Gene for killing Myzus persicae, RNA interference sequence, preparation method and application thereof

Abstract

The present invention discloses a Myzus persicae lethal gene, an RNA interference sequence, and a preparation method and application thereof. The Myzus persicae lethal gene is ATPd , ATPd , and the cDNA sequence thereof is shown in SEQ ID NO.1. The RNA interference sequence is dsRNA synthesized from the ATPd gene fragment, and the RNA interference sequence is shown in SEQ ID NO.6. By introducing the RNA interference sequence into the body of Myzus persicae, the expression level of the ATPd gene in its body can be inhibited, thereby resulting in a decrease in the survival rate, a shortening of the lifespan, and a weakening of the reproductive capacity of Myzus persicae, and it can be used for the control of Myzus persicae in agricultural production. The present invention is designed for the specific gene of Myzus persicae, and has the advantages of strong specificity, high safety, high lethality rate, environmental friendliness, etc., can achieve the green and precise control of Myzus persicae, and can effectively reduce the damage of chemical pesticides to the ecological environment.

Description

Technical Field

[0001] The present invention relates to a lethal gene applicable to the control of Myzus persicae and a preparation method and application of an RNA interference sequence thereof, belonging to the technical field of Myzus persicae control. Background Art

[0002] Myzus persicae belongs to the Aphididae family of the Hemiptera order and is widely distributed around the world. It is one of the main pests of hundreds of crops such as cruciferous vegetables, tobacco, chili peppers, potatoes, and eggplants. It can also transmit a variety of viral diseases, causing great losses to agricultural production. At present, the control of Myzus persicae and the viral diseases it transmits mainly rely on the large-scale use of pesticides, which brings serious safety problems to China's agricultural production. Therefore, there is an urgent need in production for a safe and green prevention and control technology or strategy that can make up for the disadvantages of chemical control.

[0003] RNA interference (RNAi) refers to the phenomenon in which double-stranded RNA, either endogenous or exogenous, triggers the highly specific degradation of mRNA, resulting in the specific inhibition of target gene expression. According to the principle of RNA interference, screening for lethal genes of Myzus persicae and preparing RNA interference preparations can safely and efficiently control Myzus persicae, 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 technology. Summary of the Invention

[0004] Based on the above, the present invention provides a gene that can cause the death of Myzus persicae through RNA interference, its RNA interference sequence, a preparation method, and an application. The lethal gene for Myzus persicae is APTd. The RNA interference sequence designed according to the lethal gene can inhibit the expression of the APTd gene in Myzus persicae, thereby causing the death of Myzus persicae. The RNA interference sequence of the APTd gene can be applied to the control of Myzus persicae in agriculture. This technology is designed for the specific genes of Myzus persicae, does not change the genome of Myzus persicae, and only has a killing effect on Myzus persicae. It has the advantages of strong specificity, high safety, high lethality, environmental friendliness, etc., can achieve the green and precise control of Myzus persicae, and can effectively reduce the damage of chemical pesticides to the ecological environment.

[0005] The technical solution of the present invention is as follows:

[0006] In the first aspect, the present invention provides a lethal gene for Myzus persicae, the lethal gene for Myzus persicae being APTd, and the cDNA sequence of APTd being as shown in SEQ ID NO.1.

[0007] Second aspect, the present invention provides an RNA interference sequence of the Myzus persicae lethal gene ATPd, and the RNA interference sequence is dsRNA synthesized according to the ATPd gene fragment, and the RNA interference sequence is as shown in SEQ ID NO.6.

[0008] Third aspect, the present invention provides a preparation method for preparing the RNA interference sequence, and the preparation method includes the following steps:

[0009] S1. Cloning of the full-length cDNA of the ATPd gene to obtain the Myzus persicae ATPd gene sequence;

[0010] S2. Select the target region of RNAi in the Myzus persicae ATPd gene sequence, and design the target region amplification primers ATPd-F2 / ATPd-R2;

[0011] S3. Extract the total RNA of Myzus persicae, reverse transcribe to synthesize cDNA, and then perform PCR amplification on the ATPd gene contained in the cDNA with the primers ATPd-F2 / ATPd-R2, and purify the product to obtain the target gene;

[0012] S4. Clone the target gene into a plasmid vector, transform it into competent cells, and extract the plasmid;

[0013] S5. Add the T7 promoter sequence to the 5'-end of the target region amplification primer, use the extracted plasmid as a template for PCR amplification, purify the product to obtain the target gene with the T7 sequence, and then use the MEGAscript T7 Transcription Kit to synthesize the RNA anti-interference preparation.

[0014] Optionally, in the step S1, according to the ATPd gene sequence obtained from the Myzus persicae transcriptome database, the primer pair ATPd-F1 / ATPd-R1 is designed and synthesized, using Myzus persicae cDNA as a template for PCR amplification, the amplified product is recovered and cloned into a plasmid vector, and then transformed into competent cells, and the ATPd gene sequence is obtained by sequencing.

[0015] Optionally, 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.

[0016] Optionally, the reaction system of the PCR amplification in S1 is: 12.5 μL of Premix Taq enzyme, 1 μL of cDNA template, 1 μL of each of the forward and reverse primers, 9.5 μL of ddH2O; the reaction procedure is: 95°C for 30 s; 94°C for 30 s, 57°C for 30 s, 72°C for 1 min, 35 cycles, 72°C for 10 min.

[0017] Optionally, 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

[0018] Fourthly, the present invention provides an application of the RNA interference sequence in controlling Myzus persicae

[0019] Compared with the prior art, the advantages of the present invention are as follows: firstly, a gene that can cause the death of Myzus persicae through RNA interference is provided. The gene causing the death of Myzus persicae is ATPd, and the ATPd gene encodes the V0 domain of V-ATPase (Vacuolar-type proton ATPase). V-ATPase is a cation-transporting ATPase in eukaryotic cells and plays an important role in maintaining the acidic environment of cell organelles, and plays an important function in insect intestinal alkalization, nutrient absorption and ion regulation; secondly, a preparation method of the RNA interference sequence of Myzus persicae is provided. Based on the lethal specific gene ATPd, dsRNA is designed, and this dsRNA can efficiently induce the specific degradation of homologous mRNA, silence the expression of the ATPd gene in Myzus persicae, and ultimately lead to the reduction of the reproductive capacity and the death of Myzus persicae. This invention provides a new way for using RNA interference technology to control pests. Description of the Drawings

[0020] Figure 1 The obtained ATPd gene sequence of Myzus persicae and the deduced amino acid sequence

[0021] Figure 2 The silencing effect of dsRNA with different concentrations mediated by nanocarriers on the ATPd gene of Myzus persicae

[0022] Figure 3 The silencing effect of the ATPd gene of Myzus persicae after being interfered by dsRNA mediated by nanocarriers for different times

[0023] Figure 4 The influence of RNA interference on the survival rate of Myzus persicae

[0024] Figure 5 The influence of RNA interference on the mortality rate of Myzus persicae

[0025] Figure 6 The influence of RNA interference on the survival days of Myzus persicae

[0026] Figure 7 The influence of RNA interference on the reproductive capacity of Myzus persicae Detailed Embodiments

[0027] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following detailed description of the specific embodiments of the present invention will be provided in conjunction with the accompanying drawings. Many specific details are set forth in the following description to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0028] Example 1:

[0029] A tobacco aphid lethal gene ATPd, whose cDNA sequence is shown in SEQ ID NO.1, is specifically as follows:

[0030]

[0031] Example 2:

[0032] A Myzus persicae RNA interference sequence, and its preparation method includes the following steps:

[0033] (1) Cloning of the full-length cDNA of the ATPd gene to obtain the Myzus persicae ATPd gene sequence.

[0034] Extract the total RNA of Myzus persicae using the TRIzol method and synthesize the first strand of cDNA. Based on the ATPd gene sequence obtained from the Myzus persicae transcriptome database, design the upstream and downstream primers ATPd-F1 / ATPd-R1 for the ATPd gene.

[0035] The DNA sequence of the upstream primer ATPd-F1 is shown in SEQ ID NO.2, specifically:

[0036] ATTCAAATTGCGGGCGGACAG

[0037] The DNA sequence of the downstream primer ATPd-R1 is shown in SEQ ID NO.3, specifically:

[0038] CCGGTACCCTGCACAAATAAC

[0039] Using the cDNA as a template, perform PCR amplification using the designed primers ATPd-F1 / ATPd-R1. The PCR reaction system is: 12.5 μL of Premix Taq enzyme, 1 μL of cDNA template, 1 μL of each of the forward and reverse primers, and 9.5 μL of ddH2O. The reaction program is: 30 s at 95 °C; 30 s at 94 °C, 30 s at 57 °C, 1 min at 72 °C, 35 cycles, and 10 min at 72 °C. Separate the PCR products obtained by PCR amplification by agarose gel electrophoresis, purify and recover the target fragment. Clone the recovered target fragment into the pMDTM 19-T vector and then transform it into the competent cell DH5α. After verification by colony PCR, perform sequencing.

[0040] (2) Select the target region for RNAi in the Myzus persicae ATPd gene sequence and design primers for amplifying the target region.

[0041] According to the Myzus persicae ATPd gene sequence cloned in the above step, select the target region for RNAi, and use the SnapDragon-dsRNA Design online tool to design the PCR amplification primers ATPd-F2 / ATPd-R2 for the target region.

[0042] The DNA sequence of the upstream primer ATPd-F2 is shown in SEQ ID NO.4, specifically:

[0043] GGAACACTTCACCAGCGCCC

[0044] The DNA sequence of the downstream primer ATPd-R2 is shown in SEQ ID NO.5, specifically:

[0045] ACCAGCGCCATCAAACAGAGC

[0046] (3) Extract the total RNA of Myzus persicae, reverse transcribe it to synthesize cDNA, and then perform PCR amplification on the ATPd gene contained in the cDNA using the primers ATPd-F2 / ATPd-R2. Purify the product to obtain the target gene.

[0047] (4) Clone the target gene into a plasmid vector and transform it into competent cells, then extract the plasmid.

[0048] Clone the ATPd gene fragment into pMDTM 19-T vector (Takara) and transform it into competent cells DH5α. Extract the plasmid and verify it by sequencing.

[0049] (5) Add the T7 promoter sequence to the 5'-end of the primers in the target region:

[0050] dsATPd-F: TAATACGACTCACTATAGGGGGAACACTTCACCAGCGCCC

[0051] dsATPd-R: TAATACGACTCACTATAGGGACCAGCGCCATCAAACAGAGC

[0052] Use the extracted plasmid as a template for PCR amplification. Purify the product to obtain the target gene with the T7 sequence, and then use the MEGAscript T7 Transcription Kit to synthesize dsRNA containing the Myzus persicae lethal gene ATPd fragment. Its sequence is shown in SEQ ID NO.6, specifically:

[0053] GGAACACTTCACCAGCGCCCCATATCTGAATTGATACCAAAGTGTCATCCATTGGGTAG

[0054] TTTCGAACAAATGGAAGCTATACATGTTGCTGCTACTCCAGCTGAGCTCTACAATGCTG

[0055] TGTTGGTCGACACACCCTTGGCTCCATTCTTTGTTGACTGCATTAGCGAACAGGATCTT

[0056] GACGAAATGAATATTGAAATTATTCGTAACACTCTATATAAGGCGTACTTGGAATCTTTT

[0057] TATGATTTTTGTAAAAAGCTTGGAGGTATCACGGCTGATACTATGTGTGAAATATTATCA

[0058] TTTGAAGCTGATAGACGAGCAATTAATATAACGATCAACTCTTTTGGAACAGAATTGAC

[0059] TAAAGATGATAGAGCTAAGCTGTATCCAAGATGTGGTAAACTCTATCCAGATGGTCTGG

[0060] CAGCTTTAGCTAGAGCTGATGATTATGATCAGGTTAAAGCTGTAGCTGAATACTTTGCT

[0061] GAATACAGTGCTCTGTTTGATGGCGCTGGT

[0062] Example 3:

[0063] Detection of RNA interference efficiency and application of the Myzus persicae lethal gene ATPd

[0064] Control design:

[0065] The green fluorescent protein (GFP) gene was used as a control, and PCR amplification primers GFP-F / GFP-R for the target region were designed in the same way.

[0066] The DNA sequence of the upstream primer GFP-F is shown in SEQ ID NO.7, specifically:

[0067] GCCAACACTTGTCACTACTT

[0068] The DNA sequence of the downstream primer GFP-R is shown in SEQ ID NO.8, specifically:

[0069] GGAGTATTTTGTTGATAATGGTCTG

[0070] Extract the total RNA of Myzus persicae, reverse transcribe to synthesize cDNA, and then use this primer pair to perform PCR amplification on the cDNA. Purify the product to obtain the control gene. Clone this control gene fragment into the pMDTM 19-T vector (Takara) and transform it into the competent cell DH5α. Extract the plasmid and add the T7 promoter sequence to the 5'-end of the primers GFP-F / GFP-R:

[0071] dsGFP-F: TAATACGACTCACTATAGGGGCCAACACTTGTCACTACTT

[0072] dsGFP-R: TAATACGACTCACTATAGGGGGAGTATTTTGTTGATAATGGTCTG

[0073] Use the extracted plasmid as a template for PCR amplification. Purify the product to obtain the target gene with the T7 sequence, and then use the MEGAscript T7 Transcription Kit to synthesize dsRNA as a control.

[0074] Experimental steps:

[0075] (1) Mix dsRNA with the nanocarrier at a mass ratio of 1:1 (the final concentrations of the nanocarrier and dsRNA are 500 ng / μL), and add 0.5% of the detergent to form the dsRNA / nanocarrier / detergent complex. The control is prepared in the same manner as described above to obtain the dsGFP / nanocarrier / detergent complex.

[0076] (2) Drop 0.3 μL of dsRNA / nanocarrier / detergent and dsGFP / nanocarrier / detergent (control) onto the ventral and dorsal sides of the 3rd instar nymphs of Myzus persicae. After 24, 48, and 72 h of interference treatment, randomly select 10 live insects for detecting the effect of RNA interference on the silencing of the ATPd gene.

[0077] (3) Use NCBI Primer-BLAST to design qRT-PCR primers for the ATPd gene. Use cDNA as a template to perform qRT-PCR amplification to detect the gene expression level. The PCR reaction system is: TB Premix Ex Taq II 10 μL, cDNA template 1 μL, forward and reverse primers 1 μL each, ddH2O 7 μL. Select the β-actin and 18s of Myzus persicae as internal reference genes. Use 2 -ΔΔCTThe relative transcription levels were measured by the method, and the geometric mean of two internal control genes was selected for normalization.

[0078] The DNA sequence of the upstream primer qATPd-F is shown in SEQ ID NO.9, specifically:

[0079] GGAAGGATTATGTCGCGGCT

[0080] The DNA sequence of the downstream primer qATPd-R is shown in SEQ ID NO.10, specifically:

[0081] TAACAAGCTTCTCGCGCAAC

[0082] The DNA sequence of the upstream primer β-actin-F is shown in SEQ ID NO.11, specifically:

[0083] AGTGCGACGTTGACATCAGA

[0084] The DNA sequence of the downstream primer β-actin-R is shown in SEQ ID NO.12, specifically:

[0085] GCTTGGAGCTAAGGCAGTGA

[0086] The DNA sequence of the upstream primer 18s-F is shown in SEQ ID NO.13, specifically:

[0087] TCAACACGGGAAACCTCACCA

[0088] The DNA sequence of the downstream primer 18s-R is shown in SEQ ID NO.14, specifically:

[0089] CACCACCCACCGAATCAAGAA

[0090] (4) Using the RNA interference method in (2), the 3rd instar nymphs of Myzus persicae were selected for RNAi treatment, and their molting amount and survival amount were counted every day for 7 consecutive days. The GFP treatment was used as a control, with 30 Myzus persicae in each treatment, and repeated 3 times.

[0091] (5) One-way analysis of variance was performed using IBM SPSS Statistics for Windows, version 19.0, and multiple comparisons were performed using the Tukey method. p < 0.05 was considered statistically significant.

[0092] Result analysis:

[0093] (1) Sequence analysis of the ATPd gene of Myzus persicae

[0094] A 1519-bp sequence was obtained by clone sequencing. Analysis using the Blast tool on NCBI revealed that its nucleotide sequence had a high similarity with the ATPd sequences of other homologous insects, and this sequence was determined to be the ATPd gene of aphids. The open reading frame (ORF) of the ATPd gene was 1056 bp, encoding 351 amino acids. The 5' untranslated region (5'-UTR) was 201 bp, and the 3' untranslated region (3'-UTR) was 262 bp. There was a polyadenylation signal AATAA. The relative molecular mass of the encoded protein was 40.21 kD, the isoelectric point (pI) was 4.93, and the molecular formula was C 1811 H 2778 N 464 O 535 S 19 ; The N-terminal amino acid was methionine (M, Met). The predicted protein half-life was 30 h; it contained 53 negatively charged amino acid residues (Asp + Glu) and 34 positively charged amino acid residues (Arg + Lys); the stability coefficient was 36.29, indicating that the protein was stable; the total average hydrophobicity index (GRAVY) was -0.160, suggesting that it was a hydrophilic protein; the aliphatic amino acid index was 90.34( Figure 1 ).

[0095] (2) The silencing effect of RNA interference on the ATPd gene of Myzus persicae

[0096] The effects of different concentrations of dsRNA (20, 40, 80, 160, 320, 500 ng / μL) mediated by nanocarriers on silencing the ATPd gene of Myzus persicae were determined. The results showed that dsATPd could effectively reduce the expression level of the ATPd gene. After 48 h of interference, the silencing effects reached 27.12%, 56.76%, 46.33%, 71.55%, 62.34%, and 71.97% respectively. The silencing effects were the best with 160 ng / μL and 500 ng / μL dsRNA( Figure 2 ).

[0097] After the 3rd instar nymphs of Myzus persicae were interfered with 500 ng / μL dsRNA mediated by nanocarriers for 24, 48, and 72 h, the expression levels of the ATPd gene were measured respectively. The results showed that compared with the control GFP, the silencing effect was not significant after 24 h, and the silencing effects reached 71.97% and 72.68% after 48 h and 72 h respectively( Figure 3 ).

[0098] (3) The effects of RNA interference on the survival rate and lifespan of Myzus persicae

[0099] The survival rate and lifespan of Myzus persicae were measured for 7 consecutive days after RNA interference of the ATPd gene. The results showed that silencing the ATPd gene could effectively reduce the survival rate of M. persicae. The survival rate of M. persicae decreased rapidly on the 3rd and 4th days after silencing the ATPd gene, and the survival rate was only 21.11% on the 7th day ( Figure 4 ), the mortality rate of M. persicae within 7 days increased significantly, which was 6.45 times that of the control ( Figure 5 ); the survival days of M. persicae after RNA interference (6.82 d) were significantly shorter than those of the control (15.17 d) ( Figure 6 ).

[0100] (4) Effect of RNA interference on the fecundity of M. persicae

[0101] The fecundity of M. persicae was measured after RNA interference of the ATPd gene. The results showed that silencing the ATPd gene could effectively reduce the reproductive ability of M. persicae. The total number of nymphs produced per adult M. persicae within 7 days after RNA interference (7.7) was significantly lower than that of the control (36.2) ( Figure 7 ). SEQUENCE LISTING Sequence Listing <110> Guizhou Academy of Tobacco Sciences <120> Lethal gene of M. persicae, RNA interference sequence, and its preparation method and application <160> 4 <210> 1 <211> 1519 <212> cDNA <213> Lethal gene ATPd of M. persicae <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 TAATACCCTT TATCATACAG TTTTGAGGTA TAATTTAGAT TGTTCATAGA 1380 TTGCATAGTA TATTAATTCA TTGTTTTTAT TTATCATTCC AACGGTATTA ATATGTCTGG 1440 AGCATTGTTG ATAGCTTTTT CAGAATATTA TGCATGTTTT TGTTTTTTAT TAGATTTTAA 1500 GTGTCCAAAA 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 GGAACACTTC ACCAGCGCCC 20 <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 lethal gene ATPd fragment of Myzus persicae <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 TCAACACGGG AAACCTCACC A 21 <210> 14 <211> 21 <212> DNA <213> Artificial sequence <400>4 CACCACCCAC CGAATCAAGA A 21

Claims

1. Application of RNA interference molecule in the control of Myzus persicae, wherein, The RNA interference molecule is dsRNA synthesized according to the lethal gene of Myzus persicae ATPd gene fragment, the nucleotide sequence of the RNA interference molecule is shown in SEQ ID NO.6, and the ATPd cDNA sequence is shown in SEQ ID NO.

1.

2. A method for preparing the RNA interference molecule described in claim 1, characterized in that, The preparation method includes the following steps: S1. According to ATPd the full-length cDNA clone of the gene, obtain the ATPd gene sequence of Myzus persicae; S2. Select Myzus persicae ATPd The target region of RNAi in the gene sequence, and design primers for amplifying the target region ATPd -F2 / ATPd -R2; S3. Extract the total RNA of Myzus persicae, reverse transcribe to synthesize cDNA, and then use the 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; S4. Clone the target gene into a plasmid vector, transform it into competent cells, and extract the plasmid. S5. Add a 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 a T7 sequence, and then synthesize the RNA interference molecule using the MEGAscript T7 TranscriptionKit.

3. The preparation method according to claim 2, wherein In the step S1, according to ATPd the DNA sequence, synthesize the primer pair ATPd -F1 / ATPd -R1. Using the cDNA of Myzus persicae as a template, perform PCR amplification. After recovering the amplification product, clone it into a plasmid vector, then transform it into competent cells, and sequence to obtain ATPd the gene sequence.

4. The preparation method according to claim 3, characterized in that, The said ATPd - The DNA sequence of F1 is shown in SEQ ID NO.2, and the said ATPd - The DNA sequence of R1 is shown in SEQ ID NO.

3.

5. The preparation method according to claim 3, wherein, The reaction system for the PCR amplification in S1 is as follows: 12.5 μL of Premix Taq enzyme, 1 μL of cDNA template, 1 μL each of forward and reverse primers, and 9.5 μL of ddH2O; the reaction procedure is: 30 s at 95°C; 30 s at 94°C, 30 s at 57°C, 1 min at 72°C, for 35 cycles, and 10 min at 72°C.

6. The preparation method according to claim 2, characterized in that, The said ATPd - The DNA sequence of F2 is as shown in SEQ ID NO. 4, and the said ATPd - The DNA sequence of R2 is as shown in SEQ ID NO. 5.

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