家蚕Bmiap基因在BmNPV增殖过程中的作用机制研究

发布时间：2018-03-08 12:21

  本文选题：家蚕　切入点：Bmiap　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：家蚕是泌丝昆虫,蚕业是我国重要的传统产业。然而,蚕病的发生导致蚕业产值大大降低,其中尤以家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)所引发的血液型脓病危害最为严重。细胞凋亡指细胞受基因调控的自主的细胞死亡过程,是细胞为维持内环境稳态的一种手段。细胞凋亡在机体的生长发育、细胞免疫、器官形成中具有重要的意义,同时在宿主免疫反应中也发挥着重要作用。家蚕细胞凋亡在其先天性免疫反应中具有重要作用,对家蚕细胞凋亡机理的深入解析,可为家蚕先天性免疫反应机制的进一步完善及家蚕抗病毒品种培育提供理论依据。本研究对家蚕细胞凋亡相关基因Bmiap进行了克隆鉴定及功能分析,并初步解析了Bmiap基因在杆状病毒BmNPV增殖过程中可能存在的作用机制。主要研究结果如下:1.家蚕Bmiap基因的克隆鉴定及功能分析我们成功克隆了Bmiap基因,获得包括其完整ORF的cDNA序列,该基因CDS长度为1041bp,编码346aa,蛋白预测分子量大小为38.86kDa,等电点为6.49。Bmiap具有保守的C-端BIR结构域和N-端RING结构域;构建了Bmiap的真核表达载体和CRISPR/Cas9敲除载体,Bmiap真核表达载体转染家蚕BmN-SWU1细胞后,免疫荧光结果显示该基因定位于细胞质中,western blot结果在40kDa附近检测到特异性条带;经CRISPR/Cas9基因编辑技术敲除内源性Bmiap后,细胞核皱缩并且有凋亡小体形成,Tunel染色结果绿色荧光增多,caspase酶活上升,细胞发生凋亡,说明Bmiap具有抑制细胞凋亡的作用。2.家蚕Bmiap与BmNPViaps的相互作用研究家蚕核型多角体病毒BmNPV中鉴定到两个IAP家族同源蛋白BmNPViap1和BmNPViap2。通过免疫荧光和免疫共沉淀分析发现Bmiap与BmNPViap1、BmNPViap2均存在相互作用;qRT-PCR分析发现,在BmNPV侵染细胞72h后Bmiap和BmNPViap2表达下调,而BmNPViap1表达上调;过表达Bmiap后,BmNPV侵染宿主细胞,BmNPViap1表达上调,而BmNPViap2表达下调,Vp39表达上调,敲除Bmiap后,BmNPViap1表达下调,BmNPViap2表达上调,VP39表达下调;分别过表达Bm NPViap1和BmNPViap2后以BmNPV侵染宿主细胞,结果发现,Bmiap表达上调,同时,过表达BmNPViap2会促进Vp39的上调表达,而过表达BmNPViap1后侵染BmNPV,发现Vp39仅在BmNPV侵染后48h表达发生轻微上调,72h后无显著差异。以上结果说明,在BmNPV增殖早期,Bmiap及BmNPViaps表达都呈上调趋势,而在BmNPV侵染晚期Bmiap及BmNPViap2表达发上下调,BmNPViap1表达进一步上调;Bmiap能促进BmNPViap1表达,抑制BmNPViap2的表达,对Bm NPV的增殖具有促进作用,而BmNPViap1和BmNPViap2都能促进Bmiap的上调表达,但是仅BmNPViap2对BmNPV的增殖具有促进作用,而BmNPViap1对病毒的增殖无显著作用。3.Bmiap基因在BmNPV侵染过程中作用机制研究本部分以Bmiap为诱饵蛋白通过免疫共沉淀的方法对Bmiap的相互作用蛋白进行了筛选,鉴定到一个热激蛋白家族成员Bmhsc70-4和一个蛋白磷酸酶Bmpp5,通过免疫荧光和免疫共沉淀验证了Bmiap与Bmhsc70-4、Bmpp5存在相互作用关系;进一步的研究发现,Bmhsc70-4与Bmpp5都存在抑凋亡作用;本部分的RT-PCR数据分析发现:BmNPV侵染过程中,Bmhsc70-4和Bmpp5的mRNA水平呈上调趋势;过表达Bmhsc70-4和Bmpp5会促进Bmiap和Vp39上调表达,敲除Bmhsc70-4、Bmpp5,Bmiap和Vp39表达下调;过表达Bmiap,Bmhsc70-4和Bmpp5的表达上调,敲除Bmiap,Bmhsc70-4和Bmpp5的表达下调。以上结果说明:Bmiap与Bmhsc70-4、Bmpp5之间具有相互促进作用。
[Abstract]:Silkworm is a silk spinning insect, sericulture is an important traditional industries in our country. However, lead to the occurrence of silkworm diseases in sericulture production is greatly reduced, especially in the Bombyx mori nuclear polyhedrosis virus (Bombyx mori, nucleopolyhedrovirus, BmNPV) blood pus disease harm caused by the most serious. The apoptosis cell death process by means of gene regulation independent cells, cells as a means of internal environment homeostasis. Cells in the body's growth and development, cellular immunity plays an important role in organ formation, while the host immune response also plays an important role. Silkworm apoptosis plays an important role in the innate immune response, in-depth analysis of the the mechanism of apoptosis of Bombyx mori, and provide a theoretical basis for cultivating mechanism of silkworm innate immune response and further improve the silkworm resistant varieties. The research on silkworm apoptosis related Bmiap gene cloning and functional analysis, and preliminary analysis of the mechanism of Bmiap gene may exist in the process of baculovirus BmNPV proliferation. The main results are as follows: molecular cloning and functional analysis of Bmiap gene of 1. silkworm we successfully cloned Bmiap sequence of cDNA ORF including the complete, the length of the CDS gene 1041bp, encoding 346aa protein, the predicted molecular mass of 38.86kDa and isoelectric point is 6.49.Bmiap with C- terminal BIR domain and N- terminal conserved RING domain; constructed the eukaryotic expression vector of Bmiap and CRISPR/Cas9 knockout vector, Bmiap eukaryotic expression vector was transfected into silkworm BmN-SWU1 cells, immunofluorescence results showed that the gene located in the cytoplasm, Western blot results in 40kDa detected near the specific band; after CRISPR/Cas9 knockdown of endogenous Bmiap gene editing technology, nuclear shrinkage and The formation of apoptotic bodies, Tunel staining showed green fluorescence increased, increased caspase activity, cell apoptosis, the karyotype study of interaction of Bombyx mori polyhedrosis virus BmNPV Bmiap.2. and BmNPViaps Bmiap of silkworm has the effect of inhibiting apoptosis in the identification of the two IAP family with BmNPViap1 and BmNPViap2. protein by immunofluorescence and immunogold analysis Bmiap and BmNPViap1 co precipitation, there were BmNPViap2 interaction; qRT-PCR analysis showed that the expression of Bmiap and BmNPViap2 downregulated in BmNPV infected cells after 72h, the increased expression of BmNPViap1; overexpression of Bmiap after BmNPV infected host cells, upregulation of BmNPViap1 expression and BmNPViap2 expression, Vp39 expression, Bmiap knockdown BmNPViap1 expression the expression of BmNPViap2 was up-regulated, down regulated expression of VP39; expression of Bm and BmNPViap2 NPViap1 respectively in BmNPV infected host cells, the expression of Bmiap. At the same time, increase, overexpression of BmNPViap2 can promote the expression of Vp39 and overexpression of BmNPViap1 up-regulated after infection of BmNPV, Vp39 was found only in BmNPV after the infection of 48h expression was slightly up-regulated, 72h had no significant difference. These results indicated that the proliferation of BmNPV in early, the expression of Bmiap and BmNPViaps were up-regulated, while BmNPV infection in the late Bmiap and BmNPViap2 expression in hair cut, the expression of BmNPViap1 further increased; Bmiap can promote the expression of BmNPViap1 and inhibit the expression of BmNPViap2 can promote the proliferation of NPV and Bm, BmNPViap1 and BmNPViap2 can promote the expression of Bmiap is upregulated, but only BmNPViap2 on the proliferation of BmNPV and BmNPViap1 can promote the proliferation of the virus, no significant the role of.3.Bmiap gene in the BmNPV infection process mechanism of this part for Bmiap on the interaction of Bmiap protein by immunoprecipitation of bait protein The screening, identification of a member of the heat shock protein family Bmhsc70-4 and a protein phosphatase Bmpp5, Bmiap and Bmhsc70-4 was verified by immunofluorescence and immunoprecipitation, there is interaction between Bmpp5; further study found that Bmhsc70-4 and Bmpp5 have anti apoptosis by RT-PCR; this part of the data analysis showed that BmNPV infection in the process of Bmhsc70-4, Bmpp5 and mRNA levels were up-regulated; overexpression of Bmhsc70-4 and Bmpp5 can promote the expression of Bmiap and upregulation of Vp39, knockdown of Bmhsc70-4, Bmpp5, Bmiap and Vp39 expression; overexpression of Bmiap upregulated Bmhsc70-4 and Bmpp5 expression and knockdown of Bmiap, down regulate the expression of Bmhsc70-4 and Bmpp5. The results indicated: Bmiap and Bmhsc70-4, with positive interaction between Bmpp5.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S884.51

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