阅读说明：本技术 CvBV3-3基因在降低昆虫体液免疫反应中的应用 (Application of CvBV3-3 gene in reducing humoral immune response of insect ) 是由 陈学新 王泽华 黄健华 时敏 王知知 于 2019-10-16 设计创作，主要内容包括：本发明公开了CvBV3-3基因在降低果蝇体液免疫反应中的应用,该CvBV3-3基因是菜蛾盘绒茧蜂多分DNA病毒的35个基因组片段中的第3个环上的第3个基因,碱基序列如SEQ ID NO.1所示。本发明利用转基因技术,将菜蛾盘绒茧蜂多分DNA病毒基因CvBV3-3转入果蝇基因组,获得了稳定遗传、纯合体的转基因果蝇品系；利用UAS/GAL4系统,纯合体的CvBV3-3转基因果蝇与BS8700果蝇品系进行杂交,得到的品系表达多分DNA病毒基因后,发现能够抑制昆虫血淋巴的黑化活力以及降低果蝇幼虫体内对寄生蜂幼虫的包囊作用,在受到病原物侵染时,具有明显的免疫力低的性状,在对研究昆虫免疫相关机制、增强人类免疫类药剂的筛选等方面有重要意义。(The invention discloses an application of a CvBV3-3 gene in reducing the humoral immune response of drosophila melanogaster, wherein the CvBV3-3 gene is the 3 rd gene on the 3 rd ring in 35 genome segments of plutella xylostella coilia 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 CvBV3-3 into the drosophila melanogaster genome, so as to obtain a transgenic drosophila melanogaster strain with stable heredity and homozygote; by utilizing a UAS/GAL4 system, the homozygous CvBV3-3 transgenic drosophila melanogaster is hybridized with the BS8700 drosophila melanogaster strain, and after the obtained strain expresses multi-component DNA virus genes, the blackening activity of insect hemolymph can be inhibited, the encapsulation effect of the drosophila melanogaster larva in vivo on parasitic wasp larva is reduced, and the drosophila melanogaster has the obvious character of low immunity when being infected by pathogens, and has important significance in the aspects of researching insect immunity related mechanisms, enhancing the screening of human immunity medicaments and the like.)

The application of the CvBV3-3 gene in reducing the humoral immune response of insects is characterized in that the base sequence of the CvBV3-3 gene is shown as SEQ ID NO. 1; the insect is fruit fly or diamondback moth.

The application of the CvBV3-3 gene in inhibiting the black forming activity of insect hemolymph is characterized in that the base sequence of the CvBV3-3 gene is shown as SEQ ID NO. 1; the insect is fruit fly or diamondback moth.

The application of the CvBV3-3 gene in reducing the encystment of drosophila larvae on parasitic wasp larvae is characterized in that the base sequence of the CvBV3-3 gene is shown as SEQ ID NO. 1.

The application of the CvBV3-3 gene in preparing a drosophila melanogaster model with low immunity is characterized in that the base sequence of the CvBV3-3 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 CvBV3-3 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 CvBV3-3 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 CvBV3-3 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-; CvBV3-3/CvBV 3-3; TM2/TM 6B.

Technical Field

The invention relates to the technical field of molecular biology and genetic engineering, in particular to application of a CvBV3-3 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', Tsakas, s.and v.j.marmarsaas (2010) 'institute immunity and issues: An animal Biology.' inventebrate and specific journal7(2) '228. 238; grignard, e.v., i.m.d. virus, m.a.stem v. infection, m.a.barrier v. strain and strain, 2014. throttle and strain, 18, 9. host, etc.' throttle and strain, 119. fig. 46. fig. 1. 12, 9. host, strain, and strain, 2014. 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). "Innateimemtnity and its evolution and preservation by hypothermiptorparasities", "BioEssays 23(4): 344. 351; Wu, S.and E.J.Ling (2009)" Phagocytosis, nodal and cellular immunity in insects 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 Biochemistry and Molecular biology 31(3):219 and 229, Blandin, s.and e.a.levanshina (2004)." thio-conjugation proteins immunity and immunity "Molecular immunity 40(12):903 and 908, Imler, j.l.and p.buble (2005. antibiotic peptides: 21. tissue J.21. expression: chemical expression: 1.86).

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 CvBV3-3 gene in reducing the humoral immune response of insects and preparing low-immune drosophila.

The specific technical scheme is as follows:

the CvBV3-3 gene is the 3 rd gene on the 3 rd 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 polypeptide can code 95 amino acids, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 2.

The invention provides an application of a CvBV3-3 gene in reducing insect humoral immune response, wherein the base sequence of the CvBV3-3 gene is shown as SEQ ID NO. 1; the insect is fruit fly or diamondback moth.

Experiments show that the CvBV3-3 gene can inhibit the blackening activity of insect hemolymph, and the base sequence of the CvBV3-3 gene is shown in SEQ ID NO. 1; the insect is fruit fly or diamondback moth.

Experiments show that the CvBV3-3 gene can reduce the encystment of drosophila larvae on parasitic wasp larvae, and the base sequence of the CvBV3-3 gene is shown in SEQ ID NO. 1.

The invention also provides application of the CvBV3-3 gene in preparing a drosophila melanogaster model with low immunity, wherein the base sequence of the CvBV3-3 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 CvBV3-3 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 CvBV3-3 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 CvBV3-3 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-; CvBV3-3/CvBV 3-3; 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 CvBV3-3 into the drosophila melanogaster genome, so as to obtain a transgenic drosophila melanogaster strain with stable heredity and homozygote; by utilizing a UAS/GAL4 system, the homozygous CvBV3-3 transgenic drosophila melanogaster is hybridized with the BS8700 drosophila melanogaster strain, and after the obtained strain expresses multi-component DNA virus genes, the blackening activity of insect hemolymph can be inhibited, the encapsulation effect of the drosophila melanogaster larva in vivo on parasitic wasp larva is reduced, and the drosophila melanogaster has the obvious character of low immunity when being infected by pathogens, and has important significance in the aspects of researching insect immunity related mechanisms, enhancing the screening of human immunity medicaments and the like.

(2) According to the invention, after recombinant CvBV3-3 virus particles are injected into a diamondback moth body, the blackening activity of the hemolymph of the diamondback moth is obviously inhibited, which shows that the CvBV3-3 gene can inhibit the blackening activity of the hemolymph of the diamondback moth.

Drawings

FIG. 1 shows the PCR assay of CvBV3-3 transgenic Drosophila in example 1.

FIG. 2 shows the results of the semi-quantitative PCR assay of CvBV3-3 expression in transgenic Drosophila in example 1.

FIG. 3 shows the results of testing the influence of the CvBV3-3 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 results of the examination of the influence of the CvBV3-3 gene on 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 CvBV3-3 gene on the melanogenesis activity of Drosophila melanogaster larvae in example 1.

FIG. 7 is the result of testing the influence of the CvBV3-3 gene on the blackening activity of the larvae of plutella xylostella in example 2;

wherein CK represents untreated plutella xylostella; Bac-GFP refers to plutella xylostella injected with recombinant baculovirus (GFP gene); Bac-CvBV3-3 shows a plutella xylostella injected with recombinant baculovirus (CvBV3-3 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.