed by unicellular prokaryotes such as myxobacteria and cyanobacteria. Genomic sequence analysis results indicate that archaea, metazoans and dinoflagellates also have the potential to synthesize non-ribosomal peptides (Wang H, Fewer D P, Holm L, et al. Atlas of nonribosomal peptides and polyketide biochemical pathway reactions 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 antibiosis, antivirus and anticancer, and are widely used at present. NRP is mainly formed by catalysis of non-ribosomal peptide synthetase (NRPS), the first identified NRPS is L-aminoadipoyl-L-cysteine-D-valine synthase, which catalyzes the first step of biosynthesis of Beta-lactam antibiotics, and 3 amino acids are condensed into a tripeptide compound to form penicillin and other Beta-lactam antibiotics (Smith D J, Burnham M K R, Bull J H, et al.Embo Journal, 1990,9(3)): 741-747.). NRPS has multiple physiological functions and can be used as antibiotics (antibiotics) to inhibit competitors competing with nutrients per se; can also be used as toxin (toxins) and plays an important role in the process of invading host and colonizing by pathogenic bacteria; still others may be used as siderophores (siderophores) involved in microbial growth, as a site for storage of nitrogenous substances or as a signaling factor to regulate microbial growth, proliferation and differentiation. Some NRPSs have been completed with other enzyme systems such as polyketide or terpenoid synthase systemsSynthesis of hybrid molecules. A typical NRPS consists of a plurality of modules (modules) arranged in a specific spatial order, each module consisting of a plurality of functional domains (domains) or catalytic units, on which modules and domains structurally diverse non-ribosomal peptides are sequentially synthesized. Recently corn leaf spot bacteria: (Cochliobolus heterostrophus) Whole genome sequencing data show that 12 genes encoding NRPS exist in the maize small leaf spot pathogen, wherein NPS6 is used as a typical virulence determinant in the maize small leaf spot and is involved in not only pathogenesis but also H₂O₂Is a maize pathogen (C)Cochliobolus miyabeanus) Virulence determinants of (1), and H₂O₂The tolerance of (2) is concerned. Pathogenic bacteria of rice (Sporonella gondii.) (Cochliobolus miyabeanus) Fusarium graminearum (F. graminearum) of the wheat scab pathogenFusarium graminearum) And Arabidopsis thaliana pathogenic bacteria: (Alternaria brassicicola) Neutralization ofNPS6Deletion of homologous genes also results in their corresponding decreased virulence and reduced virulence for H₂O₂(iii) a poor tolerance (oxide S, Moeder W, Krasnoff S, et al, NPS6, encoding a nonribosomal peptide synthesized in a siderophore-mediated iron method, is a conserved virus detector of plant pathogenic microorganisms.Plant Cell, 2006, 18(10): 2836- 2853. ）。

The genome sequence analysis of Verticillium dahliae shows that 6 verticillium dahliae coexistNRPSGene, gene expression quantity of different infection period is analyzed and foundVdNRPS2The expression is obviously up-regulated in the early infection stage, and is probably closely related to the pathogenicity of verticillium dahliae. Accordingly, fromVdNRPS2The screening of the gene to obtain the target disease-resistant gene segment and the construction of the corresponding interference vector have important application prospects for improving the disease resistance of cotton.

Disclosure of Invention

One object of the present invention is to provide Verticillium dahliaeVdNRPS2Gene resistance target gene fragment;

another object of the present invention is to provide a microorganism containing the Verticillium dahliaeVdNRPS2RNA interference vector of gene anti-disease target gene fragment；

Another object of the present invention is to use the Verticillium dahliae as a microorganismVdNRPS2The gene disease-resistant target gene fragment and the RNA interference vector containing the target gene fragment are applied to plant disease resistance or are used for constructing new varieties of disease-resistant transgenic plants.

In order to achieve the purpose, the invention adopts the main technical scheme that:

the invention firstly discloses verticillium dahliae capable of improving plant pathogenic bacteria resistanceVdNRPS2Target gene fragment, the nucleotide sequence of which is shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 respectively; preferably, the nucleotide sequence of the target gene fragment is shown as SEQ ID No. 1.

The invention also discloses a verticillium dahliae containing strainVdNRPS2An RNA interference vector of a target gene segment and a host cell containing the RNA interference vector.

In addition, dsRNA transcribed from the target gene fragment shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 is also included in the scope of the present invention.

The Verticillium dahliae of the inventionVdNRPS2The target gene fragment can be applied to improving the disease resistance of plants to diseases caused by verticillium dahliae, and comprises the following steps: (1) construction of a microorganism containing the Verticillium dahliaeVdNRPS2RNA interference vector of target gene fragment; (2) transforming the constructed RNA interference vector into a plant or plant cells; (3) screening to obtain the transgenic plant with improved disease resistance to verticillium dahliae.

Preferably, a method for constructing the RNA interference vector comprises: the verticillium dahliae is processed by BP reactionVdNRPS2The target gene fragment is connected into pDONR207, and is constructed into pK7GWIWG2(I),0 through LR reaction to obtain the Gateway interference vector.

The RNA interference vector can be applied to improving the disease resistance of plants to diseases caused by verticillium dahliae, and comprises the following steps: (1) transforming the RNA interference vector into a plant or plant cell; (2) screening to obtain the transgenic plant with improved disease resistance to verticillium dahliae.

The disease caused by the verticillium dahliae is preferably cotton verticillium wilt.

The invention further discloses a method for cultivating a new variety of transgenic plants resisting verticillium dahliae, which comprises the following steps: (1) construction of a microorganism containing the Verticillium dahliaeVdNRPS2RNA interference vector of target gene fragment; (2) transforming the constructed RNA interference vector into a plant or plant cells; (3) screening to obtain a new transgenic plant variety with improved disease resistance to verticillium dahliae.

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

The plant of the invention is a host plant of verticillium dahliae, preferably a crop or a vegetable, and comprises the following components: tobacco, cotton, tomato, potato, melon, watermelon, cucumber or peanut.

The invention adopts Host-induced gene silencing technology (HIGS) and takes a highly pathogenic verticillium dahliae strain V991 as an experimental material to construct a plurality of tobacco fragile split virus (TRV) interference plasmids aiming at a non-ribosomal peptide synthetase 2 (NRPS 2, VDAG _ 03964) target gene of verticillium dahliae. Transformation of Nicotiana benthamiana by Agrobacterium injectionNicotina benthamiana) And inoculating Verticillium dahliae. Constructing Gateway interference vector with the target segment capable of obviously reducing disease index of plant to obtain transgenic plant with stable inheritance. And (3) detecting the fungal biomass and the transcription level of the target gene through disease indexes and molecular biology means, and screening the interference fragment with the best effect. Verticillium dahliae screened by the inventionVdNRPS2The target gene fragment and the RNA interference vector constructed by applying the target gene fragment can be applied to improving the disease resistance of plants to diseases caused by verticillium dahliae and cultivating new varieties of transgenic plants resisting the verticillium dahliae.

Detailed description of the invention

According to the invention, Verticillium dahliae isVdNRPS2Coding sequence information, designing primers, amplifying to obtain 5 different segments aiming at target genes, wherein the 5 target genes are respectivelyVdNRPS2-1、VdNRPS2-2、VdNRPS2-3、VdNRPS2-4 andVdNRPS2-5, the nucleotide sequences of which are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, respectively. By passingBamH I andEcor I the cloned target fragment is cut by enzyme and then constructed into TRV2 vector, becoming VIGS series RNAi vector. After the bacterial liquid amplification and DNA sequencing verification, the sequence is found to be completely consistent with the sequence of the target fragment. The positive material, which was verified to be correct, was then transformed into agrobacterium GV3101 for injection into nicotiana benthamiana.

From day 7 after injection, the albinism of the young shoots of Nicotiana benthamiana began to appear. By day 10, the newly grown leaves are all white, and this phenomenon of leaf whitening may last for 45 days. This indicates that the VIGS vector in Nicotiana benthamiana produced a large amount of dsRNA and acted as an interference at day 7 after inoculation. Thus, 10 days from the 7 th day after the injection of VIGS series vectors were selected⁶Inoculation by root dipping of cfu/mL spore suspension. The disease index of Nicotiana benthamiana is counted for 10 dpi (days post-infection), 11 dpi and 12 dpi after inoculation. The results show that the disease indexes of the Nicotiana benthamiana injected with the fungal target gene fragment are all reduced compared with the disease indexes of the Nicotiana benthamiana injected with the fungal target gene fragment in an unloaded state; the disease index gradually increases with the number of days. Wherein, the injection is carried outVdNRPS2-1、VdNRPS2-3、VdNRPS2The disease index of tobacco with three groups of target gene segments is always kept at a lower level, which preliminarily shows that the introduction of the three groups of target gene segments can effectively reduce the disease index of plants.

The invention obtains the target section capable of improving the resistance of plants to pathogenic bacteria by a VIGS screening method. To further verifyVdNRPS2The relation with pathogenicity of pathogenic bacteria and the section which can obviously reduce the pathogenicity of the pathogenic bacteria after interference are obtained stablyThe experiment designs a primer aiming at a target gene, wherein BP loci are arranged at two ends of the primer, and a target gene segment is obtained by amplification. 3 clones obtained by BP reaction and LR reactionVdNRPS2Target fragment (a)VdNRPS2-1、VdNRPS2-3、VdNRPS25) respectively connecting to Gateway interference vectors pK7GWIWG2(I) and 0 to construct a plant transformation vector containing the fungal target gene.

To obtain a gene capable of stably inheriting a target gene for pathogenic bacteriaVdNRPS2The constructed Gateway interference vector is transformed into Nicotiana benthamiana by the dsRNA of (double-stranded ribonucleic acid) through an agrobacterium-mediated and tissue culture method, and finally, the transgenic tobacco is obtained. To the obtained product containingdsVdNRPS2The positive transgenic tobacco is inoculated with Verticillium dahliae, and then disease index analysis is carried out at 10 dpi, 11 dpi and 12 dpi after inoculation. According to the experimental result, the resistance of the transgenic tobacco to pathogenic bacteria is obviously improved, and the disease index is reduced by about 50-85%. And (3) extracting transgenic tobacco root DNA, and performing fungal biomass analysis by utilizing qRT-PCR. The fungal biomass of the transgenic positive tobacco is obviously reduced and is about 20-40% of that of the wild type. Disease index statistics and fungal biomass analysis can obviously observe that the transgenic positive tobacco has stronger resistance to pathogenic bacteria.

To further verify the relationship between the decrease in disease index and the expression of the target gene, the expression level of the target gene in transgenic plants was observed to decrease by about 55-80% compared to wild-type plants by analyzing the expression level of the target gene of the pathogenic bacteria at the roots of the plants. At the same time, RNAi-VdNRPS2The disease resistance of the transgenic tobacco is obviously superior to that of wild tobacco. And the detection according to the disease index, the fungal quantity and the target gene expression quantity of the three materials shows thatVdNRPS2Segment 1 of a Gene (VdNRPS2-1) dsRNA is designed as a target fragment, so that the optimal interference effect can be achieved, and the pathogenicity of pathogenic bacteria can be effectively reduced.

Definitions of terms related to the invention

Unless defined otherwise, 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 either single-or double-stranded form. Unless specifically limited, the term encompasses 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, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues.

The term "recombinant host cell" or "host cell" means a cell comprising a nucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell. The host cell may be a prokaryotic cell or a eukaryotic cell.

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

Drawings

FIG. 1 is a schematic view of the VIGS vector.

FIG. 2 shows the amplification results of different sections of VIGS.

FIG. 3 shows the results of the amplification and verification of VIGS interference vector bacterial liquid; m is marker, N is a negative control, 1-4 (fragment 1), 5-8 (fragment 2), 9-12 (fragment 3), 13-16 (fragment 4) and 17-20 (fragment 5) are directed againstVdNRPS2And (3) gene construction of a bacterial liquid amplification result of the VIGS plasmid.

FIG. 4 shows the statistical results of disease index.

FIG. 5 shows the result of RNAi amplification; m is marker, 1, 2 and 3 are respectively aimed atVdNRPS2-1、VdNRPS2-3、VdNRPS2-5 strips.

FIG. 6 is a schematic diagram of an RNAi vector.

FIG. 7 is a PCR assay of transgenic positive tobacco; m: marker, 1-4 (segment 1), 5-8 (segment 3) and 9-12 (segment 5) are transgenic positive tobacco, WT is wild type tobacco.

FIG. 8 shows the result of index analysis of the disease state of transgenic tobacco.

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

FIG. 10 shows the results of analysis of the expression level of a fungal target gene in plants.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the embodiments are illustrative only and are not to be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 Verticillium dahliaeVdNRPS2Screening of target gene fragment against pathogenic bacteria, construction of RNA interference vector and verification of tobacco resistance

Processing materials and methods

First material

Tobacco: nicotiana benthamiana (B)Nicotiana benthamiana) And (5) strain.

The culture conditions are as follows: planting the seeds in the mixed nutrient soil (aromatic clean nutrient soil: vermiculite = 1: 1) sterilized at high temperature and high pressure, wherein the temperature is 23 +/-2 ℃, the relative humidity is 75 +/-5%, and the light cycle L: d is 16 h: and 8 h.

Two strains and plasmids

Cotton verticillium wilt pathogens: verticillium dahliae (C.), (Verticillium dahliae) V991, a highly pathogenic defoliating strain, which was a gift from the Jianguilian researcher, the institute of plant protection, the national academy of agricultural sciences.

Viral vector (b): tobacco rattle virus: (Tobacco rattle virusTRV) binary vectors (TRV 1 and TRV 2) were gifted by professor liuyule, university of qinghua.

And (3) agrobacterium: strains GV3101 and LBA4404, were stored in the inventors' laboratory.

Plant stable genetic vectors: pDONR207 and pK7GWIWG2(I),0 vectors were stored in the inventors' laboratory.

The culture and plant inoculation mode of the fungus

Culturing Verticillium dahliae spores in liquid CM culture medium, and performing shake culture at 25 deg.C for 5-7 days. Filtering with 5 layers of gauze, centrifuging to collect spore, diluting with distilled water and observing under microscope, adjusting spore concentration to 10⁶And the cfu/mL is reserved.

When the leaf of the Nicotiana benthamiana grows to 6-8 true leaves, selecting the seedlings with consistent growth vigor for inoculation. The seedlings were dug out from the roots with tweezers and the soil of the roots was washed in distilled water. Completely soaking the roots of the seedlings in the diluted 10⁶And (3) after 2 min in cfu/mL spore suspension or distilled water, moving the seedlings back to the original plastic pots as soon as possible, watering to moisten the soil, and carrying out disease index statistics.

Fourth, plant disease index statistics

According to the related literature (Wang HM, Lin ZX, Zhang XL, et al, Mapping and qualitative train location analysis ofverticillium wilt resistance genes in cotton. Journal of Integrative Plant Biology2008, 50(2): 174-. The disease index calculation formula is as follows (formula 1):

TABLE 1 index of disease statistics

Formula 1 disease index = [ Ʃ (number × level)/(total plant × highest level) ] × 100

Construction of interference vector for fifthly VIGS

In order to screen and obtain a target gene segment with the best interference effect, 2 pairs of specific primers are designed according to the coding sequence of non-ribosomal peptide synthetase 2 (NRPS 2, VDAG _ 03964) of Verticillium dahliae, wherein two ends of each primer containEcoR I andBamh I (see Table 2), respectively amplifying the target fragments. Then, the PCR amplification product is detected by l% agarose gel electrophoresis, and fragments are recovered. And (3) carrying out enzyme digestion reaction on the target fragment and the vector respectively, and constructing the target fragment and the vector into a TRV2 vector by utilizing T4 ligase. Finally, the verified positive plasmid is transformed into agrobacterium GV3101 by enzyme digestion and sequencing analysis.

TABLE 2VdNRPS2Information on primers in different regions

Note: the bold italics are the restriction sites.

Sixthly, VIGS conversion method

Agrobacterium monoclonals containing positive plasmids were plated in LB liquid medium (25. mu.g/mL Rif and 50. mu.g/mL Kan) and shake-cultured overnight at 28 ℃. Adding the bacterial liquid (2% in proportion) into LB liquid culture medium for culturing again the next day, and performing shaking culture until OD is reached₆₀₀When the concentration is 0.5-0.6, the cells are collected by low-temperature centrifugation. The waste was discarded, and the cells were resuspended in an injection medium (10 mM MES, 10 mM MgCl)₂100 μ M acetosyringone), adjusting OD₆₀₀To 0.8-1.0. Two agrobacterium strains (TRV 1 and TRV2+ target gene fragments) were grown at 1: 1 mixing and standing at room temperature for 3-5 h without shaking. And finally, injecting the agrobacterium tumefaciens mixed solution into the tender leaves by using an injector.

Construction of a Stable genetic vector

In order to obtain stably inherited Nicotiana benthamiana containing target gene dsRNA, primers (both ends contain partial BP sites) are redesigned for DNA segments capable of obviously improving the resistance of plants to pathogenic bacteria, and then attb primers are used for amplification (shown in Table 3) for construction of stable genetic interference vectors. (ii) ligation of the target sequence into pDONR207 by BP reaction; it was then constructed into pK7GWIWG2(I),0 by LR reaction; finally, the constructed vector is transformed into agrobacterium LBA 4404.

TABLE 3 Stable genetic interference primer information

And in the transformation of Nicotiana benthamiana

Tobacco leaves of Nicotiana benthamiana planted on MS minimal medium were cut into 0.4X 0.6 cm-sized pieces (with edges and main veins removed) and placed in OD₆₀₀0.1-0.2, soaking in the bacterial liquid of agrobacterium LBA4404 containing the positive plasmid for 5 min, and sucking the bacterial liquid on the surface of the plant material by using sterile filter paper. Then, the small leaves were placed on a tobacco bud differentiation medium (MS + NAA 0.2 mg/L +6-BA 2 mg/L) on which a layer of filter paper was laid, and cultured in a dark room at 25 ℃ for 3 days. The tobacco explants which are subjected to co-culture are transferred to a screening culture medium (MS + NAA 0.2 mg/L +6-BA 2 mg/L + Kan 100 mg/L + Carb 500 mg/L) containing corresponding antibiotics for culture, and the illumination period is 16 h illumination/8 h darkness. After 2-3 weeks, when the resistant bud grows to 1-2 cm high, the bud cut by a sterile scalpel is transferred into a rooting culture medium (MS + Kan 100 mg/L + Carb 500 mg/L) to induce rooting, and adventitious roots are formed after 1-2 weeks. Then, DNA of the transgenic plant is extracted and PCR detection is carried out (as shown in Table 4), and a transgenic positive plant is obtained.

TABLE 4 detection of primer information

Detection of fungal biomass

In order to compare the biomass change of pathogenic bacteria in the transgenic Nicotiana benthamiana and the wild type Nicotiana benthamiana, the biomass of the root verticillium dahliae of different plant genotypes is determined by utilizing qRT-PCR. Extracting the total DNA of the root of the inoculated 12-day indigenous tobacco, taking the ITS of the internal transcribed spacer region of the verticillium dahliae as a target fragment, and simultaneously taking the housekeeping base of the indigenous tobaccoDue to the fact thatactinFor housekeeping fragments, relative quantification was performed (table 5). The qRT-PCR reaction was completed on ABI7500, and the result was 2^-∆∆CtThe method carries out result analysis. The unimodal property and amplification efficiency of the primer meet the experimental requirements.

TABLE 5 fluorescent quantitation primer information

Analysis of expression level of the target Gene

In order to determine that the improvement of the resistance of the Nicotiana benthamiana has a certain relation with the reduction of the target gene, the transcription level of the target gene in the Nicotiana benthamiana is further determined by utilizing qRT-PCR. Extracting total RNA from 12-day-old Nicotiana benthamiana root, and performing reverse transcription analysis. In pathogenic bacteriaVdNRPS2Primers were designed for the coding sequence of the gene as fragments of interest (Table 6) with pathogenic bacteriaactinAs housekeeping fragments, relative quantification of transcript levels was performed.

The qRT-PCR reaction was completed on ABI7500, and the result was 2^-∆∆CtThe method carries out result analysis. The unimodal property and amplification efficiency of the primer meet the experimental requirements.

TABLE 6 fluorescent quantitative primer information

Test results of the wall thickness of the wall

Verticillium dahliaeVdNRPS2Construction of an interference vector

The experiment used Tobacco Rattle Virus (TRV) vectors provided by the qinghua liuyule teacher (fig. 1). The cDNA for TRV is located between the double 35S promoter and nopaline synthase terminator (NOSt). TRV1 contains other elements such as the viral RNA-dependent RNA polymerase (RdRp), the Mobile Protein (MP), and the 16 kDa cysteine-rich region. TRV2 contains other elements such as the Capsid Protein (CP) of the virus, the Multiple Cloning Site (MCS), and the like. The introduction of multiple cloning sites facilitates the insertion of foreign genes.

Verticillium dahliaeVdNRPS2Coding sequence information, designing primers, amplifying to obtain 5 different segments of the target gene (FIG. 2), wherein the 5 target genes are respectivelyVdNRPS2-1、VdNRPS2-2、VdNRPS2-3、VdNRPS2-4 andVdNRPS2-5, the nucleotide sequences of which are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5.

By passingBamH I andEcor I the cloned target fragment is cut by enzyme and then constructed into TRV2 vector, becoming VIGS series RNAi vector. After verification by bacterial fluid amplification and DNA sequencing, the sequence was found to be identical to the target fragment sequence (fig. 3). The positive material, which was verified to be correct, was then transformed into agrobacterium GV3101 for injection into nicotiana benthamiana.

Analysis of disease index of Nicotiana benthamiana

From day 7 after injection, the albinism of the young shoots of Nicotiana benthamiana began to appear. By day 10, the newly grown leaves are all white, and this phenomenon of leaf whitening may last for 45 days. This indicates that the VIGS vector in Nicotiana benthamiana produced a large amount of dsRNA and acted as an interference at day 7 after inoculation. Thus, 10 days from the 7 th day after the injection of VIGS series vectors were selected⁶Inoculation by root dipping of cfu/mL spore suspension. The disease index of Nicotiana benthamiana is counted for 10 dpi, 11 dpi and 12 dpi after inoculation. The results show (fig. 4) that the disease index was decreased compared to that of tobacco benthamiana injected with fungal target gene fragments; the disease index gradually increases with the number of days. Wherein, the injection is carried outVdNRPS2-1、VdNRPS2-3、VdNRPS25 in the tobacco with three groups of target gene segments, the disease index is always kept at a lower level, which preliminarily shows that the introduction of the three groups of target gene segments can effectively reduce the disease index of the plant.

Obtaining of transgenic plants

Screening by VIGSThe method obtains 3 target segments capable of improving the resistance of plants to pathogenic bacteria. To further verifyVdNRPS2The relation between the primer and pathogenicity of pathogenic bacteria and the section which can obviously reduce the pathogenicity of the pathogenic bacteria after interference to obtain the transgenic Nicotiana benthamiana with stable inheritance. Bright bands can be found from electrophoretic figure 5. After further DNA sequencing and alignment, the sequence is found to be completely consistent with the target sequence.

3 clones obtained by BP reaction and LR reactionVdNRPS2Target fragment (a)VdNRPS2-1、VdNRPS2-3、VdNRPS2-5) respectively connecting to Gateway interference vector pK7GWIWG2(I),0 (figure 6) to construct a plant transformation vector containing a fungal target gene.

To obtain a gene capable of stably inheriting a target gene for pathogenic bacteriaVdNRPS2The constructed Gateway interference vector is transformed into Nicotiana benthamiana by an agrobacterium-mediated and tissue culture method, and finally, the transgenic tobacco is obtained (figure 7).

Fourth transgenic tobacco disease resistance analysis

To the obtained product containingdsVdNRPS2Positive transgenic tobacco of-1, 3, 5 genes, and inoculating Verticillium dahliae. Disease index analysis was then performed from 10 dpi, 11 dpi, and 12 dpi post-inoculation.

As can be seen from FIG. 8, the resistance of the transgenic tobacco to pathogenic bacteria is obviously improved, and the disease index is reduced by about 50-85%. And (3) extracting transgenic tobacco root DNA, and performing fungal biomass analysis by utilizing qRT-PCR. FIG. 9 shows that the fungal biomass of transgenic positive tobacco is significantly reduced, about 20-40% of wild type. Disease index statistics and fungal biomass analysis can obviously observe that the transgenic positive tobacco has stronger resistance to pathogenic bacteria.

In order to further verify the relationship between the reduction of disease index and the expression of target gene, the expression level of the target gene in the transgenic plant can be observed by analyzing the expression level of the target gene of pathogenic bacteria at the root of the plant compared with that of the wild plantA decrease of about 55-80% (fig. 10). At the same time, the picture shows RNAi-VdNRPS2The disease resistance of the transgenic tobacco is obviously superior to that of wild tobacco. And the detection according to the disease index, the fungal quantity and the target gene expression quantity of the three materials shows thatVdNRPS2Segment 1 of a Gene (VdNRPS2-1) dsRNA is designed as a target fragment, so that the optimal interference effect can be achieved, and the pathogenicity of pathogenic bacteria can be effectively reduced.

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> Verticillium dahliae VdNRPS2 gene antipathogen target gene fragment, interference vector and application thereof

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gaccttctgg agggagaggc tgcagggcgc caacggccct cagttccctg ctctgccata 60

tgaaggctac cagacgcagg cggactccct tcttgagatc cacgtaccgc tgtctggtcg 120

accggcgtcc aacacgacag tggcgactgt cattcgtggc gcgtgggcct atgttgcctc 180

gcggtacagc gcaacaacgg acgtcgtctt tggcgagacg ctcacggggc gcaatgctgc 240

cctcaggggc gccgaagaga ttgaaggccc catgatcacc accattccgt tccgtgtcca 300

gatccacgac gagagcagtg tcacagagta catccaggag atccaagact tgactgccca 360

gcagattccc tacgagcaca cgggcctgca acacattcgc cgtctcagcc ccgacgccta 420

cgaggcctgc gaactgcgga caggccttgt tctgcatccc agtgccgagg gcgaagcgca 480

acccaat 487

<210> 3

<211> 446

<212> DNA

<213> Verticillium dahliae

<400> 3

ctgttgtaca agacgggtga tctggtcagg tatgaccctg acggcaccgg cgccatcgcc 60

tttgtcggcc gcaaggacca gcaagtcaag ctccgcggac agcgcatcga gctggccgag 120

gtggagcacc atctccgggg taagctcccg tccagtgtca agcttgcggc cgaggtcatc 180

aagccgggcg gcacggagcc gacgctggtg gccttcatcg tcgagcaggc atcagccgcg 240

agcgatgacg gagagggcga cctcacgact ctttccgcgg agctcagcca aatcgtcgcc 300

gagattgata cagctcttgg agccgagatt cctcgataca tggtcccgtc gtcttacatt 360

cctctgcgca aaatgccctc gcttgtttct ggaaagatcg accgcaagcg tctccgcgag 420

ctgggatcgt cgatgagcag ggagga 446

<210> 4

<211> 448

<212> DNA

<213> Verticillium dahliae

<400> 4

caagaagccg tcgagccctt ctcgttgctt gacaaggact ggacccgtga ggctgccgtt 60

gctgacgtcg cgaaactgtg cgagattgag gaggccacgg ttgaggacgt gtatccctgc 120

acgcctctac aggaagcttt gatggccttg tcggcaaagg tcaaagaggc ctacgttgcg 180

cagcgagtgg tggacctcga cgatgcagcg atggcggaca agctgaggtt ggctttcgat 240

gcagccgcca cagattgccc cattttgagg acgaggattg tccaggtgcc ccagcacggc 300

ctcgttcaag tcgttgtcaa cgagaagatt gcctggcacc ttggcgatga cctccagggc 360

taccttgtga aggaccgcga cgaggcaatg gacctcggca agccccttgt gcgatacgcc 420

ctcatcacca gccccgaatc gtcaaagg 448

<210> 5

<211> 439

<212> DNA

<213> Verticillium dahliae

<400> 5

atcccctcag atgaggatcg catgaatgac ctcagtgacg ccatccgcaa gagcaagtct 60

aacatggccc acatgacgcc ctccgtcgct agagttctgg acccgaatgt catcccgtcc 120

ctcgaggtcc tcggtctcgg cggtgaggct gtttctgctg gtgatgcctc agcctggagt 180

cagagtgcca aggtcatcat cgcgtacggc ccctccgagt gcacagttgg ctgcaccatc 240

aacggcagcg tcagcagcac gtcaacaaat atcggcaaag gcaccggcgg cctcacatgg 300

atcgtcgatc ctgacgacca cgaccgcctg atgcccgtcg gcgccgtcgg tgagcttctc 360

atcgaggggc ccgtcgtggg cctgggctat ctcaacgacc ccacaaagac ggccgaagtc 420

ttcatcaagg atcctacat 439

Claims (10)

1. Verticillium dahliae (A. dahliae) ((A. dahliae))Verticillium dahliae）VdNRPS2The gene is resistant to pathogenic bacteria target gene fragments, and is characterized in that: the nucleotide sequence is shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 5.

2. The Verticillium dahliae as claimed in claim 1VdNRPS2The gene is resistant to pathogenic bacteria target gene fragments, and is characterized in that: the nucleotide sequence is shown as SEQ ID No. 1.

3. The Verticillium dahliae strain of claim 1 or 2VdNRPS2dsRNA transcribed from a target gene fragment of the gene against pathogenic bacteria.

4. Comprising the Verticillium dahliae as claimed in claim 1 or 2VdNRPS2RNA interference vector of gene antipathogen target gene segment; wherein the RNA interference vector is a Gateway interference vector.

5. A method for constructing the RNA interference vector of claim 4, comprising: the Verticillium dahliae strain of claim 1 or 2VdNRPS2Inserting the target gene fragment of the gene antipathogen into a Gateway interference vector to obtain the gene antipathogen.

6. The Verticillium dahliae strain of claim 1 or 2VdNRPS2The application of the gene anti-pathogenic bacteria target gene segment in improving the disease resistance of plants to verticillium dahliae.

7. Use according to claim 6, characterized in that it comprises the following steps: (1) construction of a culture medium containing the Verticillium dahliae strain of claim 1 or 2VdNRPS2Gateway interference vector of gene antipathogen target gene fragment; (2) transforming the constructed Gateway interference vector into a plant or plant cell;(3) screening to obtain transgenic plants with improved disease resistance caused by verticillium dahliae.

8. The use of the RNA interference vector of claim 4 for increasing the resistance of a plant to a disease caused by Verticillium dahliae, comprising: (1) transforming the RNA interference vector into a plant or plant cell; (2) screening to obtain transgenic plant with raised disease resistance caused by verticillium dahliae.

9. A method for breeding a new variety of transgenic plants resistant to diseases caused by Verticillium dahliae is characterized by comprising the following steps: (1) construction of a microorganism containing the Verticillium dahliae of claim 1 or 2VdNRPS2RNA interference vector of gene antipathogen target gene segment; (2) transforming the constructed RNA interference vector into a plant or plant cells; (3) screening to obtain a new transgenic plant variety with improved resistance to diseases caused by Verticillium dahliae.

10. The method of claim 9, wherein the plant is a host plant of verticillium dahliae, including but not limited to any one of tobacco, cotton, tomato, potato, melon, watermelon, cucumber, or peanut.

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