CN113549635A

CN113549635A - Application of verticillium dahliae VdPRMT1 gene in improving disease resistance of crops or vegetables

Info

Publication number: CN113549635A
Application number: CN202111096745.9A
Authority: CN
Inventors: 苏晓峰; 吴思源; 刘璐; 王�琦; 程红梅
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Baoding Branch Biochemical Technology Co ltd
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2021-10-26
Anticipated expiration: 2041-09-18
Also published as: CN113549635B

Abstract

The invention discloses Verticillium dahliaeVdPRMT1The application of the gene in improving the disease resistance of crops. The invention adopts host-induced gene silencing technology and takes verticillium dahliae strains as materials to construct a plurality of verticillium dahliaeVdPRMT1The tobacco brittle crack virus interference plasmid of the gene is transformed into the Nicotiana benthamiana and inoculated with the verticillium dahliae, and the verticillium dahliae is preliminarily determinedVdPRMT1Genes are associated with pathogenicity. The invention further uses Verticillium dahliaeVdPRMT1As a target gene, a target gene fragment capable of remarkably improving the disease resistance of the plant is obtained by screening by combining a transgene technology and an RNAi technology. The target gene fragment screened by the invention and the Gateway interference vector constructed by applying the target gene fragment can be applied to the aspects of improving the resistance of plants to diseases caused by verticillium dahliae, cultivating new varieties of transgenic plants resisting the verticillium dahliae and the like.

Description

Application of verticillium dahliae VdPRMT1 gene in improving disease resistance of crops or vegetables

Technical Field

The invention relates to a verticillium dahliae protein arginine methyltransferase 1 gene (VdPRMT1) In particular to Verticillium dahliaeVdPRMT1The application of the gene in improving the disease resistance of crops or vegetables, belonging to verticillium dahliaeVdPRMT1The field of new application of gene.

Background

Cotton Verticillium Wilt (Verticillium Wilt of cotton) is called as cotton cancer, which causes 10-35% of average cotton yield reduction in many countries, seriously harms cotton production, and causes huge economic loss (Wangxiaoling; Wangchun; Xiebing construction; Yang star courage, Verticillium Wilt pathogenicity and plant Verticillium Wilt resistance molecular mechanism research progress.Agricultural science of Henan. 2014, 43 (01), 1-6.). 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 wide, and the plant species capable of being infected can be more than six hundred, including annual herbaceous plants, perennial herbaceous plants and woody plants, wherein the plants are not as rich in cruciferous plants, solanaceae plants, compositae plants, rosaceae plants and the like and have important economic benefitsCrops, seedlings and flowers of value, and the range of host infectable is expanding (Sun L.; Qin J.; Rong W., cell surface-induced gene, VdCSIN1, regulation of photoperiod formation and pathogenesis via cAMP-mediated signalling inVerticillium dahliae. Molecular plant pathology 2019,20 (3), 323-33.). The verticillium dahliae is a soil-borne plant pathogenic fungus, so that the control is very difficult, and no medicament with good effect exists in the current production.

Methylation of arginine residues of proteins is a very important type of posttranslational modification of proteins involved in a variety of biological processes including pre-mRNA splicing processing, mRNA translation, DNA damage repair, protein interactions, autophagy, etc. (Guccione E.; Richard S., The regulation, functions and clinical research of arginine methyl).Nature Reviews Molecular Cell Biology2019, 20(10), 642-57.). The methylation of arginine residues is catalyzed by protein arginine methyltransferases (PRMT), which transfers a methyl group from S-Adenosyl-L-methionine (AdoMet/SAM) to the guanidino nitrogen atom of a protein arginine residue to react methyl arginine and S-Adenosyl-L-homocysteine (S-Adenosyl-L-homocysteine, AdoHcy/SAH) (Lee H.W.; Kim S.; Paik W.K., S-Adenosylmethionine: protein-arginine methyltransferases. Purification and catalysis of the enzyme.Biochemistry 1977, 16(1), 78-85.). In recent years, there have been a number of reports of studies of PRMT in humans, mammals and plants, and studies in fungi have been mainly reported with saccharomyces cerevisiae (r) ((r))Saccharomyces cerevisiae) Mainly, the reported protein arginine methyltransferases in Saccharomyces cerevisiae include Hmt1 (Rmt 1), Rmt2, Hsl7 and Sfm 1. Hmt1 (Rmt 1) is The major Protein arginine Methyltransferase of Saccharomyces cerevisiae, belonging to type I PRMT, and is a homolog of PRMT1 in mammals, which is responsible for catalyzing approximately 89% of omega-MMA and approximately 66% of aDMA (Gary J. D.; Lin W. J.; Yang M. C., The Predominant Protein-arginine transferase front) in Saccharomyces cerevisiaem Saccharomyces cerevisiae. Journal of Biological Chemistry 1996, 271 (21), 12585-94.). Hmt1 the reaction substrate is both histone and non-histone. Hmt1 can catalyze the generation of aDMA between the 3 rd arginine residue of histone H4 and the 2 nd arginine residue of histone H3 in Saccharomyces cerevisiae (Li H. T.; Gong T.; Zhou Z., Yeast Hmt1 catalysts amplification of histone H3 arginine 2)in vitro. Biochemical Journal 2015, 467 (3), 507-15.). Hmt1 is generally a class of proteins containing RGG/RG repeats, most of which are located at the N-or C-terminus of the protein, with arginine methylation sites located in the RGG/RG repeats (Brandizi N.A.; Zeng F.; Lam Q.N., Sbp1 modifications of Pab1 mRNA in a poly (A) -and RGG-dependent maner).RNA 2018, 24 (1), 43-55；Bedford M. T., Arginine methylation at a glance. Journal of Cell Science2007, 120(24), 4243.). Studies have shown that the U1 small ribonucleoprotein (snRNP) subunit Npl3 Protein undergoes methylation modification of Hmt1 to facilitate its binding to mRNA precursor and thus participate in the splicing process of mRNA precursor (Muddukrishna B.; Jackson C. A.; Yu M. C., Protein alignment methyl of Npl3 proteins splicing of the SUS1 intron hardbanding non-consensus 5' splice site and branch site).Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 2017, 1860 (6), 730-9.). Li Chi et al (Li Z.; Wu L.; Wu H., Arginine methyl is required for modifying pre-mRNA splicing and indexing of autophagy in edge blast funus).New Phytologist 2020, 225 (1) 413-29.) against Pyricularia oryzae causing Pyricularia oryzae (Pyricularia oryzae)Magnaporthe oryzae) The PRMT of (5) was studied, and it was found that Pyricularia oryzae has four kinds of PRMT, but only when the deletion is homologous to Saccharomyces cerevisiae Hmt1MoHMT1When the mutant is used, the growth phenotype and the pathogenicity of the mutant are obviously changed and reduced compared with the wild type, and the rest mutants have no obvious difference compared with the wild type.

The verticillium dahliae contains four verticillium dahliae PRMTs which are homologous with Saccharomyces cerevisiae PRMTsVdPRMT1（VDAG_08751）、VdPRMT2（VDAG_04075）、VdPRMT3(VDAG _ 04088) andVdPRMT4(VDAG _ 02006). Wherein,VdPRMT1with Saccharomyces cerevisiaeHMT1The total length of cDNA is 1035 bp, and the length of the coded protein is 344 aa.

The published literature indicates that HMT1 is the protein arginine methyltransferase that plays a major role in saccharomyces cerevisiae, and thus VdPRMT1, which is homologous to the yeast HMT1, is likely the protein arginine methyltransferase that is predominant in verticillium dahliae.

Hitherto, it has not been clear whether or not Verticillium dahliae VdPRMT1 has a certain correlation with verticillium wilt of cotton, and therefore, Verticillium dahliae was determinedVdPRMT1Whether the gene is related to the verticillium wilt of cotton or not and screening to obtain verticillium dahliaeVdPRMT1The gene target gene fragment has important application prospect for preventing and treating cotton verticillium wilt.

Disclosure of Invention

One of the objects of the present invention is to identify Verticillium dahliaeVdPRMT1The gene is associated with pathogenicity.

Another object of the present invention is to provide a microorganism containing the Verticillium dahliaeVdPRMT1An interference vector for a gene target gene segment;

another object of the present invention is to use the Verticillium dahliae as a microorganismVdPRMT1The gene target gene fragment and the interference vector containing the target gene fragment are applied to resisting diseases caused by verticillium dahliae or are used for constructing and obtaining a new transgenic plant variety resisting the diseases caused by verticillium dahliae.

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

the present invention first discloses Verticillium dahliae ()Verticillium dahliae）VdPRMT1The application of the gene in improving the resistance of crops or vegetables to pathogenic bacteria.

As a specific embodiment of the present invention, the Verticillium dahliae isVdPRMT1The application of the gene in improving the resistance of crops to pathogenic bacteria comprises the following steps: (1) constructing an interference vector containing a Verticillium dahliae protein arginine methyltransferase 1 gene target gene segment;(2) transforming the constructed interference vector into a plant or plant cell; (3) screening to obtain transgenic crops with improved resistance to diseases caused by verticillium dahliae.

As a specific embodiment of the present invention, the RNA interference vector is a Gateway interference vector; as a preferred embodiment of the present invention, the method for constructing the Gateway interference vector comprises: inserting the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment into a Gateway interference vector to obtain the gene; more preferably, the method for constructing the Gateway interference vector comprises the following steps: the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment is connected to pDONR207 through BP reaction, and then is constructed to pK7GWIWG2(I) and 0 through LR reaction to obtain the Gateway interference vector.

As a preferred embodiment of the invention, the nucleotide sequence of the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment is shown in SEQ ID No.19 or SEQ ID No. 20; more preferably, the nucleotide sequence of the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment is shown in SEQ ID No. 19.

As a preferred embodiment of the invention, the pathogenic bacteria is Verticillium dahliae; the crops or vegetables include but are not limited to any one or more of tobacco, cotton, tomatoes, potatoes, melons, watermelons, cucumbers or peanuts.

The invention also discloses a verticillium dahliae containing strainVdPRMT1An interference vector of a target gene segment and a host cell containing the interference vector.

Furthermore, dsRNA transcribed from the target gene segment shown in SEQ ID No.19 or SEQ ID No.20 is naturally also included in the scope of the present invention.

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

The invention further discloses a method for cultivating a new variety of transgenic plants for resisting diseases caused by verticillium dahliae, which comprises the following steps: (1) constructing a plant containing said DaliBacteriaVdPRMT1An interference vector for the target gene fragment; (2) transforming the constructed interference vector into a plant or plant cell; (3) screening to obtain a new transgenic plant variety with improved disease resistance to verticillium dahliae.

The protocol for transformation and introduction of the nucleotides into plants of the present invention may vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the nucleotide into a plant cell include: microinjection, electroporation, Agrobacterium-mediated transformation, or 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: any one or more of tobacco, cotton, tomato, potato, melon, watermelon, cucumber or peanut.

The invention adopts Host-induced gene silencing technology (Host-induced gene silencing) and takes the verticillium dahliae V991 with high pathogenicity as an experimental material to construct a plurality of verticillium dahliaeVdPRMT1Target gene of tobacco rattle virus: (tobacco rattle virusTRV) interfering plasmids. Transformation of Nicotiana benthamiana by Agrobacterium injectionNicotina benthamiana) And inoculating Verticillium dahliae. Constructing Gateway interference vector with the target gene segment capable of obviously reducing disease index of plant to obtain transgenic plant with stable inheritance. And (3) detecting the biomass of the fungus and the transcription level of a target gene through disease indexes and molecular biology means, and screening an interference section with the best effect. Verticillium dahliae screened by the inventionVdPRMT1The target gene fragment and the Gateway 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

In order to screen and obtain a target gene segment with the best interference effect, the invention is based on the coding sequence of verticillium dahliae protein arginine methyltransferase 1 (PRMT 1, VDAG _ 08751)In the following, primers are designed, 2 different sections aiming at a target gene are obtained through amplification and are named as VdPRMT1-1 and VdPRMT1-2 respectively, and the nucleotide sequences are shown as SEQ ID No.19 and SEQ ID No.20 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 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, at day 7 after inoculation, the VIGS vector in nicotiana benthamiana has produced a large amount of dsRNA and has exerted an interfering effect. Thus, 10 days from the 7 th day after the injection of VIGS series vectors were selected⁶Root dipping inoculation of individual/mL spore suspension. And (4) counting the disease index of the Nicotiana benthamiana at 10 dpi, 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. Part of tobacco disease indexes are kept at a lower level all the time, and the introduction of the target fragment can reduce the disease indexes 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 verifyVdPRMT1The relation between the primer and pathogenicity of pathogenic bacteria and the fragment which can obviously reduce the pathogenicity of the pathogenic bacteria after interference are obtained, the genetically modified Nicotiana benthamiana with stable inheritance is obtained, a primer aiming at a target gene is designed, BP loci are arranged at two ends of the primer, and a target gene fragment is obtained through amplification. 1 obtained clone was subjected to BP reaction and LR reactionVdPRMT1The target fragment was ligated to the Gateway interference vector pK7GWIWG2(I),0 to form a plant transformation vector containing the fungal target gene.

To obtain a gene capable of stably inheriting a target gene for pathogenic bacteriaVdPRMT1The 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.

For the obtained content dsVdPRMT1The positive transgenic tobacco of (2) is inoculated with Verticillium dahliae. Disease index analysis was then performed from day 10, day 11 and day 12 post inoculation. The test results show that the resistance of the transgenic tobacco to pathogenic bacteria is obviously improved, and the disease index is reduced by about 60-85%. And (3) extracting transgenic tobacco root DNA, and performing fungal biomass analysis by utilizing qRT-PCR. Test results show that the fungal biomass of the transgenic positive tobacco is obviously reduced and is about 10 to 25 percent 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, analysis of the expression level of the target gene of pathogenic bacteria at the root of the plant showed that the expression level of the target gene in the transgenic plants was decreased by about 65% compared to wild-type plants. Meanwhile, the picture shows that the disease resistance of the RNAi-VdPRMT1 transgenic tobacco is obviously superior to that of wild tobacco. This means thatVdPRMT1Segment 1 of a Gene (VdPRMT1-1The nucleotide sequence of which is shown in SEQ ID No. 19) as a target fragment to design dsRNA, can achieve the best interference effect, thereby effectively reducing the pathogenicity of verticillium dahliae pathogenic bacteria.

Definitions of terms to which the invention relates

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 (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. biol. chem. 260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell. Probes 8:91-98 (1994)).

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 VIGS vector information.

FIG. 2 amplification results of different sections of VIGS; m: marker, 1-2 is directed toVdPRMT1Amplification of different segments of the gene.

FIG. 3 shows the results of the amplification verification of VIGS interference vector bacterial liquid; m is marker, 1-12 are directed toVDPRMT1And (3) gene construction of a bacterial liquid amplification result of the VIGS plasmid.

FIG. 4 index of disease statistics.

FIG. 5 RNAi amplification results; m is marker, 1 is forVdPRMT1The amplified band of (3).

FIG. 6 RNAi vector information.

FIG. 7 PCR detection of transgenic positive tobacco; m: marker, 1-9 is RNAi-VdPRMT1 transgenic tobacco, WT is wild type tobacco.

FIG. 8 transgenic tobacco disease index analysis.

FIG. 9 in vivo fungal biomass analysis of plants.

FIG. 10 analysis of expression level of 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 described embodiments are exemplary only and are not limiting upon the scope of the invention. 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 dahliaeVdPRMT1Screening of gene anti-pathogenic bacterium target gene fragment, construction of interference vector and application of gene anti-pathogenic bacterium target gene fragment in tobacco transformation

Working test material

First material

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

The culture conditions are as follows: planting the seeds in high-temperature and high-pressure sterilized mixed nutrient soil (aromatic clean nutrient soil: vermiculite = 1: 1), 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.

Method of testing a wall of a duct

First method for cultivating fungi and inoculating plants

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, and centrifuging to collect spores. Diluting with distilled water, observing under microscope, and adjusting spore concentration to 10⁶The cells are ready for use after being treated with one/mL.

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 spore suspension/mL or distilled water, moving the seedlings back to the original plastic pots as soon as possible, watering the seedlings to moisten the soil, and carrying out disease index statistics.

Animal disease index statistics of plants

The disease level of tobacco infection with Verticillium dahliae in this experiment was determined according to the relevant literature (Wang HM, Lin ZX, Zhang XL, Chen W, Guo XP, Nie YC, Li YH. 2008. Mapping and qualitative train Biology analysis of Verticillium wilting resistance genes in cotton. Journal of Integrated Plant Biology 50: 174) and with appropriate adjustments (see Table 1). The disease index calculation formula is shown in the following formula 1:

TABLE 1 index of disease statistics

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

Construction of the interference vector for the three-step 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 protein arginine methyl transferase 1 (PRMT 1, VDAG _ 08751) of verticillium dahliae protein, wherein the two ends of each primer containEcoR I andBamh I (see Table 2), the target fragments were amplified separately. Then detecting the PCR amplification product by using l% agarose gel electrophoresis, and carrying out fragment recoveryAnd (6) harvesting. Respectively carrying out enzyme digestion reaction on the target fragment and the vector, and utilizing T₄Ligase it was constructed into the TRV2 vector. Finally, the verified positive plasmid is transformed into agrobacterium GV3101 by enzyme digestion and sequencing analysis.

TABLE 2 VdPRMT1 different segment primer information

Note: the bold italics are the restriction sites.

Fourth 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 for 3-5 h at room temperature without shaking. And finally, injecting the agrobacterium tumefaciens mixed solution into the tender leaves by using an injector.

Construction of 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; then, it was constructed into pK7GWIWG2(I),0 by LR reaction, and finally the constructed vector was transformed into Agrobacterium LBA 4404.

TABLE 3 Stable genetic interference primer information

Hexagon conversion of tobacco

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 primer information

Detection of fungal biomass

In order to compare the biomass change of pathogenic bacteria in transgenic Nicotiana benthamiana and wild type Nicotiana benthamiana, qRT-PCR was used in the test to determine the biomass of Verticillium dahliae of different plant genotypes. 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 gene of the indigenous tobaccoactinFor housekeeping fragments, relative quantification was performed (see 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

And analysis of expression amount of target gene

To determine that there is a relationship between the improvement of nicotiana benthamiana resistance and the decrease of the target gene, we further determined the transcription level of the target gene in nicotiana benthamiana by using qRT-PCR. Extracting total RNA from 12-day-old Nicotiana benthamiana root, and performing reverse transcription analysis. In pathogenic bacteriaVdPRMT1Primers were designed to target fragments (see Table 6) based on the coding sequence of the gene, and pathogenic bacteria were used simultaneouslyactinAs housekeeping fragments, relative quantification of transcript levels was performed.

TABLE 6 fluorescent quantitation primer information

⒊ test results

Construction of interference carrier of Verticillium dahliae VDPRPRM 1

The experiment used Tobacco Rattle Virus (TRV) vectors provided by the mr of qinghua liuyule (see fig. 1). The cDNA of 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 dahliaeVdPRMT1Coding sequence information, designing primers, amplifying to obtain 2 different segments (figure 2) aiming at target gene, respectively named as VdPRMT1-1 and VdPRMT1-2, and the nucleotide sequences are respectively SEQ IDNo.19 and SEQ ID No. 20.

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 sequence of the target fragment (see 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, at day 7 after inoculation, the VIGS vector in nicotiana benthamiana has produced a large amount of dsRNA and has exerted an interfering effect. Thus, 10 days from the 7 th day after the injection of VIGS series vectors were selected⁶Root dipping inoculation of individual/mL spore suspension. The disease index of Nicotiana benthamiana was counted at day 10 (days post-inoculation, dpi), 11 dpi, and 12 dpi after inoculation. The results are shown in fig. 4, compared with the unloaded tobacco, the disease index of the Nicotiana benthamiana injected with the fungal target gene fragment is reduced; the disease index gradually increases with the number of days. Part of tobacco disease indexes are kept at a lower level all the time, and the introduction of the target fragment can reduce the disease indexes of plants.

Obtaining of transgenic plants

By the VIGS screening method, a target segment capable of improving the resistance of plants to pathogenic bacteria is obtained. To further verifyVdPRMT1The relation between the primer and pathogenicity of pathogenic bacteria and the section which can obviously reduce the pathogenicity of the pathogenic bacteria after interference, the transgenic Nicotiana benthamiana which is stably inherited is obtained, a primer aiming at a target gene is designed, BP loci are arranged at two ends of the primer, and a target gene segment is obtained through amplification. From the electrophoretogram 5, a bright band can be found, and after further DNA sequencing and alignment, the sequence is completely consistent with the target sequence.

1 obtained clone was subjected to BP reaction and LR reactionVdPRMT1The target fragment was ligated to the Gateway interference vector pK7GWIWG2(I),0 (see FIG. 6) to form a plant transformation vector containing the fungal target gene.

To obtain a gene capable of stably inheriting a target gene for pathogenic bacteriaVdPRMT1The constructed Gateway interference vector is transformed into Nicotiana benthamiana by an agrobacterium-mediated and tissue culture method, and finally, the transgenic tobacco is obtained (as shown in figure 7).

Fourth transgenic tobacco disease resistance analysis

For the obtained content dsVdPRMT1The positive transgenic tobacco of (2) is inoculated with Verticillium dahliae. Disease index analysis was then performed from day 10, day 11 and day 12 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 60-85%. 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 10-25% of wild type. Disease index statistics and fungal biomass analysis obviously observe that transgenic positive tobacco has stronger resistance to pathogenic bacteria.

To further verify the relationship between the reduction of disease index and the expression of the target gene, a decrease of about 65% in the expression level of the target gene in transgenic plants compared to wild-type plants was observed by analyzing the expression level of the target gene in pathogenic bacteria at the roots of the plants (fig. 10). Meanwhile, the picture shows that the disease resistance of the RNAi-VdPRMT1 transgenic tobacco is obviously superior to that of wild tobacco. This means thatVdPRMT1Segment 1 (VdPRMT 1-1, the nucleotide sequence of which is shown in SEQ ID No. 19) of the gene is used as a target fragment to design dsRNA, 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

Application of <120> verticillium dahliae VdPRMT1 gene in improving disease resistance of crops or vegetables

<130> BJ-2002-210714A-L

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 31

<212> DNA

<213> Artifical sequence

<400> 1

atagaattcg gagcactctg agcagcacta c 31

<210> 2

<211> 30

<212> DNA

<213> Artifical sequence

<400> 2

ataggatcca ggtgtcacga gcgtagagga 30

<210> 3

<211> 31

<212> DNA

<213> Artifical sequence

<400> 3

ataggatccc gctcgtgaca cctacctgaa c 31

<210> 4

<211> 29

<212> DNA

<213> Artifical sequence

<400> 4

ataggatcca atctcctcgc cgtgctgga 29

<210> 5

<211> 35

<212> DNA

<213> Artifical sequence

<400> 5

aaaaaagcag gctggagcac tctgagcagc actac 35

<210> 6

<211> 34

<212> DNA

<213> Artifical sequence

<400> 6

aagaaagctg ggtaggtgtc acgagcgtag agga 34

<210> 7

<211> 29

<212> DNA

<213> Artifical sequence

<400> 7

ggggacaagt ttgtacaaaa aagcaggct 29

<210> 8

<211> 29

<212> DNA

<213> Artifical sequence

<400> 8

ggggaccact ttgtacaaga aagctgggt 29

<210> 9

<211> 19

<212> DNA

<213> Artifical sequence

<400> 9

gagcagcact acttcaaga 19

<210> 10

<211> 20

<212> DNA

<213> Artifical sequence

<400> 10

ccactcggag atgataatgt 20

<210> 11

<211> 30

<212> DNA

<213> Artifical sequence

<400> 11

ccgccggtcc atcagtctct ctgtttatac 30

<210> 12

<211> 30

<212> DNA

<213> Artifical sequence

<400> 12

cgcctgcggg actccgatgc gagctgtaac 30

<210> 13

<211> 28

<212> DNA

<213> Artifical sequence

<400> 13

ggacctttat ggaaacattg tgctcagt 28

<210> 14

<211> 27

<212> DNA

<213> Artifical sequence

<400> 14

ccaagataga acctccaatc cagacac 27

<210> 15

<211> 19

<212> DNA

<213> Artifical sequence

<400> 15

cggcactgcc attctgtcc 19

<210> 16

<211> 19

<212> DNA

<213> Artifical sequence

<400> 16

tgtcgacctt ggggaaggg 19

<210> 17

<211> 22

<212> DNA

<213> Artifical sequence

<400> 17

ggcttcctca aggtcggcta tg 22

<210> 18

<211> 23

<212> DNA

<213> Artifical sequence

<400> 18

gctgcatgtc atcccacttc ttc 23

<210> 19

<211> 413

<212> DNA

<213> Artifical sequence

<400> 19

ggagcactct gagcagcact acttcaagag ttacgaccac catggcattc acgaggagat 60

gctgaaagac gaggttcgca cccgctctta catgaacgct attgttcaga acaagcacct 120

gttcaaggac aaggtcgtcc ttgacgttgg ctgcggcact gccattctgt ccatgttcgc 180

cgccaaggcc ggcgctaagc acgtcattgg tgttgacatg tcgaccatta ttcacaaggc 240

ccgcgagatc gttgaggtca acggcctctc tgacaagatt accctcatcc agggcaagat 300

ggaggaggtc cagctcccct tccccaaggt cgacattatc atctccgagt ggatgggcta 360

cttcctgctg tacgagagca tgctcgacac ggtcctctac gctcgtgaca cct 413

<210> 20

<211> 457

<212> DNA

<213> Artifical sequence

<400> 20

cgctcgtgac acctacctga acaaggacgg cctcatcttc cccgacaagg ccaccatttt 60

cgccgccggc atcgaggacg gcgagtacaa ggacgagaag attggcttct gggacaacgt 120

gtacggcttc aactactccc ccctcaaggc cacggcgctg tccgagcccc tcgtcgacac 180

ggtcgaggtc aaggccgtcg tgacggagcc cgtgcccatc ctgacactgg acctctacaa 240

gtgccaggtc tccgacctcg ccttcaacac gaccttcaag ctgcccgtgc gccgcgacga 300

ctttgtgcac gccgtcgtcg cctggttcga cattgacttc accgccgccc acaagcccat 360

ccgcttctcc acgggccccc acacaaaata cacccactgg aagcagaccg tcttctacct 420

caaggagatg ctcactgtcc agcacggcga ggagatt 457

Claims

1. Verticillium dahliae (C.), (Verticillium dahliae）VdPRMT1The application of the gene in improving the resistance of crops or vegetables to pathogenic bacteria.

2. Use according to claim 1, characterized in that it comprises the following steps: (1) constructing an RNA interference vector containing a Verticillium dahliae protein arginine methyltransferase 1 gene target gene segment; (2) transforming the constructed RNA interference vector into a plant or plant cells; (3) screening to obtain transgenic crops with improved resistance to diseases caused by verticillium dahliae.

3. The use according to claim 2, wherein the RNA interference vector is a Gateway interference vector.

4. The use according to claim 3, wherein said Gateway interference vector is constructed by a method comprising: and (3) inserting the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment into a Gateway interference vector to obtain the gene.

5. The use according to claim 4, characterized in that the Gateway interference vector is obtained by ligating the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment into pDONR207 by BP reaction and constructing it into pK7GWIWG2(I),0 by LR reaction.

6. The use according to claim 2 or 4, wherein the nucleotide sequence of the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment is shown as SEQ ID No.19 or SEQ ID No. 20.

7. The use according to claim 6, wherein the nucleotide sequence of the verticillium dahliae protein arginine methyltransferase 1 gene target gene fragment is shown as SEQ ID No. 19.

8. Use according to claim 1, wherein the pathogenic bacterium is Verticillium dahliae; the crops or vegetables include but are not limited to any one or more of tobacco, cotton, tomatoes, potatoes, melons, watermelons, cucumbers or peanuts.

9. A method for breeding a new variety of disease-resistant transgenic plants is characterized by comprising the following steps: (1) constructing an RNA interference vector containing a Verticillium dahliae protein arginine methyltransferase 1 gene 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 according to claim 9, wherein the nucleotide sequence of the verticillium dahliae protein arginine methyltransferase 1 gene antipathogen target gene fragment is shown as SEQ ID No.19 or SEQ ID No. 20; the RNA interference vector is a Gateway interference vector; the plant is a host plant of verticillium dahliae, and comprises any one or more of but not limited to tobacco, cotton, tomatoes, potatoes, melons, watermelons, cucumbers or peanuts.