CN115558669A - Myzus persicae lethal gene, RNA interference sequence, preparation method and application thereof - Google Patents

Abstract

The invention discloses a myzus persicae lethal gene, an RNA interference sequence, a preparation method and application thereof, wherein the myzus persicae lethal gene isATPd，ATPdThe cDNA sequence of (A) is shown in SEQ ID NO. 1. The RNA interference sequence isATPddsRNA and RNA interference sequence synthesized by the gene segment is shown as SEQ ID NO. 6. The RNA interference sequence is introduced into the body of myzus persicae to inhibit the myzus persicaeATPdThe expression level of the gene causes the reduction of the survival rate of the myzus persicae, the shortening of the service life and the weakening of the reproductive capacity, and can be used for the prevention and the treatment of the myzus persicae in agricultural production. The invention is designed aiming at the specific gene of the myzus persicae and has strong specificity,The method has the advantages of high safety, high lethality rate, environmental friendliness and the like, can realize green accurate prevention and control of myzus persicae, and can effectively reduce the damage of chemical pesticides to the ecological environment.

Description

Aphid lethal gene, RNA interference sequence, preparation method and application thereof

technical field

The invention relates to a lethal gene suitable for preventing and controlling aphids and its RNA interference sequence preparation method and application, belonging to the technical field of preventing and controlling aphids.

Background technique

The tobacco aphid Myzuspersicae belongs to Hemiptera (Hemiptera) Aphididae (Aphididae), widely distributed all over the world, is one of the main pests of hundreds of crops such as cruciferous vegetables, tobacco, pepper, potato, eggplant, etc. A virus disease caused great losses to agricultural production. At present, the control of aphids and the viral diseases they spread mainly depends on the large-scale use of pesticides, which has brought serious safety problems to agricultural production in our country. Therefore, there is an urgent need for a safe and green control technology or strategy in production, which can make up for the disadvantages of chemical control.

RNA interference (RNAi) refers to the efficient and specific degradation of mRNA triggered by endogenous or exogenous double-stranded RNA, resulting in the phenomenon of specifically hindering the expression of target genes. According to the principle of RNA interference, screening the lethal genes of aphids and preparing RNA interference preparations can safely and efficiently control aphids, which is a good way to solve the problem of chemical control under the current development trend, and is known as a new generation of pest control new technology.

Contents of the invention

Based on the above, the present invention provides a gene capable of killing aphids through RNA interference and its RNA interference sequence, preparation method and application. The lethal gene of tobacco aphid is APTd, and the RNA interference sequence designed according to the lethal gene can inhibit the expression of the APTd gene in the tobacco aphid, thereby leading to the death of the tobacco aphid. The APTd gene RNA interference sequence can be applied to the prevention and control of the tobacco aphid in agriculture. This technology is a specific gene design for aphids, will not change the genome of aphids, only has a killing effect on aphids, and has the advantages of strong specificity, high safety, high lethality, and environmental friendliness. The green precision control of aphids can effectively reduce the damage to the ecological environment caused by chemical pesticides.

Technical scheme of the present invention is:

In the first aspect, the present invention provides a lethal gene of A. persicae, said A. persicae lethal gene is ATPd, and the cDNA sequence of said ATPd is shown in SEQ ID NO.1.

In a second aspect, the present invention provides an RNA interference sequence of the lethal gene ATPd of aphids, the RNA interference sequence is a dsRNA synthesized according to the ATPd gene fragment according to claim 1, and the RNA interference sequence is such as SEQ ID NO.6 shown.

In a third aspect, the present invention provides a preparation method for preparing the RNA interference sequence, the preparation method comprising the following steps:

S1, the cloning of the full-length cDNA of the ATPd gene to obtain the ATPd gene sequence of Aphids aphids;

S2. Select the target region of RNAi in the ATPd gene sequence of Aphid aphid, and design the target region amplification primers ATPd-F2/ATPd-R2;

S3, extracting the total RNA of Aphis persicae, reverse-transcribing and synthesizing cDNA, then using primers ATPd-F2/ATPd-R2 to perform PCR amplification on the ATPd gene contained in the cDNA, and purifying the product to obtain the target gene;

S4. Cloning the target gene into a plasmid vector, transforming it into competent cells, and extracting the plasmid;

S5. Add T7 promoter sequence to the 5'-end of the amplification primer in the target region, use the extracted plasmid as a template for PCR amplification, purify the product to obtain the target gene with T7 sequence, and then use MEGAscript T7Transcription Kit to synthesize RNA anti-interference preparation.

Optionally, in the S1 step, a primer pair ATPd-F1/ATPd-R1 is designed and synthesized according to the ATPd gene sequence obtained from the Aphidia tabacum transcriptome database, PCR amplification is performed using the Aphidia persicae cDNA as a template, and the amplified product is recovered and cloned to a plasmid vector, then transformed into competent cells, and sequenced to obtain the ATPd gene sequence.

Optionally, the DNA sequence of the upstream primer ATPd-F1 is shown in SEQ ID NO.2, and the DNA sequence of the downstream primer ATPd-R1 is shown in SEQ ID NO.3.

Optionally, the reaction system for PCR amplification described in S1 is: 12.5 μL of Premix Taq enzyme, 1 μL of cDNA template, 1 μL of forward and reverse primers, 9.5 μL of ddH ₂ O; the reaction program is: 95°C for 30s; 94 30s at ℃, 30s at 57℃, 1min at 72℃, 35 cycles, 10min at 72℃.

Optionally, the DNA sequence of the upstream primer ATPd-F2 is shown in SEQ ID NO.4, and the DNA sequence of the downstream primer ATPd-R2 is shown in SEQ ID NO.5

In a fourth aspect, the present invention provides an application of the RNA interference sequence in the control of Nicotiana tabaci.

Compared with the prior art, the present invention has the following advantages: one, it provides a gene that can make the aphids lethal by RNA interference, the lethal gene of aphids is ATPd, and the ATPd gene encodes V-ATPase (Vacuolar-type protonATPase) V0 domain, V-ATPase is a kind of cation transporting ATPase in eukaryotic cells, which plays an important role in maintaining the acidic environment of organelles, and plays an important role in the alkalinization of insect intestines, nutrient absorption and ion regulation; the second is to provide A method for the preparation of RNA interference sequences for Aphids aphids was developed, and dsRNA was designed based on the lethal specific gene ATPd, which can efficiently induce the specific degradation of homologous mRNAs, silence the expression of the ATPd gene in A. , the invention provides a new way to use RNA interference technology to control pests.

Description of drawings

The ATPd gene sequence and the deduced amino acid sequence of Fig. 1 cloned obtained;

Fig. 2 The silencing effect of nanocarriers mediating different concentrations of dsRNA on the ATPd gene of Mysia aphids;

Fig. 3 Silencing effect of nanocarrier-mediated dsRNA interference on ATPd gene of aphids at different times;

The impact of Fig. 4 RNA interference on the survival rate of Mysia aphid;

The influence of Fig. 5 RNA interference on the death rate of aphids;

The impact of Fig. 6 RNA interference on the survival days of Aphids aphids;

Fig. 7 Effect of RNA interference on the fecundity of aphids.

detailed description

In order to make the above objects, features and advantages of the present invention more comprehensible, specific implementations of the present invention will be described in detail below in conjunction with the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described here, and those skilled in the art can make similar improvements without departing from the connotation of the present invention, so the present invention is not limited by the specific implementations disclosed below.

Example 1:

A tobacco aphid lethal gene ATPd, its cDNA sequence is shown in SEQ ID NO.1, specifically as follows:

CCTATTATCTGTGACTCTCGATGATTGTCAATTGATAAAAATTAAGGTGTGTACTATAATTTGTACTTGTAGACACAGTGTACTGTGTGCTGTATGTGTGCTCTAACGTAATTTCGTTTTCTGTAGTGTGCTAAGTATTCAAATTGCGGCGGACAGTTACGTTTTAATCTTCAGAACCCTCTCAGGTCGACGAAAGAAATCATGGTGGATACCGGTTGTTTCTTCAACATTGACGGTGGTTACTTGGAAGGATTATGTCGCGGCTTTAAGTGTGGCATACTCAGACATGCCGATTACTTGAACCTTGAGCAGTGCGAGACCTTGGACGATCTCAAATTGCATCTGCAATCCACAGACTATGGCCAGTTCTTAGCCAATGAACCCAGCCCGCTGGCAGTATCCGTTATCGATGACAAGTTGCGCGAGAAGCTTGTTATTGAATTTCAACACATGCGCAATCATGCCGTGGAACCCCTCAGTACGTTCCTCGATTACATCACGTACAGTTACATGATTGACAATATTATATTGTTGATCACCGGAACACTTCACCAGCGCCCCATATCTGAATTGATACCAAAGTGTCATCCATTGGGTAGTTTCGAACAAATGGAAGCTATACATGTTGCTGCTACTCCAGCTGAGCTCTACAATGCTGTGTTGGTCGACACACCCTTGGCTCCATTCTTTGTTGACTGCATTAGCGAACAGGATCTTGACGAAATGAATATTGAAATTATTCGTAACACTCTATATAAGGCGTACTTGGAATCTTTTTATGATTTTTGTAAAAAGCTTGGAGGTATCACGGCTGATACTATGTGTGAAATATTATCATTTGAAGCTGATAGACGAGCAATTAATATAACGATCAACTCTTTTGGAACAGAATTGACTAAAGATGATAGAGCTAAGCTGTATCCAAGATGTGGTAAACTCTATCCAGATGGTCTGGCAGCTTTAGCTAGAGCTGATGATTATGATCAGGTTAAAGCTGTAG CTGAATACTTTGCTGAATACAGTGCTCTGTTTGATGGCGCTGGTACTAATCCTGGTGAAAAAACTCTTGAAGATCGTTTCTTTGAACATGAAGTGAAGTTGAATGTCAATGCATTCATGCGCCAATTCCATTATGGCGTTTTTTACTCGTATTTGAAACTTAAAGAACAAGAATGCAGAAATGTTGTGTGGATATCTGAGTGTGTGTCGCAAAAGCATCGGGCCCGCATGGATAACTACATTCCCATTTTCAAGTAACTTATAAATCGATGGTTATTTGTGCAGGTACCGGTATATTATCAATTATCATCAATTTAACATAATAAATTATTAATACCCTTTATCATACAGTTTTGAGGTATAATTTAGATTGTTCATAGATTGCATAGTATATTAATTCATTGTTTTTATTTATCATTCCAACGGTATTAATATGTCTGGAGCATTGTTGATAGCTTTTTCAGAATATTATGCATGTTTTTGTTTTTTATTAGATTTTAAGTGTCCAAAAGGAAAAAAA

Example 2:

A kind of RNA interference sequence of aphids, its preparation method comprises the following steps:

(1) Cloning of the full-length cDNA of the ATPd gene to obtain the sequence of the ATPd gene of A. persicae.

The total RNA was extracted by TRIzol method, and the first strand of cDNA was synthesized. The upstream and downstream primers ATPd-F1/ATPd-R1 of the ATPd gene were designed based on the ATPd gene sequence obtained from the Aphid sativa transcriptome database.

The DNA sequence of the upstream primer ATPd-F1 is shown in SEQ ID NO.2, specifically:

ATTCAAATTGCGGGCGGACAG

The DNA sequence of the downstream primer ATPd-R1 is shown in SEQ ID NO.3, specifically:

CCGGTACCCTGCACAAATAAC

Using cDNA as a template, PCR amplification was performed using the above-mentioned designed primers ATPd-F1/ATPd-R1. The PCR reaction system was: 12.5 μL of Premix Taq enzyme, 1 μL of cDNA template, 1 μL of forward and reverse primers, and 9.5 μL of ddH ₂ O. The reaction program is: 95°C for 30s; 94°C for 30s, 57°C for 30s, 72°C for 1min, 35 cycles, 72°C for 10min. The PCR product obtained by PCR amplification is separated by agarose gel electrophoresis, and the target fragment is purified and recovered. The recovered target fragment was cloned into pMDTM 19-T vector and then transformed into competent cell DH5α. Sequencing was performed after verification by colony PCR.

(2) Select the target region of RNAi in the ATPd gene sequence of Aphid aphid, and design primers for amplification of the target region.

The obtained ATPd gene sequence was cloned according to the above steps, the target region of RNAi was selected, and the PCR amplification primers ATPd-F2/ATPd-R2 of the target region were designed using the SnapDragon-dsRNA Design online tool.

The DNA sequence of the upstream primer ATPd-F2 is shown in SEQ ID NO.4, specifically:

GGAACACTTCACCAGCGCCC

The DNA sequence of the downstream primer ATPd-R2 is shown in SEQ ID NO.5, specifically:

ACCAGCGCCATCAACAGAGC

(3) Extract total RNA from Aphids persicae, reverse transcribe to synthesize cDNA, then use primers ATPd-F2/ATPd-R2 to perform PCR amplification on the ATPd gene contained in the cDNA, and purify the product to obtain the target gene.

(4) Cloning the target gene into a plasmid vector, transforming it into competent cells, and extracting the plasmid.

The ATPd gene fragment was cloned into pMDTM 19-T vector (Takara), and transformed into competent cell DH5α, and the plasmid was extracted and verified by sequencing.

(5) Add the T7 promoter sequence to the 5'-end of the amplification primer in the target region:

dsATPd-F: TAATACGACTCACTATAGGGGGAACACTTCACCAGCGCCC

dsATPd-R: TAATACGACTCACTATAGGGACCAGCGCCATCAAACAGAGC

The extracted plasmid was used as a template to carry out PCR amplification, and the purified product was obtained to obtain the target gene with T7 sequence, and then the dsRNA containing the ATPd fragment of the Aphids aphidifera lethal gene was synthesized by using the MEGAscript T7 Transcription Kit, and its sequence was shown in SEQ ID NO.6. Specifically:

GGAACACTTCACCAGCGCCCCATATCTGAATTGATACCAAAGTGTCATCCATTGGGTAGTTTCGAACAAATGGAAGCTATACATGTTGCTGCTACTCCAGCTGAGCTCTACAATGCTGTGTTGGTCGACACACCCTTGGCTCCATTCTTTGTTGACTGCATTAGCGAACAGGATCTTGACGAAATGAATATTGAAATTATTCGTAACACTCTATATAAGGCGTACTTGGAATCTTTTTATGATTTTTGTAAAAAGCTTGGAGGTATCACGGCTGATACTATGTGTGAAATATTATCATTTGAAGCTGATAGACGAGCAATTAATATAACGATCAACTCTTTTGGAACAGAATTGACTAAAGATGATAGAGCTAAGCTGTATCCAAGATGTGGTAAACTCTATCCAGATGGTCTGGCAGCTTTAGCTAGAGCTGATGATTATGATCAGGTTAAAGCTGTAGCTGAATACTTTGCTGAATACAGTGCTCTGTTTGATGGCGCTGGT

Example 3:

RNA interference efficiency and application detection of ATPd lethal gene ATPd

Control design:

Using the green fluorescent protein (GFP) gene as a control, the PCR amplification primers GFP-F/GFP-R of the target region were also designed.

The DNA sequence of the upstream primer GFP-F is shown in SEQ ID NO.7, specifically:

GCCAACACTTGTCACTACTT

The DNA sequence of the downstream primer GFP-R is shown in SEQ ID NO.8, specifically:

GGAGTATTTTGTTGATAATGGTCTG

Extract the total RNA of Aphis persicae, reverse transcribe and synthesize cDNA, then use the primers to amplify the cDNA by PCR, purify the product to obtain the control gene, clone the control gene fragment into pMDTM 19-T vector (Takara), and transform it into a sensory In the state cell DH5α, extract the plasmid, and add the T7 promoter sequence to the 5'-end of the primer GFP-F/GFP-R:

dsGFP-F: TAATACGACTCACTATAGGGGCCAACACTTGTCACTACTT

dsGFP-R: TAATACGACTCACTATAGGGGGAGTATTTTGTTGATAATGGTCTG

Use the extracted plasmid as a template for PCR amplification, purify the product to obtain the target gene with T7 sequence, and then use MEGAscript T7 Transcription Kit to synthesize dsRNA as a control.

experiment procedure:

(1) dsRNA and nanocarrier (nanocarrier) were mixed at a mass ratio of 1:1 (the final concentration of nanocarrier and dsRNA was 500 ng/μL), and 0.5% surfactant (detergent) was added to form a dsRNA/nanocarrier/detergent complex. The control dsGFP/nanocarrier/detergent complex was prepared by the same method as above.

(2) Drop 0.3 μL of dsRNA/nanocarrier/detergent and dsGFP/nanocarrier/detergent (control) onto the ventral back of the 3rd instar nymph of Aphis aphids, and after 24, 48, and 72 hours of interference treatment, 10 live worms were randomly selected for RNA detection Effect of interference on ATPd gene silencing.

Premix Ex Taq II 10μL、cDNA模板1μL、正反向引物各1μL、ddH₂O 7μL。选用烟蚜β-actin和18s作为内参基因。采用2^-ΔΔCT法测定相对转录水平，选取两个内控基因的几何平均值进行归一化处理。(3) Use NCBI Primer-BLAST to design qRT-PCR primers for the ATPd gene, use cDNA as a template, and perform qRT-PCR amplification to detect gene expression levels. The PCR reaction system is: TB

Premix Ex Taq II 10 μL, cDNA template 1 μL, forward and reverse primers 1 μL each, ddH ₂ O 7 μL. Aphid β-actin and 18s were selected as internal reference genes. Relative transcript levels were determined by the 2 ^-ΔΔCT method, and the geometric mean of two internal control genes was selected for normalization.

The DNA sequence of the upstream primer qATPd-F is shown in SEQ ID NO.9, specifically:

GGAAGGATTATGTCGCGGCT

The DNA sequence of the downstream primer qATPd-R is shown in SEQ ID NO.10, specifically:

TAACAAGCTTCTCGCGCAAC

The DNA sequence of the upstream primer β-actin-F is shown in SEQ ID NO.11, specifically:

AGTGCGACGTTGACATCAGA

The DNA sequence of the downstream primer β-actin-R is shown in SEQ ID NO.12, specifically:

GCTTGGAGCTAAGGCAGTGA

The DNA sequence of the upstream primer 18s-F is shown in SEQ ID NO.13, specifically:

TCAACACGGGAAACCTCACCA

The DNA sequence of the downstream primer 18s-R is shown in SEQ ID NO.14, specifically:

CACCACCCCACCGAATCAAGAA

(4) Using the RNA interference method in (2), select the 3rd instar nymphs of A. tabaciformis for RNAi treatment, and count the amount of molt and survival every day for 7 days. GFP treatment is used as a control, and 30 aphids per treatment are repeated. 3 times.

(5) IBM SPSS Statistics for Windows, version 19.0 was used for single factor analysis of variance, and Tukey's method was used for multiple comparisons, p<0.05 was considered statistically significant.

Result analysis:

(1) Sequence analysis of the ATPd gene of Aphid persicae

The 1519bp sequence was obtained by cloning and sequencing, and its nucleotide sequence was found to be highly similar to the ATPd sequence of other homologous insects by NCBI Blast tool analysis, and the sequence was determined to be the ATPd gene of aphids. The open reading frame (ORF) of the ATPd gene is 1056bp, encoding 351 amino acids, the 5'untranslated region is 201bp (5'-UTR), and the 3'untranslated region (3'-UTR) is 262bp. There is a tailing signal AATAA, the relative molecular weight of the encoded protein is 40.21kD, the isoelectric point (pI) is 4.93, and the molecular formula is C ₁₈₁₁ H ₂₇₇₈ N ₄₆₄ O ₅₃₅ S ₁₉ ; the N-terminal amino acid is methionine (M, Met). The half-life of the protein is 30h; it contains 53 negatively charged amino acid residues (Asp+Glu) and 34 positively charged amino acid residues (Arg+Lys); the stability coefficient is 36.29, which means the protein is stable; the total average The hydrophobicity index (GRAVY) was -0.160, indicating that it was a hydrophilic protein; the aliphatic amino acid index was 90.34 (Figure 1).

(2) Silencing effect of RNA interference on ATPd gene

The experiment measured the effect of nano-carriers mediating different concentrations of dsRNA (20, 40, 80, 160, 320, 500 ng/μL) to silence the ATPd gene of the aphid. The effects reached 27.12%, 56.76%, 46.33%, 71.55%, 62.34% and 71.97%, respectively, and the silencing effects of 160ng/μL and 500ng/μL dsRNA were the best (Figure 2).

Nanocarriers mediated 500ng/μL dsRNA to interfere with the 3rd instar nymphs of Aphids aphids for 24, 48, and 72 hours, respectively, and measured the expression of ATPd gene. The results showed that compared with the control GFP, the silencing effect was not significant after 24 hours, and the silencing was silenced after 48 and 72 hours. The effects reached 71.97% and 72.68% respectively (Fig. 3).

(3) The effect of RNA interference on the survival rate and lifespan of Aphids persicae

The experiment measured the survival rate and lifespan of Aphids persicae for 7 consecutive days after RNA interference with the ATPd gene. The results showed that silencing the ATPd gene can effectively reduce the survival rate of A. persicae, and the survival rate of A. persicae decreased rapidly on the 3rd and 4th day after silencing the ATPd gene. , the survival rate at the 7th day was only 21.11% (Fig. 4), and the death rate of Aphis persicae significantly increased within 7 days, which was 6.45 times that of the control (Fig. 5); (15.17d) was significantly shortened (Fig. 6).

(4) The effect of RNA interference on the fecundity of Aphis persicae

The experiment measured the fecundity of A. persicae after RNA interference of ATPd gene. The results showed that silencing the ATPd gene could effectively reduce the fecundity of A. persicae. 36.2 heads) was significantly reduced (Figure 7).

SEQUENCE LISTING

sequence listing

<110> Guizhou Academy of Tobacco Science

<120> Aphid lethal gene, RNA interference sequence, preparation method and application thereof

<160> 4

<210> 1

<211> 1519

<212> cDNA

<213> Aphid lethal gene ATPd

<400>1

CCTATTATCT GTGACTCTCG ATGATTGTCA ATTGATAAAA ATTAAGGTGT GTACTATAAT 60

TTGTACTTGT AGACACAGTG TACTGTGTGC TGTATGTGTG CTCTAACGTA ATTTCGTTTT 120

CTGTAGTGTG CTAAGTATTC AAATTGCGGC GGACAGTTAC GTTTTAATCT TCAGAACCCT 180

CTCAGGTCGA CGAAAGAAAT CATGGTGGAT ACCGGTTGTT TCTTCAACAT TGACGGTGGT 240

TACTTGGAAG GATTATGTCG CGGCTTTAAG TGTGGCATAC TCAGACATGC CGATTACTTG 300

AACCTTGAGC AGTGCGAGAC CTTGGACGAT CTCAAATTGC ATCTGCAATC CACAGACTAT 360

GGCCAGTTCT TAGCCAATGA ACCCAGCCCG CTGGCAGTAT CCGTTATCGA TGACAAGTTG 420

CGCGAGAAGC TTGTTATTGA ATTTCAACAC ATGCGCAATC ATGCCGTGGA ACCCCTCAGT 480

ACGTTCCTCG ATTACATCAC GTACAGTTAC ATGATTGACA ATATTATATT GTTGATCACC 540

GGAACACTTC ACCAGCGCCC CATATCTGAA TTGATACCAA AGTGTCATCC ATTGGGTAGT 600

TTCGAACAAA TGGAAGCTAT ACATGTTGCT GCTACTCCAG CTGAGCTCTA CAATGCTGTG 660

TTGGTCGACA CACCCTTGGC TCCATTCTTT GTTGACTGCA TTAGCGAACA GGATCTTGAC 720

GAAATGAATA TTGAAATTAT TCGTAACACT CTATATAAGG CGTACTTGGA ATCTTTTTAT 780

GATTTTTGTA AAAAGCTTGG AGGTATCACG GCTGATACTA TGTGTGAAAT ATTATCATTT 840

GAAGCTGATA GACGAGCAAT TAATATAACG ATCAACTCTT TTGGAACAGA ATTGACTAAA 900

GATGATAGAG CTAAGCTGTA TCCAAGATGT GGTAAACTCT ATCCAGATGG TCTGGCAGCT 960

TTAGCTAGAG CTGATGATTA TGATCAGGTT AAAGCTGTAG CTGAATACTT TGCTGAATAC 1020

AGTGCTCTGT TTGATGGCGC TGGTACTAAT CCTGGTGAAA AAACTCTTGA AGATCGTTTC 1080

TTTGAACATG AAGTGAAGTT GAATGTCAAT GCATTCATGC GCCAATTCCA TTATGGCGTT 1140

TTTTACTCGT ATTTGAAACT TAAAGAACAA GAATGCAGAA ATGTTGTGTG GATATCTGAG 1200

TGTGTGTCGC AAAAGCATCG GGCCCGCATG GATAACTACA TTCCCATTTT CAAGTAACTT 1260

ATAAATCGAT GGTTATTTGT GCAGGTACCG GTATATTATC AATTATCATC AATTTAACAT 1320

AATAAATTAT TAATACCCCTT TATCATACAG TTTTGAGGTA TAATTTAGAT TGTTCATAGA 1380

TTGCATAGTA TATTAATTCA TTGTTTTTAT TTATCATTCC AACGGTATTA ATATGTCTGG 1440

AGCATTGTTG ATAGCTTTTT CAGAATATTA TGCATGTTTT TGTTTTTTAT TAGATTTTAA 1500

GTGTCCAAAAA GGAAAAAAA 1519

<210> 2

<211> 21

<212>DNA

<213> Artificial sequence

<400>2

ATTCAAATTG CGGGCGGACA G 21

<210> 3

<211> 21

<212>DNA

<213> Artificial sequence

<400>3

CCGGTACCCT GCACAAATAA C 21

<210> 4

<211> 20

<212>DNA

<213> Artificial sequence

<400>4

GGAACACTTCACCAGCGCCC20

<210> 5

<211> 21

<212>DNA

<213> Artificial sequence

<400>4

ACCAGCGCCA TCAAACAGAG C 21

<210> 6

<211> 504

<212> dsRNA

<213> dsRNA of the ATPd fragment of the lethal gene ATPd

<400>4

GGAACACTTC ACCAGCGCCC CATATCTGAA TTGATACCAA AGTGTCATCC ATTGGGTAGT 60

TTCGAACAAA TGGAAGCTAT ACATGTTGCT GCTACTCCAG CTGAGCTCTA CAATGCTGTG 120

TTGGTCGACA CACCCTTGGC TCCATTCTTT GTTGACTGCA TTAGCGAACA GGATCTTGAC 180

GAAATGAATA TTGAAATTAT TCGTAACACT CTATATAAGG CGTACTTGGA ATCTTTTTAT 240

GATTTTTGTA AAAAGCTTGG AGGTATCACG GCTGATACTA TGTGTGAAAT ATTATCATTT 300

GAAGCTGATA GACGAGCAAT TAATATAACG ATCAACTCTT TTGGAACAGA ATTGACTAAA 360

GATGATAGAG CTAAGCTGTA TCCAAGATGT GGTAAACTCT ATCCAGATGG TCTGGCAGCT 420

TTAGCTAGAG CTGATGATTA TGATCAGGTT AAAGCTGTAG CTGAATACTT TGCTGAATAC 480

AGTGCTCTGT TTGATGGCGC TGGT 504

<210> 7

<211> 20

<212>DNA

<213> Artificial sequence

<400>4

GCCAACACTT GTCACTACTT 20

<210> 8

<211> 25

<212>DNA

<213> Artificial sequence

<400>4

GGAGTATTTT GTTGATAATG GTCTG 25

<210> 9

<211> 20

<212>DNA

<213> Artificial sequence

<400>4

GGAAGGATTA TGTCGCGGCT 20

<210> 10

<211> 20

<212>DNA

<213> Artificial sequence

<400>4

TAACAAGCTT CTCGCGCAAC 20

<210> 11

<211> 20

<212>DNA

<213> Artificial sequence

<400>4

AGTGCGACGT TGACATCAGA 20

<210> 12

<211> 20

<212>DNA

<213> Artificial sequence

<400>4

GCTTGGAGCT AAGGCAGTGA 20

<210> 13

<211> 21

<212>DNA

<213> Artificial sequence

<400>4

TCAACACGGGAAACCTCACC A 21

<210> 14

<211> 21

<212>DNA

<213> Artificial sequence

<400>4

CACCACCCAC CGAATCAAGA A 21

Claims (8)

1. A lethal gene that can be used for preventing and treating aphids by RNA interference, characterized in that the lethal gene is ATPd, and the cDNA sequence of ATPd is shown in SEQ ID NO.1. 2. An RNA interference sequence, characterized in that, the RNA interference sequence is dsRNA synthesized according to the ATPd gene fragment according to claim 1, and the RNA interference sequence is as shown in SEQ ID NO.6. 3. a preparation method for preparing the RNA interference sequence described in claim 2, characterized in that, the preparation method comprises the following steps: S1, the cloning of the full-length cDNA of the ATPd gene to obtain the ATPd gene sequence of Aphids aphids; S2. Select the target region of RNAi in the ATPd gene sequence of Aphid aphid, and design the target region amplification primers ATPd-F2/ATPd-R2; S3, extracting the total RNA of Aphis persicae, reverse-transcribing and synthesizing cDNA, then using primers ATPd-F2/ATPd-R2 to perform PCR amplification on the ATPd gene contained in the cDNA, and purifying the product to obtain the target gene; S4. Cloning the target gene into a plasmid vector, transforming it into competent cells, and extracting the plasmid; S5. Add T7 promoter sequence to the 5'-end of the amplification primer in the target region, use the extracted plasmid as a template for PCR amplification, purify the product to obtain the target gene with T7 sequence, and then use MEGAscript T7 TranscriptionKit to synthesize the RNA interference sequence . 4. The preparation method according to claim 3, characterized in that, in the S1 step, the primers are synthesized according to the DNA sequence of ATPd to ATPd-F1/ATPd-R1, and the cDNA of Aphid aphid is used as a template for PCR amplification, The amplified product was recovered and cloned into a plasmid vector, then transformed into competent cells, and sequenced to obtain the ATPd gene sequence. 5. The preparation method according to claim 4, wherein the DNA sequence of the upstream primer ATPd-F1 is as shown in SEQ ID NO.2, and the DNA sequence of the downstream primer ATPd-R1 is as shown in SEQ ID NO.3 shown. 6. The preparation method according to claim 4, characterized in that the reaction system for PCR amplification in S1 is: 12.5 μL of Premix Taq enzyme, 1 μL of cDNA template, 1 μL of forward and reverse primers, ddH ₂ O 9.5 μL; the reaction program is: 95°C for 30s; 94°C for 30s, 57°C for 30s, 72°C for 1min, 35 cycles, 72°C for 10min. 7. The preparation method according to claim 3, wherein the DNA sequence of the upstream primer ATPd-F2 is as shown in SEQ ID NO.4, and the DNA sequence of the downstream primer ATPd-R2 is as shown in SEQ ID NO.5 shown. 8. The application of the RNA interference sequence described in claim 2 in the control of tobacco aphid.

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