CN112899291B - ATP synthetase of rice tip nematode and application thereof - Google Patents

Abstract

The invention discloses a rice tip nematode ATP synthetase and application thereof, wherein the amino acid sequence of the rice tip nematode ATP synthetase is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the rice tip nematode ATP synthetase gene is shown as SEQ ID NO: 1 is shown. After RNAi treatment is adopted, the reproductive capacity of the rice apicultus nematode can be inhibited, the rice defense gene expression is promoted, the plant immune response can be activated, and a new strategy is provided for nematode prevention and control in agricultural production. The invention provides a new target for cultivating the nematode-resistant variety plants and a new way for preventing and treating the aphelenchoides besseyi.

Description

ATP synthetase of rice tip nematode and application thereof

Technical Field

The invention relates to the technical field of plant disease and insect pest control, and particularly relates to a rice tip nematode ATP synthetase and application thereof.

Background

Rice is one of the most important food crops, and the plant parasitic nematodes cause economic losses of over 160 hundred million dollars of rice all year around the world, wherein the most important nematodes parasitizing the upper part of the soil are Aphelenchoides besseyi. The rice aphelenchoides besseyi infects rice, and the economic loss can reach 50 percent. The nematodes are distributed in the main rice areas of China and are seriously damaged locally.

The ATPase gene has important relation with plant disease resistance induced allergic necrosis. It is found that the ATPase protein gene PDE1 of Magnaporthe grisea is closely related to the formation of attachment cells when rice blast fungus is infected; another ATPase gene MgAPT2 of Magnaporthe grisea can induce resistance of plants and cause allergic necrosis. Plants can also trigger self-defense responses by modulating ATPase through self-secretion. The tobacco ATPase NtAA 1 participates in the self-disease-resistant reaction, and after the gene is silenced, the tobacco inoculated with the Pseudomonas syringae (Pseudomonas syringae) loses the defense reaction of the allergic necrosis. The ATPase AtOM66 of arabidopsis located on the outer mitochondrial membrane plays an important role in inducing allergic necrosis of arabidopsis against pathogen infection. ATPase is reported to be widely present in nematodes, and plays a considerable role in the life activities of nematodes.

Among Plant nematodes, only the ATPase gene of Meloidogyne incognita (Meloidogyne incognita) is currently successfully cloned, and it has been reported that silencing of the ATP synthase gene of Meloidogyne incognita leads to a significant increase in nematode larval mortality, and that both in vitro soaking and Plant-mediated RNAi results indicate that silencing of this gene leads to a significant decrease in pathogenicity and a significant decrease in host root knot number (Huang Y H, Mei M, Shen B M, et al. RNAi MiASB captured high motility of Meloidogyne incognita jveniles and inhibited the root knot number of host [ J. Agricultural science, 2013,9(4):483 42; Huang Y H, Mei M, Mao Z C, et al. molecular cloning and viral-induced growth of microorganism of wheat J.193, J. 2014,138. J.. However, no report on ATP synthetase and functions of aphelenchoides besseyi exists so far.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the defects of the existing prevention and control technology of aphelenchoides besseyi and the defects of the research on the effect protein ATP synthetase of aphelenchoides besseyi, and provides an ATP synthetase for aphelenchoides besseyi and the application thereof.

The first purpose of the invention is to provide a rice tip nematode ATP synthase gene.

The second purpose of the invention is to provide a rice tip nematode ATP synthetase.

The third purpose of the invention is to provide the application of the inhibitor of the rice tip nematode ATP synthase gene and/or the inhibitor of the rice tip nematode ATP synthase in the preparation of the drugs for preventing and treating the rice tip nematode.

The fourth purpose of the invention is to provide the application of the rice tip nematode ATP synthase gene and/or the rice tip nematode ATP synthase as a drug screening target point for resisting rice tip nematodes.

The fifth purpose of the invention is to provide the rice tip nematode ATP synthase gene and/or the inhibitor of the rice tip nematode ATP synthase.

It is a sixth object of the present invention to provide a gene encoding the dsRNA described in the above.

The seventh purpose of the invention is to provide an expression vector, a transgenic cell line or a host bacterium containing the gene.

The eighth purpose of the invention is to provide the application of any one or more of the gene, the expression vector, the transgenic cell line or the host bacterium in the preparation of the pesticide for preventing and treating the rice apicomplexa.

In order to achieve the purpose, the invention is realized by the following scheme:

the invention claims and protects a rice tip nematode ATP synthase gene, SEQ ID NO: 1 is the whole length of the ATP synthetase gene of the rice tip nematode.

And, a rice nematode ATP synthase, the amino acid sequence of which is as shown in SEQ ID NO: 2, respectively.

The application of the inhibitor of the rice tip nematode ATP synthase gene and/or the inhibitor of the rice tip nematode ATP synthase in the preparation of the drugs for preventing and treating the rice tip nematodes also belongs to the protection range of the invention.

Preferably, the medicament inhibits the reproductive capacity of Aphelenchoides besseyi.

Preferably, the drug inhibits the growth of Aphelenchoides besseyi.

Preferably, the inhibitor is dsRNA.

More preferably, the dsRNA is double-stranded RNA consisting of a nucleotide sequence shown as SEQ ID No.3 as a sense strand and a nucleotide sequence reverse-complementary to the nucleotide sequence shown as SEQ ID No.3 as an antisense strand.

The application of the rice tip nematode ATP synthase gene and/or the rice tip nematode ATP synthase as a rice tip nematode resistant drug screening target also belongs to the protection scope of the invention.

The invention also claims a rice tip nematode ATP synthetase gene and/or an inhibitor of the rice tip nematode ATP synthetase, wherein the inhibitor is dsRNA, and the dsRNA is double-stranded RNA which takes a nucleotide sequence shown as SEQ ID NO.3 as a sense strand and takes a nucleotide sequence which is reversely complementary with the nucleotide sequence shown as SEQ ID NO.3 as an antisense strand.

The invention also claims genes encoding the dsRNA;

and an expression vector, a transgenic cell line or a host bacterium containing the gene.

The application of any one or more of the gene, the expression vector, the transgenic cell line or the host bacterium in the preparation of the pesticide for preventing and treating the rice apicomplexa also belongs to the protection scope of the invention.

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

the invention provides ATP synthetase of rice tip nematodes and application thereof, after RNAi treatment is adopted, the reproductive capacity of the rice tip nematodes can be inhibited, rice defense gene expression can be promoted, plant immune response can be activated, apoptosis can be induced, and a new strategy is provided for nematode prevention and control in agricultural production. The invention provides a new target for cultivating the nematode-resistant variety plants and a new way for preventing and treating the aphelenchoides besseyi.

Drawings

FIG. 1 is a phylogenetic tree of APS synthetases from Aphelenchoides besseyi and other nematodes; Ab-ATPS of the rice nematode stem tip is underlined; the gene number of each sequence is indicated in parentheses.

FIG. 2 shows the expression level of Ab-atps in aphelenchoides besseyi eggs, larvae, females and males; "i" indicates the standard error of the mean number (n ═ 3), and different letters indicate significant differences between treatments (p < 0.05).

FIG. 3 is the tissue localization of mRNA encoded by the Ab-atps gene from Aphelenchoides besseyi; a: ab-atps mRNA localizes to the esophagus; c: ab-atps s mRNA localises to the reproductive system; B. d: a sense probe control; oesophagus: food passage; mb: a middle esophageal bulb; v: a vulva; productive system: the reproductive system.

FIG. 4 shows the detection of the silencing efficiency of Ab-atps gene in aphelenchoides besseyi after different treatments; g12, G24, G36 and G48: treating nematodes for 12h, 24h, 36h and 48h by using gfp dsRNA; a12, a24, a36 and a 48: ab-atps dsRNA treated nematodes for 12h, 24h, 36h and 48 h; "i" indicates the standard error of the mean (n-3), and different letters indicate significant differences between treatments (p < 0.05).

FIG. 5 shows the interaction between OsRLK3 and Ab-ATPS; a: the interaction of OsRLK3 and Ab-atps; pGBKT7-OsRLK3(BD) co-transformed with pGADT7-Ab-ATPS (AD) grown as white colonies on YDPA on QDO/XA (SD/-Leu/-Trp/-Ade/-His/X- α -gal/AbA) medium, which turned blue; co-transformation of pGADT7-Lam and pGBKT7-53 yeast as positive controls; cotransforming pGADT7 empty vector and pGBKT7-OsRLK3 yeast, pGADT7-Ab-ATPS and pGBKT7 empty vector yeast as negative controls; b: the yeast liquid co-transformed by pGBKT7-OsRLK3(BD) and pGADT7-Ab-ATPS (AD) is diluted by 1, 10 degrees of gradient ^-1 ，10 ^-2 Growth was carried out on QDO/XA medium.

FIG. 6 shows the amount of nematode reproduction and the expression change of OsRLK3 gene after different treatments; g12, G24, G36 and G48: after 12h, 24h, 36h and 48h of nematode treatment by gfp dsRNA, inoculating a culture medium for 35d and separating; a12, a24, a36 and a 48: the Ab-atps gene RNAi is used for treating the number of nematodes isolated after the aphelenchoides besseyi 12h, 24h, 36h and 48h are inoculated into a culture medium 35d, the 'I' represents the standard error of the mean (n is 9), and different letters represent that the difference between treatments is obvious (p is less than 0.05); b: the column height is the average of the ratios of treated samples and blank (n-3, each replicate consisting of three rice plants, the technique replicated twice). The internal reference gene is OsUBQ5, the 'I' represents the standard error of the average (n is 3), different capital letters represent that the OsRLK3 gene of aphelenchoides besseyi rice inoculated with Ab-atps dsRNA for 48h soaking is remarkably different among treatments (p is less than 0.05), different lower case letters represent that the OsRLK3 gene of aphelenchoides besseyi rice inoculated with gfp dsRNA for 48h soaking is remarkably different among treatments (p is less than 0.05), and the internal reference gene comprises: the gene expression levels of aphelenchoides besseyi rice inoculated with Ab-atps dsRNA for soaking for 48h and aphelenchoides besseyi rice inoculated with gfp dsRNA for soaking for 48h at the same time are significantly different (p <0.05), and the gene expression levels of ns: the gene expression level of the rice stem tip nematode rice inoculated with Ab-atps dsRNA and soaked for 48h at the same time has no significant difference (p is more than 0.05) with the rice stem tip nematode rice inoculated with gfp dsRNA and soaked for 48h, and the gene expression level of the rice stem tip nematode rice inoculated with Ab-atps dsRNA has the following characteristics that: days post inoculation, gfp: inoculating gfp dsRNA to soak the rice of aphelenchoides besseyi for 48h, Ab-atps, inoculating Ab-atps dsRNA to soak the rice of aphelenchoides besseyi for 48 h.

FIG. 7 shows subcellular localization and transient expression of Ab-ATPS in tobacco for Aphelenchoides besseyi; a: Ab-ATPS localizes to cell membranes, cytoplasm, and nucleus in tobacco; b: western-blot detection Ab-ATPS is expressed in tobacco, and M: low molecular weight protein Marker, 1: transient expression of GFP in tobacco, 2: transient expression of Ab-ATPS-GFP in tobacco; c: Ab-ATPS causes tobacco lamina cell death, 1: MES buffer, 2: pCAMBIA1300-GFP, 3: pCAMBIA1300-Ab-ATPS-GFP, 4: pCAMBIA1300 empty vector plasmid.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 cloning and sequence analysis of the full-Length sequence of the Gene of ATP synthetase Ab-ATPs of Aphelenchoides oryzae

First, experiment method

A key defense gene OsRLK3 of rice and a cDNA library of aphelenchoides besseyi are utilized to obtain a gene through a yeast two-hybrid experiment, and a partial sequence of an Ab-Atps gene of aphelenchoides besseyi is obtained through sequencing.

Further, RACE primers are designed according to the sequence, and then the full-length gene of the Ab-Atps of the Aphelenchoides besseyi is obtained by using BD SMARTTM PCR cDNA Synthesis Kit (Takara, Japan) by taking the cDNA of the Aphelenchoides besseyi as a template, and the specific steps are as follows:

1. 5' RACE amplification:

(1) first round PCR primers:

forward primer UPM: 5'-CTAATACGACTCACTCACTATAGGGC-3'

Reverse primer D2RACER 1: 5'-ATCGCCAATCTGAACGATCAAGCCGCCCAT-3'

Reaction system:

the reaction conditions are as follows: reacting at 94 ℃ for 30s, reacting at 72 ℃ for 3min, and performing 5 cycles; reacting at 94 ℃ for 30s, at 70 ℃ for 30s, at 72 ℃ for 3min, and performing 5 cycles; reacting at 94 ℃ for 30s, at 68 ℃ for 30s and at 72 ℃ for 3min for 25 cycles; reacting at 72 ℃ for 10 min; storing at 4 ℃.

After the reaction is finished, detecting the reaction product by using 1% agarose gel electrophoresis, and storing the residual PCR product at the temperature of-20 ℃.

(2) Second round nested (nest) PCR primers:

forward primer NUP: 5'-AAGCAGTGGTATCAACGCAGAGT-3'

Reverse primer IA-D2R: 5'-CCGACTGTTTCAAGTTGCGAT-3'

Reaction system:

reaction conditions are as follows: reacting at 94 ℃ for 30s, reacting at 72 ℃ for 3min, and performing 5 cycles; reacting at 94 ℃ for 30s, at 70 ℃ for 30s, at 72 ℃ for 3min, and performing 5 cycles; reacting at 94 ℃ for 30s, at 68 ℃ for 30s, at 72 ℃ for 3min, and performing 25 cycles; reacting at 72 ℃ for 10 min; storing at 4 ℃.

After the reaction is finished, detecting the reaction product by using 1% agarose gel electrophoresis, and storing the residual PCR product at the temperature of-20 ℃.

2. Ligation transformation of PCR products

The 5' Race amplification product was purified and ligated with pMD18-T vector (Takara, Japan) to obtain a recombinant plasmid, transformed into Escherichia coli (Escherichia coli) competent cell DH5 α, and positive clones were picked and sent to an industrial biology institute for sequencing (Sangon Biotech, China).

3. Amplification of the full-Length sequence of the Ab-atps Gene

According to the 3' poly A sequence of the target gene obtained by the sequencing result, a primer is designed for carrying out full-length amplification on Ab-Atps gene cDNA of the aphelenchoides besseyi.

PCR primer sequences:

D2F：5’-TTTGAGCGAAATGTCCGCAAG-3’

D2R：5’-AGTCTACGAGTTCCTACTGACGA-3’

reaction system:

reaction conditions are as follows: reacting at 94 ℃ for 2 min; reacting at 98 ℃ for 10s, at 55 ℃ for 30s and at 72 ℃ for 15s for 30 cycles; the reaction was carried out at 72 ℃ for 10 min.

Phylogenetic trees of the amino acid sequences of the ATP synthetases of Ab-ATPS and of the other 23 representative parasites in the database were constructed by the neighbor-join method using MEGA (Molecular evolution Genetics Analysis, USA) software.

Second, experimental results

Extracting positive clone plasmid and sequencing to obtain 301bp PolyA-containing fragment. RACE amplification is carried out on the gene to obtain a cDNA sequence (shown as SEQ ID NO: 1) with the full length of 1341bp, ORF identification is carried out by using ORF finder of NCBI to obtain a complete ORF, and the gene codes 183 amino acids (shown as SEQ ID NO: 2).

The nucleotide sequences of Ab-ATPS of Aphelenchoides besseyi and ATP synthetase of 23 representative nematodes in a protein database are compared to construct a phylogenetic tree (figure 1), and the result shows that the affinity relationship between Ab-ATPS of Aphelenchoides besseyi and ATP synthetase of round nematodes of Lamibo (Strongyloides rati) is the closest, which is consistent with the homology analysis result obtained by BLASTX.

Example 2 measurement of expression amounts of Ab-atps Gene in different insect states of Aphelenchoides besseyi

First, experiment method

1. The MicroElute total RNA kit (OMEGA) was used to extract the RNAs of 500 females, males, larvae and eggs, respectively, of the test Aphelenchus oryzae. The method comprises the following specific steps:

the MicroElute total RNA kit is used, and the micro nematode RNA extraction is carried out according to the instruction operation, and the steps are as follows:

(1) nematodes (<500 rods) were placed into DEPC-treated 1.5mL centrifuge tubes as needed and washed 3 times with DEPC water. The centrifuge tube was placed in a pre-cooled mortar, liquid nitrogen was added to the mortar and the nematode in the tube was immediately ground into a fine powder with a grinding rod, and 350. mu.L of reagent TRK and 7. mu.L of beta-mercaptoethanol were rapidly added.

(2) The vortex shaker shaken for 30s, centrifuged at 1000rpm for 1min, and the supernatant was pipetted into another fresh DEPC-treated 1.5mL centrifuge tube.

(3) To the new tube containing the supernatant was added 350. mu.L of 70% ethanol and mixed well.

(4) The mixture was added to the silica gel filtration column cannula provided in the kit and centrifuged at 1000rpm for 1 min.

(5) The filtrate was decanted, the filter column was transferred to a new 2mL centrifuge tube, 400. mu.L of RWC solution was added, and centrifugation was carried out at 10000rpm for 1 min.

(6) mu.L of DNase-I, 7. mu.L of buffer and 33. mu.L of DEPC water were mixed well and applied to the center of the filter column at 37 ℃ for 30 min. Whether this is done is determined according to whether it is necessary to remove DNA from RNA.

(7) The filter column was transferred to a new 2mL centrifuge tube, 500. mu.L of the solution was added and centrifuged at 10000rpm for 1 min.

(8) And repeating the previous step.

(9) The empty tube of the filter column was centrifuged at full speed (13000rpm) and the filter on the column was dried.

(10) Add 15-20. mu.L DEPC water carefully to the center of the filter, leave at room temperature for 2min, centrifuge at full speed for 1min, and collect RNA. It is used immediately or stored at-80 deg.C.

2. Reverse transcription of the extracted RNA into cDNA

The extracted RNA was reverse transcribed into cDNA using HiScriptTM Q RT Supermix for qPCR (+ gDNA wiper) (Vazyme, China) kit.

3. Ab-atps expression level detection

The Ab-atps expression quantity of eggs, larvae, females and males is respectively determined by taking the synthesized corresponding cDNA as a template according to the operation of a SYBR Green Real-time PCR Master Mix-plus kit instruction, and three repeats are arranged in total.

Ab-atps gene expression level detection primers were used as follows:

QD2F：5’-ACTTCGAGGACATTCTCCGTT-3’

QD2R：5’-CATGATCTCCGGTCGAACCTG-3’

a pair of specific primers Ab18sF and Ab18sR are designed according to an 18S rRNA gene sequence (AY508035) of rDNA of the aphelenchoides besseyi, and a 140bp fragment is obtained by amplification and is used as an internal reference gene, wherein the primer sequences are as follows:

Ab18sF：5’-CTCGTGGTGGCTGGTATGCTG-3’

Ab18sR：5’-GTTTCCCGTGTTGAGTCAAATTAAG-3’

the qPCR was performed using an instrument CFX-96(Bio-Rad), and data analysis for real-time fluorescence quantification was performed using CFX anger software provided by Bio-Rad.

Second, experimental results

The qPCR detection results of the expression quantities of Ab-atps in different insect states of Aphelenchoides besseyi show that the expression quantities of Ab-atps are larval, egg, female and male from high to low, and the expression quantities of the Ab-atps in the first three insect states are 26.79, 13.10 and 4.86 times of the expression quantities of the male (figure 2). Except that the expression amount difference between the female worm and the male worm is not significant (p >0.05), the expression amount difference between other worm states is significant (p < 0.05).

Example 3 in situ hybridization of Ab-atps Gene

First, experiment method

And (3) fixing about 10000 mixed nematode state nematode liquid to a constant volume of 30-50 mu L, adding 3% paraformaldehyde solution, fixing for 18 h at 5 ℃, and fixing for 4h at 22 ℃. Primers were designed based on the full-length cDNA of Ab-atps and used as template for the synthesis of DIG-labeled RNA probe (Roche, Germany) by the following steps:

1. amplification primer of RNA probe in vitro transcription template (DNA)

In the test, an RNA probe of Ab-atps is synthesized by an in vitro transcription method, template DNA required by the in vitro transcription RNA probe is obtained by PCR, and an amplification primer of the template DNA is designed as follows:

A. amplification primers for antisense strand in vitro transcription template:

IA-D2F：5′-TAATACGACTCACTATAGGGAAATACGCGACCAGTTTGTATCAGG-3′

IA-D2R：5′-CCGACTGTTTCAAGTTGCGAT-3′

B. amplification primers for sense strand in vitro transcription template:

IS-D2F：5′-TAATACGACTCACTATAGGGCCGACTGTTTCAAGTTGCGAT-3′

IS-D2R：5′-AAATACGCGACCAGTTTGTATCAGG-3′

2. obtaining of RNA Probe in vitro transcription template (DNA)

Recombinant plasmids containing the full-length Ab-Atps gene were used as DNA templates for transcription of amplified RNA probes. Sense and antisense strand template DNA was amplified using primers IA-D2F and IA-D2R, PCR reaction system formulation and reaction conditions were performed as described in example 1. The elution is carried out by dissolving in RNase-free water to determine the concentration, which should be in the range of 0.5-1. mu.g/. mu.L. The obtained transcription template of the positive and negative strand was stored at 4 ℃.

3. Synthesis of RNA Probe

Sense and antisense RNA probes were synthesized separately, in the following reaction system (20. mu.L total):

reaction conditions are as follows: incubation was carried out at 37 ℃ for 2h and the products were checked by electrophoresis.

4. Purification of probes

10U DNase I and 5 mu L of 10 multiplied DNase I Buffer are added into the prepared probe, and the probe is subjected to water bath at 37 ℃ for 15min to remove DNA, then purification and concentration are carried out, and the RNA probe is added into a hybridization solution containing nematodes. After hybridization was complete, the samples were examined and photographed using a differential interference microscope.

Second, experimental results

The results of in situ hybridization showed that Ab-atps mRNA was localized in the esophagus and reproductive system of female Aphelenchoides besseyi (FIG. 3), and no hybridization signal was detected in the nematodes hybridized with the sense probe.

Example 4 Ab-atps Gene RNAi interference

First, experiment method

RNAi of Ab-atps gene adopts double-stranded RNA (dsRNA) soaking method. The synthesis of double-stranded RNA adopts an in vitro transcription method. The ORF of the Ab-atps gene was cloned into the pMD18-T vector (Takara, Japan) and the cloning was verified by sequencing. dsRNA for silencing Ab-atps gene and control gfp gene was transcribed in vitro by referring to Script MaxTM Thermo T7 Transcription kit (TOYOBO, Japan), purified as dsRNA synthesis product, and analyzed by electrophoresis for integrity, Nano-drop spectrophotometer for quality and concentration, and stored at-80 deg.C. The method comprises the following specific steps:

1. obtaining of in vitro transcribed Single-stranded RNA (Single strand RNA) with positive and negative strands

ssRNA Synthesis methods for Ab-atps and gfp ssRNA for the sense and antisense strands of the partial Ab-atps fragment was obtained as described in example 3. The template for gfp ssRNA synthesis is a plasmid containing the gfp full-length gene, which is stored in the laboratory.

The ssRNA synthesis primer sequences for the sense and antisense strands of the gfp partial fragment are as follows:

antisense strand template primer:

G-T7A：5′-GGATCCTAATACGACTCACTATAGGG CGATGCGGTTCACCAGGGTGTCG-3′

G-S：5′-CACAAGTTCAGCGTGTCCGGCG-3′

sense strand template primer:

G-T7S： 5′-GGATCCTAATACGACTCACTATAGGGCACAAGTTCAGCGTGTCCGGCG-3′

G-A：5′-CGATGCGGTTCACCAGGGTGTCG-3′

2. in vitro transcription Synthesis of double-stranded RNA (dsRNA)

(1) After the synthesis of ssRNA is finished, 5U of RQ1 RNase-free DNase is added, and the reaction is carried out for 15min at the temperature of 37 ℃ so as to digest DNA in the reaction solution;

(2) detecting the quality and concentration of the ssRNA by electrophoresis;

(3) adding equal proportion of sense strand RNA and antisense strand RNA into a new 1.5mL centrifugal tube treated by DEPC, and lightly mixing and shaking up;

(4) incubating at 94 ℃ for 10min, naturally cooling to room temperature, and annealing to form dsRNA;

(5) 5 μ L of RnaseT1 was added using a pipette and incubated at 37 ℃ for 30 min.

(6) 10U DNase I, 5 mu L of 10 XDNase I Buffer and water bath at 37 ℃ are added into the prepared probe for 15min to remove DNA, then purification and concentration are carried out, the concentration is detected, and the prepared probe is stored at-80 ℃ for later use.

3. dsRNA soaking treatment

The aphelenchoides besseyi cultured on carrot callus is separated and collected in DEPC treated centrifuge tubes, 500 mixed entomorphus nematodes are collected in each centrifuge tube, 50 mu L of Ab-atps dsRNA (2 mu g/mu L) is added for soaking for 12h, 24h, 36h and 48h respectively, 50 mu L of gfp dsRNA (2 mu g/mu L) is used for soaking as treatment contrast, 8 treatments are carried out, and each treatment is repeated for 3 times. The soaking was carried out at 25 ℃.

4. Ab-atps silencing efficiency detection after soaking treatment

The above nematodes were treated, washed three times with DEPC water, RNA was extracted, and Ab-atps expression level was measured by qPCR, in the same manner as in example 2. Each treatment was repeated 3 times and each replicate sample technique was repeated 2 times, with the final results averaging 6 tests.

Second, Experimental methods

The obtained Ab-atps dsRNA is double-stranded RNA consisting of a nucleotide sequence shown as SEQ ID NO.3 as a sense strand and a nucleotide sequence which is reversely complementary to the nucleotide sequence shown as SEQ ID NO.3 as an antisense strand.

After soaking Aphelenchoides besseyi dsRNA with Ab-atps, the silencing efficiency of Ab-atps gene is detected by qPCR. The results show (fig. 4) that the relative expression of Ab-atps is reduced by 86.7%, 80.7%, 86.3% and 75.4% respectively compared with corresponding gfp dsRNA soaked for the same time for nematodes treated by Ab-atps dsRNA soaking for 12h, 24h, 36h and 48h, and the difference is significant (p is less than 0.05); the silencing efficiency of the Ab-atps gene RNAi treatment differed insignificantly over time (p > 0.05); the difference of the expression amount of gfp dsRNA soaked in the control group at each treatment time is not significant (p > 0.05).

Example 4 interaction of Ab-atps Gene with Rice OsRLK3 Gene

First, experiment method

The Ab-atps gene was constructed in full length into the AD domain of the vector pGADT7 according to the Cloneexpress II One Step Cloning Kit (Vazyme, China).

The sequence of the amplification primer is as follows:

ADD2F：5’-CAGCTCGAGCTCGATGGATCCTCCGCAAGTCGTGCCGCC-3’

ADD2R：5’-GCCATGGAGGCCAGTGAATTCTGATGGTCTCGTTGAGTTTCTTGA-3’

reaction system:

reaction conditions are as follows: reacting at 94 ℃ for 2 min; reacting at 98 ℃ for 10s, at 55 ℃ for 30s and at 72 ℃ for 15s for 30 cycles; the reaction was carried out at 72 ℃ for 10 min.

The double cleavage sites are NcoI/BamHI. After the sequence was confirmed, the plasmid was ligated to obtain a recombinant plasmid and transformed into a competent cell of Escherichia coli (DH 5. alpha.); positive clones were picked for sequencing (Sangon Biotech, China) and recombinant plasmids were extracted. The ligated vector was transformed into Y187 yeast competent cells, followed by small scale co-transformation experiments with Y2H yeast containing OsRLK3 gene, and the whole of the bacterial solution was spread on QDO/XA/AbA plates, which were sealed with a sealing membrane, and cultured in an inverted state at 30 ℃ for 3-5d, to generate blue colonies indicating the interaction between AD and BD, according to the instructions of Matchmaker two-hybrid system (Clontech, USA).

Second, experimental results

Reconstructing the coding region of the gene Ab-ATPS to a Gal4-AD (pGADT7) structural domain, carrying out small-scale co-transformation on the Gal4-BD (pGBKT7) structural domain and an OsRLK3 sequence fragment containing a signal peptide removed, carrying out positive clone screening by using a QDO culture medium, after culturing for 3-7 days, selecting a monoclonal blot, culturing on the QDO/XA culture medium, wherein colonies within 48h show blue consistent with a positive control, and the co-transformed pGADT7 empty vector and pGBKT7-OsRLK3 yeast, pGADT7-Ab-ATPS and pGBKT7 empty vector yeast which are used as negative controls do not grow on the QDO/XA culture medium. This result indicates that there is an interaction between OsRLK3 and Ab-atps (FIG. 5).

Example 5 Effect of Ab-atps Gene silencing on the Productivity of Aphelenchoides besseyi and on the expression level of OsRLK3 Gene in Rice

First, experiment method

1. Mixed-worm Aphelenchus aphrodisiae were treated with Ab-atps dsRNA of example 3 by soaking for 12h, 24h, 36h and 48h, respectively, and with gfp dsRNA of example 3 for 12h, 24h, 36h and 48h as controls. 30 females were picked for each treatment and inoculated on carrot callus, and each treatment was repeated 9 times. And (4) placing the inoculated carrot callus in an incubator at 25 ℃ for dark culture for 35d, and separating and counting nematodes on the carrot callus.

2. Mixed-pest aphelenchoides besseyi is soaked and treated with Ab-atps dsRNA and gfp dsRNA of example 3 for 48h, rice not inoculated with aphelenchoides besseyi is treated as a blank control, the RNA Extraction of the overground parts of rice plants of DAI0.5, DAI1, DAI2, DAI3 and DAI7 is carried out by using General RNA Extraction Kit (Dongsheng, China), and rice (CK) not inoculated with nematodes at the same time point is used for detecting the expression quantity of OsRLK3 genes of rice, and the qPCR and data analysis methods are the same as example 2. Each treatment was performed in 3 replicates, each replicate sample consisted of 3 rice plants, and each sample was PCR replicated twice.

Second, experimental results

After soaking Aphelenchoides besseyi dsRNA with Ab-atps, the silencing efficiency of Ab-atps gene is detected by qPCR. The results show (fig. 6A) that the relative expression of Ab-atps, which is decreased by 86.7%, 80.7%, 86.3% and 75.4% compared to the corresponding gfp dsRNA soaked for the same time in nematodes soaked for 12h, 24h, 36h and 48h by Ab-atps dsRNA, respectively, is significantly different (p < 0.05); the silencing efficiency of the Ab-atps gene RNAi treatment was not significantly different with time (p > 0.05); the difference of the expression amount of gfp dsRNA soaked in the control group at each treatment time is not significant (p > 0.05). The breeding results after Ab-atps dsRNA soaking treatment of aphelenchoides besseyi at different time and culturing on carrot slices for 35d show (figure 6A): ab-atps were RNAi treated with 12h, 24h, 36h and 48h of Aphelenchoides oryzae with significantly lower numbers of colonies (p <0.05) than control nematodes treated with gfp dsRNA soaked for the same time period, and with increasing soaking time the number of colonies decreased, except that the difference between treatments 12h and 24h and 36h and 48h was not significant (p >0.05), and the difference between other treatments was significant (p < 0.05). The breeding amount of each treated nematode soaked by gfp dsRNA has no obvious difference (p is more than 0.05) along with the increase of soaking time, and is not obviously reduced along with the increase of the treating time within 48 h.

After Ab-atps dsRNA and gfp dsRNA are selected to soak for 48h and the aphelenchoides besseyi is inoculated on rice, the detection result of the expression quantity of the OsRLK3 gene of the rice shows that the expression quantity of the OsRLK3 gene is obviously lower than that of gfp dsRNA treatment (p <0.05) in DAI0.5 Ab-atps dsRNA treatment, is obviously higher than that of gfp dsRNA treatment (p <0.05) in DAI 1Ab-atps dsRNA treatment, and has no obvious difference (p >0.05) in DAI 2-7 treatment (FIG. 6B). In the Ab-atps dsRNA treatment, the expression level of OsRLK3 is remarkably highest in DAI1 (p <0.05), and in the gfp dsRNA treatment, the expression level of OsRLK3 is remarkably highest in DAI 0.5. Therefore, the aphelenchoides besseyi after the Ab-atps gene silencing has influence on the expression of the OsRLK3 gene of the rice, and the interaction relationship between the Ab-atps and the OsRLK3 is also shown.

Example 6 transient expression of Ab-atps Gene

First, experiment method

1. Plant expression vector construction

The plant transient expression vector used was pCAMBIA1300, and Ab-atps and gfp genes were ligated to the same pCAMBIA1300 vector in full length according to Clonexpress II One Step Cloning Kit (Vazyme, China), respectively. Separately, pCAMBIA1300 vector linked only to gfp gene was constructed as a control by the same method. The method comprises the following specific steps:

pCAMBIA1300-GFP vector construction:

the primer sequence is as follows:

GF：5’-CGCGTCGACATGTCCGCAAGTCGT-3’

GR：5’-TGGCTGCAGTTAAACGGCCTCTTTGAT-3’

the PCR reaction system and conditions were as in example 1.

pCAMBIA1300-Ab-ATPS-GFP vector construction primers:

PD2F：5’-ATCGAGCTCATGGTGAGCAAGGGC-3’

PD2R：5’-CGCGGATCCCTTGTACAGCTCGTC-3’

the PCR reaction system and conditions were as in example 1. The template is a full-length sequence fragment of Ab-atps gene.

The double enzyme cutting sites connecting the Ab-atps gene sequence are SalI/PstI; the green fluorescent protein (gfp) gene sequence double-enzyme cutting site is SacI/BamHI; ab-atps and gfp genes were ligated to the same pCAMBIA1300 vector in full length according to Clonexpress II One Step Cloning Kit (Vazyme, China), respectively. Separately, pCAMBIA1300 vector linked only to gfp gene was constructed as a control by the same method, and the constructed vector was transformed into E.coli DH5 alpha. And selecting positive clones, sequencing and verifying, collecting bacterial liquid, and extracting plasmids for later use.

2. Agrobacterium GV3101 transformation

The ligated vector is transformed into Agrobacterium GV3101 competent cells, as described in the following text. Positive clones were selected and verified by PCR for future use. Adding 50% glycerol with equal volume times into the engineering agrobacterium liquid with correct identification, freezing with liquid nitrogen, and storing at-80 ℃.

3. Tobacco injection

The positive clones were selected and cultured at 28 ℃ and 250rpm for 24-48 hours in LB liquid medium containing 50. mu.g/mL of Rif and 50. mu.g/mL of Kan. The cells were concentrated by centrifugation at 6000rpm for 5min and washed 3 times with 10mM MES (pH 5.5). Then diluting the thallus with MES to OD600 ═ 0.5, adding acetosyringone with final concentration of 100 μ M, and standing at 28 deg.C in dark for 4 h; slightly pricking a box of small holes on the leaves of the four-week-old tobacco by using a needle of a 1m L syringe, and then taking a proper amount of bacteria liquid by using the syringe without the needle to pour the bacteria liquid into the tobacco leaves at the small holes. Each leaf was punched with 4 wells and injected with engineered Agrobacterium and MES carrying pCAMBIA1300-Ab-ATPS-GFP, pCAMBIA1300 empty vector plasmid, respectively, to observe whether the leaf generates cellular necrosis and to observe the gene expression using a fluorescence microscope.

4. Tobacco Total protein extraction

(1) The extraction of the total protein of the tobacco leaves is carried out according to the instruction of a plant protein extraction kit (purchased from Kaikyi biology Co., Ltd.), and the specific steps are as follows:

(2) respectively collecting tobacco leaf discs injected with engineering agrobacterium with pCAMBIA1300-Ab-ATPS-GFP and pCAMBIA1300-GFP plasmids and MES, adding liquid nitrogen for full grinding, and avoiding repeated freeze thawing;

(3) transferring the ground powder into a 1.5mL centrifuge tube, and adding 300 mu L of lysate;

(4) adding appropriate amount of protease inhibitor into the above liquid, and placing on ice for 30min, and inverting the centrifuge tube several times;

(5) centrifuging at 14000rpm for 20min at 4 deg.C to obtain supernatant as tobacco protein solution, and storing at-20 deg.C.

5. SDS electrophoresis

6、Western blot

The target protein is subjected to western blot detection by using a GFP antibody (purchased from Beijing Quanyu gold Biochemical Co., Ltd.), and the specific steps are as follows:

(1) after electrophoresis is finished, the gel is gently taken out of the glass plate and is placed into a container filled with a membrane transferring liquid for soaking for 10 min;

(2) the PVDF film was cut to the size of a business card, and gloves were used to prevent contamination of the protein components on the hands. Soaking the PVDF membrane in a methanol solution for 10-30s, soaking the PVDF membrane in distilled water, oscillating for 5min, and finally soaking the PVDF membrane in a membrane transferring solution, oscillating for 10 min;

(3) cutting filter paper into the size of a business card, wherein 8 pieces of filter paper are needed, and soaking the business card in a membrane transferring solution. Stacking the filter paper 4 pieces → SDS-PAGE gel → PVDF membrane → filter paper 4 pieces → anode in the order from the cathode to avoid bubbles between the gel and the membrane;

(4) area (cm) of PVDF membrane ² ) Electrifying for 1h under the current intensity calculated by a formula of multiplied by 0.8mA, immersing the PVDF membrane into a plastic container containing PBST after the membrane is rotated, and oscillating for 5min to clean the membrane;

(5) immersing the PVDF membrane into a 5% skimmed milk powder solution, and oscillating for 1h at room temperature;

(6) the primary antibody was diluted with PBS at a ratio of 1: 5000, and the PVDF membrane was immersed in the primary antibody dilution solution and shaken at room temperature for 2 hours to bring the PVDF membrane and the primary antibody into full contact.

(7) Wash 3 times with PBST shaking for 10min each time.

(8) The secondary antibody was diluted with PBS at a ratio of 1: 5000, and the PVDF membrane was immersed in the secondary antibody dilution solution and shaken at room temperature for 1 hour to bring the PVDF membrane and the secondary antibody into full contact.

(9) Wash 3 times with PBST shaking for 10min each time.

(10) The gel imager photographed.

Second, experimental results

To further confirm the location of the Ab-ATPS in plant cells, it was fused with GFP and transiently expressed in tobacco epidermal cells (FIG. 7). In tobacco cells expressing GFP protein alone and Ab-ATPS-GFP fusion expression, fluorescence is distributed to the nucleus, cytoplasm and cell membrane. Transient expression tobacco leaf protein is extracted and used for Western blot detection, and the result shows that a GFP protein control band appears at about 40kDa (the size is about 37kDa), and an Ab-ATPS-GFP protein band appears between 50 kDa and 70kDa (the size is about 57kDa), which indicates that the Ab-ATPS-GFP fusion protein is expressed correctly. Cell death occurred at the site of Agrobacterium containing pCAMBIA1300-Ab-ATPS-GFP plasmid injection 5d after Agrobacterium injection into transient expression leaf, whereas there was no change at the site of Agrobacterium containing pCAMBIA1300-GFP plasmid injection, MES buffer and pCAMBIA1300 empty vector plasmid injection, thus Ab-ATPS protein was thought to trigger apoptosis at the inoculation site (FIG. 7C).

It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the scope of the present invention, and those skilled in the art can make other variations or modifications based on the above description and idea, and it is not necessary or necessary to exhaust all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> south China university of agriculture

<120> ATP synthetase of rice tip nematode and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1311

<212> DNA

<213> Aphelenchoides besseyi

<400> 1

aagcagtggt atcaacgcag agtattggtg tcacagaacc gttgcaatgc gttgacattc 60

ggtaacttct cttcgaccaa attcaaactc accaaatgcg atgcaatccg gagaatgtaa 120

gattttcgag agtcggctgc atcttttgcc gaactcatga cctccgatga aaactgaaat 180

tccgaacgtt aacagtagtg atgagaggac caattaacag atttcaaacg gaatcctgat 240

gataactcgt tgttcacgac cctcgaacgg catggcaaca agccgcaaag aggccccaac 300

aactcctata actctatggc aaccgtcgaa ccagctagct gtttggtttt cttgctgttt 360

ctttcagtaa tttcgaaatt agcaatttga ttctacttga actgtgagat gactttcgaa 420

acgtgagaat tttcaacctt ttttgttcta aactaaattt tcagtgagaa gctgagaagc 480

gagaagagaa actctcggag cgatcgtgcc tgttcggcac gttgctcttc tccgcgtccg 540

tatggtcgat cggctttcgt ccttttctcg tggacaagaa tttcgttgag ttagacgtga 600

ttttgttttc tgatttccga ctttctgtga ccaaaaattt gcgaaacgtg attccacacc 660

ttctttacga ccgttttcag cgaaatgtcc gcaagtcgtg ccgccggtgt tccgcgcaaa 720

tacgcgacca gtttgtatca ggcggcaaaa aaactgaaca agttggacgc tgtggaaaag 780

gacgtgaaga tcgtaaaaga tttgtacgcg tctgatcaga agttttcggc gtttgtcaag 840

aatccgacgt tgaatcgcaa cttgaaacag tcggcgttga cgagtgttct gaagtcaatt 900

ggcgtttcct cggaaacgca aaaattcttc ggagttttgg ctgaaaatgg acgacttgga 960

tttttaaacg aggtgctcgt caacttcgag gacattctcc gttccaaccg cggcgatttg 1020

accgtcgaag ttgtttcggc cgacgcgttg aacgacgcca ccaaacgctc catcagcgat 1080

gcactcggaa agagtagtaa gagcgtctcg atcacgtatc aggttcgacc ggagatcatg 1140

ggcggcttga tcgttcagat tggcgatcgt cgtttggatc tgtcgatcgc atcgcgtgtc 1200

aagaaactca acgagaccat caaagaggcc gtttaatgat agggatgttc gtcagtagga 1260

actcgtagac tgtgtgatta tttggagaaa taaaaatata aacttgtcgt g 1311

<210> 2

<211> 183

<212> PRT

<213> Aphelenchoides besseyi

<400> 2

Met Ser Ala Ser Arg Ala Ala Gly Val Pro Arg Lys Tyr Ala Thr Ser

1 5 10 15

Leu Tyr Gln Ala Ala Lys Lys Leu Asn Lys Leu Asp Ala Val Glu Lys

20 25 30

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

35 40 45

Ala Phe Val Lys Asn Pro Thr Leu Asn Arg Asn Leu Lys Gln Ser Ala

50 55 60

Leu Thr Ser Val Leu Lys Ser Ile Gly Val Ser Ser Glu Thr Gln Lys

65 70 75 80

Phe Phe Gly Val Leu Ala Glu Asn Gly Arg Leu Gly Phe Leu Asn Glu

85 90 95

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

100 105 110

Thr Val Glu Val Val Ser Ala Asp Ala Leu Asn Asp Ala Thr Lys Arg

115 120 125

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

130 135 140

Tyr Gln Val Arg Pro Glu Ile Met Gly Gly Leu Ile Val Gln Ile Gly

145 150 155 160

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

165 170 175

Glu Thr Ile Lys Glu Ala Val

180

<210> 3

<211> 157

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaatacgcga ccagtttgta tcaggcggca aaaaaactga acaagttgga cgctgtggaa 60

aaggacgtga agatcgtaaa agatttgtac gcgtctgatc agaagttttc ggcgtttgtc 120

aagaatccga cgttgaatcg caacttgaaa cagtcgg 157

Claims (8)

1. An ATP synthase gene of aphelenchoides besseyi is characterized in that the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

2. An ATP synthetase for aphelenchoides besseyi, characterized in that the amino acid sequence is shown as SEQ ID NO: 2, respectively.

3. The use of the inhibitor of an ATP synthase gene of aphelenchoides besseyi according to claim 1 in the preparation of a medicament for the control of aphelenchoides besseyi, wherein the inhibitor is dsRNA consisting of a sequence as shown in SEQ ID NO: 3 as a sense strand and consisting of a nucleotide sequence identical to SEQ ID NO: 3 as a double-stranded RNA consisting of an antisense strand.

4. The use of the Aphelenchoides besseyi ATP synthase gene according to claim 1 as a target for screening drugs against Aphelenchoides besseyi.

5. An inhibitor of the Aphelenchoides besseyi ATP synthase gene of claim 1, wherein the inhibitor is a dsRNA consisting of a sequence as set forth in SEQ ID NO: 3 as a sense strand and consisting of a nucleotide sequence identical to SEQ ID NO: 3 as a double-stranded RNA consisting of an antisense strand.

6. A gene encoding the dsRNA of claim 5.

7. An expression vector, transgenic cell line or host bacterium comprising the gene of claim 6.

8. Use of any one or more of the gene of claim 6 or the expression vector, the transgenic cell line or the host bacterium of claim 7 in the preparation of a drug for controlling aphelenchoides besseyi.

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