CN110669790B - Application of CvBV12-7 gene in reducing humoral immune response of insect - Google Patents

Application of CvBV12-7 gene in reducing humoral immune response of insect Download PDF

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CN110669790B
CN110669790B CN201910981869.1A CN201910981869A CN110669790B CN 110669790 B CN110669790 B CN 110669790B CN 201910981869 A CN201910981869 A CN 201910981869A CN 110669790 B CN110669790 B CN 110669790B
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陈学新
王泽华
黄健华
时敏
胡荣敏
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Zhejiang University ZJU
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Abstract

The invention discloses an application of a CvBV12-7 gene in reducing insect humoral immune response, wherein the CvBV12-7 gene is the 7 th gene on the 12 th ring in 35 genome segments of plutella xylostella cocoon bee multi-component DNA virus, and the base sequence is shown as SEQ ID NO. 1. The invention uses the transgenic technology to transfer plutella xylostella cotesia xylostella multi-DNA virus gene CvBV12-7 into the drosophila melanogaster genome, thus obtaining a transgenic drosophila melanogaster strain with stable heredity and homozygote; by utilizing the UAS/GAL4 system, the homozygous CvBV12-7 transgenic drosophila melanogaster is hybridized with the BS8700 drosophila melanogaster strain, and after the obtained strain expresses multiple DNA virus genes, the melanogenesis activity of insect hemolymph can be inhibited, and the melanogenesis activity is obviously low in immunity when infected by pathogens, so that the method has important significance in the aspects of researching insect immunity related mechanisms, enhancing the screening of human immunity medicaments and the like.

Description

Application of CvBV12-7 gene in reducing humoral immune response of insect
Technical Field
The invention relates to the technical field of molecular biology and genetic engineering, in particular to application of a CvBV12-7 gene in reducing humoral immune response of insects.
Background
Insects belong to arthropods of invertebrates and are the largest group of animals on earth. At present, more than 100 species of insects are known, which are varied in shape and large in number, and are closely related to agricultural production and human health. The research on insects not only can enrich the human knowledge of the nature, but also is helpful to solve the important problems in the actual production and the human disease prevention and control. Especially, basic research and application research on insect immunity are always hot spots, and the problems of mechanisms, signals and the like related to insect immunity are researched through experiments, so that the method has very important practical significance for pest control, insect benefiting and disease preventing, drug development, human immunity mechanism research and the like.
Insects form a unique set of natural immune systems during long-term evolution, including cellular immunity (cellular immunity) and humoral immunity (humoral immunity), both distinct and interrelated (Lavine, m.d. and m.r. strand (2002) ' institute and the same role in immunity, ' institute Biochemistry and Molecular Biology 32(10) ' 1295. 1309. ' tsakasakas, s.and v.j.marmaraas (2010) ' institute immunity and analysis: An ecological environment. ' investebrate and environment Journal 7(2): 228. 238. ' great. e.v. useful, i.m.structural, m.m.a.v. environmental immunity, and g.v. supplement, cell infection, v.content, 119. supplement and sample of 40. Journal of strain, 201446. host organism. Cellular immunity in insects is mediated by insect blood cells and mainly includes Phagocytosis, cyst and nodulation (Schmidt, O., U.S. and M.Strand (2001). "input immunity and its evolution and preservation by hypodiplonteran endoprasitics", "BioEssays 23(4): 344. cndot. 351; Wu, S.and E.J.Ling (2009)" Phagocytosis, differentiation and encystation in cellular immunity in insects 52(7): 791. cndot. 798). Humoral immunity and cellular immunity in insects are often accompanied. Humoral immunity in insects is from the recognition of pathogens to the production of Antimicrobial peptides (AMPs) and melanin (melanin) and ultimately destruction of foreign pathogens or foreign substances by the production of Antimicrobial peptides or blackening reactions (Lowenberger, C. (2001). "origin immunity of insects physiology 31(3):219 and 229, Blandin, s.and e.a.levanshina (2004)." thio-related antigens immunity "immunity 40(12):903 and 908, Imler, j.l.and p.buble (2005. antibiotic peptides: amplifiers: 21.86, chemical expression j.21.21.21.21).
Currently, studies on insect immunity are mainly focused on the development of insect resistance and the immunosuppression of natural enemies against insects. Insect resistance refers to the ability to develop in an insect population to tolerate doses that kill most individuals in the normal population. Many insects have long developed varying degrees of resistance to insecticides due to their selective action. And natural enemy organisms in nature, particularly parasitic wasps can effectively control pests, and parasitism is completed by inhibiting the immune system of host insects. The deep research on the natural enemy for inhibiting the host immunity can not only develop effective measures and ways for preventing and controlling insects, optimize biological prevention and control of pests and greatly reduce the use of chemical pesticides, but also has profound practical significance for ensuring the steady development of sustainable agriculture in China.
Polypeptides of DNA Viruses (PDV) are classified into bracon Bee Virus (BV) and ichneumoniae (Ichnorus, IV) viruses (Turnbull, M.and B.Webb (2002), Perspectives on polydiviruses orientations and evolution. Advances in Virus Research, Academic Press.58: 203-. PDV is a specific virus of obligate symbiosis of parasitic wasps, injected into lepidopteran host larvae at the time of oviposition of the parasitic wasps, and is an important parasitic factor that helps the parasitic wasps to successfully parasitize (Bai, S., X.Chen, J.Cheng, W.Fu and J.He (2005). "Effects of water-associated factors of Cotesia Plutella on growth and maintenance of Plutella xylostella larvae." Acta Phytophylactica site 32 (235-240). Its main physiological function is to suppress the immunity of the host (Dupuy, C., E.Huguet and J.M.Drezen (2006) 'Unfolding the evolution store of polynavities.' Virus Research 117(1): 81-89; Strand, M.R.and G.R.Burke (2012) 'Polynaviruses as systems and gene delivery systems.' PLoS Pathologens 8 (7)). Plutella xylostella cocoon bee multi-component DNA virus (CvBV) is a multi-component DNA virus which is researched more in China, and consists of 35 circular DNAs and encodes 157 toxic genes (Chen, Y.F., F.Gao, X.Q.Ye, S.J.Wei, M.Shi, H.J.Zheng and X.X.Chen (2011). "Deep sequencing of Cotesia vestalis viruses the complex of a polydinavirus genome." Virology (414): 42-50.). After the CvBV enters a host diamondback moth, the CvBV gene quickly expresses toxic protein to inhibit the immune response of the diamondback moth. Therefore, there is a need for further intensive studies on the CvBV gene.
Disclosure of Invention
The invention finds a new application of the CvBV12-7 gene in reducing the humoral immune response of insects and preparing low-immune drosophila.
The specific technical scheme is as follows:
the CvBV12-7 gene belongs to a PTP (protein tyrosine dephosphorylation enzyme) gene family, is the 7 th gene on the 12 th ring in 35 genome segments of plutella xylostella cocoon bee multi-component DNA virus, and has a base sequence shown in SEQ ID NO. 1; it can code 296 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2.
The invention provides an application of a CvBV12-7 gene in reducing insect humoral immune response, wherein the base sequence of the CvBV12-7 gene is shown as SEQ ID NO. 1; the insect is fruit fly or diamondback moth.
Experiments show that the CvBV12-7 gene can inhibit the blackening activity of insect hemolymph, and the base sequence of the CvBV12-7 gene is shown in SEQ ID NO. 1; the insect is fruit fly or diamondback moth.
The invention also provides application of the CvBV12-7 gene in preparing a drosophila melanogaster model with low immunity, wherein the base sequence of the CvBV12-7 gene is shown in SEQ ID NO. 1.
The invention provides a preparation method of a low-immunity fruit fly model, which comprises the following steps:
(1) preparing a recombinant plasmid containing a CvBV12-7 gene, injecting the recombinant plasmid into white eye wild type drosophila melanogaster embryos, and obtaining red eye transgenic drosophila melanogaster after the embryos grow to adults; the base sequence of the CvBV12-7 gene is shown in SEQ ID NO. 1;
(2) performing two rounds of hybridization on the red-eye transgenic fruit fly and a balance line, and selecting homozygote transgenic fruit fly according to the phenotype of offspring;
(3) and (3) hybridizing the homozygote transgenic drosophila prepared in the step (2) with a BS8700 drosophila strain by using a UAS/GAL4 system, and specifically expressing the gene CvBV12-7 in progeny drosophila blood cells to obtain the immune hypo drosophila.
Wherein the white eye wild type fruit fly is W1118; the genotype of the equilibrium system is w-/w-; Sp/Cyo; TM2/TM 6B.
The genotype of the homozygote transgenic drosophila is w-/w-; CvBV12-7/CvBV 12-7; TM2/TM 6B.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention uses the transgenic technology to transfer plutella xylostella cotesia xylostella multi-DNA virus gene CvBV12-7 into the drosophila melanogaster genome, thus obtaining a transgenic drosophila melanogaster strain with stable heredity and homozygote; by utilizing the UAS/GAL4 system, the homozygous CvBV12-7 transgenic drosophila melanogaster is hybridized with the BS8700 drosophila melanogaster strain, and after the obtained strain expresses multiple DNA virus genes, the melanogenesis activity of insect hemolymph can be inhibited, and the melanogenesis activity is obviously low in immunity when infected by pathogens, so that the method has important significance in the aspects of researching insect immunity related mechanisms, enhancing the screening of human immunity medicaments and the like.
(2) After recombinant CvBV12-7 virus particles are injected into a diamondback moth body, the blackening activity of the hemolymph of the diamondback moth is obviously inhibited, and the CvBV12-7 gene can inhibit the blackening activity of the hemolymph of the diamondback moth.
Drawings
FIG. 1 shows the PCR assay of CvBV12-7 transgenic Drosophila in example 1.
FIG. 2 shows the results of the semi-quantitative PCR assay of CvBV12-7 expression in transgenic Drosophila in example 1.
FIG. 3 shows the results of testing the influence of the CvBV12-7 gene on Drosophila immunity in example 1.
FIG. 4 is the result of testing that the CvBV12-7 gene affected the melanogenesis activity of Drosophila larvae in example 1.
FIG. 5 shows the results of the enzyme activity assay of CvBV12-7 in example 2.
FIG. 6 shows the results of detection of double stranded RNA of CvBV12-7 in example 2.
FIG. 7 shows the results of the interference efficiency measurements of CvBV12-7 in example 2.
FIG. 8 is the result of detecting the blackening activity of the larvae of plutella xylostella after the CvBV12-7 gene is disrupted in example 2;
where GFP indicates the injection of GFP double-stranded RNA and CvBV12-7 indicates the injection of CvBV12-7 double-stranded RNA.
FIG. 9 shows the results of the detection of the influence of the CvBV12-7 gene on the blackening activity of the larvae of plutella xylostella in example 2;
wherein GFP represents plutella xylostella injected with recombinant baculovirus (GFP gene); CvBV12-7 represents a diamondback moth injected with recombinant baculovirus (CvBV12-7 gene).
Detailed Description
The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.
Example 1
1. Construction of transgenic Drosophila
According to the two end sequences of the CvBV12-7 gene open reading frame (ORF, shown in SEQ ID NO. 1) gene and the sequence of the multiple Cloning site on the pUASttb vector, the action mode of combining homologous recombinase (Clonexpress II One Step Cloning Kit, Novozam) is designed, and the primer sequences are as follows:
12-7-P：5’-TTCGTTAACAGATCTGCGGCCGCATGAGTTCTAACAAGGCGGAAAT-3’
12-7-AP：5’-TCCTCTAGAGGTACCCTCGAGTTACGTATAAAGATTCAAATATTC-3’；
total post-parasitic plutella xylostella RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and a cDNA library was constructed using the kit. The CvBV12-7 gene fragment with carrier homologous sequence is PCR amplified from the constructed cDNA library by using specific primers 3-3-P and 3-3-AP, and the fragment is recombined and cloned to the pUASttb vector by using homologous recombinase. The recombinant vector was transformed into E.coli TG1 for propagation and the recombinant plasmid was sequenced to verify the correctness of the sequence.
And microinjecting the correctly verified recombinant plasmid into the embryo of the white-eye wild type drosophila melanogaster W1118, wherein the embryo develops to an adult, namely G0 generation, and the red-eye drosophila melanogaster in G0 generation is a successful transgenic strain, and screening is carried out based on the fact.
By utilizing PCR detection, the genome DNA of the CvBV12-7 transgenic drosophila melanogaster and the genome DNA of the wild type W1118 drosophila melanogaster are extracted, and the PCR detection is carried out on the genome DNA by using specific primers of 12-7-P and 12-7-AP respectively. A fragment of size consistent with that of CvBV12-7 was amplified from the CvBV12-7 transgenic Drosophila but not in the genomic DNA of wild type W1118 Drosophila melanogaster. The plutella xylostella cotesia coides multi-component DNA virus CvBV12-7 gene is successfully inserted into the genome of the transgenic fruit fly, and the transgenic fruit fly strain is successfully constructed (as shown in figure 1).
2. Construction of CvBV12-7 transgenic drosophila homozygote strain
In this embodiment, CvBV12-7 is inserted into chromosome III, so that the transgenic line successfully constructed in step 1 is crossed with a balanced line (w-/w-; Sp/Cyo; TM2/TM6B), virgins (w-/w-; +/Cyo; TM2/CvBV12-7) with rolling wings and large balanced rods and male flies (w-/w-; +/Cyo; TM6B/CvBV12-7) with rolling wings and hairy shoulders are selected as progeny according to the offspring phenotype, and the selected flies are crossed; or selecting male drosophila melanogaster (w-/w-; +/Cyo; TM2/CvBV12-7) with rolling fin and large balancing rod as offspring and virginator drosophila melanogaster (w-/w-; +/Cyo; TM6B/CvBV12-7) with rolling fin and hairy shoulder, and crossing the selected drosophila melanogaster; the hybrid progeny selected only CvBV12-7 transgenic Drosophila (w-/w; +/+; CvBV12-7/CvBV12-7) homozygous for the orthoptera phenotype for seed protection.
3. Expression of CvBV12-7 gene in drosophila melanogaster blood cells
The homozygous CvBV12-7 transgenic Drosophila in step 2 was crossed with BS8700(FlyBase ID: FBti0064641) Drosophila strain using the UAS/GAL4 system, such that CvBV12-7 was specifically expressed in Drosophila blood cells, and the CvBV12-7 transgenic strain was crossed with wild type W1118 as a control.
Extracting total RNA of the blood cells of the filial generation, carrying out reverse transcription to obtain cDNA (the specific method is the same as the step 1 part of the embodiment), taking housekeeping gene Actin as an internal reference, and carrying out semi-quantitative detection by using a quantitative specific primer.
The quantitative specific primers were as follows:
CvBV12-7-qPCR-F：5’-TCGACAGCTTCAAACAACCC-3’
CvBV12-7-qPCR-F：5’-GTCGCCCGTTCTTCCAATT-3’
Dro-actin-qPCR–F：5’-GCTGAGCGTGAAATCGTCCG-3’
Dro-actin-qPCR-R：5’-GGAGTTGTAGGTGGTCTCGTGGA-3’
the results in FIG. 2 show that at mRNA level, the CvBV12-7 transgenic line and the filial generation of W1118 have no expression of CvBV12-7, while the CvBV12-7 transgenic line and the filial generation of BS8700 have high expression level. Thus, it was demonstrated that transgenic Drosophila expressing CvBV12-7 specifically in blood cells was obtained.
4. Influence of specific expression of CvBV12-7 gene in blood cells of drosophila on drosophila
a) Detection result of influence of CvBV12-7 on drosophila melanogaster immunity
Virgins of wild-type W1118 and BS8700 were individually crossed with the CvBV12-7 transgenic line and postnatal drosophila males adults (approximately 60 heads) were injected in vivo with 33nl of PBS and staphylococcus aureus (OD ═ 0.4). And (4) counting the survival condition of the fruit flies every 12 hours after injection, and drawing a survival curve.
FIG. 3 results show that PBS injection does not affect the survival rate of Drosophila; after staphylococcus aureus injection, the survival rate of filial generation of the CvBV12-7 transgenic line and BS8700 is remarkably lower (p is 0.0081) than that of a control group (filial generation of the CvBV12-7 transgenic line and wild type W1118), which indicates that the CvBV12-7 can reduce the immunity of fruit flies.
b) Detection result of influence of CvBV12-7 on melanogenesis activity of drosophila melanogaster
The CvBV12-7 transgenic line was used to cross virgins with wild-type W1118 and BS8700, respectively. 30 offspring drosophila larvae hemolymph are extracted and added into 200 mul PBS, and 20 mul is taken out for protein concentration determination after vortex mixing. The total blood lymph Protein concentration was determined using the Pierce (TM) Coomassie (Bradfold) Protein Assay Kit. For the blackening activity assay, 20. mu.l of heat-inactivated E.coli suspension (suspended in PBS, boiled in boiling water at OD. about.0.5 for 10min) was added to the remaining about 180. mu.l of the hemolymph, and the mixture was reacted at room temperature for 20 min. Mu.l of the reaction solution was added to 140. mu.l of dopa solution (3g/L), and OD490 was measured every 5min for 60min in total for three mechanical repetitions. Three biological replicates per treatment were blanked with dopa solution. One unit of enzyme activity is defined as the change in OD490 per minute per mg of protein.
The results in fig. 4 show that compared with the control group (filial generation of the CvBV12-7 transgenic line and the wild-type W1118), the blackening activity of the hemolymph of the filial generation of the CvBV12-7 transgenic line and the BS8700 is significantly inhibited.
Example 2
1. Construction of recombinant vectors
According to the Open Reading Frame (ORF) of the CvBV12-7 gene (shown in SEQ ID NO. 1), the sequence of both ends of the GFP gene and the sequence of the multiple Cloning sites on the pFastBac-HTB vector, and the action mode of combining homologous recombinase (Cloneexpress II One Step Cloning Kit, Novonoprazan), specific primers are designed, and the sequences of the primers are as follows:
12-7-F：5’-ACGAGCTCACTAGTCGCGGCCGCTATGAGTTCTAACAAGGCGGAAAT-3’；
12-7-R：5’-CTTGGTACCGCATGCCTCGAGTTACGTATAAAGATTCAAATATTC-3’；
GFP-F：5’-ACGAGCTCACTAGTCGCGGCCGCTATGGTGAGCAAGGGCGAGGA-3’；
GFP-R：5’-CTTGGTACCGCATGCCTCGAGTTACTAGAGGTACCCTCGAG-3’；
total post-parasitic plutella xylostella RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and a cDNA library was constructed using the kit. Specific primers 12-7-F and 12-7-R are utilized to PCR amplify a CvBV12-7 gene fragment with a carrier homologous sequence from the constructed cDNA library, and homologous recombinase is utilized to recombine and clone the fragment to a pFastBac-HTB vector. The plasmid with a GFP label is used as a template, a GFP gene fragment with a carrier homologous sequence is amplified by using a specific primer GFP-F, GFP-R, and the fragment is recombined and cloned to a pFastBac-HTB carrier by using a homologous recombinase. The recombinant vector was transformed into E.coli TG1 for propagation and the recombinant plasmid was sequenced to verify the correctness of the sequence.
Transferring the correctly verified recombinant plasmid into DH10 BacTMIn Ecoli, unlike conventional transformation, after addition of nonreactive medium, plates were plated at 37 ℃ for 4 hours with shaking at 225 rpm. LB plates contained 50. mu.g/ml Kan, 7. mu.g/ml Gentamicin, 10. mu.g/ml tetracycline, 100. mu.g/ml Biuo-gal, 40. mu.g/ml IPTG. And after culturing for two days at 37 ℃, selecting white spots for bacterial liquid detection.
Transfer 200. mu.l of the correct DH10 Bac detected in the previous stepTMEcoli was cultured in 20ml of a resistant medium (containing 50. mu.g/ml Kan, 7. mu.g/ml Gentamicin, 10. mu.g/ml tetracyline) at 37 ℃ and 250rpm overnight. Using PureLinkTMThe HiPure Plasmid Miniprep Kit extracts recombinant baculovirus DNA. Transfecting recombinant baculovirus DNA into an Sf9 cell line by using Cellffectin II, blowing and beating the cells to a suspension state after 72 hours, transferring the cell suspension into a centrifugal tube, centrifuging for 5min at 500g, and sucking the supernatant, namely P1 generation virus particles. The virus particles should be kept in a refrigerator at 4 ℃ protected from light. Will be provided withThe collected P1 generation virus is added into a new Sf9 cell line, and P2 generation virus particles are collected after 72 hours, and the like.
Collecting P5 generation virus particles, centrifuging at 2000-3000rpm for 20-30min, and taking supernatant; centrifuging at 10000rpm for 30min, and collecting supernatant; centrifuge at full speed for 2h, leave a small amount (about 20. mu.l) of medium, and resuspend the virions by low speed vortexing. Detecting the titer of the virus particles by using the resuspended virus particles as a template and using an absolute quantitative mode, and then diluting the titer of the virus particles to be 2 x 105Mu.l/l.
3.CvBV12-7 enzyme activity assay
In the presence of 5X 106A10 cm dish of Sf9 cells was placed at 5X 10 positions7The copied GFP recombinant virus particles and CvBV12-7 recombinant virus particles are cultured at 27 ℃ for 5 days and then the cells are collected. After washing with PBS 2-3 times, add pre-cooled 250 μ l cell lysate, vortex at maximum speed for 15-30s, ice-wash for 20min, during which vortex 15s is taken out, 3-5 times in total. PTP activity was subsequently determined using the Tyrosine Phosphotase Assay System (Promega) kit.
The results in FIG. 5 show that more free phosphate was detected in lysates of Sf9 cells infected with CvBV12-7 recombinant virus compared to the control (GFP recombinant virus), suggesting that CvBV indeed has protein tyrosine dephosphorylating enzyme (PTP) activity.
4. Interference with expression of CvBV12-7
Using synthetic double strands
Designing a primer with the target fragment length of about 500bp and a T7 RNA polymerase promoter sequence at the 5' end:
12-7-dsF：5’-TAATACGACTCACTATAGGTTGTGATGCTGACAGAACTTC-3’
12-7-dsR：5’-TAATACGACTCACTATAGGCCGTAACTGGAATACTTGAAT-3’
a plasmid of a recombinant CvBV12-7 gene is used as a template, a fragment is amplified by using a high fidelity PCR MIX 2 x Phanta Master MIX (Vazyme), then a double strand is synthesized by using a T7 RNAi Transcription Kit (Novozam), and the synthesized double strand is detected by a gel electrophoresis mode.
Respectively carrying out micro-injection on 500ng CvBV12-7 double-stranded RNA to 3-year-old middle-stage diamondback moth larvae, carrying out single-head parasitism after 4h, and setting a control group to be injected with 500ng GFP double strands. Total RNA was extracted from single-headed diamond back moths 12h, 24h, 36h and 48h after parasitization for a total of 5 biological replicates. The interference efficiency was determined by qPCR detection of gene expression levels, where the quantitative primer sequences were as follows:
12-7-rtF：5’-TCGACAGCTTCAAACAACCC-3’；
12-7-rtR：5’-GTCGCCCGTTCTTCCAATT-3’；
the results in FIG. 6 show that high-quality and high-concentration CvBV12-7 double-stranded RNA is synthesized.
The results in FIG. 7 show that the interference efficiency can reach 60-70% within 36h after the parasitization.
5. Determination of cabbage moth blackening activity
To normal 3-instar middle-stage plutella xylostella larvae, 0.05. mu.l of the viral particle suspension (i.e., 10. mu.l) was injected4Copy number of recombinant virus), after 24h 20 heads of plutella xylostella hemolymph are extracted to determine the blackening activity.
500ng CvBV12-7 double-stranded RNA is injected into the bodies of the plutella xylostella larvae growing normally in the 3-instar middle period, single-head parasitism is carried out after 4h, and a control group is set to inject 500ng GFP double strands. Extracting 20 pieces of plutella xylostella hemolymph 24h after interference to determine blackening activity of the plutella xylostella hemolymph
Add 20 heads of plutella xylostella hemolymph into 200. mu.l PBS, vortex and mix evenly, and then take out 20. mu.l for determination of protein concentration. The total blood lymph Protein concentration was determined using the Pierce (TM) Coomassie (Bradfold) Protein Assay Kit. For blackening activity, 20. mu.l of heat-inactivated E.coli suspension (suspended in PBS, boiled in boiling water at OD. gtoreq.0.5 for 10min) was added to the remaining about 180. mu.l of hemolymph, and the mixture was reacted at room temperature for 20 min. Mu.l of the reaction solution was added to 140. mu.l of dopa solution (3g/L), and OD490 was measured every 5min for 60min in total for three mechanical repetitions. Three biological replicates per treatment were blanked with dopa solution. One unit of enzyme activity is defined as the change in OD490 per minute per mg of protein.
The results in FIG. 8 show that the blackening activity of the plutella xylostella is increased to about twice that of the postparasitism after the CvBV12-7 is interfered to be transcribed in the host body after the parasitism, and the result shows that the CvBV12-7 can inhibit the blackening activity of the plutella xylostella.
The results in FIG. 9 show that the blackening activity of the hemolymph of the diamondback moth is significantly inhibited after the recombinant CvBV12-7 virus particles are injected into the diamondback moth compared with the recombinant GFP virus particles.
Sequence listing
<110> Zhejiang university
Application of <120> CvBV12-7 gene in reducing insect humoral immune response
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 891
<212> DNA
<213> plutella xylostella cocoon bee multi-component DNA virus (Cotesia vestalis polydnavirus)
<400> 1
atgagttcta acaaggcgga aatcctggat tgcgaaatat caacatcaat aattgacgaa 60
aaaataatta atgaatttct ttgtcaagaa cacactaaat taatgatgaa catggaagcg 120
gacggaactt ttgtcgcccc tgcatctgct agaaacctcg gaaacggtaa acctgggaca 180
gatgaattat gcttcgacca caaccgcgta attcttaagg aagagaagga atccagcgat 240
tacataaatg cgagctacat cgacagcttc aaacaaccca aagcatatat cgtcacaaag 300
actcctgatt cagaggcgga aatccataaa ttttggaaaa tggtctggga acaacaatct 360
gaggtcattg tcatgcttaa taaacctgat caaaacgaga agggtgttct ctattggaaa 420
ttggaagaac gggcgacgct ctattgtgga aagctcaacg tagagacaat taaagttcga 480
catttacatc acagtttcga gattaccaca ctactaatca cgcacgaaga tgggggttcg 540
ttgttggttg accacttttt atacaagaac tggccgaaaa ttgattctgt gcccccaggc 600
gctgatttcc tggatttagt caacatgaca cgaacggaca ctagatatgt gcaaaagctt 660
tccaaaggct tcaagactcc agtagtagtc cattgcagtg atgggctgaa tcgatcaatt 720
gtgttttgtg taatcgacat atcgataact aaagatcaga aagttgtcga tgtaaacata 780
ttctctatcg tgtcccaact gagaaaacaa agatacaatt gcctgcacaa tgttgatcat 840
tatatttttt gttattcagc attttgtgaa tatttgaatc tttatacgta a 891
<210> 2
<211> 296
<212> PRT
<213> plutella xylostella cocoon bee multi-component DNA virus (Cotesia vestalis polydnavirus)
<400> 2
Met Ser Ser Asn Lys Ala Glu Ile Leu Asp Cys Glu Ile Ser Thr Ser
1 5 10 15
Ile Ile Asp Glu Lys Ile Ile Asn Glu Phe Leu Cys Gln Glu His Thr
20 25 30
Lys Leu Met Met Asn Met Glu Ala Asp Gly Thr Phe Val Ala Pro Ala
35 40 45
Ser Ala Arg Asn Leu Gly Asn Gly Lys Pro Gly Thr Asp Glu Leu Cys
50 55 60
Phe Asp His Asn Arg Val Ile Leu Lys Glu Glu Lys Glu Ser Ser Asp
65 70 75 80
Tyr Ile Asn Ala Ser Tyr Ile Asp Ser Phe Lys Gln Pro Lys Ala Tyr
85 90 95
Ile Val Thr Lys Thr Pro Asp Ser Glu Ala Glu Ile His Lys Phe Trp
100 105 110
Lys Met Val Trp Glu Gln Gln Ser Glu Val Ile Val Met Leu Asn Lys
115 120 125
Pro Asp Gln Asn Glu Lys Gly Val Leu Tyr Trp Lys Leu Glu Glu Arg
130 135 140
Ala Thr Leu Tyr Cys Gly Lys Leu Asn Val Glu Thr Ile Lys Val Arg
145 150 155 160
His Leu His His Ser Phe Glu Ile Thr Thr Leu Leu Ile Thr His Glu
165 170 175
Asp Gly Gly Ser Leu Leu Val Asp His Phe Leu Tyr Lys Asn Trp Pro
180 185 190
Lys Ile Asp Ser Val Pro Pro Gly Ala Asp Phe Leu Asp Leu Val Asn
195 200 205
Met Thr Arg Thr Asp Thr Arg Tyr Val Gln Lys Leu Ser Lys Gly Phe
210 215 220
Lys Thr Pro Val Val Val His Cys Ser Asp Gly Leu Asn Arg Ser Ile
225 230 235 240
Val Phe Cys Val Ile Asp Ile Ser Ile Thr Lys Asp Gln Lys Val Val
245 250 255
Asp Val Asn Ile Phe Ser Ile Val Ser Gln Leu Arg Lys Gln Arg Tyr
260 265 270
Asn Cys Leu His Asn Val Asp His Tyr Ile Phe Cys Tyr Ser Ala Phe
275 280 285
Cys Glu Tyr Leu Asn Leu Tyr Thr
290 295
<210> 3
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttcgttaaca gatctgcggc cgcatgagtt ctaacaaggc ggaaat 46
<210> 4
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcctctagag gtaccctcga gttacgtata aagattcaaa tattc 45
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tcgacagctt caaacaaccc 20
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gtcgcccgtt cttccaatt 19
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gctgagcgtg aaatcgtccg 20
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggagttgtag gtggtctcgt gga 23
<210> 9
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
acgagctcac tagtcgcggc cgctatgagt tctaacaagg cggaaat 47
<210> 10
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cttggtaccg catgcctcga gttacgtata aagattcaaa tattc 45
<210> 11
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acgagctcac tagtcgcggc cgctatggtg agcaagggcg agga 44
<210> 12
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cttggtaccg catgcctcga gttactagag gtaccctcga g 41
<210> 13
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
taatacgact cactataggt tgtgatgctg acagaacttc 40
<210> 14
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
taatacgact cactataggc cgtaactgga atacttgaat 40
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tcgacagctt caaacaaccc 20
<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gtcgcccgtt cttccaatt 19

Claims (6)

  1. The application of the CvBV12-7 gene in reducing the humoral immune response of insects is characterized in that the base sequence of the CvBV12-7 gene is shown as SEQ ID NO. 1; the insect is fruit fly or diamondback moth.
  2. The application of the CvBV12-7 gene in inhibiting the black forming activity of insect hemolymph is characterized in that the base sequence of the CvBV12-7 gene is shown as SEQ ID NO. 1; the insect is fruit fly or diamondback moth.
  3. The application of the CvBV12-7 gene in preparing a drosophila melanogaster model with low immunity is characterized in that the base sequence of the CvBV12-7 gene is shown as SEQ ID NO. 1.
  4. 4. A preparation method of a fruit fly model with low immunity is characterized by comprising the following steps:
    (1) preparing a recombinant plasmid containing a CvBV12-7 gene, injecting the recombinant plasmid into white eye wild type drosophila melanogaster embryos, and obtaining red eye transgenic drosophila melanogaster after the embryos grow to adults; the base sequence of the CvBV12-7 gene is shown in SEQ ID NO. 1;
    (2) performing two rounds of hybridization on the red-eye transgenic fruit fly and a balance line, and selecting homozygote transgenic fruit fly according to the phenotype of offspring;
    (3) and (3) hybridizing the homozygote transgenic drosophila prepared in the step (2) with a BS8700 drosophila strain by using a UAS/GAL4 system, and specifically expressing the gene CvBV12-7 in progeny drosophila blood cells to obtain the immune hypo drosophila.
  5. 5. The method of claim 4, wherein the white-eye wild type drosophila melanogaster is W1118; the genotype of the equilibrium system is w-/w-; Sp/Cyo; TM2/TM 6B.
  6. 6. The method of claim 5, wherein the homozygous transgenic drosophila has a genotype of w-/w-; CvBV12-7/CvBV 12-7; TM2/TM 6B.
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