CN116003561B - Rot stem nematode DdSX protein, coding gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant protection, and particularly discloses a rotten stem nematode DdSX protein, a coding gene and application thereof. The amino acid sequence of DdSX001,001 protein is shown as SEQ ID NO: 1. The DNA sequence of the gene DdSX001 encoding DdSX001,001 protein is shown in SEQ ID NO: 2. The infection of the rotting stem nematodes to crops such as potatoes, sweet potatoes, chinese medicinal materials and the like can be limited by inhibiting the expression of the gene DdSX, so that the control of the rotting stem nematodes of related crops is realized.

Description

Rot stem nematode DdSX protein, coding gene and application thereof

Technical Field

The invention belongs to the technical field of plant protection, and particularly discloses a rotten stem nematode DdSX protein, a coding gene and application thereof.

Background

The rotting stem nematodes (Ditylenchus destructor) are important migration type plant parasitic nematodes and are widely distributed in temperate regions, and can harm more than 120 plants such as potatoes, sweet potatoes, carrots and the like. The rot stem nematodes are the second largest nematode disease on potatoes, which is next to the potato cyst nematodes, and usually result in a yield loss of 30% -50%, which is particularly serious in temperate climatic regions. In China, the rotting stem nematodes not only harm potatoes, but also severely restrict the development of the sweet potato industry, and can cause 50% -70% yield loss to the sweet potatoes, and even kill the sweet potatoes in serious cases. In addition, in recent years, research has found that the rotten stem nematodes can also harm Chinese medicinal materials such as Chinese angelica, dangshen and the like, and have serious influence on the development of the Chinese medicinal material industry. In recent years, the occurrence area of the rotting stem nematodes is continuously enlarged in China, and the rotting stem nematodes have a further outbreak trend. However, the characteristics of strong stress resistance of the rotting stem nematodes, irregular field insect states, long survival years and the like lead to great control difficulty in production, so that new disease control technology is urgently needed to be developed at present.

The chemical nematicide is still mainly used for preventing and treating the rot stem nematode in production, but the traditional chemical nematicide is a chemical agent with high toxicity, high residue, high dosage and high cost, so that the quality safety of products can be seriously influenced, and the ecological environment safety can be seriously influenced. Currently, most conventional chemical wire killers have been disabled. In addition, the rotten stem nematodes have strong resistance to part of biological pesticides, and the prevention and control effects of the biological pesticides are very limited. Therefore, the invention aims to develop a novel pesticide target molecule and provides a target for developing a novel drug for preventing and treating the rot stem nematode.

Disclosure of Invention

In view of the above technical problems, the present invention provides the following technical solutions:

In a first aspect of the present invention, there is provided a protein DdSX a from a stem rot nematode, having the amino acid sequence as set forth in SEQ ID NO: 1.

In a second aspect of the present invention, there is provided a gene DdSX001 encoding the said stem rot nematode DdSX001,001 protein, the DNA sequence of which is as set forth in SEQ ID NO: 2.

In a third aspect of the present invention, there is provided a recombinant vector comprising the gene DdSX.

In a fourth aspect of the invention, there is provided the use of said gene DdSX001 in the control of rot stem nematode disease.

Preferably, the control of the rot stem nematode disease is inhibition of parasitic rot stem nematodes on plants and/or inhibition of pathogenic rot stem nematodes on plants.

Preferably, the plant is potato, sweet potato, carrot, radix Codonopsis or angelica.

In a fifth aspect of the present invention, there is provided a method for controlling a rot stem nematode disease by inhibiting the expression of the gene DdSX001,001 and/or reducing the DdSX001,001 protein.

Preferably, the agent used in the method is an agent that knocks out gene DdSX001, an agent that silences gene DdSX001, or an inhibitor of gene DdSX 001.

Preferably, the inhibitor comprises DdSX001 gene RNAi fragment dsEXP1 and/or dsEXP2;

The nucleotide sequence of dsEXP is shown as SEQ ID NO:13, the nucleotide sequence of dsEXP is shown as SEQ ID NO: 14.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a rotting stem nematode DdSX protein and a gene DdSX001 encoding the DdSX protein, which can limit the infection of rotting stem nematodes to dicotyledonous plants such as potatoes, sweet potatoes, chinese medicinal materials and the like by inhibiting the expression of the gene DdSX001, so as to realize the prevention and control of the rotting stem nematode diseases of related crops.

Drawings

FIG. 1 is a DdSX001 gene transcriptional expression profile;

FIG. 2 is a DdSX001 gene tissue expression profile, digoxin markers specifically appear at esophageal gland sites;

FIG. 3 shows the result of RNAi fragment amplification of DdSX001 gene; lane M is DM2000 Marker; 1. lane 2 is dsEXP1; 3. lane 4 is dsEXP2;

FIG. 4 is the silencing efficiency of DdSX001,001 genes by different dsRNA interference fragments;

FIG. 5 is a graph of inhibition DdSX001 against the effect of controlling rotting stem nematodes; GFP dsRNA as a negative control; dsEXP1, dsEXP are treatments for silencing dsSX 001.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1

DdSX001 Gene cloning

1. Extraction of total RNA of rotten stem nematodes

1) Fresh rotting stem nematodes (about ten thousand heads) were collected, the worms were washed 3 times with DEPC water, transferred into a sterile, enzyme-free 1.5mL centrifuge tube, and 300. Mu.L Trizol (Takara Co.) was added;

2) Adding 100 μL of glass powder sterilized at 121 ℃ for 90min, fully mixing, and grinding for 5min with an electric grinder at a frequency of 3 KRPM;

3) Adding 700 mu L of Trizol into the tube, and standing at room temperature for 10min;

4) Extracting total RNA of the rotten stem nematodes according to the Trizol kit operation guidelines.

2. CDNA preparation

The cDNA was obtained by reverse transcription using the extracted total RNA of the rotten stem nematode as a template using a reverse transcription kit (Takara Co.).

3. DdSX001 Gene coding sequence amplification

Using cDNA as a template, an upstream primer/downstream primer: ATGCAAACTCTCGCATTTGCG (shown as SEQ ID NO: 3)/CTAGCAATATCCATAAGTGACAGCG (shown as SEQ ID NO: 4) were subjected to PCR amplification. The amplification system was 2X Flash PCR MasterMix (century well), 25. Mu.l; 1 μl of each of the upstream primer (10 mM)/the downstream primer (10 mM); cDNA, 2. Mu.l; deionized water, 21 μl, total 50 μl. The amplification procedure was denaturation at 94℃for 5min; next 39 cycles, each cycle was 94 ℃ melting for 30sec,60 ℃ combining for 30sec,72 ℃ extension for 1min; finally, the mixture is extended for 10min at 72 ℃ and stored at 4 ℃.

4. Amplified fragment recovery

And (3) carrying out 1% agarose gel electrophoresis separation and identification on the PCR product, and carrying out gel recovery on the band with the fragment size of about 1000bp by using a gel recovery kit (TIANGEN company) to obtain a gel recovery product.

5. Sequencing of amplified fragments

6. The gel recovery product was ligated to pMD19-T simple vector (Takara Corp.) and sent to the company for sequencing. Sequencing results to obtain DdSX gene coding sequence as shown in SEQ ID NO: 2.

>DdSX001_CDs

ATGCAAACTCTCGCATTTGCGTTAATTTTTGGCGCATGTTTGAGCGCCACTGACGCCGGTCTCATCGAGGCTCAGTTGAATACGCCAGTTCCCGACAGTGTGTTCACTTTTTACGGTGCTGCTGGACGTGGGGCTTGTGGATTGGACATCAGCTCATGCTCTGCTGCAGCTTCTGGATCGCTGTTTGACCCCAACGCTCAGTGGGTGCCATCCAATTTTCCGGATGGACGTTACATTCTCAATGATCCGATATGTAATGGGATCTGCATCAAAGTGGAATATAAGGGCAAATCTGCGGTGTTCCCAATCGACAACAAGTGCCCTGAATGCCCTGTGAATCACGTGGACTTGTCAGAAACCGCCTTTCTTGTGCTGGAGCCTCTGGGCGGTACTGTGGGAAAGGCTACCGGAGCAACTCTTACATACTTACTTTGCAATCAGACCACCATTTCGAGCTGCAATGGTGCCGCTACAGCTGGACCCACTACTGCTGGACCCACATCTGCCCCTACTACAAAGCTATCTGTTACCACTACTAAGGCTCCCTCCGCTGGAAGCACAACCATCGCTTCGGGTGTCACAACCCCGAGCAATGGTGGTGGAAATTCTGGAAAATTAAGCGTATCCGCTACTGTGGCATCTAGCTGGAATGGTGGCATGCAACTGGCTCTTGTTTTCACTAACAACGACTCAAAGAGTGTTTGCTCCGTCACTTTTTCGATTGCCCTTCAGTCTGGACAAACAATCAGCAACTCGTGGAACATGGAGAATGGGTCTACTACCAATCAATACAAATTGCCATCATGGATAAACATCTCTCCATCTGGGGGTCAAAATAGCAACGCTGGACTGAATATTGATGGAGGAGGGAGCACTCCGCCGACTGTCAGCGCTGTCACTTATGGATATTGCTAG

Example 2

DdSX001,001 gene coding protein sequence

Translation of the DdSX001 gene coding sequence using ORF finder on-line software https:// www.ncbi.nlm.nih.gov/orffinder gave a DdSX protein sequence as shown in SEQ ID NO: 1.

>DdSX001_protein

MQTLAFALIFGACLSATDAGLIEAQLNTPVPDSVFTFYGAAGRGACGLDISSCSAAASGSLFDPNAQWVPSNFPDGRYILNDPICNGICIKVEYKGKSAVFPIDNKCPECPVNHVDLSETAFLVLEPLGGTVGKATGATLTYLLCNQTTISSCNGAATAGPTTAGPTSAPTTKLSVTTTKAPSAGSTTIASGVTTPSNGGGNSGKLSVSATVASSWNGGMQLALVFTNNDSKSVCSVTFSIALQSGQTISNSWNMENGSTTNQYKLPSWINISPSGGQNSNAGLNIDGGGSTPPTVSAVTYGYC

Example 3

DdSX001 Gene expression profiling

1. Transcriptional expression characterization

1) Fresh rotting stem nematodes cultured on different culture mediums of potato, sweet potato and fusarium are collected respectively, and total RNA of the rotting stem nematodes on the different culture mediums is extracted respectively according to the method in example 1.

2) CDNA was obtained by reverse transcription using total RNA derived from the rotten stem nematodes in different culture media as templates, respectively, using HISCRIPT III ALL-in-one RT SuperMix Perfect for qPCR kit (Vazyme).

3) The relative transcriptional expression level of DdSX001 gene in different treatments was detected using REAL TIME PCR quantification technique with different cdnas as templates.

For the same cDNA template, the actin gene is used as reference gene, and the method adoptsSELECT MASTER Mix kit (Takara), real-time RT-PCR detection is carried out on an ABI7500 fluorescent quantitative PCR instrument, three biological repeated experiments are respectively carried out, and the analysis result of the 2-delta Ct method is adopted, so that the expression difference of DdSX001 genes when rotten stem nematodes eat different culture mediums is clear. The reaction system was SYBR Premix Ex Taq II, 10. Mu.l; ROX REFENCE DYE II,0.4 μl; 0.3. Mu.l each of the upstream primer (10. Mu.M) and the downstream primer (10. Mu.M); template, 2 μl; ddH ₂ O makes up 20. Mu.l. The reaction procedure is denaturation at 95 ℃ for 5min; next 39 cycles, each cycle was 95 ℃ melt 10sec,60 ℃ combined 20sec,72 ℃ extended 30sec; and finally, running a melting curve amplification program. Wherein the sequences of the upstream primer and the downstream primer are respectively: ATCGCTGTTTGACCCCAAC (shown as SEQ ID NO: 5) and AGTAGTGGGTCCAGCTGTAG (shown as SEQ ID NO: 6).

The results are shown in FIG. 1. Taking the expression level of the gene when the rotting stem nematodes take fusarium as a reference, and up-regulating the expression level of the gene by 2.19 times when the rotting stem nematodes take potatoes; when the rotting stem nematodes feed on sweet potatoes, the gene expression is up-regulated by 2.61 times. The results show that the gene can significantly up-regulate expression in the process of damaging plants by rotting stem nematodes, and the functions of the gene can be related to damaging plant hosts.

2. Tissue expression characterization

1) Preparation of hybridization fragments

The pMD19-T simple vector containing DdSX gene coding sequence is used as a template, and an upstream primer/a downstream primer are used as the template: AATACGCCAGTTCCCGAC (SEQ ID NO: 7)/GTACCGCCCAGAGGCTC (SEQ ID NO: 8) amplified a DdSX001 gene fragment in a 2X Flash PCR MasterMix (century well) amplification system of 25. Mu.l; 1 μl of each of the upstream primer (10 mM)/the downstream primer (10 mM); cDNA, 2. Mu.l; deionized water, 21 μl, total 50 μl. The amplification procedure was denaturation at 94℃for 5min; next 39 cycles, each cycle was 94 ℃ melting for 30sec,54 ℃ combining 30sec,72 ℃ extending for 30sec; finally, the mixture is extended for 10min at 72 ℃ and stored at 4 ℃. The PCR product was identified by 1% agarose gel electrophoresis, and a band with a fragment size of about 300bp was subjected to gel recovery using a gel recovery kit (TIANGEN Co.) to obtain a DdSX001 gene hybridization fragment.

2) In situ hybridization

The method comprises the steps of preparing a reverse strand probe/forward strand probe by using DdSX001 gene hybridization fragment gel recovery product as a template, respectively using an upstream primer or a downstream primer used for hybridization fragment preparation as a linker, and using a digoxin probe labeling kit (Roche company). The amplification system was 10XEx Taq Buffer,2.5. Mu.l; PCR DIG probe synthesis. Mu.l; 1 μl of the upstream primer (10 mM) or the downstream primer (10 mM); the product was recovered with a gel of 0.5. Mu.l; exTaq,0.5 μl; deionized water, 18.5 μl, total 25 μl. The amplification procedure was denaturation at 94℃for 5min; next 28 cycles, each cycle was 94 ℃ melting for 30sec,60 ℃ combining 30sec,72 ℃ extending for 20sec; finally, the mixture is extended for 10min at 72 ℃ and stored at 4 ℃. Hybridization of the forward and reverse probes with the pre-treated worms, respectively, using digoxin in situ hybridization kit (Roche Co.) resulted in the position-specific expression of DdSX001 in the esophageal glands of the rotting stem nematodes, see FIG. 2.

Example 4

DdSX001 RNAi fragment sequence of 001 gene

1. The coding sequence of DdSX gene is predicted and analyzed by using siRNA online prediction software http:// sidirect2.RNAi. Jp/design. Cgi and http:// biodev. Extra. Cea. Fr/DSIR/DSIR. Php, fragments which can be used for RNAi are screened, and RNAi fragment specific primers are designed.

2. The invention designs dsEXP F/dsEXP1R by using a pMD19-T simple vector containing DdSX001 gene coding sequence as a template: TAATACGACTCACTATAGGGATCGCTGTTTGACCCCAAC (shown as SEQ ID NO: 9)/TAATACGACTCACTATAGGGAGTAGTGGGTCCAGCTGTAG (shown as SEQ ID NO: 10) and dsEXP F/dsEXP2R: TAATACGACTCACTATAGGGGGAAGCACAACCATCGCTTC (shown as SEQ ID NO: 11)/TAATACGACTCACTATAGGGTATTCAGTCCAGCGTTGCTAT (shown as SEQ ID NO: 12) were subjected to PCR amplification. The amplification system was 2X Flash PCR MasterMix (century well), 25. Mu.l; 1 μl of each of the upstream primer (10 mM)/the downstream primer (10 mM); cDNA, 2. Mu.l; deionized water, 21 μl, total 50 μl. The amplification procedure was denaturation at 94℃for 5min; next 39 cycles, each cycle was 94 ℃ melting for 30sec,60 ℃ combining 30sec,72 ℃ extension for 30sec; finally, the mixture is extended for 10min at 72 ℃ and stored at 4 ℃. The PCR products were identified by 1% agarose gel electrophoresis, and the bands with the fragment size of about 300bp were subjected to gel recovery using a gel recovery kit (Tianmo biosystems), to obtain gel recovery products of DdSX001 gene RNAi fragments dsEXP and dsEXP2, see FIG. 3.

3. DsRNA interference fragments were synthesized by in vitro transcription using HiScribe T Quick HIGH YIELD RNA SYNTHESIS kit (NEB) with RNAi fragment gel recovery as template.

The rotting stem nematode DdSX gene RNAi system was formulated as follows: 0.3M spermidine, 3 μl;0.5M octopamine, 30 μl;5% gelatin, 3 μl; dsRNA interference fragment, 600mg; the above reagents were formulated and added to the nematode suspension to a final volume of 300 μl. The reaction system was placed on a shaker at 26℃and the dark treatment at 120rpm was performed for 24h, 48h, 72h, and the silencing efficiencies of the different fragments for the different treatment times were compared.

As a result, as shown in FIG. 4, after the treatment of the rotting stem nematodes with dsEXP and dsEXP interfering fragments for 24h, 48h and 72h, respectively, the expression level of the rotting stem nematode DdSX001 gene was significantly reduced in each of the treated groups, as compared with the control group rotting stem nematodes treated with GFP dsRNA. When the expression level of DdSX gene in the control group at each treatment time (24 h, 48h, 72 h) was defaulted to 100%, the expression level of DdSX gene in the dsEXP treatment group at each time point was only 50.1%, 41.6% and 14.1% of the control group, and the expression level of DdSX gene in the dsEXP treatment group at each time point was only 62.6%, 51.1% and 23.1% of the control group. The results show that the dsEXP gene interference fragments and the dsEXP gene interference fragments designed by the invention can obviously inhibit the expression of DdSX001 genes.

Example 5

Application of DdSX001 gene expression inhibition in prevention and control of rotten stem nematodes

The interfering fragments dsEXP and dsEXP2 were synthesized by the procedure of example 4, and the rotting stem nematodes were treated for 72 hours with the above reaction system to create a rotting stem nematode treated group dsEXP and dsEXP2 population with silencing DdSX001 gene. The dsRNA interference fragment in example 4, 600mg, was replaced with equal amounts of GFP dsRNA and equal volumes of ddH ₂ O, and the rotting stem nematodes were treated under the same conditions to establish a negative control group (GFP dsRNA) and a blank control group (water control) of rotting stem nematode populations. And (3) inoculating the same amount of the rotting stem nematodes of different groups to the vicinity of root systems of sweet potato seedlings, placing the seedlings in a 25 ℃ illumination incubator for 20 days, and investigating the number of the nematodes of different groups penetrating into plants.

As shown in FIG. 5, the numbers of rotten stem nematodes invaded into plants in the blank control group and the negative control group are 73 and 75 respectively, while the numbers of nematodes invaded into plant tissues in the two treatment groups of dsEXP and dsEXP, which silence DdSX001 gene, are 23 and 34 respectively. The result shows that inhibiting DdSX001,001 gene expression can effectively inhibit the infection of the sweet potato plant by the rotting stem nematodes.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (7)

1. A rotting stem nematode DdSX protein, which is characterized in that the amino acid sequence is shown in SEQ ID NO: 1.

2. A gene DdSX001 encoding the nematode DdSX001,001 protein of claim 1, which has a DNA sequence as set forth in SEQ ID NO: 2.

3. A recombinant vector comprising the gene DdSX001,001 of claim 2.

4. Use of the gene DdSX001 according to claim 2 for controlling rot stem nematode diseases, wherein the gene DdSX001 is used for controlling rot stem nematode diseases of potatoes or sweet potatoes by inhibiting expression of the gene DdSX001 and/or reducing the protein DdSX001 encoded by the gene DdSX 001.

5. The use according to claim 4, wherein the control of the rot stem nematode disease is inhibition of parasitic rot stem nematodes on plants and/or inhibition of pathogenic rot stem nematodes on plants.

6. A method for controlling rot stem nematode disease of potato or sweet potato by inhibiting expression of gene DdSX001 according to claim 2 and/or reducing DdSX001 protein according to claim 1.

7. The method of claim 6, wherein the agent used in the method is an agent that knocks out gene DdSX001 or an inhibitor of gene DdSX001, said inhibitor of gene DdSX001 effecting inhibition of DdSX001 by silencing DdSX001 expression;

agents that silence DdSX001 expression comprise DdSX001 gene RNAi fragments dsEXP1 and/or dsEXP2;

The nucleotide sequence of dsEXP is shown as SEQ ID NO:13, the nucleotide sequence of dsEXP is shown as SEQ ID NO: 14.