CN109666675B - Nilaparvata lugens NlAtg3 gene, encoding protein and application thereof - Google Patents

Abstract

The invention discloses a brown planthopperNlAtg3Genes, encoded proteins and uses thereof.NlAtg3The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the gene is necessary for the normal survival of the brown planthopper, and the survival rate of the brown planthopper is reduced due to the inhibition of the function of the gene.NlAtg3The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 2. SaidNlAtg3The application of the gene or the protein is used for researching and developing pesticides and biologically preventing brown planthopper. The invention successfully implements RNA interference on the gene, and the result shows that the survival rate of pests is reduced, and the invention is expected to fully play the function of ecological prevention and control while realizing the inhibition of the pests.

Description

Nilaparvata lugens NlAtg3 gene, encoding protein and application thereof

Technical Field

The invention relates to a Nilaparvata lugens NlAtg3 gene, a coding protein and application thereof.

Background

Brown planthopper (Nilaparvata lugensSt å l) is a monophagic rice pest, which mainly damages rice by eating rice phloem juice. At present, the chemical control is mainly used for emergency control of brown planthoppers, but the chemical control easily causes the problems of the rise of the drug resistance of pests, the residue of harmful substances on rice, the rampant pests, environmental pollution and the like. In the pesticides for controlling brown planthoppers, effective pesticides which once have better effects, such as fipronil, imidacloprid, buprofezin and the like, are eliminated or limited in use, and for the reasons, the chemical pesticides mostly aim at killing pests in a large scale and high strength, but due to the practical reasons that the genetic diversity of brown planthoppers is very complex and the like, part of individuals can survive, and the formation of populations with stronger adaptability can be finally caused as a result of the action of high selective pressure. On the other hand, chemical pesticides can also endanger non-target organisms including natural enemies while killing pests, and inevitably cause negative effects on the ecological system of the farmland, so that the ecological control function cannot be exerted. Therefore, screening and discovering a new target for controlling the brown planthopper and adjusting the current strategy for controlling the brown planthopper have important practical significance.

CN107988244A discloses an ATPSb gene related to the survival of brown planthopper, a coded protein and application thereof, wherein the gene plays an important role in maintaining the normal survival of the brown planthopper, and the survival rate of the brown planthopper is reduced due to the inhibition of the gene.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a brown planthopper NlAtg3 gene, a coding protein and application thereof. The invention is based onNlAtg3Gene-encoded proteins that are relatively conserved but nucleic acid sequences and methods of making the sameThe method has the characteristic of low homology of other organisms, RNA interference is carried out on a target gene, the suppression of the brown planthopper at a nucleic acid level is realized, the survival rate of the brown planthopper is obviously reduced, and the possible killing of non-target organisms caused by pesticide spraying and the like at a protein level is avoided.

The technical scheme of the invention is as follows:

brown planthopperNlAtg3The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the gene codes ATG3 protein which is necessary for normal survival of brown planthopper, and the survival rate of the brown planthopper is reduced due to the inhibition of the function of the gene.

The brown planthopperNlAtg3The gene coded protein has an amino acid sequence shown in SEQ ID NO. 2, and the inhibition of the gene function can cause the survival rate of brown planthopper to be reduced.

The brown planthopperNlAtg3The application of the gene is used for researching and developing pesticides and biologically controlling brown planthopper.

Aiming at the brown planthopperNlAtg3The application of the RNA interference technology of the gene in controlling the brown planthopper leads the survival rate of the brown planthopper to be reduced.

The brown planthopperNlAtg3The application of the gene coded protein is used for researching and developing pesticides and biologically preventing brown planthopper.

The invention has the beneficial effects that: (1) The survival rate of the brown planthopper is reduced, and the direct damage of the pests on rice crops due to feeding can be reduced. (2) The invention utilizes the characteristic of lower homology of the nucleotide sequence of the target gene and the nucleotide sequence of the natural enemy, can carry out RNA interference on the level of nucleic acid, avoids the damage to non-target organisms such as the natural enemy and the like due to the conservative protein structure, and is expected to fully play the function of ecological prevention and control while realizing the inhibition of pests.

Drawings

FIG. 1 shows brown planthopperNlAtg3mRNA expression level of the gene. Wherein, 1-2N is 1-2 years nymph; 3-4N for 3-4 years nymphs; 5N, 5-year-old nymphs; e1-9, eclosion of female brown planthopper for 1-9 days.

FIG. 2 shows RNA interference against Nilaparvata lugensNlAtg3Influence of the amount of gene expression;

data in the figure are mean ± standard deviation of 3 replicates, asterisks indicate very significant difference between treatment and control on statistical analysis: (TAnd (4) checking the test result,P<0.01). dsGFP: a control group;dsNlAtg3：dsNlAtg3fed RNA interference group.

FIG. 3 is a schematic view ofNlAtg3Influence of RNA interference of the gene on survival rate of brown planthopper;

data in the figure are mean ± standard deviation of 3 replicates, asterisks indicate significant difference between treatment and control groups on statistical analysis: (TAnd (4) checking the test result,P<0.05 Double asterisks indicate significant differences between treatment and control groups on statistical analysis: (TAnd (4) checking the test result,P<0.01）。

Detailed Description

The ATPSb gene is a subunit of ATP synthase in mitochondria, which is responsible for ATP synthesis in mitochondria, and the function of the ATP synthase is interfered to influence ATP synthesis in mitochondria (CN 107988244 a).

NlAtg3The gene is a gene which codes ATG3 protein in brown planthopper, and ATG3 protein is a kind of ubiquitination E2 protein in the process of cell Autophagy (Autophagy), which catalyzes ATG8 protein Phosphatidylethanolamine (PE). Autophagy is a cell autophagy action that is ubiquitous in organisms and is an evolutionarily conserved important process in eukaryotes for turnover of intracellular material. In the process, some damaged proteins or organelles are wrapped by autophagic vesicles (autophagosomes) with a double-layer membrane structure and then are delivered into lysosomes for degradation and recycling. Many Autophagy proteins (ATGs) are involved in the formation of Autophagy bodies and are finally fused with lysosomes to complete the degradation of target proteins. Autophagy of cells plays an important role in the development process of insects,NlAtg3the gene is interfered, and the autophagy process of insect cells can be influenced, so that the normal growth and development of the insects are influenced.

Therefore, the ATPSb gene is involved in the synthesis of ATP in the mitochondria of cells,NlAtg3the gene is involved in the autophagy process of cells, and the two genesThe two genes belong to different cell physiological processes and are different in nature as different targets for controlling the brown planthopper.

The invention is further described with reference to the following figures and detailed description.

Example 1

1. Materials and methods

1.1 Test brown planthopper

The test brown planthopper population is a Tn population raised on an insect-susceptible rice variety TN1, and is continuously raised on TN1 for more than 60 generations by the laboratory, wherein the raising temperature is 26 +/-2 ℃, the relative humidity is 80% +/-5%, and the light cycle is 12L.

Primary reagent

TaKaRa MiniBEST Universal RNA Extraction Kit，TaKaRa MiniBEST Agarose Gel DNA Extraction Kit，PrimeScript RT reagent Kit With gDNA Eraser，DNA 2000 Marker，Premix Taq™（TaKaRa TaqVersion 2.0 plus dye), SYBR Premix Ex Taq were purchased from TaKaRa, SMARTer RACE 5'/3' Kit User Manual from Clontech, MEGAscript T7 High Yield transformation Kit from Ambion, sequencing and primer synthesis were accomplished by Shanghai Sangni Biotechnology Limited.

Nilaparvata lugens (Nilaparvata lugens)NlAtg3Cloning of full-Length cDNA of Gene

Collecting mixed materials of the brown planthopper nymphs and adults of different ages on TN1 rice, and immediately putting the mixed materials into liquid nitrogen for freezing and storing. When sampling, 30-50 nymphs and 5 adults are taken. Total RNA was extracted using TaKaRa MiniBEST Universal RNA Extraction Kit, and the detailed procedures were performed according to the instructions. RNA integrity and purity was checked by agarose gel electrophoresis and Nanodrop 2000 (Thermo). Using 1. Mu.g of total RNA as a template, cDNA was synthesized by reverse transcription using PrimeScript RT reagent Kit With gDNA Eraser Kit, and stored at-20 ℃ for future use.

Obtaining a partial core sequence of the Nilaparvata lugens NlAtg3 gene according to transcriptome sequencing sequence information of the laboratory, and comparing and identifying the sequence through an NCBI website. Primer design Using Primer Premier 5.0 softwareNlAtg3-F andNlAtg3-R (Table 1), performed on core sequencesAnd (6) verifying. And (3) verifying the core sequence by taking mixed brown planthopper cDNA of different ages on TN1 as a template.NlAtg3Cloning of full-length cDNA was performed according to Hao et al (2015) using a 50. Mu.L PCR amplification reaction system including 25. Mu.L PCR Mix, 2. Mu.L each 10. Mu. Mol/L forward and reverse primers, 2. Mu.L cDNA template, and 19. Mu.L ddH 2O. The PCR reaction program is: 4 min at 94 ℃; 30 cycles of 94 ℃ for 30 s,55 ℃ for 30 s,72 ℃ for 3 min; 10 min at 72 ℃; storing at 4 deg.C. The PCR amplification product is detected by 1% Agarose Gel electrophoresis, a target fragment is recovered by TaKaRa MiniBEST Agarose Gel DNA Extraction Kit, the target fragment is connected to a carrier pMD-18T for overnight at 4 ℃, the target fragment is transformed into JM109 competent cells, 1mL LB liquid culture medium is added into the JM109 competent cells for shake culture at 37 ℃ and 2 h is taken, 200 mu L of bacterial liquid is taken and coated in an LB solid culture medium containing 1% Amp for shake culture at 37 ℃ and 9 h is taken for carrying out shake culture at 37 ℃ in a 1.5 mL centrifugal tube of an LB liquid culture medium 1mL containing 1% Amp for 12 h at 37 ℃,1 mu L of bacterial liquid is taken for PCR identification of a positive clone strain and sent to Shanghai Sangny biological technology Limited for sequencing. And (3) comparing and verifying the sequencing result by using DNAMAN software and the original sequence.

Selecting an RNA sample meeting the requirement, and synthesizing a template of 5'-RACE and 3' -RACE by using SMARTer RACE 5'/3' Kit User Manual. Design of outer primers separatelyNlAtg3-5O andNlAtg3-3O (Table 1), and inner primerNlAtg3-5I andNlAtg3-3I (Table 1). According to the RACE kit specification, the 2-end of the target gene is amplified by adopting nested PCR, subjected to electrophoresis, gel recovery, connection, transformation, sequencing and sequencing results are compared and spliced by using DNAMAN software to obtainNlAtg3The full-length cDNA of (1). Designing a full-length verification primer according to the obtained full-length cDNA sequenceNlAtg3-FL-F andNlAtg3FL-R (Table 1) verified the spliced full-length sequence.

TABLE 1 Gene cloning, fluorescent quantitative PCR and primers for synthesizing dsRNA

1.4 Nilaparvata lugens (Nilaparvata lugens)NlAtg3Sequence analysis of genes

Based on DNAMMAN spellingObtained byNlAtg3Full-length cDNA sequence information, open reading frame and protein translation conditions are predicted by using open reading frame analysis software (ORF finder https:// www.ncbi.nlm.nih.gov/orffinder /), amino acid sequence homology alignment is carried out by using NCBI Blastx, molecular weight, theoretical isoelectric point and the like of protein are predicted by using online tool ExPASY (http:// web. Expass. Org/protparam /), signal peptide is predicted by using SignalP 4.1 server (http:// www.cbs.dtu.dk/services/SignalP /), and functional domain of protein is predicted by using online tool ProScan (http:// www.ebi.ac.uk/interpro/search/sequence-search).

Nilaparvata lugens (Nilaparvata lugens)NlAtg3Analysis of expression regularity of gene

The relative expression amounts of the NlAtg3 gene in brown planthopper populations at different ages on TN1 rice are detected by a fluorescent quantitative PCR technology, wherein the relative expression amounts comprise nymphs at 1-2 ages, 3-4 ages and 5 ages, and female adults and males at 1, 3, 5, 7 and 9 days after eclosion. The specific primer of the fluorescent quantitative PCR is QNlAtg3-F and QNlAtg3-R (Table 1), using RPS11 gene as internal reference (Yuan et al, 2014), detecting brown planthopperNlAtg3Relative expression level of gene. The fluorescent quantitative PCR refers to a reaction system and a method of Ma Yan and the like (2013), wherein the annealing temperature is changed to 53 ℃.

Feeding method for RNAi

Design of primer ds for synthesizing dsRNA interference fragment based on cDNA full-length sequenceNlAtg3-F and dsNlAtg3-R (table 1) and a protecting base (GGATCC) and T7 promoter (TAATACGACTCACTATA) at the 5' end of the specific primer. In order not to affect the transcript level detection after RNAi, the interference fragment does not include the fragment detected by fluorescent quantitative PCR. dsRNA was synthesized according to MEGAscript T7 High Yield transformation Kit (Ambion) Kit instructions. The synthesized dsRNA is purified by a LiCl precipitation method: adding 30ul ddH2O and 30ul LiCl Precipitation Solution into a dsRNA reaction system, standing at-20 ℃ for about 1h, centrifuging at 11000rpm at 4 ℃ for 15min, removing supernatant, adding 1ml of 70% ethanol prepared by DEPC water, washing, centrifuging, removing ethanol, adding 20ul ddH ₂ Dissolving O, and standing at-20 ℃ for use.

The method comprises the steps of taking 2-year brown planthopper nymphs bred on TN1 rice varieties to carry out artificial breeding, wherein breeding conditions refer to a nutrient solution formula of Fu et al (2001), a feeding device is a double-pass glass tube (2.5 cm X15 cm) with two ends covered with Parafilm (containing feeding solution), carrying out RNA interference treatment after the feeding solution is fed for 5 days for adaptation, and preparing purified dsRNA (double-stranded ribonucleic acid) with the feeding solution to a final concentration of 0.5 mu g/mu L. During treatment, the feeding liquid is added with dsRNA diluent with a corresponding volume to serve as a control group (CK), 20 brown planthopper nymphs which are subjected to pretreatment and basically consistent in development are connected to each feeding device, 3 times of treatment are set, the feeding liquid is replaced every day, dead brown planthoppers are cleaned and counted, and the survival rate is calculated.

In addition, parallel RNA interference treatment is arranged, 25 brown planthopper nymphs which are grown uniformly are placed in each feeding device, two tubes are used as one repeat, each group is sampled every 2 days, 6 nymphs are taken in each group, and RNA extraction and follow-up are carried outNlAtg3And (3) carrying out fluorescent quantitative PCR detection on the gene expression quantity.

Data statistics and analysis

Data arrangement is carried out by adopting WPS Excel software and SPSS16.0 independent sample is appliedTThe test was conducted for significant difference analysis.

Results and analysis

2.1 Nilaparvata lugens (Nilaparvata lugens)NlAtg3cDNA full-Length cloning and sequence analysis of Gene

Analysis of obtained brown planthopper autophagy related gene by using biological softwareNlAtg3The full-length cDNA sequence of the gene. Using the ORF Finder analysis it was found that,NlAtg3the ORF of the gene is 990 bp (SEQ ID NO: 1) in total and encodes 329 amino acids (SEQ ID NO: 2). ExPASy software analysis findingNlAtg3The gene-encoded protein has a molecular weight of 37.78 kDa and an isoelectric point (pI) of 4.46. Protein domain prediction discoveryNlAtg3The gene has a typical N-terminal (amino acids 8-158) and C-terminal structure (amino acids 300-323).

Nilaparvata lugens (Nilaparvata lugens)NlAtg3Analysis of expression regularity of gene

Quantitative PCR detection Using fluorescenceNlAtg3The expression rule of the gene in different development stages of the brown planthopper is found,NlAtg3the genes are expressed in different development stages of brown planthopper, nymph of 1-2 years is relatively high, nymph of 3-4 years is intermediate, and nymph of 5 years is lowest (figure 1, left); the female did not change significantly in each developmental stage (figure 1, right).

Interference on Nilaparvata lugensNlAtg3Effect of Gene expression level

The fluorescent quantitative PCR detects the brown planthopper injected with 5 mug/mul dsRNA 4d, and the result shows that,NlAtg3the expression level after gene injection of dsRNA is greatly reduced, and compared with a dsGFP control group, the expression level achieves the extremely obvious difference (P)<0.01 (FIG. 2).

2.4 NlAtg3Effect of RNA interference of genes on survival rate of brown planthopper

RNA interference results found, injection ds NlAtg3The lethal effect is significant, on day 4 of RNA interference, ds is injectedNlAtg3The survival rate of the treated group was significantly different from that of the control group (P)<0.05 Very significant difference (P) from the control group was achieved from day 5 onwards<0.01 Only 6.67% survival by day 7 (figure 3).

Sequence listing

<110> China metering university

<120> brown planthopper NlAtg3 gene, encoding protein and application thereof

<141> 2018-11-21

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<212> DNA

<213> Brown planthopper (Nilaparvata lugens Stal)

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atgcaaagtg ttattaatac tgtaaaaggg actgctctcg gtgttgctga gtatttaaca 60

cctgttttaa aggaatcgaa atttcgtgag acgggagtga tcactcctga agagtttgtg 120

gcggccggcg atcacctggt gcatcactgc cccacctggc agtgggcgtc cggggacgaa 180

gccagggcca agacctactt gcccaagacc aaacagtttc tcatcaccaa gaatgtgccc 240

tgctcacgaa ggtgcaaaga gattgagtat tgtgacgagc aagagaaaat tctagagcct 300

gatgatcctg acggtggctg ggtggacact caccgcgtgg accccactag tggcctcgac 360

gaaaatgtct ccggaatgac attagactca aacacaactg ttcagagggt caatgattca 420

cctccatctg gagactttca gacgaacacc acaaacgcca ataataatgt cgatgatgat 480

gatgatgacg atgatgataa cgaagaagca gccgacatgg aaatgtttga ggaaagtggt 540

cttctagacg aggaggatga ggcgactgct gaggatgtca aaatcgaaaa agatgaaaga 600

aatgctgcag gtgacggcga gatagtcaaa accagaacgt atgacttaca tatcacatac 660

gacaaatatt accagactcc gcgattatgg ctttttggtt atgatgagaa ccataagcca 720

ctgaatgttg aacagatgta tgaagatgtg aatcaagatt atgcgaagaa aactgtgaca 780

atggaaacac atccgcacgt tccagggcct cccatggctt ctgtccatcc ttgcaggcac 840

gctgaagtga tgcagaagat aattcagacc gtattggaag gaggaggcga gctaggtgtt 900

catatgtatt tgataatttt tctgaaattt gttcaatcag tcattccaac tatagagtat 960

gattacacac aaaattttac aatgtggtct aaaacgctgt ga 1002

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Met Gln Ser Val Ile Asn Thr Val Lys Gly Thr Ala Leu Gly Val Ala

1 5 10 15

Glu Tyr Leu Thr Pro Val Leu Lys Glu Ser Lys Phe Arg Glu Thr Gly

20 25 30

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

35 40 45

His Cys Pro Thr Trp Gln Trp Ala Ser Gly Asp Glu Ala Arg Ala Lys

50 55 60

Thr Tyr Leu Pro Lys Thr Lys Gln Phe Leu Ile Thr Lys Asn Val Pro

65 70 75 80

Cys Ser Arg Arg Cys Lys Glu Ile Glu Tyr Cys Asp Glu Gln Glu Lys

85 90 95

Ile Leu Glu Pro Asp Asp Pro Asp Gly Gly Trp Val Asp Thr His Arg

100 105 110

Val Asp Pro Thr Ser Gly Leu Asp Glu Asn Val Ser Gly Met Thr Leu

115 120 125

Asp Ser Asn Thr Thr Val Gln Arg Val Asn Asp Ser Pro Pro Ser Gly

130 135 140

Asp Phe Gln Thr Asn Thr Thr Asn Ala Asn Asn Asn Val Asp Asp Asp

145 150 155 160

Asp Asp Asp Asp Asp Asp Asn Glu Glu Ala Ala Asp Met Glu Met Phe

165 170 175

Glu Glu Ser Gly Leu Leu Asp Glu Glu Asp Glu Ala Thr Ala Glu Asp

180 185 190

Val Lys Ile Glu Lys Asp Glu Arg Asn Ala Ala Gly Asp Gly Glu Ile

195 200 205

Val Lys Thr Arg Thr Tyr Asp Leu His Ile Thr Tyr Asp Lys Tyr Tyr

210 215 220

Gln Thr Pro Arg Leu Trp Leu Phe Gly Tyr Asp Glu Asn His Lys Pro

225 230 235 240

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

245 250 255

Lys Thr Val Thr Met Glu Thr His Pro His Val Pro Gly Pro Pro Met

260 265 270

Ala Ser Val His Pro Cys Arg His Ala Glu Val Met Gln Lys Ile Ile

275 280 285

Gln Thr Val Leu Glu Gly Gly Gly Glu Leu Gly Val His Met Tyr Leu

290 295 300

Ile Ile Phe Leu Lys Phe Val Gln Ser Val Ile Pro Thr Ile Glu Tyr

305 310 315 320

Asp Tyr Thr Gln Asn Phe Thr Met Trp Ser Lys Thr Leu

325 330

Claims (2)

1. Brown planthopperNlAtg3The application of the gene is characterized in that the RNA interference technology is utilized to inhibit brown planthopperNlAtg3The function of the gene can reduce the survival rate of the brown planthopperNlAtg3The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. Use as claimed in claim 1 for the biological control of brown planthopper.

Images