CN111808832A - Gene of cation transfer ATP enzyme of rhizoctonia solani, fragment Rscta thereof and application - Google Patents

Abstract

The invention discloses a rice sheath blight bacterium cation transfer ATP enzyme gene, a fragment Rscta thereof and application. The invention provides a cation transfer ATPase gene of Rhizoctonia solani and a fragment Rscta thereof, wherein the cation transfer ATPase gene is significantly induced and expressed in the early stage process that the Rhizoctonia solani infects rice; according to dsRNA synthesized by the gene fragment Rscta of the cation transfer ATPase of rhizoctonia solani, the gene of the cation transfer ATPase of rhizoctonia solani is silenced through in vitro treatment, so that the pathogenicity of the rhizoctonia solani is obviously reduced; the dsRNA can be used for preparing a rice sheath blight bacterium prevention and control preparation, and a recombinant vector containing a segment Rscta is transformed into a plant to obtain a rice sheath blight bacterium resistant transgenic plant; therefore, the gene or the fragment Rscta of the cation transfer ATPase of Rhizoctonia solani has wide application prospects in preventing and treating Rhizoctonia solani, preparing a Rhizoctonia solani prevention and treatment preparation, and constructing transgenic plants resistant to Rhizoctonia solani.

Description

Gene of cation transfer ATP enzyme of rhizoctonia solani, fragment Rscta thereof and application

Technical Field

The invention belongs to the technical field of biological control. More particularly, relates to a rice sheath blight bacterium cation transport ATP enzyme gene and a fragment Rscta thereof and application.

Background

Rice sheath blight (Rice sheath blight height) caused by rhizoctonia solani is one of the important diseases of Rice, and the damage degree is second to Rice blast. The disease area of the rice sheath blight disease in China per year exceeds 2 hundred million mu, and the rice sheath blight disease is the rice disease with the widest disease area. Rice areas with severe disease development can result in severe yield losses. The pathogenic bacteria of the disease can not only infect rice, but also infect plants as many as 260 plants such as cruciferae and the like. However, the studies on the resistance of plants to Rhizoctonia solani and the gene functions of the rhizoctonia solani are few, and no rice varieties with high resistance to banded sclerotial blight are developed; the reason for this is that the fungus is a fungus with strong saprophytic property, and the cell has a plurality of cell nucleuses.

Host-induced gene silencing technology is an RNA interference (RNAi) -based strategy that involves expressing appropriate RNAi constructs in a host plant, targeting essential genes of pathogenic bacteria, and transferring double-stranded RNA (dsrna) or small interfering RNA (sirna) into the pathogenic bacteria during the interaction process, thereby silencing the gene of interest and thereby inhibiting virulence; at present, the technology is proved to be an effective strategy for silencing exogenous pathogenic genes in hosts, and is widely applied to plant disease resistance and pest control. The Virus-induced gene silencing technology (VIGS technology) is short for VIGS technology, is based on the RNAi principle of homologous RNA complementary combination degradation, is a technology for instantly and quickly identifying the plant gene function by utilizing the natural defense mechanism of plants against viruses, and is a reverse genetics method for verifying the gene function by using a gene sequence. In 2010, Nowara silences genes GTF1 and GTF2 of wheat powdery mildew in wheat bodies by using a VIGS vector based on BSMV, and has obvious influence on the growth of infection nails of the powdery mildew; therefore, a new approach to identify plant pathogenic gene functions using VIGS was proposed.

Cation-transporting atpases are a class of proteins that catalyze cation conversion and convert ATP to ADP and phosphate, which can transport cations from one side of the membrane to the other. Cation-transporting ATPases are involved in important processes in eukaryotes, such as active transport, ATP metabolism, cell motility, growth and development, and protein selection. At present, the research on the function of the cation-transporting ATPase gene is mainly focused on human and animals, and the research on plant pathogenic fungi is very rare. Sudundong et al analyzed the information of P-type ATPase gene family in anthrax graminearum, anthrax sewise and anthrax colletotrichum by bioinformatics method, but no studies on gene function were made (Sudung et al, analysis of P-type ATPase gene family in 3 anthrax, proceedings for tropical crops 2015). Pengchen researches the function of a MoCTA1 gene in a academic paper, and finds that deletion mutants of the gene have certain influence on pathogenicity of pathogenic bacteria (analysis of gene families of P-type ATP enzyme of rice blast fungus, researches on genes MoCTA1 and MoCTA3 of the P-type ATP enzyme, university of agriculture in Anhui, 2012), but through gene sequence comparison, the gene of a cation transport ATP enzyme of Rhizoctonia solani has no homology with the gene of MoCTA1, and the evolution relationship is far away.

Although The whole genome sequence of Rhizoctonia solani has been published (Zheng A et al, The evolution and genetic mechanisms of The rice sheath height gene, nat. Commun, 2013), The method provides abundant information for The gene function research of The Rhizoctonia solani, and plays an important role in creating varieties resistant to rice sheath blight. However, the currently known gene information for controlling the disease is very little, a single gene is lacked for breeding resistant varieties, and the pathogenic bacteria have the property of multinuclear and lack a stable genetic transformation system. Therefore, a new molecular biology technology is urgently needed to develop research on rice sheath blight resistance and culture new disease-resistant varieties, and the research is one of the key measures for improving crop yield, using less pesticides, protecting ecological environment and promoting agricultural sustainable development by using high technology in modern agricultural production.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the prior art in the research of pathogenic related genes of rice sheath blight disease, and provides a gene of cation transport ATPase of rhizoctonia solani, a fragment Rscta thereof and application.

The first object of the present invention is to provide a cation-transporting ATPase gene of Rhizoctonia solani.

The second purpose of the invention is to provide a protein coded by the gene of the cation transport ATP enzyme of rhizoctonia solani.

The third purpose of the invention is to provide a gene fragment Rscta of the cation transfer ATPase of Rhizoctonia solani.

The fourth purpose of the invention is to provide the application of the ATPase gene or the fragment Rscta in preventing and treating Rhizoctonia solani or preparing a Rhizoctonia solani preventing and treating preparation.

The fifth purpose of the invention is to provide the application of the ATPase gene or the fragment Rscta in constructing transgenic plants resisting rice sheath blight germ.

The sixth object of the present invention is to provide a Rhizoctonia solani control preparation.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a rice sheath blight bacterium cation transfer ATP enzyme gene, wherein the full-length cDNA sequence of the gene is shown as SEQ ID NO:1, the length of the full-length cDNA sequence is 2484 bp.

The full-length DNA sequence of the gene is shown as SEQ ID NO:2, the length of the full-length DNA sequence is 3026bp, and the sequence comprises 10 introns respectively located at positions 94-142, 181-282, 410-463, 1137-1186, 1334-139, 1529-1578, 1848-1904, 2084-2137, 2427-2485, and 2692-2740.

The coding sequence of the gene is shown as SEQ ID NO: 3, the length of the coding sequence is 2484bp, and the coding sequence encodes 827 amino acids.

The invention also provides a protein coded by the gene of the cation transfer ATP enzyme of rhizoctonia solani, and the amino acid sequence of the protein is shown as SEQ ID NO: 4, consisting of 827 amino acids, the molecular weight of the protein is 93.12 kDa.

The research of the invention finds that the gene of the cation transfer ATP enzyme of the rice sheath blight bacterium is the gene related to the pathogenicity of the rice sheath blight bacterium, the gene is induced in the early stage process of rice infected by the rice sheath blight bacterium, and the expression level is continuously increased; therefore, the following applications should be within the scope of the present invention:

the ATP enzyme gene or the fragment Rscta is applied to the prevention and treatment of rhizoctonia solani or the preparation of a prevention and treatment preparation of rhizoctonia solani.

The ATPase gene or the fragment Rscta is applied to the construction of a rice sheath blight disease resistance transgenic plant.

The invention also provides a rice sheath blight bacterium prevention and treatment preparation containing a substance capable of inhibiting the expression of the ATPase gene.

Preferably, the substance is dsRNA or a recombinant vector or recombinant bacterium comprising a fragment Rscta.

Preferably, the nucleic acid sequence of the dsRNA is as set forth in SEQ ID NO: and 6.

In addition, the invention also provides a method for constructing a rice sheath blight bacterium resistant transgenic plant, wherein dsRNA containing the rice sheath bacterium cation transfer ATPase gene segment Rscta is synthesized in vitro and is used for treating and silencing the rice sheath bacterium cation transfer ATPase gene in vitro;

or constructing the gene fragment Rscta of the cation transfer ATPase of rhizoctonia solani into a TRV2 vector, and injecting the TRV2-Rscta silent vector into a plant by an injection method to obtain a transgenic plant expressing the gene fragment Rscta of the cation transfer ATPase of rhizoctonia solani;

or dsRNA is generated in the transgenic plant and used for silencing the cationic transfer ATP enzyme gene of the inoculated rice sheath blight bacterium, so that the pathogenicity of the rice sheath bacterium on the plant is reduced.

According to the sequence information of the rice sheath blight bacterium cation transport ATPase gene or the fragment Rscta thereof provided by the present invention, a person skilled in the art can easily obtain a gene equivalent to the fragment Rscta by the following method:

(1) obtaining through database retrieval; (2) screening other rhizoctonia genome libraries or cDNA libraries by taking the fragment Rscta as a probe to obtain the fragment Rscta; (3) designing oligonucleotide primers according to sequence information of the fragment Rscta, and obtaining the oligonucleotide primers from genomes, mRNAs and cDNAs of rhizoctonia or other closely-related fungi by a PCR (polymerase chain reaction) amplification method; (4) is obtained by modifying a gene engineering method on the basis of the fragment Rscta; (5) the gene is obtained by a chemical synthesis method.

The invention has the following beneficial effects:

the invention obtains a rice sheath blight bacterium cation transfer ATP enzyme gene, which is a pathogenicity key gene of rice sheath blight bacterium (soil-borne fungal disease), wherein the gene is obviously induced and expressed in the early stage process that the rice is infected by the rice sheath blight bacterium, and dsRNA synthesized according to a rice sheath blight bacterium cation transfer ATP enzyme gene segment Rscta can be used for carrying out in-vitro treatment to silence the rice sheath blight bacterium cation transfer ATP enzyme gene so as to obviously reduce the pathogenicity of the rice sheath blight bacterium;

the invention also provides a rice sheath blight bacterium control preparation, which contains a substance capable of inhibiting the expression of the gene of the cation transfer ATP enzyme of the rice sheath blight bacterium, wherein the substance is dsRNA or a recombinant vector or a recombinant bacterium containing a fragment Rscta; the recombinant vector of the gene for efficiently silencing the cation transfer ATP enzyme of the rhizoctonia solani is transformed into a plant to obtain a transgenic plant resisting the rhizoctonia solani, the plant has obvious disease resistance to the rhizoctonia solani, the problem of soil-borne fungal diseases caused by the rhizoctonia solani in production is effectively solved, and the soil-borne fungal diseases are effectively prevented and controlled;

in addition, the method only expresses a small fragment of gene in the plant, does not produce products such as protein and the like, and does not produce potential risks brought by transgenic food; therefore, the gene or the fragment Rscta of the cation transfer ATPase of Rhizoctonia solani has wide application prospects in preventing and treating Rhizoctonia solani, preparing a Rhizoctonia solani prevention and treatment preparation, and constructing transgenic plants resistant to Rhizoctonia solani.

Drawings

FIG. 1 is a graph showing the amplification result of a gene fragment Rscta of a cation-transporting ATPase of Rhizoctonia solani; wherein, M: DNA molecular weight standard; 1: and (3) PCR products.

FIG. 2 is a diagram showing the results of gene expression analysis of a gene encoding a cation-transporting ATPase for Rhizoctonia solani in the infection process of Rhizoctonia solani.

FIG. 3 is a graph showing RNA extraction results of dsRNA treatment in vitro on Rhizoctonia solani for 24h, 48h and 72 h; wherein, M: DNA molecular weight standard; 1: RNA extracted for 24 hours; 2: RNA extracted for 48 hours; 3: 72h of extracted RNA; 4: 96h of extracted RNA.

FIG. 4 is a graph showing the results of dsRNA treatment on rice sheath blight bacterium at 0h, 24h, 48h and 72h in vitro.

FIG. 5 is a VIGS vector information map.

FIG. 6 is a map of a TRV2-Rscta silencing vector constructed according to the present invention.

FIG. 7 is a diagram showing the result of colony validation of the TRV2-Rscta silent vector constructed according to the present invention transformed into Agrobacterium tumefaciens GV 3101; wherein, M: DNA molecular weight standard; 1. 2: and (5) bacterial colonies.

FIG. 8 is a phenotypic drawing of Nicotiana benthamiana after inoculation of Rhizoctonia solani 6 d.

FIG. 9 is a graph showing the results of analysis of fungal biomass and target gene transcription levels of Nicotiana benthamiana after inoculation of Rhizoctonia solani 6 d; wherein, the graph (A) is a result graph of the fungal biomass analysis of the Nicotiana benthamiana after 6d inoculation of Rhizoctonia solani; (B) the graph is the result of analysis of the transcription level of the target gene of Nicotiana benthamiana inoculated with Rhizoctonia solani 6 d.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 cloning of Gene fragment Rscta of cation-transporting ATPase of Rhizoctonia solani

1. Experimental methods

The cloning of the gene fragment Rscta of the cation transfer ATP enzyme of Rhizoctonia solani comprises the following steps:

s1, extracting RNA of rhizoctonia solani

Extraction of Rhizoctonia solani RNA Using TaKaRa Total RNA extraction kit (code No. 9769). 0.1g of Rhizoctonia solani hyphae is weighed, quickly ground into powder in liquid nitrogen, added into a 1.5mL sterilized centrifuge tube containing Buffer RL lysate, and the lysate is centrifuged at 12000rpm and 4 ℃ for 5 min. The supernatant was carefully pipetted into a fresh 1.5mL sterile centrifuge tube. The 1/2 volumes of absolute ethanol from the supernatant from the sample lysis step were added and the mixture (containing the pellet) was immediately transferred to the RNase Column (containing 2mL Collection Tube). Centrifuge at 12000rpm for 1min, and discard the filtrate. mu.L of Buffer RWA, 600. mu.L of Buffer RWB, and 600. mu.L of Buffer RWB were added to the RNA Spin Column in this order, and centrifuged at 12000rpm for 30 seconds, and the filtrate was discarded. The tube was then emptied and centrifuged at 12000rpm for 1 min. The RNA Spin Column was mounted on a 1.5mL RNase Free Collection Tube, and 125. mu.L of RNase Free ddH was added to the center of the RNA Spin Column membrane₂O (preheated at 65 ℃) and stands for 5min at room temperature. Centrifuging at 12000rpm for 2min to elute RNA, and obtaining the RNA of the rice sheath blight bacterium.

S2. Synthesis of first Strand of cDNA

cDNA was synthesized using the RNA from Rhizoctonia solani obtained in step S1 as a template, using TARAKA PrimeScript reverse transcription kit (code No. 6210A). 2000ng of Total RNA, 1. mu.L of Oligo dTprimer, 1. mu.L of dNTP mix (10mM each), supplemented with RNase Free ddH, were added to the microcentrifuge tube₂O to 10. mu.L. Mixing, centrifuging for several seconds, keeping the temperature at 65 ℃ for 5min, and rapidly cooling on ice. Adding 4 μ L of 5 XPrimeScript II Buffer, 0.5 μ L of RNaseInhibitor (40U/. mu.L), 1 μ L of PrimeScript II RTase (200U/. mu.L), and RNase Free ddH₂O to 20. mu.L, and slowly mix. Keeping the temperature at 42 deg.C for 50min, keeping the temperature at 95 deg.C for 5min (enzyme deactivation) to obtain Rhizoctonia solani cDNA, and cooling on iceAfter cooling, the mixture is stored at-20 ℃ for later use.

S3 cloning of fragment Rscta

Cloning primer of fragment Rscta, 0493F:5'-tgaagaaggagaggagagg-3', shown as SEQ ID NO: 7; 0493R:5'-ctggtcggcggtattatc-3', shown in SEQ ID NO: 8.

The rice sheath blight bacterium cDNA obtained in step S2 is used as a template, and a high fidelity polymerase of code No. TP001 is used for PCR amplification reaction. The reaction conditions are as follows: at 98 ℃ for 2 min; 40 cycles of 98 ℃, 10s, 58 ℃, 15 s; finally 72 ℃ for 10 min. A1.0% agarose gel was prepared using TAE buffer, and the PCR product was subjected to agarose gel electrophoresis. PCR product purification was performed using the Axygen PCR clean-up kit.

2. Results of the experiment

The full-length cDNA sequence of the gene for transferring ATP from cations of rhizoctonia solani is shown as SEQ ID NO:1, and the full-length DNA sequence is shown as SEQ ID NO:2, and the coding sequence is shown as SEQ ID NO: 3, the amino acid sequence of the encoded protein is shown as SEQ ID NO: 4, respectively.

The nucleotide sequence of the gene fragment Rscta of the cation transfer ATPase of Rhizoctonia solani is shown as SEQ ID NO: 5, respectively.

The amplification result of the gene fragment Rscta for transferring the positive ions of Rhizoctonia solani is shown in FIG. 1, and it can be seen that the gene fragment Rscta for transferring the positive ions of Rhizoctonia solani is successfully amplified by the method and has a size of 286 bp.

Example 2 expression analysis of the Gene of cation-transporting ATP for Rhizoctonia solani in Rice infection

1. Experimental methods

Expression analysis of a rice sheath blight bacterium cation transfer ATP enzyme gene in a rice infection process by rice sheath blight bacterium comprises the following steps:

s1. preparation of inoculation spray

Firstly, a strain GD-118 (Shu-Can-Wei, and the like, an optimized method for extracting proteins of rhizoctonia solani suitable for dimensional electrophoresis, academic newspaper of university of China agriculture, 2017) of rhizoctonia solani stored in a laboratory is activated by using a PDA (personal digital assistant) plate (culture dish diameter is 90mm, 8mL of PDA culture medium), and the rhizoctonia solani is cultured for 2 days at 28 ℃ under the dark condition. Then cutting the PDA plate with the fungi into square mycelium blocks with the side length of about 1mm by using a sterilized blade, transferring the square mycelium blocks into a conical flask (containing 200mL of PDB), culturing the square mycelium blocks at 28 ℃ for 180r/min in the dark under shaking for 2d, and homogenizing the whole flask into liquid to ensure that the OD600 is 1.0, thus being used for inoculation.

S2, planting and inoculating rice

Rice seeds (rice variety: Nipponbare) are sterilized by respectively using 10% of sodium hypochlorite and 30% of hydrogen peroxide, then the seeds are placed in a sterile culture dish, and are soaked for more than 3d by using sterile water until germination occurs, and then the germinated seeds are transferred to a square pot (length: 80 cm; width: 40 cm; height: 15cm) containing 10cm of sterile soil. The three-leaf one-heart period of rice is selected for inoculation, and the liquid homogenized in S1 is used for inoculation under the condition of no water and dry in a pot, 5mL of the liquid is inoculated to each rice plant, and the temperature and the humidity of 30 ℃ are more than 90% so as to be beneficial to disease attack.

S3. fluorescent quantitative PCR analysis of sample

In order to verify the expression quantity of different genes in the rice infection process of rhizoctonia solani, 6 time points after inoculation are selected, and after 24 hours, 48 hours, 72 hours, 96 hours and 120 hours, the whole rice is collected as a sample. The total RNA of the rice samples was extracted at different times using TaKaRa plant RNA extraction kit (code No.9769) in the same manner as "step S1 of example 1".

The RNA reverse transcription was carried out using TARAKA PrimeScript reverse transcription kit (code No.6210A) in the same manner as in "step S2 of example 1", and the cDNA product obtained by the reverse transcription was diluted 10-fold and used for real-time fluorescent quantitative PCR. Designing a fluorescent quantitative PCR Primer of the fragment Rscta by using Primer Premier 5.0 software, wherein 0493qPCR F is 5'-tcttatggccgcccagac-3' and is shown as SEQ ID NO. 9; 0493qPCR R:5'-agctggaccttcttgagc-3', shown in SEQ ID NO: 10. The specificity of the primers was verified by gel electrophoresis, sequencing and dissolution curves. The experiment used a Bio-Rad CFX real-time PCR system with a 20. mu.L reaction system containing 10. mu.L MIX, 0.2. mu.M upstream/downstream primers, 2. mu.L cDNA template and the appropriate amount of purified water. Three technical replicates were performed for each sample. Water (W)The internal reference gene of Rhizoctonia solani was subjected to homogenization treatment of gene expression between samples using GAPDH, primer GAPDH F:5'-ggtcggcaaagtcataccat-3', shown in SEQ ID NO: 11; primer GAPDH R:5'-tctgcgtccttcttggagata-3', shown in SEQ ID NO: 12. Results adopted 2^-ΔΔCtThe method is used for analysis.

2. Results of the experiment

The gene expression analysis result of the cation transport ATPase gene of Rhizoctonia solani in the rice sheath blight infection process is shown in FIG. 2, and it can be seen that the cation transport ATPase gene is induced in the early stage of rice sheath blight infection, and the expression level is continuously increased along with the time extension, which indicates that the gene may play a key role in the interaction process of Rhizoctonia solani and rice.

Example 3 Synthesis and in vitro silencing experiments of fragment Rscta dsRNA

1. Experimental methods

S1, in vitro synthesis of fragment Rscta dsRNA

dsRNA for fragment Rscta was synthesized using T7 RNA Polymerase (Thermo Fisher Scientific, Catalog number: EP 011). The T7 RNA polymerase promoter can be added to any DNA sequence using PCR by adding the T7 promoter sequence at the 5' end of either amplification primer. The minimal T7 RNA polymerase promoter sequence is: 5'-taatacgactcactatagg-3' are provided. The 5' ends of the upstream and downstream primers are respectively added with a T7 promoter sequence, namely an upstream primer T7-0493F: 5'-taatacgactcactataggtcgcccacagcatctacaa-3' shown as SEQ ID NO. 13; the downstream primer T7-0493R: 5'-taatacgactcactataggtcccaacgagcc aatcc-3' is shown as SEQ ID NO: 14. Carrying out PCR amplification by taking cDNA of rhizoctonia solani as a template to obtain a dsRNA template, and carrying out transcription and purification to obtain dsRNA of a cation transfer ATPase gene fragment Rscta.

S2. in vitro silencing experiment of dsRNA on Rhizoctonia solani

Taking square fungus cakes with the side length of 2mm from the edge of a PDA (personal digital assistant) plate for culturing rhizoctonia solani of 2d, putting the square fungus cakes into a culture dish (containing 7mLPDB) with the diameter of 60mm, standing and culturing for 48h, adding dsRNA at a ratio of 500ng/mL, collecting hypha samples when the square fungus cakes are treated for 0h, 24h, 48h, 72h and 96h, and extracting the total RNA of the rice samples at different time by using a TaKaRa plant RNA extraction kit (code No. 9769).

2. Results of the experiment

The nucleic acid sequence of the dsRNA is shown as SEQ ID NO: and 6.

The RNA extraction results of dsRNA treatment in vitro on Rhizoctonia solani for 24h, 48h and 72h are shown in FIG. 3, and it can be seen that the RNA extracted from samples in each time period is good in quality.

The results of dsRNA in vitro treatment on 0h, 24h, 48h and 72h of Rhizoctonia solani are shown in FIG. 4, and it can be seen that the transcription level of the cation transport ATPase gene is remarkably reduced in 24h and is continuously reduced along with the increase of the treatment time, and the silencing efficiency of 72h is about 80%, thus proving that the expression of the gene related to pathogenic bacteria silencing by in vitro application of dsRNA is feasible.

Example 4 TRV2-Rscta silencing vector construction and transformed tobacco Studies

1. Experimental methods

S1. construction of TRV2-Rscta silencing vector and Agrobacterium transformation

Designing specific primers with EcoRI and BamHI enzyme cutting sites at two ends according to the coding sequence of the gene fragment Rscta of the cation transfer ATP enzyme of rhizoctonia solani, wherein an upstream primer VIGS-0493F: 5'-ccggaattctgaagaaggagaggagagg-3', as shown in SEQ ID NO: 15; the downstream primer VIGS-0493R: 5'-cgcggatccctggtcggcggtattatc-3', as shown in SEQ ID NO: 16. The rice sheath blight bacterium cDNA is used as a template, PCR amplification is carried out by using high fidelity enzyme (code No. TP001) of engine company, an amplification product is recovered, and the product and the carrier pYL156 are subjected to double enzyme digestion for 5 hours at 37 ℃. Using T to cut the target fragment and the carrier₄DNA ligase, connecting overnight at 16 ℃, transforming the connected product into E.coil DH5 alpha competence, after determining no error by sequencing, carrying out propagation and extraction on plasmids, and storing for later use. The VIGS vector information is shown in FIG. 5.

S2, preparation of agrobacterium tumefaciens competence and electric shock transformation

A single colony of Agrobacterium tumefaciens was inoculated to 4mL of a plasmid containing Rif (25)μ g/mL) in LB liquid medium, and cultured at 28 ℃ for 2d with shaking at 200 r/min. Inoculating 3mL of the culture medium into 200mL of LB culture medium, and performing shaking culture at 200r/min and 28 deg.C to logarithmic phase (cell concentration OD)₆₀₀0.6). And centrifuging to remove waste liquid, and collecting thalli. The suspension was resuspended and washed 3 times with pre-chilled sterile double distilled water, after which the cells were resuspended in 2mL of sterile pre-chilled 10% w/v glycerol. 100 μ L of the bacterial suspension was put into a 1.5mL centrifuge tube and stored at-70 ℃ for further use. Adding 3 mu L of plasmid into the bacterial liquid, gently mixing uniformly and transferring the plasmid into an aseptic precooled electric shock cup, quickly adding 1mL of LB liquid culture medium after electric shock, mixing uniformly and transferring cells into a 1.5mL centrifuge tube, and culturing for 2h at the temperature of 28 ℃ in a shaking table at 200 r/min; mu.L of the suspension was spread on LB plates containing Rif (25. mu.g/mL) and Kan (50. mu.g/mL), and the plates were placed upside down in an incubator at 28 ℃ for 2 days to observe the growth of the transformants, and the results of Agrobacterium without plasmid DNA under the same conditions of electric shock were used as controls. Single colonies were picked for PCR validation using pYL156 vector primers. The upstream primer PYL 156F: 5'-aattcactgggagatgatacgctg-3' is shown as SEQ ID NO: 17; the downstream primer PYL 156R: 5'-cctatggtaagacaatgagtcggc-3' is shown as SEQ ID NO: 18.

S3, agrobacterium culture and tobacco transient transformation

Agrobacterium monoclonals were added to 5mL of liquid LB medium (25. mu.g/mL of Rif and 50. mu.g/mL of Kan), incubated overnight at 200r/min in a shaker at 28 ℃ and 1mL of the inoculum was added to 50mL of LB medium (25. mu.g/mL of Rif and 50. mu.g/mL of Kan) and incubated with shaking at 200r/min in a shaker at 28 ℃. When culture OD₆₀₀When the concentration is 0.6, the cells are collected by centrifugation at 6000rpm for 5min, and the waste liquid is discarded. The cells were resuspended in injection media (10mM MES, 10mM MgCl)₂100. mu.M acetosyringone) to OD₆₀₀0.4. The two Agrobacterium strains (TRV1 and TRV2+ target gene fragment) were mixed at 1:1 and left to stand at room temperature for 4h without shaking. Finally, the agrobacterium containing the injection matrix is injected into the tender leaf using a syringe.

S4, pathogenic force detection and biomass measurement

The albinism of Nicotiana benthamiana was very pronounced from day 10 post-injection, indicating that the VIGS vector has produced large amounts of dsRNA and has an interfering effect in Nicotiana benthamiana. Therefore, we chose to perform live vaccination from day 10 after injection of VIGS series vectors. The disease index of the Nicotiana benthamiana is counted on the 6 th day (post-inoculation day, dpi) after inoculation, and the pathogenicity is detected by counting the number of leaf scabs and withered leaves. Extracting total DNA of the middle leaf of the Nicotiana benthamiana (transgenic plant and negative control) after 6d inoculation of Rhizoctonia solani, and taking an ITS sequence of an internal transcription spacer region of Rhizoctonia solani (R. The upstream primer Rs F:5'-gccttttctaccttaatttggcag-3', shown as SEQ ID NO: 19; the downstream primer Rs R:5'-gtgtgtaaattaagtagacagcaaatg-3' is shown as SEQ ID NO: 20. Meanwhile, the tobacco actin gene is used as an internal reference gene, and an upstream primer EF1a:5'-tggtgtcctcaagcctggtat-3' is shown as SEQ ID NO: 21; the downstream primer EF1b:5'-acgcttgagatccttaaccgc-3' is shown as SEQ ID NO: 22. The specific method was the same as "step S3 in example 2", and the results were analyzed by the 2-. DELTA.Ct method (Suxiao peak, 2014).

2. Results of the experiment

The map of the TRV2-Rscta silent vector constructed by the invention is shown in FIG. 6, and it can be seen that the total length of the vector is 9930bp, which is the same as the actual sequencing result after comparison.

The colony verification result of the TRV2-Rscta silent vector constructed by the invention transformed into the Agrobacterium tumefaciens GV3101 is shown in FIG. 7, the size of the band is 717bp, the band is single and bright and has correct size, which indicates that the TRV2-Rscta silent vector has been successfully transferred into the Agrobacterium tumefaciens GV 3101.

The phenotype of the nicotiana benthamiana inoculated with the rhizoctonia solani 6d is shown in fig. 8, and it can be seen that the resistance of the transgenic tobacco to pathogenic bacteria is obviously improved, the morbidity degree is weakened, and the resistance is enhanced.

The results of analysis of the fungal biomass and the target gene transcription level of Nicotiana benthamiana inoculated with Rhizoctonia solani 6d are shown in FIG. 9, in which (A) is a diagram showing the results of analysis of the fungal biomass of Nicotiana benthamiana inoculated with Rhizoctonia solani 6 d; (B) FIG. 6d shows the results of analysis of the transcription level of the target gene of Nicotiana benthamiana after inoculation with Rhizoctonia solani; as can be seen, the fungal biomass of the transgenic positive tobacco is obviously reduced, which is only 20% of that of the wild type, and the expression level of the cation transport ATPase gene in the transgenic plants is reduced by about 60%.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> rhizoctonia solani cation transport ATP enzyme gene, fragment Rscta thereof and application

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atgcggttct gccgcaccat tgtcttgact gcctctgtcc taggctcgct ggtttcagcc 60

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gtcattaatt cggaagtgtt cctgcgcgag ctcatttcca atgcaaacga cgctttggag 180

aagcttcgtc tcatcagctt gacggacaag tcttaccaaa tcccagactc gcctctcaat 240

atcacgatca agctcgtgaa ggatggcgaa ggcactggtg gacgcgttat tattactgac 300

accggtattg gtatgactgc tgatgaactg gccaaaaacc ttggcaccct cgccaagtct 360

ggtacaagcg agttcctgaa caaggcggaa aaggatacta gcggaaacct gattggccag 420

ttcggtcttg gtttctattc tagcttcctc gtcgccgaca aagttcaggt tgcctctctc 480

ccacctgtct ccaaggcaaa cccagatcca ctccaacaca tctttacaag tggatcggat 540

gatagcaact ttgaaatctc tgttgaccct cgcggaaact cgctcggcca ttctggaacc 600

gaaattacta tgtacttgaa ggatgaggca ttggaattct tagacgagca acgtgtgcgc 660

gaccttgttt ccaaacattc ggcatttgcg acttcgttcc cactctacct ccagaccttc 720

aaaactgaag aagtccctga cgaggaagct attgctgctg ccaaggcagc tgaacccgag 780

tcgacttcca catctgccgc cgagcctgaa aagactaagg aagccgagac cgatgaggac 840

gaagctatca ttgaggatgt caaggaagat gacgagaaga aagaagaacc tgcatctcct 900

cctactccca tgaagaacgt tacgaccgag gaatgggtcc gtctgaatga ccaagctcct 960

ctatggatga gggaaccaaa agatgtaacc aaagacgagg tcaatcagtt cttcatgtcc 1020

actttcaagg agcacaacgg tcctctcgcc cacagcatct acaagggtga ctctggtcca 1080

gtttcgttcc gcaccatgtt cttcatccca tcggatctga acgagaagtt ctggcaaagc 1140

gcgaagcccg agttgaacaa catccgtctc atggttaagc gcgtcttcat taccagcgac 1200

ctcggaccca atgccatgcc taaatggctc agttggctca aggctatcgt tgatgccgat 1260

gacctccctc tcaacgtctc ccgtgaaact ctccaatcaa accggttcct tcgtcagatt 1320

cccaatatcc tcgtccgtcg cttcatcaac ttggtcgata ggatgtctaa ggacgaggag 1380

aacccagaac ttttccgtaa attcatgaag atttatggct cggttgttaa gctgggagct 1440

gttgaaagcc ccaaggaaca acaaaagctg gctggattgg ctcgttggga tactaatttg 1500

aggaatttca ccagtctgga tcagtacgtc gaaaacagaa agaagggtca gactcagatt 1560

ttctacctcg ccggaattgg ccagcgccct gatgaattgg ccaaatcttt gttcgttgaa 1620

aagcttcatg cgcgtggata tgaagtcttg ttactcaatg accctatgga cgagatcctc 1680

atgtctcacc ttcgtaactg gaagggtctg cagttccagg acgctgccaa gaagggtctg 1740

aagttcgggg acgaggatga agatgccgag gaagaaaagg cccgccagga gaagctcaag 1800

gagacgttca agcctctctt ggagtacctc aagaacgaga cctccaacgt tgttcttgat 1860

gttatcttgt ccaatcgtct tgttaccagc ccttgtgcaa ttgtcgccga ctcgtatggc 1920

tacagcgcaa acatggaacg tcttatggcc gcccagactg gaggaaagat gaaggagaat 1980

gactttatgc acgattgggc gaagaagcaa aagttccttg aaatcaaccc gaactccccg 2040

ctgattgaag gaatgctcaa gaaggtccag gctttgattg acattgaaga aggagaggag 2100

agggacagtc aacttgaaga ggaactaaag gaagttgcat ctattctcgt tgacggagcc 2160

ctcgtccgct ctggtttcga ggttcccgat tccaatctac gcaggttctt cactcgtatg 2220

gatcgcgtcc ttcgccgttc tctcggtgtc tctgagaccg ccaagggcga cgaaactgtt 2280

aagcccgctc ctccagtcga tgatactcct ctcgaagagc ctggtgtcct accggaaggt 2340

gcatttgagg acatcattga taataccgcc gaccagaaaa ccttccaacc caaagtcgat 2400

gtcaaagtcg acgcactgaa tgacgaagaa atcgaggaga tgttgaacaa gaaggagcag 2460

gaggttaagc gtgatgagtt gtga 2484

<210>2

<211>3026

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atgcggttct gccgcaccat tgtcttgact gcctctgtcc taggctcgct ggtttcagcc 60

caggatggta cttccaagaa gtttgagtac caggtaggtt gcatacatgt taccgtggtc 120

acctggtcta acttgcttcc agagcgatgt tagtcgtctg agaaacatcg tcattaattc 180

gctgtacagt cacaagtacg tatttatggc aaatttagtg gcgttcgctg accttgcttc 240

agggaagtgt tcctgcgcga gctcatttcc aatgcaaacg acgctttgga gaagcttcgt 300

ctcatcagct tgacggacaa gtcttaccaa atcccagact cgcctctcaa tatcacgatc 360

aagctcgtga aggatggcga aggcactggt ggacgcgtta ttattactgg taagcattta 420

caataaaagt tgaacggacg acagtgttga tacaatttcg cagacaccgg tattggtatg 480

actgctgatg aactggccaa aaaccttggc accctcgcca agtctggtac aagcgagttc 540

ctgaacaagg cggaaaagga tactagcgga aacctgattg gccagttcgg tcttggtttc 600

tattctagct tcctcgtcgc cgacaaagtt caggttgcct ctctcccacc tgtctccaag 660

gcaaacccag atccactcca acacatcttt acaagtggat cggatgatag caactttgaa 720

atctctgttg accctcgcgg aaactcgctc ggccattctg gaaccgaaat tactatgtac 780

ttgaaggatg aggcattgga attcttagac gagcaacgtg tgcgcgacct tgtttccaaa 840

cattcggcat ttgcgacttc gttcccactc tacctccaga ccttcaaaac tgaagaagtc 900

cctgacgagg aagctattgc tgctgccaag gcagctgaac ccgagtcgac ttccacatct 960

gccgccgagc ctgaaaagac taaggaagcc gagaccgatg aggacgaagc tatcattgag 1020

gatgtcaagg aagatgacga gaagaaagaa gaacctgcat ctcctcctac tcccatgaag 1080

aacgttacga ccgaggaatg ggtccgtctg aatgaccaag ctcctctatg gatgaggtat 1140

gtcatccacg tgaagcaatg aagtagcaat actcatcgtc tttcagggaa ccaaaagatg 1200

taaccaaaga cgaggtcaat cagttcttca tgtccacttt caaggagcac aacggtcctc 1260

tcgcccacag catctacaag ggtgactctg gtccagtttc gttccgcacc atgttcttca 1320

tcccatcgga tctgtgcgta attctcatga aattcacacc taatctcttt ctcattgttt 1380

aatttccaca ggaacgagaa gttctggcaa agcgcgaagc ccgagttgaa caacatccgt 1440

ctcatggtta agcgcgtctt cattaccagc gacctcggac ccaatgccat gcctaaatgg 1500

ctcagttggc tcaaggctat cgttgatggt aagcttacct attcgtcaaa tatgtgcaaa 1560

tggctcatcg ttttccagcc gatgacctcc ctctcaacgt ctcccgtgaa actctccaat 1620

caaaccggtt ccttcgtcag attcccaata tcctcgtccg tcgcttcatc aacttggtcg 1680

ataggatgtc taaggacgag gagaacccag aacttttccg taaattcatg aagatttatg 1740

gctcggttgt taagctggga gctgttgaaa gccccaagga acaacaaaag ctggctggat 1800

tggctcgttg ggatactaat ttgaggaatt tcaccagtct ggatcaggta agaaagcttt 1860

gctgaaattt caatgcgagt actgatgaac ggtatctgca acagtacgtc gaaaacagaa 1920

agaagggtca gactcagatt ttctacctcg ccggaattgg ccagcgccct gatgaattgg 1980

ccaaatcttt gttcgttgaa aagcttcatg cgcgtggata tgaagtcttg ttactcaatg 2040

accctatgga cgagatcctc atgtctcacc ttcgtaactg gaagtgagta cattttgtgt 2100

acacacatta ctagaatact cacctccatt tccacagggg tctgcagttc caggacgctg 2160

ccaagaaggg tctgaagttc ggggacgagg atgaagatgc cgaggaagaa aaggcccgcc 2220

aggagaagct caaggagacg ttcaagcctc tcttggagta cctcaagaac gagacctcca 2280

acgttgttct tgatgttatc ttgtccaatc gtcttgttac cagcccttgt gcaattgtcg 2340

ccgactcgta tggctacagc gcaaacatgg aacgtcttat ggccgcccag actggaggaa 2400

agatgaagga gaatgacttt atgcacgtaa gtgatatttt gcactcatcg gccccctttg 2460

atagtatctg acgcacttat ttcaggattg ggcgaagaag caaaagttcc ttgaaatcaa 2520

cccgaactcc ccgctgattg aaggaatgct caagaaggtc caggctttga ttgacattga 2580

agaaggagag gagagggaca gtcaacttga agaggaacta aaggaagttg catctattct 2640

cgttgacgga gccctcgtcc gctctggttt cgaggttccc gattccaatc tgtgagtagc 2700

aaattttaca agcacttcta ctgcaggata ctaataccag acgcaggttc ttcactcgta 2760

tggatcgcgt ccttcgccgt tctctcggtg tctctgagac cgccaagggc gacgaaactg 2820

ttaagcccgc tcctccagtc gatgatactc ctctcgaaga gcctggtgtc ctaccggaag 2880

gtgcatttga ggacatcatt gataataccg ccgaccagaa aaccttccaa cccaaagtcg 2940

atgtcaaagt cgacgcactg aatgacgaag aaatcgagga gatgttgaac aagaaggagc 3000

aggaggttaa gcgtgatgag ttgtga 3026

<210>3

<211>2484

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atgcggttct gccgcaccat tgtcttgact gcctctgtcc taggctcgct ggtttcagcc 60

caggatggta cttccaagaa gtttgagtac cagagcgatg ttagtcgtct gagaaacatc 120

gtcattaatt cggaagtgtt cctgcgcgag ctcatttcca atgcaaacga cgctttggag 180

aagcttcgtc tcatcagctt gacggacaag tcttaccaaa tcccagactc gcctctcaat 240

atcacgatca agctcgtgaa ggatggcgaa ggcactggtg gacgcgttat tattactgac 300

accggtattg gtatgactgc tgatgaactg gccaaaaacc ttggcaccct cgccaagtct 360

ggtacaagcg agttcctgaa caaggcggaa aaggatacta gcggaaacct gattggccag 420

ttcggtcttg gtttctattc tagcttcctc gtcgccgaca aagttcaggt tgcctctctc 480

ccacctgtct ccaaggcaaa cccagatcca ctccaacaca tctttacaag tggatcggat 540

gatagcaact ttgaaatctc tgttgaccct cgcggaaact cgctcggcca ttctggaacc 600

gaaattacta tgtacttgaa ggatgaggca ttggaattct tagacgagca acgtgtgcgc 660

gaccttgttt ccaaacattc ggcatttgcg acttcgttcc cactctacct ccagaccttc 720

aaaactgaag aagtccctga cgaggaagct attgctgctg ccaaggcagc tgaacccgag 780

tcgacttcca catctgccgc cgagcctgaa aagactaagg aagccgagac cgatgaggac 840

gaagctatca ttgaggatgt caaggaagat gacgagaaga aagaagaacc tgcatctcct 900

cctactccca tgaagaacgt tacgaccgag gaatgggtcc gtctgaatga ccaagctcct 960

ctatggatga gggaaccaaa agatgtaacc aaagacgagg tcaatcagtt cttcatgtcc 1020

actttcaagg agcacaacgg tcctctcgcc cacagcatct acaagggtga ctctggtcca1080

gtttcgttcc gcaccatgtt cttcatccca tcggatctga acgagaagtt ctggcaaagc 1140

gcgaagcccg agttgaacaa catccgtctc atggttaagc gcgtcttcat taccagcgac 1200

ctcggaccca atgccatgcc taaatggctc agttggctca aggctatcgt tgatgccgat 1260

gacctccctc tcaacgtctc ccgtgaaact ctccaatcaa accggttcct tcgtcagatt 1320

cccaatatcc tcgtccgtcg cttcatcaac ttggtcgata ggatgtctaa ggacgaggag 1380

aacccagaac ttttccgtaa attcatgaag atttatggct cggttgttaa gctgggagct 1440

gttgaaagcc ccaaggaaca acaaaagctg gctggattgg ctcgttggga tactaatttg 1500

aggaatttca ccagtctgga tcagtacgtc gaaaacagaa agaagggtca gactcagatt 1560

ttctacctcg ccggaattgg ccagcgccct gatgaattgg ccaaatcttt gttcgttgaa 1620

aagcttcatg cgcgtggata tgaagtcttg ttactcaatg accctatgga cgagatcctc 1680

atgtctcacc ttcgtaactg gaagggtctg cagttccagg acgctgccaa gaagggtctg 1740

aagttcgggg acgaggatga agatgccgag gaagaaaagg cccgccagga gaagctcaag 1800

gagacgttca agcctctctt ggagtacctc aagaacgaga cctccaacgt tgttcttgat 1860

gttatcttgt ccaatcgtct tgttaccagc ccttgtgcaa ttgtcgccga ctcgtatggc 1920

tacagcgcaa acatggaacg tcttatggcc gcccagactg gaggaaagat gaaggagaat 1980

gactttatgc acgattgggc gaagaagcaa aagttccttg aaatcaaccc gaactccccg 2040

ctgattgaag gaatgctcaa gaaggtccag gctttgattg acattgaaga aggagaggag 2100

agggacagtc aacttgaaga ggaactaaag gaagttgcat ctattctcgt tgacggagcc 2160

ctcgtccgct ctggtttcga ggttcccgat tccaatctac gcaggttctt cactcgtatg 2220

gatcgcgtcc ttcgccgttc tctcggtgtc tctgagaccg ccaagggcga cgaaactgtt 2280

aagcccgctc ctccagtcga tgatactcct ctcgaagagc ctggtgtcct accggaaggt 2340

gcatttgagg acatcattga taataccgcc gaccagaaaa ccttccaacc caaagtcgat 2400

gtcaaagtcg acgcactgaa tgacgaagaa atcgaggaga tgttgaacaa gaaggagcag 2460

gaggttaagc gtgatgagtt gtga 2484

<210>4

<211>827

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Met Arg Phe Cys Arg Thr Ile Val Leu Thr Ala Ser Val Leu Gly Ser

1 5 10 15

Leu Val Ser Ala Gln Asp Gly Thr Ser Lys Lys Phe Glu Tyr Gln Ser

20 25 30

Asp Val Ser Arg Leu Arg Asn Ile Val Ile Asn Ser Glu Val Phe Leu

35 40 45

Arg Glu Leu Ile Ser Asn Ala Asn Asp Ala Leu Glu Lys Leu Arg Leu

50 55 60

Ile Ser Leu Thr Asp Lys Ser Tyr Gln Ile Pro Asp Ser Pro Leu Asn

65 70 75 80

Ile Thr Ile Lys Leu Val Lys Asp Gly Glu Gly Thr Gly Gly Arg Val

85 90 95

Ile Ile Thr Asp Thr Gly Ile Gly Met Thr Ala Asp Glu Leu Ala Lys

100 105 110

Asn Leu Gly Thr Leu Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn Lys

115 120 125

Ala Glu Lys Asp Thr Ser Gly Asn Leu Ile Gly Gln Phe Gly Leu Gly

130 135 140

Phe Tyr Ser Ser Phe Leu Val Ala Asp Lys Val Gln Val Ala Ser Leu

145 150 155 160

Pro Pro Val Ser Lys Ala Asn Pro Asp Pro Leu Gln His Ile Phe Thr

165 170 175

Ser Gly Ser Asp Asp Ser Asn Phe Glu Ile Ser Val Asp Pro Arg Gly

180 185 190

Asn Ser Leu Gly His Ser Gly Thr Glu Ile Thr Met Tyr Leu Lys Asp

195 200 205

Glu Ala Leu Glu Phe Leu Asp Glu Gln Arg Val Arg Asp Leu Val Ser

210 215 220

Lys His Ser Ala Phe Ala Thr Ser Phe Pro Leu Tyr Leu Gln Thr Phe

225 230 235 240

Lys Thr Glu Glu Val Pro Asp Glu Glu Ala Ile Ala Ala Ala Lys Ala

245 250 255

Ala Glu Pro Glu Ser Thr Ser Thr Ser Ala Ala Glu Pro Glu Lys Thr

260 265 270

Lys Glu Ala Glu Thr Asp Glu Asp Glu Ala Ile Ile Glu Asp Val Lys

275 280 285

Glu Asp Asp Glu Lys Lys Glu Glu Pro Ala Ser Pro Pro Thr Pro Met

290 295 300

Lys Asn Val Thr Thr Glu Glu Trp Val Arg Leu Asn Asp Gln Ala Pro

305 310 315 320

Leu Trp Met Arg Glu Pro Lys Asp Val Thr Lys Asp Glu Val Asn Gln

325 330 335

Phe Phe Met Ser Thr Phe Lys Glu His Asn Gly Pro Leu Ala His Ser

340 345 350

Ile Tyr Lys Gly Asp Ser Gly Pro Val Ser Phe Arg Thr Met Phe Phe

355 360 365

Ile Pro Ser Asp Leu Asn Glu Lys Phe Trp Gln Ser Ala Lys Pro Glu

370 375 380

Leu Asn Asn Ile Arg Leu Met Val Lys Arg Val Phe Ile Thr Ser Asp

385 390 395 400

Leu Gly Pro Asn Ala Met Pro Lys Trp Leu Ser Trp Leu Lys Ala Ile

405 410 415

Val Asp Ala Asp Asp Leu Pro Leu Asn Val Ser Arg Glu Thr Leu Gln

420 425 430

Ser Asn Arg Phe Leu Arg Gln Ile Pro Asn Ile Leu Val Arg Arg Phe

435 440 445

Ile Asn Leu Val Asp Arg Met Ser Lys Asp Glu Glu Asn Pro Glu Leu

450 455 460

Phe Arg Lys Phe Met Lys Ile Tyr Gly Ser Val Val Lys Leu Gly Ala

465 470 475 480

Val Glu Ser Pro Lys Glu Gln Gln Lys Leu Ala Gly Leu Ala Arg Trp

485 490 495

Asp Thr Asn Leu Arg Asn Phe Thr Ser Leu Asp Gln Tyr Val Glu Asn

500 505 510

Arg Lys Lys Gly Gln Thr Gln Ile Phe Tyr Leu Ala Gly Ile Gly Gln

515 520 525

Arg Pro Asp Glu Leu Ala Lys Ser Leu Phe Val Glu Lys Leu His Ala

530 535 540

Arg Gly Tyr Glu Val Leu Leu Leu Asn Asp Pro Met Asp Glu Ile Leu

545 550 555 560

Met Ser His Leu Arg Asn Trp Lys Gly Leu Gln Phe Gln Asp Ala Ala

565 570 575

Lys Lys Gly Leu Lys Phe Gly Asp Glu Asp Glu Asp Ala Glu Glu Glu

580 585 590

Lys Ala Arg Gln Glu Lys Leu Lys Glu Thr Phe Lys Pro Leu Leu Glu

595 600 605

Tyr Leu Lys Asn Glu Thr Ser Asn Val Val Leu Asp Val Ile Leu Ser

610 615 620

Asn Arg Leu Val Thr Ser Pro Cys Ala Ile Val Ala Asp Ser Tyr Gly

625 630 635 640

Tyr Ser Ala Asn Met Glu Arg Leu Met Ala Ala Gln Thr Gly Gly Lys

645 650 655

Met Lys Glu Asn Asp Phe Met His Asp Trp Ala Lys Lys Gln Lys Phe

660 665 670

Leu Glu Ile Asn Pro Asn Ser Pro Leu Ile Glu Gly Met Leu Lys Lys

675 680 685

Val Gln Ala Leu Ile Asp Ile Glu Glu Gly Glu Glu Arg Asp Ser Gln

690 695 700

Leu Glu Glu Glu Leu Lys Glu Val Ala Ser Ile Leu Val Asp Gly Ala

705 710 715 720

Leu Val Arg Ser Gly Phe Glu Val Pro Asp Ser Asn Leu Arg Arg Phe

725 730 735

Phe Thr Arg Met Asp Arg Val Leu Arg Arg Ser Leu Gly Val Ser Glu

740 745 750

Thr Ala Lys Gly Asp Glu Thr Val Lys Pro Ala Pro Pro Val Asp Asp

755 760 765

Thr Pro Leu Glu Glu Pro Gly Val Leu Pro Glu Gly Ala Phe Glu Asp

770 775 780

Ile Ile Asp Asn Thr Ala Asp Gln Lys Thr Phe Gln Pro Lys Val Asp

785 790 795 800

Val Lys Val Asp Ala Leu Asn Asp Glu Glu Ile Glu Glu Met Leu Asn

805 810 815

Lys Lys Glu Gln Glu Val Lys Arg Asp Glu Leu

820 825

<210>5

<211>286

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tgaagaagga gaggagaggg acagtcaact tgaagaggaa ctaaaggaag ttgcatctat 60

tctcgttgac ggagccctcg tccgctctgg tttcgaggtt cccgattcca atctgttctt 120

cactcgtatg gatcgcgtcc ttcgccgttc tctcggtgtc tctgagaccg ccaagggcga 180

cgaaactgtt aagcccgctc ccccagtcga tgatactcct ctcgaagagc ctggtgtcct 240

accggaaggt gcatttgagg acatcattga taataccgcc gaccag 286

<210>6

<211>483

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

uaauacgacu cacuauaggu cgcccacagc aucuacaagg gugacucugg uccaguuucg 60

uuccgcacca uguucuucau cccaucggau cugaacgaga aguucuggca aagcgcgaag 120

cccgaguuga acaacauccg ucucaugguu aagcgcgucu ucauuaccag cgaccucgga 180

cccaaugcca ugccuaaaug gcucaguugg cucaaggcua ucguugaugc cgaugaccuc 240

ccucucaacg ucucccguga aacucuccaa ucaaaccggu uccuucguca gauucccaau 300

auccucgucc gucgcuucau caacuugguc gauaggaugu cuaaggacga ggagaaccca 360

gaacuuuucc guaaauucau gaagauuuau ggcucgguug uuaagcuggg agcuguugaa 420

agccccaagg aacaacaaaa gcuggcugga uuggcucguu gggaccuaua gugagucgua 480

uua 483

<210>7

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

tgaagaagga gaggagagg 19

<210>8

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ctggtcggcg gtattatc 18

<210>9

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

tcttatggcc gcccagac 18

<210>10

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

agctggacct tcttgagc 18

<210>11

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ggtcggcaaa gtcataccat 20

<210>12

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

tctgcgtcct tcttggagat a 21

<210>13

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

taatacgact cactataggt cgcccacagc atctacaa 38

<210>14

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

taatacgact cactataggt cccaacgagc caatcc 36

<210>15

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ccggaattct gaagaaggag aggagagg 28

<210>16

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

cgcggatccc tggtcggcgg tattatc 27

<210>17

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

aattcactgg gagatgatac gctg 24

<210>18

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

cctatggtaa gacaatgagt cggc 24

<210>19

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

gccttttcta ccttaatttg gcag 24

<210>20

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

gtgtgtaaat taagtagaca gcaaatg 27

<210>21

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

tggtgtcctc aagcctggta t 21

<210>22

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

acgcttgaga tccttaaccg c 21

Claims (10)

1. A rice sheath blight bacterium cation transfer ATP enzyme gene is characterized in that the full-length cDNA sequence of the gene is shown in SEQ ID NO:1 is shown.

2. A rice sheath blight bacterium cation transfer ATP enzyme gene is characterized in that the full-length DNA sequence of the gene is shown in SEQ ID NO:2, respectively.

3. A gene for transporting ATP to cations of rhizoctonia solani, wherein the coding sequence of the gene is shown as SEQ ID NO: 3, respectively.

4. A protein coded by a rice sheath blight bacterium cation transfer ATP enzyme gene is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO: 4, respectively.

5. A rice sheath blight bacterium cation transport ATP enzyme gene fragment Rscta is characterized in that the nucleotide sequence of the fragment is shown as SEQ ID NO: 5, respectively.

6. Use of the ATPase gene according to any one of claims 1-3 or the fragment Rscta according to claim 5 for the control of Rhizoctonia solani or for the preparation of a Rhizoctonia solani control agent.

7. Use of the ATPase gene according to any one of claims 1-3 or the fragment Rscta according to claim 5 for constructing transgenic plants resistant to Rhizoctonia solani.

8. A Rhizoctonia solani control agent comprising a substance capable of inhibiting the expression of the ATPase gene according to any one of claims 1 to 3.

9. The formulation according to claim 8, characterized in that said substance is dsRNA or a recombinant vector or a recombinant bacterium comprising a fragment Rscta.

10. The formulation of claim 9, wherein the nucleic acid sequence of said dsRNA is as set forth in SEQ ID NO: and 6.

Images