CN112695045A - Lepidoptera insect Hpx12 gene and application thereof - Google Patents

Abstract

The present invention proposes a lepidopteran Hpx12 gene and its application. The first purpose of the present invention is to provide a lepidopteran innate immune response regulation gene Hpx12 ; the second purpose is to provide the lepidopteran innate immunity A cloning method of the response regulation gene Hpx12 ; the third purpose is to provide the application of the said Lepidopteran innate immune response regulation gene Hpx12 . In the present invention, after inhibiting the expression of the lepidopteran Hpx12 gene, the innate immune response of the worm body is inhibited, the susceptibility of the worm body to pathogens is increased, the silkworm is more susceptible to BmNPV virus infection, and the mortality rate of the lepidopteran insect is greatly increased. If the expression of Hpx12 in Lepidoptera insects is inhibited, the purpose of pest control can be achieved. It has positive significance for solving ecological deterioration caused by chemical pesticides and pesticide resistance.

Description

Lepidoptera insect Hpx12 gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a lepidoptera insectHpx12Genes and applications thereof.

Background

Lepidoptera includes two kinds of insects, i.e., moth and butterfly, and belongs to the subclass pteroidea and holomorphia. About 20 thousands of species are known worldwide, and about 8000 more species are known in china. This order is the 2 nd largest order of the Insecta, second only to the Coleoptera. The distribution range is very wide, and the tropical varieties are most abundant. Most kinds of larvae are harmful to various cultivated plants, and those with larger body form usually eat leaves or bore branches completely. Smaller patients tend to suffer from leaf curl, leaf ornamentation, scabbling, silking and netting, or food intake by digging into plant tissues. There are many important pests such as peach fruit borer, apple leaf roller, cotton bollworm, cabbage butterfly, diamond back moth and many lepidoptera granary worms such as indian meal moth, etc. In addition, the famous silkworms and tussah silkworms also belong to the order of insects. Silkworm is a lepidoptera seriation insect using mulberry leaves as food, and the silkworm passes through the development stages of eggs, larvae, pupae and imagoes which are completely different in morphology and physiological function in life.

Insects are the most flourishing species group on the earth land at present and are inexhaustible resource treasury of human beings. In recent years, the immunity of insects has attracted great attention in the aspects of basic and application research, and the research on genes, signals and other problems related to the immunity of insects through experiments has very important practical significance for pest control, insect benefiting and disease prevention, development and utilization of antibacterial substances, research on human immune mechanisms and the like.

The Hpx12 protein is a multifunctional protein, has a peroxidase conserved domain, has an integrin binding site, and can be combined with transmembrane receptor integrin, thereby realizing the adhesion between cells and extracellular matrix, and leading blood cells to play the roles of phagocytosis and coating. The Hpx12 protein has been studied less in Insecta, and the function of the protein has been studied less in Bombyx mori.

Disclosure of Invention

The present invention is based on at least one of the above technical problems, and the present invention relates to lepidopteran insectsHpx12The gene can inhibit the innate immune response of the insect body after being inhibited and expressed, the susceptibility of the insect body to germs is increased, the silkworm is more easily infected with BmNPV virus, and further the death rate of lepidoptera insects is greatly increased. If inhibiting lepidopteran insectsHpx12The expression of the gene can realize the purpose of pest control. Has positive significance for solving the problems of ecological deterioration, pesticide resistance and the like caused by chemical pesticides.

In view of the above, the present invention provides a lepidopteran insectHpx12The gene(s) is (are),Hpx12the gene is lepidoptera insect innate immune response regulating gene, and the nucleotide sequence is SEQ ID No. 1.

According to the second aspect of the invention, it proposesHpx12The gene expression inhibitor and/or activity inhibitor is applied to lepidoptera insects as a pest control target.

According to the third aspect of the invention, it proposesHpx12The gene coded protein has the amino acid sequence of SEQ ID No. 2.

According to the fourth aspect of the invention, it proposesHpx12Gene-encoded proteins inhibit lepidopteran insects in vivoHpx12The ds RNA of gene expression is used for reducing the innate immune response of lepidoptera insects and increasing the susceptibility of the lepidoptera insects to pathogenic bacteria.

Further, the ds RNA sequence is SEQ ID No. 3.

Further, the sequence of the primer for amplifying the ds RNA is SEQ ID No. 6-SEQ ID No. 7;

a forward primer: 5' -GTAATACGACTCACTATAGGGAGAGTTCGCTAT

GAATCGTTTTC -3’；SEQ ID No. 6；

Reverse primer: 5' -GTAATACGACTCACTATAGGGAGATTAGTTGCAGCTT

TAAAATCATTAA -3’；SEQ ID No. 7。

Further, the lepidopteran insect is a silkworm.

According to the fourth direction of the invention, the cloning right is proposedHpx12A method of genetic engineering comprising the steps of:

(1) mosquito according to the anophelesHpx12Protein sequence of gene (Gen Bank accession number (XP _311449), searching NCBI database to obtain homologous gene in silkwormHpx12And (4) sequencing. The sequence is used for designing gene cloning primers: SEQ ID No.4 to SEQ ID No. 5.

Synthesizing lepidoptera insect body cDNA: extracting lepidopterous insect RNA, and performing reverse transcription to obtain first-strand cDNA;

extracting the RNA of the whole silkworm, and performing reverse transcription to obtain a first chain c DNA;

（2）Hpx12PCR amplification of the genes: taking the lepidoptera insect body cDNA as a template, designing a primer according to the sequence of the lepidoptera insect body, carrying out PCR amplification, recovering and purifying a PCR amplification product, and sequencing.

According toHpx12Designing and synthesizing a specific primer by a gene sequence, carrying out PCR amplification by taking a first chain c DNA obtained by reverse transcription as a template, and selecting a TransStart high-fidelity amplification enzyme reaction system, wherein the total volume of the system is 50 mu L, and the method comprises the following steps: 200ng c DNA, 5 XTransStart FastPfu reaction buffer 10 uL, 10 Md NTP 1 uL, 2U TransStart FastPfu DNA Polymerase, 1 uL of forward and reverse primers each, and water is supplemented to 50 uL. The PCR reaction was carried out on a Mastercycler pro amplimer with the following reaction program: 4 minutes at 94 ℃; 30 cycles of 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃, 1 minute; extension at 72 ℃ for 10 min; the PCR product was recovered and purified.

The purified product is connected with a vector and is connected with pEASY-Blunt Cloning Kit vectors through a Kit reaction, and a connection system and the process are as follows: uniformly mixing 4 mu L of the purified product and 1 mu L of pEASY-Blunt Cloning Kit, and carrying out water bath for 30 min at 25 ℃; and transforming the connected vector into escherichia coli DH5a by heat shock, adding a liquid culture medium for shaking culture, then coating the obtained product on an LB (Langmuir-Blodgett) plate containing 100mg/L kanamycin for overnight culture, selecting bacterial colonies for bacterial liquid culture, extracting plasmids and detecting PCR (polymerase chain reaction). Screening positive clones, and sequencing the positive clones.

The invention firstly provides a lepidoptera insect innate immune response regulation geneHpx12Regulating gene for inhibiting lepidopteran insect innate immune responseHpx12The expression of the gene can reduce the innate immune response of lepidoptera insects, further increase the susceptibility of insect bodies to germs, and lead to the theory that the death rate of the lepidoptera insects is greatly increased, thereby having positive significance for solving the problems of ecological deterioration, insecticide resistance and the like caused by chemical insecticides.

Further, the primer sequence in the step (2) is SEQ ID No.4-SEQ ID No. 5;

a forward primer: 5' -CGGGATCCGTTCGCTATGAATCGTTTTC-3’； SEQ ID No.4；

Reverse primer: 5' -GGGTTCGAATTAGTTGCAGCTTTAAAATCATTAA-3’；SEQ ID No.5。

Through the technical scheme, the invention provides a lepidoptera insectHpx12The invention relates to a gene, protein and application, wherein the invention uses homologous clone technique to obtain a lepidoptera insect innate immune response regulation gene from silkwormHpx12Put forward and utilize for the first timeHpx12Gene expressed protein, gene for suppressing lepidopteran insect innate immune response regulationHpx12Expression of (a): the innate immune response of the lepidoptera insects is reduced, so that the susceptibility of the insects to germs is increased, and the death rate of the lepidoptera insects is greatly increased.

Drawings

FIG. 1 shows the results of example 1 of the present inventionHpx12Agarose gel electrophoresis results of the genes;

wherein, M is DNA Marker, and 1 is target segment.

FIG. 2 shows the results of example 2 of the present inventionHpx12After the gene ds RNA is injected into the worm,Hpx12the expression level of the gene at different times.

FIG. 3 shows a schematic diagram of a flowchart of an embodiment 3 of the present inventionHpx12Node number of silkworm larvae treated with BmNPV virus after injection of gene ds RNA into the body.

FIG. 4 shows a schematic diagram of a display device according to example 4 of the present inventionHpx12And (3) the expression level of the antibacterial peptide related gene 48h after the gene ds RNA is injected into the worm body.

FIG. 5 shows a schematic view of a display device according to example 5 of the present inventionHpx12And the death rate of the silkworm larvae is 48h after the gene ds RNA is injected into the silkworm bodies.

Detailed Description

In order that the above objects, features and advantages of the present invention can be more clearly understood, the present invention will be described in further detail with reference to specific embodiments. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.

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 may be practiced in other ways than those specifically described herein, and therefore the scope of the present invention is not limited by the specific embodiments disclosed below.

Example 1

Hpx12Cloning of genes

(1) Mosquito according to the anophelesHpx12Protein sequence of gene (Gen Bank accession number (XP _311449), searching NCBI database to obtain homologous gene in silkwormHpx12And (4) sequencing.

Lepidoptera insect innate immune response regulating geneHpx12The nucleotide sequence of (A) is shown in SEQ ID NO. 1.

Designing gene cloning primer ID No.4-SEQ ID No.5 by using the sequence;

a forward primer: 5' -CGGGATCCGTTCGCTATGAATCGTTTTC-3’； SEQ ID No.4；

Reverse primer: 5' -GGGTTCGAATTAGTTGCAGCTTTAAAATCATTAA-3’；SEQ ID No.5。

B. Extracting the RNA of the whole silkworm, and performing reverse transcription to obtain a first chain c DNA;

C. according toHpx12The gene sequence is obtained by performing PCR amplification by using primers SEQ ID No.4-SEQ ID No.5 and a first chain c DNA obtained by reverse transcription as a template, and a TransStart high fidelity amplification enzyme reaction system is selected, wherein the total volume of the system is 50 mu L, and the method comprises the following steps: 200ng c DNA, 5 XTransStart FastPfu reaction buffer 10 uL, 10 Md NTP 1 uL, 2U TransStart FastPfu DNA Polymerase, 1 uL of forward and reverse primers each, and water is supplemented to 50 uL. The PCR reaction is carried out inMastercycler pro amplification instrument, the reaction program is: 4 minutes at 94 ℃; 30 cycles of 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃, 1 minute; extension at 72 ℃ for 10 min; the PCR product was recovered and purified, and the result is shown in FIG. 1, and the desired product gene was obtained as shown in FIG. 1.

The purified product is connected with a vector and is connected with pEASY-Blunt Cloning Kit vectors through a Kit reaction, and a connection system and the process are as follows: uniformly mixing 4 mu L of the purified product and 1 mu L of pEASY-Blunt Cloning Kit, and carrying out water bath for 30 min at 25 ℃; and transforming the connected vector into escherichia coli DH5a by heat shock, adding a liquid culture medium for shaking culture, then coating the obtained product on an LB (Langmuir-Blodgett) plate containing 100mg/L kanamycin for overnight culture, selecting bacterial colonies for bacterial liquid culture, extracting plasmids and detecting PCR (polymerase chain reaction). Screening positive clones, and sequencing the positive clones.

Example 2

Lepidoptera insectsHpx12Synthesis and microinjection of Gene ds RNA

Lepidoptera insects obtained by cloning according to example 1Hpx12Full-length sequence SEQ ID NO.1 in gene, designHpx12Ds RNA of the gene is synthesized into ds RNA with a sequence of SEQ ID No.3 by an in vitro transcription reagent, and specifically, primers SEQ ID No. 6-SEQ ID No.7 are designed by a RiboMAX Large Scale RNA Production System-T7 kit;

a forward primer: 5' -GTAATACGACTCACTATAGGGAGAGTTCGCTAT

GAATCGTTTTC -3’；SEQ ID No. 6；

Reverse primer: 5' -GTAATACGACTCACTATAGGGAGATTAGTTGCAGCTT

TAAAATCATTAA -3’；SEQ ID No. 7。

Selecting silkworm larvae of 5 th 2 days old, uniform in size and consistent in health condition, setting an injected dsGFP control group and an injected dsHpx12 experiment group, anaesthetizing the silkworm larvae on ice before injection, then injecting ds RNA synthesized in vitro into a body cavity of the silkworm larvae by using a micro-injector, setting 3 biological repetitions by injecting 60 heads in each group, and feeding the silkworm larvae in a climatic chamber after injection.

Microinjection detection using fluorescent quantitative PCRAt last 6h, 12h, 24h and 48hHpx12As shown in FIG. 2, the gene expression and effect are best shown in FIG. 2, and it is found from FIG. 2 that 24h interference effect is best, and at this time, silkworms can be efficiently silencedHpx12The expression of the gene has better silencing effect of the target gene.

Example 3

Lepidoptera insectsHpx12The ds RNA of the gene can inhibit the innate immune response of the polypide and increase the susceptibility of the polypide to pathogenic bacteria

The surface disinfection is carried out on the 5 th-day and 3-day larva of the silkworm by using 75% ethanol. 2ml of BmNPV is injected into the blood cavity of the silkworm injected with dsRNA by a microinjector. After 2h at 25 ℃ melanin and dark nodules were counted under a microscope at 50 x magnification. Control insects were injected with only 2ml of BmNPV. Each test method was repeated three times as shown in FIG. 3. As can be seen from FIG. 3, the number of nodules was significantly reduced in the ds RNA-injected treated group compared to the control group, indicating that lepidopteran insectsHpx12After the expression of the gene is inhibited, the immunity of the silkworm is greatly reduced.

The expression of the antibacterial peptide genes (Defension 1 gene and CeCA gene) at 24h after the microinjection of ds RNA is detected by utilizing fluorescent quantitative PCR, the result of the treatment group injected with ds RNA is shown in figure 4 compared with the control group, and as can be seen from figure 4, the expression level of the antibacterial peptide genes is obviously reduced, which indicates that lepidoptera insects are shownHpx12After the expression of the gene is inhibited, the immune response of the silkworm is greatly reduced.

Silkworm (Bombyx mori)Hpx12Feeding individuals with down-regulated gene expression containing 1.5X 10⁴The results of bioassay on fresh mulberry leaves of BmNPV are shown in FIG. 5, and it can be seen from FIG. 5 thatHpx12The sensitivity of the silkworm with the down-regulated expression to BmNPV is obviously enhanced, and the death rate of the silkworm at 48h is obviously higher than that of the silkworm in the control.

Therefore, the design method for reducing the drug resistance of the pests on the basis of the molecular level has important significance for improving the pesticide effect of the biological pesticide and enhancing the pest control effect.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. .

Sequence listing

<110> Zhengzhou university of light industry

<120> lepidoptera insect Hpx12 gene and application

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ttagataact gcaccaatga cattcaactc ccctgcgacc ccaatgagag acgacgtcta 180

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agattgttac ccgaagccga ctgtggagac gatgaaacct ctccatctgg agctccttta 300

aaatctgccc gggaagtcag gcagcgtatt ttacagacag ggaaggcatc agacctctca 360

tacacacaac tattggcgat tatctcgaca atcatatttg ctgaccttgg ttcaatccat 420

gattccgtga atttgttaac ggagaccaca aattgttgca cagcggaagg aaaatcaaat 480

tatatgtgca cgcctataga tattccccag gacgaccctg tccataggtt ctctggtata 540

cgctgcttga atctcactcg accgaaatcg ttccagactt atggctgcct tgctgactcg 600

aatgtagaca gaatcgaatt cacaacaccg ttatttgatt tatcgactat ctaccgatcg 660

accgaaccgg ctccagaata cagaacatac agtggcggat tactgatgac ccaagaggcc 720

gatggcacca tcttcccccc acaagaagga ccgcacagca ataaatgctt gcaaaacgac 780

gcgtctaatg gagaaacgaa atgttttggc cctgtttcaa catcgattct gccggtcaca 840

ttgttggtcg tgtggtggtg gagactacac aataagatcg ccaaagaact aaacgaaatc 900

aatcctcatt gggatgacga aacattgttc caaactgcaa gggacattaa catcgcaata 960

acaaaccaat ttgtttacta cgaattgctg ccaactctat tcggtgaaga attctgtttg 1020

aaaaatgagc tgattcactc tgaatctgga cacagagacc tttacgatga gtctatacct 1080

gctacttatc tcgaatacta cctcgctttg agatggttcc acttggtttc agagggtgat 1140

ttgaaacttt tcgacgagga tttcaagtac gtgggcaaaa aaatggtaac agacctctct 1200

ttacatactg atttcttgat gagagataag aacttggcaa aaatgacacg aggaacttac 1260

tatcaggctg gaggagataa tgatagagct gtagacccgg ccatttgtga taagggatta 1320

ggaataatgc aaaaagcatc cgaccttaca gcagcggatt tgaggaaaaa tcagcttttc 1380

aaaattccac cttacgtcga ctacgtaaaa ctgtgccatg atgtcgaaat taaaacatgg 1440

aaagacatgt tgaaattcat tgacatggat cgaattgaaa gtctgcaaga aatttacgaa 1500

acccctggtg acgtggaact cctggcagga atctggattg aaagacctat ggagggaggt 1560

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atcccgagat ggaatctcgc tgcttggaaa gaatctaagt ga 1842

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Met Leu Thr Ser Leu Val Pro Leu Ala Pro Leu Leu Leu Thr Ala Gly

1 5 10 15

Val Ala Thr Gly Ser Pro Ser Gly Leu Pro Ala Thr Ala Gly Leu Ile

20 25 30

Gly Thr Pro Ile Leu Gly Gly Ala Leu Ala Ala Cys Thr Ala Ala Ile

35 40 45

Gly Leu Pro Cys Ala Pro Ala Gly Ala Ala Ala Leu Ala Gly Ser Cys

50 55 60

Ser Ala Pro Ala Thr Pro Ser Ala Gly Thr Pro His Thr Pro Val Ile

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Ala Leu Leu Pro Gly Ala Ala Cys Gly Ala Ala Gly Thr Ser Pro Ser

85 90 95

Gly Ala Pro Leu Leu Ser Ala Ala Gly Val Ala Gly Ala Ile Leu Gly

100 105 110

Thr Gly Leu Ala Ser Ala Leu Ser Thr Thr Gly Leu Leu Ala Ile Ile

115 120 125

Ser Thr Ile Ile Pro Ala Ala Leu Gly Ser Ile His Ala Ser Val Ala

130 135 140

Leu Leu Thr Gly Thr Thr Ala Cys Cys Thr Ala Gly Gly Leu Ser Ala

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Thr Met Cys Thr Pro Ile Ala Ile Pro Gly Ala Ala Pro Val His Ala

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Pro Ser Gly Ile Ala Cys Leu Ala Leu Thr Ala Pro Leu Ser Pro Gly

180 185 190

Thr Thr Gly Cys Leu Ala Ala Ser Ala Val Ala Ala Ile Gly Pro Thr

195 200 205

Thr Pro Leu Pro Ala Leu Ser Thr Ile Thr Ala Ser Thr Gly Pro Ala

210 215 220

Pro Gly Thr Ala Thr Thr Ser Gly Gly Leu Leu Met Thr Gly Gly Ala

225 230 235 240

Ala Gly Thr Ile Pro Pro Pro Gly Gly Gly Pro His Ser Ala Leu Cys

245 250 255

Leu Gly Ala Ala Ala Ser Ala Gly Gly Thr Leu Cys Pro Gly Pro Val

260 265 270

Ser Thr Ser Ile Leu Pro Val Thr Leu Leu Val Val Thr Thr Thr Ala

275 280 285

Leu His Ala Leu Ile Ala Leu Gly Leu Ala Gly Ile Ala Pro His Thr

290 295 300

Ala Ala Gly Thr Leu Pro Gly Thr Ala Ala Ala Ile Ala Ile Ala Ile

305 310 315 320

Thr Ala Gly Pro Val Thr Thr Gly Leu Leu Pro Thr Leu Pro Gly Gly

325 330 335

Gly Pro Cys Leu Leu Ala Gly Leu Ile His Ser Gly Ser Gly His Ala

340 345 350

Ala Leu Thr Ala Gly Ser Ile Pro Ala Thr Thr Leu Gly Thr Thr Leu

355 360 365

Ala Leu Ala Thr Pro His Leu Val Ser Gly Gly Ala Leu Leu Leu Pro

370 375 380

Ala Gly Ala Pro Leu Thr Val Gly Leu Leu Met Val Thr Ala Leu Ser

385 390 395 400

Leu His Thr Ala Pro Leu Met Ala Ala Leu Ala Leu Ala Leu Met Thr

405 410 415

Ala Gly Thr Thr Thr Gly Ala Gly Gly Ala Ala Ala Ala Ala Val Ala

420 425 430

Pro Ala Ile Cys Ala Leu Gly Leu Gly Ile Met Gly Leu Ala Ser Ala

435 440 445

Leu Thr Ala Ala Ala Leu Ala Leu Ala Gly Leu Pro Leu Ile Pro Pro

450 455 460

Thr Val Ala Thr Val Leu Leu Cys His Ala Val Gly Ile Leu Thr Thr

465 470 475 480

Leu Ala Met Leu Leu Pro Ile Ala Met Ala Ala Ile Gly Ser Leu Gly

485 490 495

Gly Ile Thr Gly Thr Pro Gly Ala Val Gly Leu Leu Ala Gly Ile Thr

500 505 510

Ile Gly Ala Pro Met Gly Gly Gly Thr Val Pro Pro Thr Ala Ala Cys

515 520 525

Ile Ile Ala Leu Gly Leu Ser Leu Thr Met Leu Ala Ala Ala His Thr

530 535 540

Thr Gly Ala Ser Ala Ala Pro Thr Ala Pro Ala Val Ala Gly Leu Ala

545 550 555 560

Gly Ile Ala Leu Ala Thr Val Ala Gly Pro Leu Cys Gly Val Gly Ala

565 570 575

Gly Val Gly Ala Ala Gly Pro Leu Cys Gly Val Gly Ala Gly Val Gly

580 585 590

Ala Ile Gly Ala Gly Ala Leu Leu Ala Val Thr Ala Ser Ala Pro Leu

595 600 605

Val Ser Cys Gly Gly Ile Pro Ala Thr Ala Leu Ala Ala Thr Leu Gly

610 615 620

Ser Leu

625

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gtaatacgac tcactatagg gagagttcgc tatgaatcgt tttctggtct accagcgact 60

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caactcccct gcgaccccaa tgagagacga cgtctagatg gctcatgcag taatttcgac 180

tatccgtcaa gaggaacatt ccatacccct gtgataagat tgttacccga agccgactgt 240

ggagacgatg aaacctctcc atctggagct cctttaaaat ctgcccggga agtcaggcag 300

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accacaaatt gttgcacagc ggaaggaaaa tcaaattata tgtgcacgcc tatagatatt 480

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acaccgttat ttgatttatc gactatctac cgatcgaccg aaccggctcc agaatacaga 660

acatacagtg gcggattact gatgacccaa gaggccgatg gcaccatctt ccccccacaa 720

gaaggaccgc acagcaataa atgcttgcaa aacgacgcgt ctaatggaga aacgaaatgt 780

tttggccctg tttcaacatc gattctgccg gtcacattgt tggtcgtgtg gtggtggaga 840

ctacacaata agatcgccaa agaactaaac gaaatcaatc ctcattggga tgacgaaaca 900

ttgttccaaa ctgcaaggga cattaacatc gcaataacaa accaatttgt ttactacgaa 960

ttgctgccaa ctctattcgg tgaagaattc tgtttgaaaa atgagctgat tcactctgaa 1020

tctggacaca gagaccttta cgatgagtct atacctgcta cttatctcga atactacctc 1080

gctttgagat ggttccactt ggtttcagag ggtgatttga aacttttcga cgaggatttc 1140

aagtacgtgg gcaaaaaaat ggtaacagac ctctctttac atactgattt cttgatgaga 1200

gataagaact tggcaaaaat gacacgagga acttactatc aggctggagg agataatgat 1260

agagctgtag acccggccat ttgtgataag ggattaggaa taatgcaaaa agcatccgac 1320

cttacagcag cggatttgag gaaaaatcag cttttcaaaa ttccacctta cgtcgactac 1380

gtaaaactgt gccatgatgt cgaaattaaa acatggaaag acatgttgaa attcattgac 1440

atggatcgaa ttgaaagtct gcaagaaatt tacgaaaccc ctggtgacgt ggaactcctg 1500

gcaggaatct ggattgaaag acctatggag ggaggttacg ttcctccaac cgcagcttgt 1560

atcataaaca aacaactgtc tctcactatg aaagctgaca gacattggta tgagagatcg 1620

gacagaccct acgctttcaa tgtcgctcag ctggcggaaa taagaaaagc aacagttgca 1680

ggcttccttt gcgaagttgg tgacggtgta gagagaatcc aacgggaggc acttaaaaga 1740

gtgactgctt cgaatccatt ggtcagctgc caagaaatcc cgagatggaa tctcgctgct 1800

tggaaagaat ctaagtgaga ttaatgattt taaagctgca actaatctcc ctatagtgag 1860

tcgtattac 1869

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<213> Artificial Sequence (Artificial Sequence)

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cgggatccgt tcgctatgaa tcgttttc 28

<210> 5

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gggttcgaat tagttgcagc tttaaaatca ttaa 34

<210> 6

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtaatacgac tcactatagg gagagttcgc tatgaatcgt tttc 44

<210> 7

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gtaatacgac tcactatagg gagattagtt gcagctttaa aatcattaa 49

Claims (9)

1. Lepidoptera insectHpx12A gene characterized by: the above-mentionedHpx12The gene is the lepidoptera insect innate immune response regulating gene, and the nucleotide sequence is SEQ ID No. 1.

2. The method of claim 1Hpx12The gene expression inhibitor and/or activity inhibitor is applied to lepidoptera insects as a pest control target.

3. The method of claim 1Hpx12A gene-encoded protein characterized by: the amino acid sequence of the protein is SEQ ID No. 2.

4. The method of claim 3Hpx12Gene-encoded proteins inhibit lepidopteran insects in vivoHpx12The ds RNA of gene expression is used for reducing the innate immune response of lepidoptera insects and increasing the susceptibility of the lepidoptera insects to pathogenic bacteria.

5. Use according to claim 4, characterized in that: the ds RNA sequence is SEQ ID No. 3.

6. Use according to claim 5, characterized in that: the sequence of the primer for amplifying the ds RNA is SEQ ID No. 6-SEQ ID No. 7;

a forward primer: 5' -GTAATACGACTCACTATAGGGAGAGTTCGCTAT

GAATCGTTTTC -3’；SEQ ID No. 6；

Reverse primer: 5' -GTAATACGACTCACTATAGGGAGATTAGTTGCAGCTT

TAAAATCATTAA -3’；SEQ ID No. 7。

7. Use according to claim 2 or 4, characterized in that: the lepidopteran insect is a silkworm.

8. Cloning of claim 1Hpx12A method of genomics, characterized by: the method comprises the following steps:

(1) synthesizing lepidoptera insect body cDNA: extracting lepidopterous insect RNA, and performing reverse transcription to obtain first-strand cDNA;

（2）Hpx12PCR amplification of the genes: taking the lepidoptera insect body cDNA as a template, designing a primer according to the sequence of the lepidoptera insect body, carrying out PCR amplification, recovering and purifying a PCR amplification product, and sequencing.

9. The method of claim 6, wherein: the primer sequence in the step (2) is SEQ ID No.4-SEQ ID No. 5;

a forward primer: 5'-CGGGATCCGTTCGCTATGAATCGTTTTC-3', respectively; SEQ ID No. 4;

reverse primer: 5'-GGGTTCGAATTAGTTGCAGCTTTAAAATCATTAA-3', respectively; SEQ ID No. 5.

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