CN112391390A - Migratory locust clathrin heavy chain gene and synthesis and application of targeting dsRNA thereof - Google Patents

Migratory locust clathrin heavy chain gene and synthesis and application of targeting dsRNA thereof Download PDF

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CN112391390A
CN112391390A CN202011303949.0A CN202011303949A CN112391390A CN 112391390 A CN112391390 A CN 112391390A CN 202011303949 A CN202011303949 A CN 202011303949A CN 112391390 A CN112391390 A CN 112391390A
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chain gene
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张建珍
史学凯
王艳丽
李涛
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Shanxi University
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
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Abstract

The invention relates to the technical field of biology, in particular to a migratory locust clathrin heavy chain gene and synthesis and application of a targeting dsRNA thereof. The invention provides a migratory locust clathrin heavy chain gene, a synthetic method of a targeting dsRNA thereof and application of the gene in pest control. Specifically, a clathrin heavy chain gene fragment is obtained from a migratory locust transcriptome by a bioinformatics method, and the sequence of the clathrin heavy chain gene fragment is obtained by further calling SEQ ID NO: 1 full length gene. Then according to SEQ ID NO: 1, designing a primer and synthesizing dsRNA of the gene, wherein the nucleotide sequence of one of the dsRNA fragments is SEQ ID NO: 5, the sequence can specifically silence target genes after being injected into a locusta migratoria body cavity, so that the locusta migratoria dies during molting, and multiple experiments show that the lethality rate of the locusta reaches more than 94%. The specificity and the high-efficiency lethality rate of the dsRNA have important practical significance for pest control, and can provide a new approach for pest control.

Description

Migratory locust clathrin heavy chain gene and synthesis and application of targeting dsRNA thereof
Technical Field
The invention relates to the technical field of biology, in particular to a migratory locust clathrin heavy chain gene, a synthetic method of a targeting dsRNA thereof and application thereof in pest control.
Background
Migratory locusts are important agricultural pests in China, mainly eat gramineae plants, and cause serious harm to agricultural production. At present, chemical prevention is mainly relied on for prevention and treatment. The long-term application of chemical insecticides causes a series of problems such as environmental pollution, pesticide residues, ecological destruction, crop resistance generation and food safety hazards. Therefore, the development of a novel method capable of replacing chemical control to treat locust plague is urgently needed.
RNA interference (RNAi) is a specific post-transcriptional gene silencing phenomenon caused by double-stranded RNA molecules, and since Nobel prize was obtained in 2006, RNAi technology has become a research hotspot of life science, and provides a new direction for gene function research, clinical disease treatment and crop pest control. Due to the characteristics of high efficiency, specificity, easy operability and the like of the technology on target genes, the technology has potential application value in the fields of pest control and the like at present. The pest control by RNA interference has the following advantages: 1) the insecticidal specificity is high, and the insecticidal composition has no killing effect on non-target organisms; 2) the method is green and environment-friendly, is harmless to the environment, and has no residue, and the RNA is easy to degrade in the nature; 3) economical, low in production cost and relatively safe. Therefore, the scholars refer to it as a fourth generation new insecticide. The premise of pest control based on RNA interference is to screen a target sequence to obtain dsRNA with high lethal effect on insects.
The development of insects needs to continuously take in nutrient substances from the outside, the endocytosis is the main absorption mode, and the clathrin-dependent endocytosis is essential for the insects to take in the nutrient substances and regulate the growth and development. The clathrin (Chc) is distributed in cell membranes and cytoplasm and is responsible for trapping macromolecular substances in the extracellular environment into cells through the cell membranes, when the exogenous substances are combined with cell membrane receptors, hundreds of clathrin are assembled to form a cage structure to wrap the exogenous substances into the cells, and then the exogenous substances are transported in the cells, and finally the nutrient substances are utilized by the cells. After the target gene is silenced, the insect can not normally take in foreign matters, so that the insect can not normally develop and finally dies.
Disclosure of Invention
Aiming at the problems, the invention firstly provides a migratory locust clathrin heavy chain gene; secondly, providing a targeting dsRNA of the migratory locust clathrin heavy chain gene and a synthetic method thereof; finally, the invention also provides application of the targeting dsRNA of the migratory locust clathrin heavy chain gene in migratory locust control.
In order to achieve the purpose, the invention adopts the following technical scheme:
the length of the nucleotide of the migratory locust clathrin heavy chain gene provided by the invention is 5296bp, and the nucleotide sequence is SEQ ID NO: 1, and (b) is shown in the specification.
Further, the amino acid sequence of the locust migratory clathrin heavy chain gene code is SEQ ID NO: 2. The amino acid sequence is the amino acid sequence of SEQ ID NO: 1 is predicted after bioinformatics analysis. Bioinformatics analysis shows that the locusta migratoria clathrin heavy chain gene (LmChc) encodes 1677 amino acids, the molecular weight is 192KD, and the theoretical isoelectric point is 5.46.
Further, the migratory locust clathrin heavy chain gene is obtained by the following steps:
step 1, searching Unigene of migratory locust based on a migratory locust transcriptome database, and obtaining a fragment of a migratory locust clathrin heavy chain gene after NCBI Blastx analysis;
step 2, splicing the fragments obtained by searching in the step 1 through GeneDoc software;
and 3, designing and synthesizing a corresponding upstream primer SEQ ID NO by using primer premier5.0 software according to the splicing result of the step 2: 3 and the downstream primer SEQ ID NO: 4;
SEQ ID NO：3：CAGTTTCAGTCGTGAGTGCGATTAGT
SEQ ID NO：4：GGCTCATCACTGAAATAACAAACAACA
step 4, extracting RNA of locusta migratoria and performing reverse transcription to obtain cDNA to obtain a template required by PCR amplification;
and 5, obtaining the migratory locust clathrin heavy chain gene through PCR amplification reaction by adopting the primer synthesized in the step 3 and the template in the step 4.
Further, the process of preparing the template in the step 4 is as follows: directly freezing 5-year-old locusta migratoria that is healthy in growth, consistent in size and half in sex in liquid nitrogen, then placing the locusta migratoria into a mortar for grinding, extracting RNA according to a kit, and carrying out reverse transcription on the extracted RNA into first-strand cDNA by adopting M-MLV reverse transcriptase so as to obtain a template required by PCR reaction.
Further, the system of the PCR amplification reaction in the step 5 is as follows: PrimeSTAR Max Premix (2X) 10. mu.L, upstream and downstream primers 0.2. mu.L each, template 1. mu.L, double distilled water 8.6. mu.L, total volume 20. mu.L; the reaction procedure is as follows: pre-denaturation at 98 ℃ for 10 s; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 150s, and the cycle is repeated 35 times.
The invention provides a targeting dsRNA of a locusta migratoria clathrin heavy chain gene, wherein one nucleotide sequence of the targeting dsRNA is SEQ ID NO: 5, and (c) a sequence shown in the specification.
Further, the synthetic method of the targeting dsRNA of the migratory locust clathrin heavy chain gene comprises the following steps:
step 1, designing and synthesizing an upstream primer SEQ ID NO containing a T7 promoter (italic part) by primer premier5.0 software according to the nucleotide sequence of the migratory locust clathrin heavy chain gene: 6 and downstream primer SEQ ID NO: 7;
SEQ ID NO：6：
Figure BDA0002787742630000031
SEQ ID NO.：7：
Figure BDA0002787742630000032
step 2, obtaining a section of template with length of 538bp and both ends of which are T7 promoters through PCR amplification, purifying the template by using a kit and then adopting T7RiboMAXTMExpress RNAi System (Promega) kit description in vitro transcription synthesis of the Targeted dsRNA of the migratory locust clathrin heavy chain gene.
Further, the system of the PCR amplification reaction in step 2 is: 2 × taq PCR Master Mix II 25 μ L, upstream and downstream primers 1 μ L each, double distilled water 21 μ L, cDNA template 2 μ L, total volume of 50 μ L; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, and circulating for 35 times; final extension at 72 ℃ for 7 min.
The targeting dsRNA of the migratory locust clathrin heavy chain gene can be applied to migratory locust control.
Furthermore, the application of the target dsRNA for controlling the migratory locust is realized by injecting the synthesized dsRNA into a migratory locust body cavity through a microsyringe and knocking down the expression of a migratory locust clathrin heavy chain gene so as to influence the growth and development of the migratory locust.
Compared with the prior art, the invention has the following advantages:
the dsRNA synthesized based on the graticum heavy chain gene of the migratory locust has high specificity and lethality to the migratory locust, after the five-instar nymphs inject the targeted dsRNA of the graticum heavy chain gene the first day, the nymphs die due to being blocked during molting, and the mortality rate reaches more than 94%. Therefore, the invention can be used for controlling locusts migratoria, has important practical significance for controlling pests, and can provide a new way for controlling pests.
Drawings
FIG. 1 is the agarose gel electrophoresis detection diagram of PCR amplification, wherein M is DL5000 DNA Marker, the bands are 100, 250, 500, 750, 1000, 1500, 2000, 3000 and 5000bp from bottom to top, and 1 represents migratory locust clathrin heavy chain gene.
Fig. 2 shows the transcript profile of the migratory locust clathrin heavy chain gene 24h after dsRNA injection, where x denotes P <0.05 and x denotes P < 0.01.
FIG. 3 is the effect of dsRNA on the growth and development of 5-year migratory locust nymphs.
Detailed Description
The technical solution in the embodiments of the present invention will be specifically and specifically described below with reference to the embodiments of the present invention and the accompanying drawings. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.
Example 1
Obtaining the full-length sequence of the migratory locust clathrin heavy chain gene cDNA and analyzing the amino acid sequence:
1. obtaining a migratory locust clathrin heavy chain gene cDNA fragment:
on the basis of a transcriptome database of migratory locust, Unigene of the migratory locust is searched, and after NCBI Blastx analysis, 1 fragment of the migratory locust clathrin heavy chain gene is determined to be obtained.
2. PCR amplification to obtain the whole length sequence of the locust migratory clathrin heavy chain gene cDNA:
1) designing primers required by PCR amplification:
splicing the gene segments by GeneDoc software, and designing corresponding upstream primers SEQ ID NO: 3: CAGTTTCAGTCGTGAGTGCGATTAGT and the downstream primer SEQ ID NO: 4: GGCTCATCACTGAAATAACAAACAACA, the primers were synthesized by Shanghai Bioengineering Co., Ltd.
2) Preparing a template required by PCR amplification:
selecting 5-year-old locusta migratoria that is healthy in growth, consistent in size and half male and female, and directly freezing the locusta migratoria in liquid nitrogen. 4 first biological replicates, which were subsequently ground in a mortar, RNA was extracted according to the TaKaRa Trizol kit. The RNA is reverse transcribed into first-strand cDNA using M-MLV reverse transcriptase. Thus obtaining the template required by the PCR reaction.
3) PCR amplification reaction:
and (2) obtaining a full-length fragment of the clathrin heavy chain gene by PCR amplification by adopting the template obtained in the step 2) and the primer synthesized in the step 1).
The PCR amplification system is as follows: PrimeSTAR Max Premix (2X) 10. mu.L, upstream and downstream primers 0.2. mu.L each, template 1. mu.L, double distilled water 8.6. mu.L, total volume 20. mu.L.
The amplification reaction procedure was: pre-denaturation at 98 ℃ for 10 s; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 150s, and the cycle is repeated 35 times.
The PCR product was purified by Gel Extraction Kit (Omega), followed by detection of a band of interest using 1.5% agarose Gel electrophoresis (FIG. 1), and after detection, the purified PCR product was sent to Shanghai Bioengineering Co., Ltd for sequencing. The nucleotide length of the locust migratory clathrin heavy chain gene obtained by sequencing is 5296bp, and the nucleotide sequence is SEQ ID NO: 1, and (b) is shown in the specification.
3. Analysis of amino acid sequence of migratory locust clathrin heavy chain gene
The clathrin heavy chain gene obtained by sequencing is translated through ExPaSy online software, and 1677 amino acids are predicted to be coded by the open reading frame of the clathrin heavy chain gene, the molecular weight is 192KD, and the isoelectric point is 5.46. Functional domain prediction the clathrin heavy chain gene was found to have 7 clathrin heavy chain repeat homology domains (CLH). The coded amino acid sequence is SEQ ID NO: 2.
Example 2
Synthesizing targeting dsRNA of the migratory locust clathrin heavy chain gene:
1. design of targeting dsRNA primer of migratory locust clathrin heavy chain gene
Based on the gene sequence of the migratory locust clathrin heavy chain obtained by sequencing, primer premier5.0 software is adopted to design a primer of the migratory locust clathrin heavy chain to target dsRNA. The sequences of the obtained primers were as follows (the italic part was the T7 promoter):
the upstream primer SEQ ID NO: 6:
Figure BDA0002787742630000061
downstream primer SEQ ID NO: 7:
Figure BDA0002787742630000071
the primers were synthesized by Shanghai Bioengineering Ltd.
2. Synthesis of migratory locust clathrin heavy chain gene specific dsRNA
Taking the clathrin heavy chain gene extracted from the plasmid as a template, and carrying out PCR amplification by using the synthesized upstream and downstream primers containing the T7 promoter sequence to obtain a gene fragment (SEQ ID NO: 7) with the length of 538 bp.
The PCR amplification system is as follows: 2 Taq PCR Master Mix II 25 uL, upstream and downstream primers 1 uL, double distilled water 21 uL, cDNA template 2 uL, total volume of 50 uL.
The reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, and circulating for 35 times; final extension at 72 ℃ for 7 min.
The PCR product was purified using Gel Extraction Kit (Omega) Kit and then subjected to T7RiboMAXTMExpress RNAi System (Promega) kit description in vitro transcription Synthesis of dsRNA (ds LmChc). It was quantified using NaNoDrop 2000(Thermo scientific) to a final concentration of 2.5. mu.g/. mu.L. Storing in a super low-temperature refrigerator at-80 deg.C for use.
Example 3
Targeted dsRNA lethal locust test of locust migratory locust clathrin heavy chain gene
1. Injection of targeted dsRNA
And selecting 33 healthy and consistent-size 5 th day migratory locust nymphs for experiment. Treatment groups 4 μ L (10 μ g) of synthetic dsRNA was gently injected into the nymph between the two and three abdominal segments of the flank using a 25 μ L format microinjector, setting 3 biological replicates, 11 beetles per biological replicate; meanwhile, 33 nymphs are selected to be set as a control group, dsGFP with the same volume and concentration as the dsRNA of the treatment group is injected, 3 biological replicates are also set, and 11 nymphs are repeated in each biological replicate. And (3) feeding the locusta migratoria after injection in a constant-temperature culture room at the temperature of 30 ℃ (the illumination time is 14 h: 10h, the temperature is 30 +/-2 ℃, and the humidity is 60%). The control group and the treatment group were fed with fresh wheat seedlings and wheat bran every day.
2. Migratory locust clathrin heavy chain gene silencing detection
Collecting 9 nymphs of 24h nymphs after injection of dsGFP and dsRNA (dsLmChc), extracting total RNA according to a TaKaRa Trizol kit, performing reverse transcription on the total RNA into first-strand cDNA by adopting M-MLV reverse transcriptase, and respectively detecting the relative expression amounts of a target gene (LmChc) and a housekeeping gene (EF1a) by adopting a Real-time PCR method, thereby adopting 2-ΔCtEfficient silencing of gene LmChcAnd (5) line calculation. Each group was set with 3 biological replicates, each biological replicate 3 nymphs.
The reaction system of the Real-time PCR method is as follows: SYBR Green mix 10. mu.L, upstream and downstream primers 0.8. mu.L each, cDNA template 4. mu.L, deionized water 4.4. mu.L, 20. mu.L total.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 1min, and melting at 95 ℃ for 15 s; annealing and extending at 60 ℃ for 31s, circulating for 40 times, carrying out fluorescence detection at 60 ℃, carrying out DNA melting analysis at 60-95 ℃, and increasing by 1 ℃ in each step.
The results in fig. 2 show that the expression of the graticum heavy chain gene (LmChc) of the treated locusta migratoria is significantly reduced after injection of dsrna (dslmchc) compared to the control group. The result shows that the dsRNA segment can obviously silence the expression level of a target gene, is effective and can be used for subsequent research.
Influence of dsRNA on growth and development of 5-year migratory locust nymphs
Five-year-old nymphs of the treated and control groups were phenotypically observed after injection of dsRNA. From figure 3, it can be seen that after the five-instar nymphs injected with the dsRNA, the control group worms all successfully molted to adults on day 7 of 5 th instar, and the adult development state after molting was good. After the treatment group is injected with dsLmChc, nymphs cannot normally molt and have death phenotype, and the mortality rate reaches more than 94%. The result shows that the clathrin heavy chain gene plays an important role in maintaining the normal growth and development process of the migratory locust, and the migratory locust cannot normally develop due to the silencing of the clathrin heavy chain gene. Therefore, the dsRNA targeting the clathrin heavy chain gene can be applied to locust control work.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Sequence listing
SEQ ID NO：1
cagtttcagtcgtgagtgcgattagttataaggtggacatccagaaaggatctgttattaacagaataatatccctgtggactctatgcagctgtaaccaccatgacgcagatgttaccgatacggtttcaagagcacctacagctcacaagtgtaggaatcaaccaggccagcgtgagcttcaacacgctcacaatggaatcggacaaattcatatgtgtgagggagaaagttggtgacacagcacaggtagtgataatagacatggcagacccatcaaatcctatcagacgacccatatctgcagactcagctataatgaacccagcaagcaaagtaatagctcttaaaggaaaggctggtgttgaagcccagaaaacccttcagattttcaacattgagatgaagagcaaaatgaaagcacatacaatgacggaagacgttatattttggaaatggatttcacttaacaccttagctcttgtcacagagacatctgtgtatcactggagtatggagggtgactccgttccacaaaagatgttcgataggcactcaagcttaaacggttgtcagattattaattatcgaactgatccaaagcagtcatggctgctacttattggaatatctgctcaacaaagcagagtagttggagccatgcagctgtactcagttgaacgcaagtgttcgcaaccaattgaagggcatgctgcatcttttgcacagtttaagatggaaggcaatgctgagccatcgactcttttctgttttgcagtaagaacactacagggtggaaagctgcacataatagaagttggtcaccctccagcggggaatcaaccatttccaaagaaagctgtagacgttttcttcccaccagaagcacagaatgactttcctgtagccatgcaggtcagtgcaaagtatgatgttatctacctgataacaaaatatgggtacatccacttgtatgacatcgaaactgcagtatgcatatacatgaacagaataagtggtgatacaatattcgtaactgcaccacatgaatcatcgggtggaataatcggagttaaccgtaaaggacaggtgctgtctgtcagtgttgaagaagacaacataataccttacataaatagtgtacttcagaatccagatttggctttacgtatggctgtacgtaacaaccttgctggtgcagaagacttgtttgttaggaagtttaatctgctgttccaaaatgggcaatatgcagaggctgcaaaggttgctgccaatgcgccaaaaggaattctgcgaactccacaaactattcagcgattccagcaagtacctacaccacagggtcagacttcacctctgttgcaatattttggcattctattggatcagggacaattaaacaaatacgaaagtctggaactttgtcggcccgtcctgcagcaaggccgtaagcagttactggaaaaatggctaaaagaagacaagttagagtgctcggaagaacttggagatctagtcaagcaagcagaccccacattagcattgtcagtttatctgagagctaatgttcctaacaaggttattcagtgtttcgccgagactggtcagttccagaagatagtattatatgccaaaaaagttggctatacccctgactacatctttctgctgcgaaatgttatgcgaataaatcctgaccagggtgtaacttttgcccaaatgttagtccaagatgatgaacctcttgcagatataaatcagatagtggatatcttcatggaacaaaacatggtaccacagtgcacagctttcttactagatgccttgaaaaataataggcctgctgaaggaccattgcagacaagattattggaaatgaatttgatgtcggctccacaggttgctgatgcgatactaagcaatcagatgttcacccattatgatagagcccacatagcccagctctgtgagaaagctggccttctacaacgagcattggagcattatacagatctgtacgatataaaacgagctgttgttcatacacatctgctcaacccagaatggttagtgggatactttggtactctttctgtggaagattcactggaatgcttaaaggcaatgctgactgctaacattaggcagaatttgcagatctgtgtccagattgccacaaaataccatgagcagttaacaacaaaagctcttatagacttgtttgaatcattcaagagctatgaaggtcttttctacttcctgggttcaatagtaaatttcagccaggatcaagaagtgcacttcaaatacatacaggcggcatgcaagactggtcagataaaagaagtggagcgcatctgcagagaatctaattgttacaacgcagaacgtgtaaagaacttcttgaaagaagcaaagcttacagatcagctaccattaatcattgtttgtgatagatttgattttgtccacgatttggtgctatacctgtatcgcaacaacttacagaagtacattgaaatttatgtccaaaaggtgaatccatcaaggttaccagttgttgttggagggcttttagatgtggactgctcagaagatattataaagaacttaatattagttgtgcgtgggcagttttccacagatgagctggtggaagaagttgaaaaacggaacagactgaaattgcttctgccgtggctcgagtctcgtgtacatgagggctgtgtagaaccagctacacacaatgctctggcaaagatctacatagacagcaacaataatccagaaagattcttaaaggagaatcagttctatgatagccgtgttgtaggaaaatactgcgagaagagagatcctcatctagcttgtgttgcatatgaacggggccagtgtgacagagagcttattaatgtttgtaatgagaattcactgttcaaatcggaagccaggtatctagttcgaagaagggatcctgaactttgggctgaagtcctaaatgaaaacaatccctataagaggcctctcatagaccaagtggtgcaaactgcattgtcggaaacccaggatccagaagatatatcagtaactgtaaaagccttcatgactgctgatctccctaatgaacttatagaactgctagaaaaaatagtattggataattctgtatttagtgatcatagaaaccttcaaaatttgttgatcctgacggcaataaaagctgatcgcagccgtgtgatggaatacattaaccgcctagataactatgatgcaccagatattgctaatattgccattaataatcagttgtatgaagaagccttcgcaattttcaaaaaatttgatgtgaacacatcagcaatccaggtgctaattgacaatgttcaaaatcttgaccgagcatatgaatttgctgaaagatgtaatgagcctgctgtttggagccagcttgcaaaagctcagttgcagcagggaatggttaaagaggcaatagactcattcataaaagctgatgatccatcagcatatatcgatgttgtagaaacggctcacaaaacaggaagctgggaagatcttgtaagatatctgcagatggctcgtaagaaggcgagggaatcgtatatagaatctgaattaatctatgcatatgcacgcacaaatagactagctgacttggaagaattcatttctggaccaaaccatgctgacattcagaagattggtgatcgctgcttcgatgatgggatgtatgaggctgcaaaactgttgtacaataatgtttcaaattttgctcggctggcaattactttagtacacctgaaagaattccaaggagctgtggatggtgcaagaaaagcaaatagcacaagaacatggaaagaagtgtgtttcgcctgtgtcgacagtgaagagttcagattagcacagatgtgtggccttcacattgttgttcatgccgatgaactggaagatctgataaactattaccaggacagaggctattttgaagaactgatcaacttgttagaagctgctcttggcttagaaagagcacacatggggatgtttacagaactggcaattttgtattcaaagtacaagcctgccaaaatgcgtgagcacttagaattattctggtctcgtgtaaacataccaaaggtattgcgggcagcagaacaagctcacctttgggcagaactagtattcctgtatgataagtatgaagaatatgataatgctgtaatagccatgatgaatcatccaacagaagcctggcgtgaagggcactttaaagacattattaccaaggttgctaacattgaactgtactacaaagctatccagttttatctagattacaaaccacttctgctgaatgatgtgttgctggtgctggcaccacgtatggatcacacgcgatcagttaacttcttcactaaggttaatcatttacaattagtgaagccataccttcgttctgttcagagcctgaataataaagctatcaatgaagcactaaataatcttttaatagaagaagaagattaccagggtttgcggacatcgatagatgcattcgacaacttcgataatattgcgcttgcgcaaaagttggagaagcatgagctaattgaattcaggcgcatagcagcatacttgtacaagggcaacaaccgttggaaacaatctgtggagctttgcaaaaaggatagactcttcaaggacgcaatggagtatgcagcagaatcgaagaatgccgaggtagcagaagagttacttgcatggtttctggagaaaggaaatcatgattgctttgcagcttgcttattccagtgttatgacctgcttcatccagatgtcatcctggaattagcttggaggcacaacattttagattttgctatgccttacctgatacaagttgtgcgtgaatacataacaaaggttgataaactggaagaggcagagtcacagcggttggaagagacggcgcagcaagatcataaacctatgataatgccggaaccccaactaatgctgacggctggtcctggaatggttggccccggatatgttcctgggtacacaaatgcctacgcacctggagtgccctatcaaggatatggaatgtaaatggctgctatgaagttttcgtgaaagggatcaaaagaaatggttttaattgtggtgtctataacatccatgtctgattagttactgttaaatgttgacgcatttagagaggattatggtaaagagattaatctgttgtttgttatttcagtgatgagcc
SEQ ID NO：2
MetThrMetIleArgHisThrSerValGlyIleAsnAlaSerValSerAsnThrThrMetSerAspLysIleCysValArgLysValGlyAspThrAlaValValIleIleAspMetAlaAspSerAsnIleArgArgIleSerAlaAspSerAlaIleMetAsnAlaSerLysValIleAlaLysGlyLysAlaGlyValAlaLysThrIleAsnIleMetLysSerLysMetLysAlaHisThrMetThrAspValIleTrpLysTrpIleSerAsnThrAlaValThrThrSerValTyrHisTrpSerMetGlyAspSerValLysMetAspArgHisSerSerAsnGlyCysIleIleAsnTyrArgThrAspLysSerTrpIleGlyIleSerAlaSerArgValValGlyAlaMetTyrSerValArgLysCysSerIleGlyHisAlaAlaSerAlaLysMetGlyAsnAlaSerThrCysAlaValArgThrGlyGlyLysHisIleIleValGlyHisAlaGlyAsnLysLysAlaValAspValAlaAsnAspValAlaMetValSerAlaLysTyrAspValIleTyrIleThrLysTyrGlyTyrIleHisTyrAspIleThrAlaValCysIleTyrMetAsnArgIleSerGlyAspThrIleValThrAlaHisSerSerGlyGlyIleIleGlyValAsnArgLysGlyValSerValSerValAspAsnIleIleTyrIleAsnSerValAsnAspAlaArgMetAlaValArgAsnAsnAlaGlyAlaAspValArgLysAsnAsnGlyTyrAlaAlaAlaLysValAlaAlaAsnAlaLysGlyIleArgThrThrIleArgValThrGlyThrSerTyrGlyIleAspGlyAsnLysTyrSerCysArgValGlyArgLysLysTrpLysAspLysCysSerGlyAspValLysAlaAspThrAlaSerValTyrArgAlaAsnValAsnLysValIleCysAlaThrGlyLysIleValTyrAlaLysLysValGlyTyrThrAspTyrIleArgAsnValMetArgIleAsnAspGlyValThrAlaMetValAspAspAlaAspIleAsnIleValAspIleMetAsnMetValCysThrAlaAspAlaLysAsnAsnArgAlaGlyThrArgMetAsnMetSerAlaValAlaAspAlaIleSerAsnMetThrHisTyrAspArgAlaHisIleAlaCysLysAlaGlyArgAlaHisTyrThrAspTyrAspIleLysArgAlaValValHisThrHisAsnTrpValGlyTyrGlyThrSerValAspSerCysLysAlaMetThrAlaAsnIleArgAsnIleCysValIleAlaThrLysTyrHisThrThrLysAlaIleAspSerLysSerTyrGlyTyrGlySerIleValAsnSerAspValHisLysTyrIleAlaAlaCysLysThrGlyIleLysValArgIleCysArgSerAsnCysTyrAsnAlaArgValLysAsnLysAlaLysThrAspIleIleValCysAspArgAspValHisAspValTyrTyrArgAsnAsnLysTyrIleIleTyrValLysValAsnSerArgValValValGlyGlyAspValAspCysSerAspIleIleLysAsnIleValValArgGlySerThrAspValValLysArgAsnArgLysTrpSerArgValHisGlyCysValAlaThrHisAsnAlaAlaLysIleTyrIleAspSerAsnAsnAsnArgLysAsnTyrAspSerArgValValGlyLysTyrCysLysArgAspHisAlaCysValAlaTyrArgGlyCysAspArgIleAsnValCysAsnAsnSerLysSerAlaArgTyrValArgArgArgAspTrpAlaValAsnAsnAsnTyrLysArgIleAspValValThrAlaSerThrAspAspIleSerValThrValLysAlaMetThrAlaAspAsnIleLysIleValAspAsnSerValSerAspHisArgAsnAsnIleThrAlaIleLysAlaAspArgSerArgValMetTyrIleAsnArgAspAsnTyrAspAlaAspIleAlaAsnIleAlaIleAsnAsnTyrAlaAlaIleLysLysAspValAsnThrSerAlaIleValIleAspAsnValAsnAspArgAlaTyrAlaArgCysAsnAlaValTrpSerAlaLysAlaGlyMetValLysAlaIleAspSerIleLysAlaAspAspSerAlaTyrIleAspValValThrAlaHisLysThrGlySerTrpAspValArgTyrMetAlaArgLysLysAlaArgSerTyrIleSerIleTyrAlaTyrAlaArgThrAsnArgAlaAspIleSerGlyAsnHisAlaAspIleLysIleGlyAspArgCysAspAspGlyMetTyrAlaAlaLysTyrAsnAsnValSerAsnAlaArgAlaIleThrValHisLysGlyAlaValAspGlyAlaArgLysAlaAsnSerThrArgThrTrpLysValCysAlaCysValAspSerArgAlaMetCysGlyHisIleValValHisAlaAspAspIleAsnTyrTyrAspArgGlyTyrIleAsnAlaAlaGlyArgAlaHisMetGlyMetThrAlaIleTyrSerLysTyrLysAlaLysMetArgHisTrpSerArgValAsnIleLysValArgAlaAlaAlaHisTrpAlaValTyrAspLysTyrTyrAspAsnAlaValIleAlaMetMetAsnHisThrAlaTrpArgGlyHisLysAspIleIleThrLysValAlaAsnIleTyrTyrLysAlaIleTyrAspTyrLysAsnAspValValAlaArgMetAspHisThrArgSerValAsnThrLysValAsnHisValLysTyrArgSerValSerAsnAsnLysAlaIleAsnAlaAsnAsnIleAspTyrGlyArgThrSerIleAspAlaAspAsnAspAsnIleAlaAlaLysLysHisIleArgArgIleAlaAlaTyrTyrLysGlyAsnAsnArgTrpLysSerValCysLysLysAspArgLysAspAlaMetTyrAlaAlaSerLysAsnAlaValAlaAlaTrpLysGlyAsnHisAspCysAlaAlaCysCysTyrAspHisAspValIleAlaTrpArgHisAsnIleAspAlaMetTyrIleValValArgTyrIleThrLysValAspLysAlaSerArgThrAlaAspHisLysMetIleMetMetThrAlaGlyGlyMetValGlyGlyTyrValGlyTyrThrAsnAlaTyrAlaGlyValTyrGlyTyrGlyMet
SEQ ID NO：3
cagtttcagtcgtgagtgcgattagt
SEQ ID NO：4
ggctcatcactgaaataacaaacaaca
SEQ ID NO：5
taatacgactcactatagggttgctggtgcagaagacttgtttgttaggaagtttaatctgctgttccaaaatgggcaatatgcagaggctgcaaaggttgctgccaatgcgccaaaaggaattctgcgaactccacaaactattcagcgattccagcaagtacctacaccacagggtcagacttcacctctgttgcaatattttggcattctattggatcagggacaattaaacaaatacgaaagtctggaactttgtcggcccgtcctgcagcaaggccgtaagcagttactggaaaaatggctaaaagaagacaagttagagtgctcggaagaacttggagatctagtcaagcaagcagaccccacattagcattgtcagtttatctgagagctaatgttcctaacaaggttattcagtgtttcgccgagactggtcagttccagaagatagtattatatgccaaaaaagttggctatacccctgactacatctttctgctgcgaaatgttatgctaatacgactcactataggg
SEQ ID NO：6
taatacgactcactatagggttgctggtgcagaagacttg
SEQ ID NO：7
taatacgactcactataggggcataacatttcgcagcaga
Sequence listing
<110> university of Shanxi
<120> migratory locust clathrin heavy chain gene and synthesis and application of targeting dsRNA thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5296
<212> DNA
<213> migratory locust (Locusa migratoria)
<400> 1
cagtttcagt cgtgagtgcg attagttata aggtggacat ccagaaagga tctgttatta 60
acagaataat atccctgtgg actctatgca gctgtaacca ccatgacgca gatgttaccg 120
atacggtttc aagagcacct acagctcaca agtgtaggaa tcaaccaggc cagcgtgagc 180
ttcaacacgc tcacaatgga atcggacaaa ttcatatgtg tgagggagaa agttggtgac 240
acagcacagg tagtgataat agacatggca gacccatcaa atcctatcag acgacccata 300
tctgcagact cagctataat gaacccagca agcaaagtaa tagctcttaa aggaaaggct 360
ggtgttgaag cccagaaaac ccttcagatt ttcaacattg agatgaagag caaaatgaaa 420
gcacatacaa tgacggaaga cgttatattt tggaaatgga tttcacttaa caccttagct 480
cttgtcacag agacatctgt gtatcactgg agtatggagg gtgactccgt tccacaaaag 540
atgttcgata ggcactcaag cttaaacggt tgtcagatta ttaattatcg aactgatcca 600
aagcagtcat ggctgctact tattggaata tctgctcaac aaagcagagt agttggagcc 660
atgcagctgt actcagttga acgcaagtgt tcgcaaccaa ttgaagggca tgctgcatct 720
tttgcacagt ttaagatgga aggcaatgct gagccatcga ctcttttctg ttttgcagta 780
agaacactac agggtggaaa gctgcacata atagaagttg gtcaccctcc agcggggaat 840
caaccatttc caaagaaagc tgtagacgtt ttcttcccac cagaagcaca gaatgacttt 900
cctgtagcca tgcaggtcag tgcaaagtat gatgttatct acctgataac aaaatatggg 960
tacatccact tgtatgacat cgaaactgca gtatgcatat acatgaacag aataagtggt 1020
gatacaatat tcgtaactgc accacatgaa tcatcgggtg gaataatcgg agttaaccgt 1080
aaaggacagg tgctgtctgt cagtgttgaa gaagacaaca taatacctta cataaatagt 1140
gtacttcaga atccagattt ggctttacgt atggctgtac gtaacaacct tgctggtgca 1200
gaagacttgt ttgttaggaa gtttaatctg ctgttccaaa atgggcaata tgcagaggct 1260
gcaaaggttg ctgccaatgc gccaaaagga attctgcgaa ctccacaaac tattcagcga 1320
ttccagcaag tacctacacc acagggtcag acttcacctc tgttgcaata ttttggcatt 1380
ctattggatc agggacaatt aaacaaatac gaaagtctgg aactttgtcg gcccgtcctg 1440
cagcaaggcc gtaagcagtt actggaaaaa tggctaaaag aagacaagtt agagtgctcg 1500
gaagaacttg gagatctagt caagcaagca gaccccacat tagcattgtc agtttatctg 1560
agagctaatg ttcctaacaa ggttattcag tgtttcgccg agactggtca gttccagaag 1620
atagtattat atgccaaaaa agttggctat acccctgact acatctttct gctgcgaaat 1680
gttatgcgaa taaatcctga ccagggtgta acttttgccc aaatgttagt ccaagatgat 1740
gaacctcttg cagatataaa tcagatagtg gatatcttca tggaacaaaa catggtacca 1800
cagtgcacag ctttcttact agatgccttg aaaaataata ggcctgctga aggaccattg 1860
cagacaagat tattggaaat gaatttgatg tcggctccac aggttgctga tgcgatacta 1920
agcaatcaga tgttcaccca ttatgataga gcccacatag cccagctctg tgagaaagct 1980
ggccttctac aacgagcatt ggagcattat acagatctgt acgatataaa acgagctgtt 2040
gttcatacac atctgctcaa cccagaatgg ttagtgggat actttggtac tctttctgtg 2100
gaagattcac tggaatgctt aaaggcaatg ctgactgcta acattaggca gaatttgcag 2160
atctgtgtcc agattgccac aaaataccat gagcagttaa caacaaaagc tcttatagac 2220
ttgtttgaat cattcaagag ctatgaaggt cttttctact tcctgggttc aatagtaaat 2280
ttcagccagg atcaagaagt gcacttcaaa tacatacagg cggcatgcaa gactggtcag 2340
ataaaagaag tggagcgcat ctgcagagaa tctaattgtt acaacgcaga acgtgtaaag 2400
aacttcttga aagaagcaaa gcttacagat cagctaccat taatcattgt ttgtgataga 2460
tttgattttg tccacgattt ggtgctatac ctgtatcgca acaacttaca gaagtacatt 2520
gaaatttatg tccaaaaggt gaatccatca aggttaccag ttgttgttgg agggctttta 2580
gatgtggact gctcagaaga tattataaag aacttaatat tagttgtgcg tgggcagttt 2640
tccacagatg agctggtgga agaagttgaa aaacggaaca gactgaaatt gcttctgccg 2700
tggctcgagt ctcgtgtaca tgagggctgt gtagaaccag ctacacacaa tgctctggca 2760
aagatctaca tagacagcaa caataatcca gaaagattct taaaggagaa tcagttctat 2820
gatagccgtg ttgtaggaaa atactgcgag aagagagatc ctcatctagc ttgtgttgca 2880
tatgaacggg gccagtgtga cagagagctt attaatgttt gtaatgagaa ttcactgttc 2940
aaatcggaag ccaggtatct agttcgaaga agggatcctg aactttgggc tgaagtccta 3000
aatgaaaaca atccctataa gaggcctctc atagaccaag tggtgcaaac tgcattgtcg 3060
gaaacccagg atccagaaga tatatcagta actgtaaaag ccttcatgac tgctgatctc 3120
cctaatgaac ttatagaact gctagaaaaa atagtattgg ataattctgt atttagtgat 3180
catagaaacc ttcaaaattt gttgatcctg acggcaataa aagctgatcg cagccgtgtg 3240
atggaataca ttaaccgcct agataactat gatgcaccag atattgctaa tattgccatt 3300
aataatcagt tgtatgaaga agccttcgca attttcaaaa aatttgatgt gaacacatca 3360
gcaatccagg tgctaattga caatgttcaa aatcttgacc gagcatatga atttgctgaa 3420
agatgtaatg agcctgctgt ttggagccag cttgcaaaag ctcagttgca gcagggaatg 3480
gttaaagagg caatagactc attcataaaa gctgatgatc catcagcata tatcgatgtt 3540
gtagaaacgg ctcacaaaac aggaagctgg gaagatcttg taagatatct gcagatggct 3600
cgtaagaagg cgagggaatc gtatatagaa tctgaattaa tctatgcata tgcacgcaca 3660
aatagactag ctgacttgga agaattcatt tctggaccaa accatgctga cattcagaag 3720
attggtgatc gctgcttcga tgatgggatg tatgaggctg caaaactgtt gtacaataat 3780
gtttcaaatt ttgctcggct ggcaattact ttagtacacc tgaaagaatt ccaaggagct 3840
gtggatggtg caagaaaagc aaatagcaca agaacatgga aagaagtgtg tttcgcctgt 3900
gtcgacagtg aagagttcag attagcacag atgtgtggcc ttcacattgt tgttcatgcc 3960
gatgaactgg aagatctgat aaactattac caggacagag gctattttga agaactgatc 4020
aacttgttag aagctgctct tggcttagaa agagcacaca tggggatgtt tacagaactg 4080
gcaattttgt attcaaagta caagcctgcc aaaatgcgtg agcacttaga attattctgg 4140
tctcgtgtaa acataccaaa ggtattgcgg gcagcagaac aagctcacct ttgggcagaa 4200
ctagtattcc tgtatgataa gtatgaagaa tatgataatg ctgtaatagc catgatgaat 4260
catccaacag aagcctggcg tgaagggcac tttaaagaca ttattaccaa ggttgctaac 4320
attgaactgt actacaaagc tatccagttt tatctagatt acaaaccact tctgctgaat 4380
gatgtgttgc tggtgctggc accacgtatg gatcacacgc gatcagttaa cttcttcact 4440
aaggttaatc atttacaatt agtgaagcca taccttcgtt ctgttcagag cctgaataat 4500
aaagctatca atgaagcact aaataatctt ttaatagaag aagaagatta ccagggtttg 4560
cggacatcga tagatgcatt cgacaacttc gataatattg cgcttgcgca aaagttggag 4620
aagcatgagc taattgaatt caggcgcata gcagcatact tgtacaaggg caacaaccgt 4680
tggaaacaat ctgtggagct ttgcaaaaag gatagactct tcaaggacgc aatggagtat 4740
gcagcagaat cgaagaatgc cgaggtagca gaagagttac ttgcatggtt tctggagaaa 4800
ggaaatcatg attgctttgc agcttgctta ttccagtgtt atgacctgct tcatccagat 4860
gtcatcctgg aattagcttg gaggcacaac attttagatt ttgctatgcc ttacctgata 4920
caagttgtgc gtgaatacat aacaaaggtt gataaactgg aagaggcaga gtcacagcgg 4980
ttggaagaga cggcgcagca agatcataaa cctatgataa tgccggaacc ccaactaatg 5040
ctgacggctg gtcctggaat ggttggcccc ggatatgttc ctgggtacac aaatgcctac 5100
gcacctggag tgccctatca aggatatgga atgtaaatgg ctgctatgaa gttttcgtga 5160
aagggatcaa aagaaatggt tttaattgtg gtgtctataa catccatgtc tgattagtta 5220
ctgttaaatg ttgacgcatt tagagaggat tatggtaaag agattaatct gttgtttgtt 5280
atttcagtga tgagcc 5296
<210> 2
<211> 1136
<212> PRT
<213> migratory locust (Locusa migratoria)
<400> 2
Met Thr Met Ile Ala His Thr Ser Val Gly Ile Ala Ala Ser Val Ser
1 5 10 15
Ala Thr Thr Met Ser Ala Leu Ile Cys Val Ala Leu Val Gly Ala Thr
20 25 30
Ala Val Val Ile Ile Ala Met Ala Ala Ser Ala Ile Ala Ala Ile Ser
35 40 45
Ala Ala Ser Ala Ile Met Ala Ala Ser Leu Val Ile Ala Leu Gly Leu
50 55 60
Ala Gly Val Ala Leu Thr Ile Ala Ile Met Leu Ser Leu Met Leu Ala
65 70 75 80
His Thr Met Thr Ala Val Ile Thr Leu Thr Ile Ser Ala Thr Ala Val
85 90 95
Thr Thr Ser Val Thr His Thr Ser Met Gly Ala Ser Val Leu Met Ala
100 105 110
Ala His Ser Ser Ala Gly Cys Ile Ile Ala Thr Ala Thr Ala Leu Ser
115 120 125
Thr Ile Gly Ile Ser Ala Ser Ala Val Val Gly Ala Met Thr Ser Val
130 135 140
Ala Leu Cys Ser Ile Gly His Ala Ala Ser Ala Leu Met Gly Ala Ala
145 150 155 160
Ser Thr Cys Ala Val Ala Thr Gly Gly Leu His Ile Ile Val Gly His
165 170 175
Ala Gly Ala Leu Leu Ala Val Ala Val Ala Ala Ala Val Ala Met Val
180 185 190
Ser Ala Leu Thr Ala Val Ile Thr Ile Thr Leu Thr Gly Thr Ile His
195 200 205
Thr Ala Ile Thr Ala Val Cys Ile Thr Met Ala Ala Ile Ser Gly Ala
210 215 220
Thr Ile Val Thr Ala His Ser Ser Gly Gly Ile Ile Gly Val Ala Ala
225 230 235 240
Leu Gly Val Ser Val Ser Val Ala Ala Ile Ile Thr Ile Ala Ser Val
245 250 255
Ala Ala Ala Ala Met Ala Val Ala Ala Ala Ala Gly Ala Ala Val Ala
260 265 270
Leu Ala Ala Gly Thr Ala Ala Ala Leu Val Ala Ala Ala Ala Leu Gly
275 280 285
Ile Ala Thr Thr Ile Ala Val Thr Gly Thr Ser Thr Gly Ile Ala Gly
290 295 300
Ala Leu Thr Ser Cys Ala Val Gly Ala Leu Leu Thr Leu Ala Leu Cys
305 310 315 320
Ser Gly Ala Val Leu Ala Ala Thr Ala Ser Val Thr Ala Ala Ala Val
325 330 335
Ala Leu Val Ile Cys Ala Thr Gly Leu Ile Val Thr Ala Leu Leu Val
340 345 350
Gly Thr Thr Ala Thr Ile Ala Ala Val Met Ala Ile Ala Ala Gly Val
355 360 365
Thr Ala Met Val Ala Ala Ala Ala Ile Ala Ile Val Ala Ile Met Ala
370 375 380
Met Val Cys Thr Ala Ala Ala Leu Ala Ala Ala Ala Gly Thr Ala Met
385 390 395 400
Ala Met Ser Ala Val Ala Ala Ala Ile Ser Ala Met Thr His Thr Ala
405 410 415
Ala Ala His Ile Ala Cys Leu Ala Gly Ala Ala His Thr Thr Ala Thr
420 425 430
Ala Ile Leu Ala Ala Val Val His Thr His Ala Thr Val Gly Thr Gly
435 440 445
Thr Ser Val Ala Ser Cys Leu Ala Met Thr Ala Ala Ile Ala Ala Ile
450 455 460
Cys Val Ile Ala Thr Leu Thr His Thr Thr Leu Ala Ile Ala Ser Leu
465 470 475 480
Ser Thr Gly Thr Gly Ser Ile Val Ala Ser Ala Val His Leu Thr Ile
485 490 495
Ala Ala Cys Leu Thr Gly Ile Leu Val Ala Ile Cys Ala Ser Ala Cys
500 505 510
Thr Ala Ala Ala Val Leu Ala Leu Ala Leu Thr Ala Ile Ile Val Cys
515 520 525
Ala Ala Ala Val His Ala Val Thr Thr Ala Ala Ala Leu Thr Ile Ile
530 535 540
Thr Val Leu Val Ala Ser Ala Val Val Val Gly Gly Ala Val Ala Cys
545 550 555 560
Ser Ala Ile Ile Leu Ala Ile Val Val Ala Gly Ser Thr Ala Val Val
565 570 575
Leu Ala Ala Ala Leu Thr Ser Ala Val His Gly Cys Val Ala Thr His
580 585 590
Ala Ala Ala Leu Ile Thr Ile Ala Ser Ala Ala Ala Ala Leu Ala Thr
595 600 605
Ala Ser Ala Val Val Gly Leu Thr Cys Leu Ala Ala His Ala Cys Val
610 615 620
Ala Thr Ala Gly Cys Ala Ala Ile Ala Val Cys Ala Ala Ser Leu Ser
625 630 635 640
Ala Ala Thr Val Ala Ala Ala Ala Thr Ala Val Ala Ala Ala Thr Leu
645 650 655
Ala Ile Ala Val Val Thr Ala Ser Thr Ala Ala Ile Ser Val Thr Val
660 665 670
Leu Ala Met Thr Ala Ala Ala Ile Leu Ile Val Ala Ala Ser Val Ser
675 680 685
Ala His Ala Ala Ala Ile Thr Ala Ile Leu Ala Ala Ala Ser Ala Val
690 695 700
Met Thr Ile Ala Ala Ala Ala Thr Ala Ala Ala Ile Ala Ala Ile Ala
705 710 715 720
Ile Ala Ala Thr Ala Ala Ile Leu Leu Ala Val Ala Thr Ser Ala Ile
725 730 735
Val Ile Ala Ala Val Ala Ala Ala Ala Thr Ala Ala Cys Ala Ala Val
740 745 750
Thr Ser Ala Leu Ala Gly Met Val Leu Ala Ile Ala Ser Ile Leu Ala
755 760 765
Ala Ala Ser Ala Thr Ile Ala Val Val Thr Ala His Leu Thr Gly Ser
770 775 780
Thr Ala Val Ala Thr Met Ala Ala Leu Leu Ala Ala Ser Thr Ile Ser
785 790 795 800
Ile Thr Ala Thr Ala Ala Thr Ala Ala Ala Ala Ile Ser Gly Ala His
805 810 815
Ala Ala Ile Leu Ile Gly Ala Ala Cys Ala Ala Gly Met Thr Ala Ala
820 825 830
Leu Thr Ala Ala Val Ser Ala Ala Ala Ala Ile Thr Val His Leu Gly
835 840 845
Ala Val Ala Gly Ala Ala Leu Ala Ala Ser Thr Ala Thr Thr Leu Val
850 855 860
Cys Ala Cys Val Ala Ser Ala Ala Met Cys Gly His Ile Val Val His
865 870 875 880
Ala Ala Ala Ile Ala Thr Thr Ala Ala Gly Thr Ile Ala Ala Ala Gly
885 890 895
Ala Ala His Met Gly Met Thr Ala Ile Thr Ser Leu Thr Leu Ala Leu
900 905 910
Met Ala His Thr Ser Ala Val Ala Ile Leu Val Ala Ala Ala Ala His
915 920 925
Thr Ala Val Thr Ala Leu Thr Thr Ala Ala Ala Val Ile Ala Met Met
930 935 940
Ala His Thr Ala Thr Ala Gly His Leu Ala Ile Ile Thr Leu Val Ala
945 950 955 960
Ala Ile Thr Thr Leu Ala Ile Thr Ala Thr Leu Ala Ala Val Val Ala
965 970 975
Ala Met Ala His Thr Ala Ser Val Ala Thr Leu Val Ala His Val Leu
980 985 990
Thr Ala Ser Val Ser Ala Ala Leu Ala Ile Ala Ala Ala Ala Ile Ala
995 1000 1005
Thr Gly Ala Thr Ser Ile Ala Ala Ala Ala Ala Ala Ile Ala Ala Leu
1010 1015 1020
Leu His Ile Ala Ala Ile Ala Ala Thr Thr Leu Gly Ala Ala Ala Thr
1025 1030 1035 1040
Leu Ser Val Cys Leu Leu Ala Ala Leu Ala Ala Met Thr Ala Ala Ser
1045 1050 1055
Leu Ala Ala Val Ala Ala Thr Leu Gly Ala His Ala Cys Ala Ala Cys
1060 1065 1070
Cys Thr Ala His Ala Val Ile Ala Thr Ala His Ala Ile Ala Ala Met
1075 1080 1085
Thr Ile Val Val Ala Thr Ile Thr Leu Val Ala Leu Ala Ser Ala Thr
1090 1095 1100
Ala Ala His Leu Met Ile Met Met Thr Ala Gly Gly Met Val Gly Gly
1105 1110 1115 1120
Thr Val Gly Thr Thr Ala Ala Thr Ala Gly Val Thr Gly Thr Gly Met
1125 1130 1135
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cagtttcagt cgtgagtgcg attagt 26
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggctcatcac tgaaataaca aacaaca 27
<210> 7
<211> 538
<212> DNA
<213> migratory locust (Locusa migratoria)
<400> 7
taatacgact cactataggg ttgctggtgc agaagacttg tttgttagga agtttaatct 60
gctgttccaa aatgggcaat atgcagaggc tgcaaaggtt gctgccaatg cgccaaaagg 120
aattctgcga actccacaaa ctattcagcg attccagcaa gtacctacac cacagggtca 180
gacttcacct ctgttgcaat attttggcat tctattggat cagggacaat taaacaaata 240
cgaaagtctg gaactttgtc ggcccgtcct gcagcaaggc cgtaagcagt tactggaaaa 300
atggctaaaa gaagacaagt tagagtgctc ggaagaactt ggagatctag tcaagcaagc 360
agaccccaca ttagcattgt cagtttatct gagagctaat gttcctaaca aggttattca 420
gtgtttcgcc gagactggtc agttccagaa gatagtatta tatgccaaaa aagttggcta 480
tacccctgac tacatctttc tgctgcgaaa tgttatgcta atacgactca ctataggg 538
<210> 5
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
taatacgact cactataggg ttgctggtgc agaagacttg 40
<210> 6
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
taatacgact cactataggg gcataacatt tcgcagcaga 40

Claims (9)

1. The migratory locust clathrin heavy chain gene is characterized in that: the full-length nucleotide sequence of the gene is SEQ ID NO: 1, and (b) is shown in the specification.
2. The method for obtaining the migratory locust clathrin heavy chain gene as claimed in claim 1, which comprises the following steps:
step 1, searching Unigene of migratory locust based on a migratory locust transcriptome database, and obtaining a fragment of a migratory locust clathrin heavy chain gene after NCBI Blastx analysis;
step 2, splicing the fragments obtained by searching in the step 1 through GeneDoc software;
and 3, designing and synthesizing a corresponding upstream primer SEQ ID NO by using primer premier5.0 software according to the splicing result of the step 2: 3 and the downstream primer SEQ ID NO: 4;
step 4, extracting RNA of locusta migratoria and performing reverse transcription to obtain cDNA to obtain a template required by PCR amplification;
and 5, obtaining the migratory locust clathrin heavy chain gene through PCR amplification reaction by adopting the primer synthesized in the step 3 and the template in the step 4.
3. The locust tree network protein heavy chain gene targeting dsRNA as claimed in any one of claims 1 to 2, wherein: one of the target dsRNA has a nucleotide sequence of SEQ ID NO: 5, and (c) a sequence shown in the specification.
4. The synthetic method of the locust fly clathrin heavy chain gene targeting dsRNA of claim 3, characterized by comprising the following steps:
step 1, designing an upstream primer SEQ ID NO: 6 and downstream primer SEQ ID NO: 7;
and 2, obtaining a template DNA fragment with two ends both being T7 promoters through PCR amplification, and obtaining the target dsRNA of the migratory locust clathrin heavy chain gene through in vitro transcription synthesis after the template DNA fragment is purified by a kit.
5. The use of the locust tree network protein heavy chain gene targeting dsRNA of any one of claims 3 to 4, wherein: is used for controlling locusta migratoria.
6. The use of the locust fly clathrin heavy chain gene targeting dsRNA as claimed in claim 5, wherein: the target dsRNA is used for controlling the migratory locust by injecting the synthesized dsRNA into a migratory locust body cavity through a microsyringe to knock down the expression of a migratory locust clathrin heavy chain gene, thereby influencing the growth and development of the migratory locust.
7. The method for obtaining the migratory locust clathrin heavy chain gene as claimed in claim 2, wherein the template preparation process in step 4 is as follows: directly freezing 5-year-old locusta migratoria that is healthy in growth, consistent in size and half in sex in liquid nitrogen, then placing the locusta migratoria into a mortar for grinding, extracting RNA according to a kit, and carrying out reverse transcription on the extracted RNA into first-strand cDNA by adopting M-MLV reverse transcriptase so as to obtain a template required by PCR reaction.
8. The method for obtaining the migratory locust clathrin heavy chain gene as claimed in claim 2, wherein the PCR amplification reaction system in the step 5 is as follows: PrimeSTAR Max Premix (2X) 10. mu.L, upstream and downstream primers 0.2. mu.L each, template 1. mu.L, double distilled water 8.6. mu.L, total volume 20. mu.L; the reaction procedure is as follows: pre-denaturation at 98 ℃ for 10 s; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 150s, and the cycle is repeated 35 times.
9. The synthetic method of the locust fly clathrin heavy chain gene targeting dsRNA as claimed in claim 4, wherein the system of PCR amplification reaction in step 2 is: 2 × taq PCR Master Mix II 25 μ L, upstream and downstream primers 1 μ L each, double distilled water 21 μ L, cDNA template 2 μ L, total volume of 50 μ L; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, and circulating for 35 times; final extension at 72 ℃ for 7 min.
CN202011303949.0A 2020-11-19 2020-11-19 Migratory locust clathrin heavy chain gene and synthesis and application of targeting dsRNA thereof Pending CN112391390A (en)

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