CN112695045B - Lepidoptera insect Hpx12 gene and application thereof - Google Patents

Lepidoptera insect Hpx12 gene and application thereof Download PDF

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Publication number
CN112695045B
CN112695045B CN202110104880.7A CN202110104880A CN112695045B CN 112695045 B CN112695045 B CN 112695045B CN 202110104880 A CN202110104880 A CN 202110104880A CN 112695045 B CN112695045 B CN 112695045B
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hpx12
gly
gene
leu
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CN112695045A (en
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时桂芹
任菲
郭法谋
周园
沈佳鑫
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Zhengzhou University of Light Industry
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Zhengzhou University of Light Industry
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Abstract

The invention provides a lepidoptera insectHpx12The invention provides a gene and application thereof, and aims to provide a lepidoptera insect innate immune response regulation geneHpx12(ii) a The second purpose is to provide the lepidopteran insect innate immune response regulation geneHpx12The cloning method of (4); the third purpose is to provide the lepidopteran insect innate immune response regulation geneHpx12The use of (1). The invention relates to lepidopteran insectsHpx12After the gene is inhibited and expressed, the innate immune response of the polypide can be inhibited, the susceptibility of the polypide to germs is increased, the silkworms are more easily infected with BmNPV viruses, and further, the death rate of lepidoptera insects is greatly increased. If inhibiting lepidopteran insectsHpx12Can 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.

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 types 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 often suffer from leaf curl, leaf ornamentation, scabbing, silking and netting, or drilling into plant tissues to eat. 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 between the 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. For solving the problems caused by chemical pesticidesThe problems of ecological deterioration, pesticide resistance and the like have positive significance.
In view of this, 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 a fourth aspect of the present invention, there is providedHpx12Gene-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' -GTAATACGACTCACTATAGGGAGTTCGCTAT
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 aspect of the invention, cloning rights are 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-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, selecting a TransStart high-fidelity amplification enzyme reaction system, and ensuring that the total volume of the system is 50 mu L, wherein the specific primer comprises the following components: 200ng c DNA,5 × TransStart FastPfu reaction buffer 10 μ L,10m Md NTP 1 μ L,2U TransStart FastPfu DNA Polymerase,1 μ L of 1 μ M each of the forward and reverse primers, and water to 50 μ L. The PCR reaction is carried out on a Mastercycler pro amplification instrument, and the reaction program is as follows: 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 ℃; transforming the connected vector into escherichia coli DH5a by heat shock, adding a liquid culture medium, performing shake culture, coating the cultured product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting bacterial colonies, performing bacterial liquid culture, extracting plasmids and performing PCR (polymerase chain reaction) detection. 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, and has 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 silkwormHpx12First proposed and utilizedHpx12Gene 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 display device according to embodiment 3 of the present inventionHpx12Number of nodes 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 in example 4 of the present inventionHpx12Expression level of the antimicrobial peptide related gene 48h after injection of the gene ds RNA into the worm.
FIG. 5 shows a flowchart of example 5 of the present inventionHpx12The 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 and features of the embodiments of the present invention 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 × TransStart FastPfu reaction buffer 10 μ L,10m Md NTP 1 μ L,2U TransStart FastPfu DNA Polymerase,1 μ L of 1 μ M each of the forward and reverse primers, and water to 50 μ L. The PCR reaction is carried out on a Mastercycler pro amplification instrument, and the reaction program is as follows: 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 ℃; transforming the connected vector into escherichia coli DH5a by heat shock, adding a liquid culture medium, performing shake culture, coating the cultured product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting bacterial colonies, performing bacterial liquid culture, extracting plasmids and performing PCR (polymerase chain reaction) detection. 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' -GTAATACGACTCACTATAGGGAGTTCGCTAT
GAATCGTTTTC -3’;SEQ ID No. 6;
Reverse primer: 5' -GTAATACGACTACACTATAGGGAGATTAGTTGCAGCTT
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 experimental 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.
Fluorescence quantitative PCR is used for detecting 6h, 12h, 24h and 48h after microinjectionHpx12As shown in FIG. 2, the expression and effect of the gene are the best interference effect in 24h, and the silkworm can be efficiently silenced in the case of the optimal interference effect in 24hHpx12The 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 was injected into the blood chamber of the silkworms which had been injected with dsRNA by a microinjector. At 25 deg.CAfter standing for 2h, melanin and dark nodules were counted under a microscope at 50-fold 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 to a feed containing 1.5X 10 4 The 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 mortality rate of the silkworm at 48h is obviously higher than that of 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 thereof
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atgctctggt cattagtttt cttggcgttt ctgaagctga ccgccggtgt tcgctatgaa 60
tcgttttctg gtctaccagc gactgatgaa aagatccaat acttcatcaa acaggaaaat 120
ttagataact gcaccaatga cattcaactc ccctgcgacc ccaatgagag acgacgtcta 180
gatggctcat gcagtaattt cgactatccg tcaagaggaa cattccatac ccctgtgata 240
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
tacgttcctc caaccgcagc ttgtatcata aacaaacaac tgtctctcac tatgaaagct 1620
gacagacatt ggtatgagag atcggacaga ccctacgctt tcaatgtcgc tcagctggcg 1680
gaaataagaa aagcaacagt tgcaggcttc ctttgcgaag ttggtgacgg tgtagagaga 1740
atccaacggg aggcacttaa aagagtgact gcttcgaatc cattggtcag ctgccaagaa 1800
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
65 70 75 80
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
145 150 155 160
Thr Met Cys Thr Pro Ile Ala Ile Pro Gly Ala Ala Pro Val His Ala
165 170 175
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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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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tcgacaatca tatttgctga ccttggttca atccatgatt ccgtgaattt gttaacggag 420
accacaaatt gttgcacagc ggaaggaaaa tcaaattata tgtgcacgcc tatagatatt 480
ccccaggacg accctgtcca taggttctct ggtatacgct gcttgaatct cactcgaccg 540
aaatcgttcc agacttatgg ctgccttgct gactcgaatg tagacagaat cgaattcaca 600
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
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgggatccgt tcgctatgaa tcgttttc 28
<210> 5
<211> 34
<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)
<400> 7
gtaatacgac tcactatagg gagattagtt gcagctttaa aatcattaa 49

Claims (3)

1. A kind ofHpx12The gene expression inhibitor and/or activity inhibitor is applied to pest control; characterized in that the aboveHpx12The gene is the silkworm innate immune response regulation gene, and the nucleotide sequence is SEQ ID No.1; the pests are silkworms.
2. Inhibiting the said in vivo of silkwormHpx12The application of the gene expression ds RNA in reducing the innate immune response of silkworms and increasing the susceptibility of the silkworms to germs is characterized in that the sequence of the ds RNA is SEQ ID No.3.
3. The use according to claim 2, wherein the primer sequence for amplifying the ds RNA is SEQ ID No.6 to SEQ ID No.7;
a forward primer: 5' -GTAATACGACTCACTATAGGGAGTTCGCTAT
GAATCGTTTTC -3’;SEQ ID No. 6;
Reverse primer: 5' -GTAATACGACTCACTATAGGGAGATTAGTTGCAGCTT
TAAAATCATTAA -3’;SEQ ID No. 7。
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WO2010026489A1 (en) * 2008-09-05 2010-03-11 The University Of British Columbia Innate immunity modulators
CN110106175A (en) * 2019-05-14 2019-08-09 中国农业科学院植物保护研究所 A kind of dsRNA and its application in control of insect

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WO2010026489A1 (en) * 2008-09-05 2010-03-11 The University Of British Columbia Innate immunity modulators
CN110106175A (en) * 2019-05-14 2019-08-09 中国农业科学院植物保护研究所 A kind of dsRNA and its application in control of insect

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Annotation and evolution of the antioxidant genes in the silkworm, Bombyx mori;Gui-Qin Shi et al.;《Arch Insect Biochem Physiol》;20120228;87-103 *
PREDICTED: Bombyx mori peroxidase (LOC101744078), mRNA;None;《NCBI Reference Sequence: XM_012690169.2》;20201202;1-2 *
家蚕免疫系统相关基因的鉴定及其诱导表达模式研究;程廷才;《中国博士学位论文全文数据库基础科学辑》;20080915;A006-64 *
家蚕抗氧化基因注释及功能研究;时桂芹;《中国博士学位论文全文数据库基础科学辑》;20140215;A006-72 *

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