CN110724708A

CN110724708A - Application of CvBV7-1 gene in reducing humoral immune response of drosophila melanogaster

Info

Publication number: CN110724708A
Application number: CN201910981868.7A
Authority: CN
Inventors: 陈学新; 王泽华; 黄健华; 时敏; 叶熹骞
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-10-16
Filing date: 2019-10-16
Publication date: 2020-01-24
Anticipated expiration: 2039-10-16
Also published as: CN110724708B

Abstract

The invention discloses an application of a CvBV7-1 gene in reducing the humoral immune response of drosophila melanogaster, wherein the CvBV7-1 gene is the 1 st gene on the 7 th ring in 35 genome segments of plutella xylostella discodermatidae multi-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 CvBV7-1 into the drosophila melanogaster genome, thus obtaining a transgenic drosophila melanogaster strain with stable heredity and homozygote; by utilizing a UAS/GAL4 system, the homozygous CvBV7-1 transgenic drosophila melanogaster is hybridized with the BS8700 drosophila melanogaster strain, and after the obtained strain expresses multi-component DNA virus genes, the melanogenesis activity of the hemolymph of the drosophila melanogaster can be inhibited, the cyst effect of the drosophila melanogaster larva in vivo on parasitic bee larvae is reduced, and the drosophila melanogaster has the obvious characteristic of low immunity when being infected by pathogens, and has important significance in the aspects of researching relevant mechanisms of drosophila melanogaster immunity, enhancing the screening of human immunity medicaments and the like.

Description

Application of CvBV7-1 gene in reducing humoral immune response of drosophila melanogaster

Technical Field

The invention relates to the technical field of molecular biology and genetic engineering, in particular to application of a CvBV7-1 gene in reducing the humoral immune response of drosophila.

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; Tsakas, s.and v.j.marmarsaas (2010) ' institute immunity and issues: An animal Biology. ' inventor. surgery. environment Journal 7(2): 228. 238; grignard, e.v., i.m. complete, m.m.a.environment. v. culture. supplement, of strain, 201440. culture of strain, and strain, 201440). 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 suppression bymenoptorparasitids", "BioEssays 23(4): 344. 351; Wu, S.and E.J.Ling (2009)" Phagocytosis, probability and expression in cellular immunity in insects "Acta Entomogica 52(7): 791. 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 and Molecular Biology 31(3):219 and 229, Blandin, s.and e.a.levanshina (2004)." thio-conditioner proteins immunity "Molecular immunity 40(12):903 and 908, Imler, j.l.and p.buildingj.2005 (antibiotic peptides) and melanin: 21.86. tissue j.21.21. Molecular Biology and 86. expression).

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 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 successfully parasitize (Bai, S., X.Chen, J.Cheng, W.Fu and J.He (2005). "efficiencies of water-associated factors of Cotesia plutellae on growing and evaluation of Plutella xylostella larvae." Acta Phytophylactica site 32(3): 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 Research117(1): 81-89; Strand, M.R. and G.R. Burke (2012)", "Polydnaviruses as systems and field 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 vitamins 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 new application of the CvBV7-1 gene in reducing the humoral immune response of drosophila melanogaster and preparing hypoimmunity drosophila melanogaster.

The specific technical scheme is as follows:

the CvBV7-1 gene is the 1 st gene on the 7 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 encoding sequence can encode 237 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2.

The invention provides an application of a CvBV7-1 gene in reducing drosophila humoral immune response, wherein the base sequence of the CvBV7-1 gene is shown as SEQ ID No. 1.

Experiments show that the CvBV7-1 gene can inhibit the melanization activity of drosophila haemolymph, and the base sequence of the CvBV7-1 gene is shown in SEQ ID No. 1.

Experiments show that the CvBV7-1 gene can reduce the encystment of drosophila larvae on parasitic wasp larvae, and the base sequence of the CvBV7-1 gene is shown in SEQ ID No. 1.

The invention also provides an application of the CvBV7-1 gene in preparing a drosophila melanogaster model with low immunity, wherein the base sequence of the CvBV7-1 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 CvBV7-1 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 CvBV7-1 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 CvBV7-1 gene 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/TM6B。

the genotype of the homozygote transgenic drosophila is w-/w-; CvBV7-1/CvBV 7-1; TM2/TM 6B.

Compared with the prior art, the invention has the following beneficial effects:

the invention uses the transgenic technology to transfer plutella xylostella cotesia xylostella multi-DNA virus gene CvBV7-1 into the drosophila melanogaster genome, thus obtaining a transgenic drosophila melanogaster strain with stable heredity and homozygote; by utilizing a UAS/GAL4 system, the homozygous CvBV7-1 transgenic drosophila melanogaster is hybridized with the BS8700 drosophila melanogaster strain, and after the obtained strain expresses multi-component DNA virus genes, the melanogenesis activity of the hemolymph of the drosophila melanogaster can be inhibited, the cyst effect of the drosophila melanogaster larva in vivo on parasitic bee larvae is reduced, and the drosophila melanogaster has the obvious characteristic of low immunity when being infected by pathogens, and has important significance in the aspects of researching relevant mechanisms of drosophila melanogaster immunity, enhancing the screening of human immunity medicaments and the like.

Drawings

FIG. 1 shows the PCR assay of CvBV7-1 transgenic Drosophila in example 1.

FIG. 2 shows the results of the semi-quantitative PCR assay of CvBV7-1 expression in transgenic Drosophila in example 1.

FIG. 3 shows the results of the detection of the influence of the CvBV7-1 gene on Drosophila immunity in example 1.

FIG. 4 shows the encystment of Drosophila larvae on parasitic wasp larvae in example 1.

FIG. 5 is the result of the examination that the CvBV7-1 gene affected the encystment of Drosophila larvae on parasitic wasp larvae in example 1;

where N denotes that no black block is generated, S denotes that a small black block is generated, and L denotes that a large black block is generated.

FIG. 6 shows the results of testing the influence of the CvBV7-1 gene on the melanogenesis activity of Drosophila melanogaster larvae in example 1.

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 Open Reading Frame (ORF) of the CvBV7-1 gene (shown in SEQ ID NO. 1) and the sequence of the multiple Cloning site on the pUASttb vector, the action mode of homologous recombinase (Clonexpress II One Step Cloning Kit, Novozan) is combined, and specific primers are designed, wherein the primer sequences are as follows:

7-1-P：5’-TTCGTTAACAGATCTGCGGCCGCATGTTCAACAATATACTGATAGCT C-3’；

7-1-AP：5’-TCCTCTAGAGGTACCCTCGAGTTAATTGGCCTGTAAAGATATC-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 CvBV7-1 gene fragment with carrier homologous sequence is PCR amplified from the constructed cDNA library by using specific primers 7-1-P and 7-1-AP, and the fragment is recombined and cloned to pUA Sttb 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 CvBV7-1 transgenic drosophila melanogaster and the genome DNA of wild type W1118 drosophila melanogaster are extracted, and the specific primers 7-1-P and 7-1-AP are respectively used for carrying out PCR detection on the genome DNA. A fragment of size consistent with CvBV7-1 was amplified from the CvBV7-1 transgenic Drosophila but not in the genomic DNA of wild-type W1118 Drosophila melanogaster. The plutella xylostella cotesia coides multi-component DNA virus CvBV7-1 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 CvBV7-1 transgenic drosophila homozygote strain

In this embodiment, CvBV7-1 is inserted into chromosome II, so the transgenic line successfully constructed in step 1 is crossed with a balanced line (w-/w-; Sp/Cyo; TM2/TM6B), virginator fruit flies (w-/w-; CvBV 7-1/Cyo; TM2/+) with rolling wings and large balanced rods and male fruit flies (w-/w-; CvBV 7-1/Cyo; TM6B/+) with rolling wings and shoulder hair are selected according to the offspring phenotype, and the selected fruit flies are crossed; or selecting male drosophila melanogaster (w-/w-; CvBV 7-1/Cyo; TM2/+) with rolling fin and large balancing rod as progeny and virgin drosophila melanogaster (w-/w-; CvBV 7-1/Cyo; TM6B/+) with rolling fin and shoulder hair, and hybridizing the selected drosophila melanogaster; the filial generation selects the CvBV7-1 gene-transferred fruit fly (w-/w-; CvBV7-1/CvBV 7-1; TM2/TM6B) of straight wing, large balance rod, shoulder hairy homozygote for seed protection.

3. Expression of CvBV7-1 gene in drosophila melanogaster blood cells

The homozygous CvBV7-1 transgenic Drosophila in step 2 was crossed with BS8700(FlyBaseID: FBti0064641) Drosophila strain using the UAS/GAL4 system, such that CvBV7-1 was specifically expressed in Drosophila blood cells, and CvBV7-1 transgenic strain 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:

CvBV7-1-qPCR-F：5’-TCTTGCCTCTAACGAGCCAC-3’

CvBV7-1-qPCR-F：5’-TTTCGAATCAGCAGACACCC-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 CvBV7-1 transgenic line and the filial generation of W1118 have no expression of CvBV7-1, while the CvBV7-1 transgenic line and the filial generation of BS8700 have high expression level. Thus, it was demonstrated that transgenic Drosophila expressing CvBV7-1 specifically in blood cells was obtained.

4. Influence of specific expression of CvBV7-1 gene in blood cells of drosophila on drosophila

a) Detection result of influence of CvBV7-1 on drosophila melanogaster immunity

Virgins of wild-type W1118 and BS8700 were individually crossed with the CvBV7-1 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 CvBV7-1 transgenic line and BS8700 is remarkably lower (p is 0.00267) than that of the control group (filial generation of the CvBV7-1 transgenic line and the wild type W1118), which shows that the CvBV7-1 can reduce the immunity of fruit flies.

b) CvBV7-1 influences the encystment of fruit flies on young bees of Boladia catenulata

The drosophila larvae are parasitized by the baladi small-ring cecrophycidae, because of humoral immune response, the parasitized larvae often can generate black tissue masses (actually, the drosophila larvae can be encapsulated by the brood, and the size of the black masses reflects the strength of humoral immune response), and the parasitized larvae are divided into three types, namely, no black masses are generated, small black masses are generated, and large black masses are generated (fig. 4).

The CvBV7-1 transgenic line was used to hybridize with wild type virginator Drosophila W1118 and BS8700, respectively, and the number of eggs was counted after spawning. And when the eggs are hatched to 2 years old, putting the female parasitic wasps which are fully mated according to the ratio of the number of the eggs to the parasitic wasps to be 20:1, taking out the parasitic wasps after 2-3h of parasitization, and counting the black mass of the drosophila larvae after 48 h.

The results in fig. 5 show that the progeny of the cross of the CvBV7-1 transgenic line with BS8700 produced a significantly reduced number of large black blocks (where n represents a statistical number) compared to the control (progeny of the cross of the CvBV7-1 transgenic line with wild-type W1118). This further demonstrates that overexpression of CvBV7-1 in Drosophila blood cells suppresses the humoral immune response in Drosophila.

c) Detection result of influence of CvBV7-1 on melanogenesis activity of drosophila melanogaster

The CvBV7-1 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. 6 show that compared with the control group (filial generation of the CvBV7-1 transgenic line and the wild-type W1118), the blackening activity of the hemolymph of the filial generation of the CvBV7-1 transgenic line and the BS8700 is significantly inhibited.

Sequence listing

<110> Zhejiang university

Application of <120> CvBV7-1 gene in reducing humoral immune response of drosophila

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>714

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<213> plutella xylostella cocoon bee multi-component DNA virus (Cotesia vestalis polydnavirus)

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attttggcag caatacataa cggagtaaaa caaatagaaa gcaggaatca tcgaggctta 480

gaccaaatat ttcatgagac tactgaagag gaatcgaata tgtcattaaa actaaatact 540

ttataccgca agtgtggctc gttagaggca agacttgact accaagaact gcgtcttaga 600

cattttaacg atgagagttt gccgatgcaa gacattttcg acggagattt agctcattta 660

aaaacaatat tagaattagc taatttggag ttgatatctt tacaggccaa ttaa 714

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His Arg Arg Ala Ile Asp Glu Gln Ser Lys Trp Val Asn Phe Gly Ser

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Asp Gln Ile Phe His Glu Thr Thr Glu Glu Glu Ser Asn Met Ser Leu

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Asp Tyr Gln Glu Leu Arg Leu Arg His Phe Asn Asp Glu Ser Leu Pro

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gctgagcgtg aaatcgtccg 20

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Claims

The application of the CvBV7-1 gene in reducing the humoral immune response of drosophila melanogaster is characterized in that the base sequence of the CvBV7-1 gene is shown as SEQ ID NO. 1.
The application of the CvBV7-1 gene in inhibiting the melanogenesis activity of drosophila haemolymph is characterized in that the base sequence of the CvBV7-1 gene is shown in SEQ ID NO. 1.
The application of the CvBV7-1 gene in reducing the encystment of drosophila larvae on parasitic wasp larvae is characterized in that the base sequence of the CvBV7-1 gene is shown as SEQ ID No. 1.
The application of the CvBV7-1 gene in preparing a drosophila melanogaster model with low immunity is characterized in that the base sequence of the CvBV7-1 gene is shown as SEQ ID NO. 1.
5. 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 CvBV7-1 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 CvBV7-1 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 CvBV7-1 gene in progeny drosophila blood cells to obtain the immune hypo drosophila.
6. The method of claim 5, wherein the white-eye wild type drosophila melanogaster is W1118; the genotype of the equilibrium system is w-/w-; Sp/Cyo; TM2/TM 6B.
7. The method of claim 6, wherein the homozygous transgenic drosophila has a genotype of w-/w-; CvBV7-1/CvBV 7-1; TM2/TM 6B.