本文選題：家蠶　切入點：Bmiap　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：家蠶是泌絲昆蟲,蠶業(yè)是我國重要的傳統(tǒng)產(chǎn)業(yè)。然而,蠶病的發(fā)生導(dǎo)致蠶業(yè)產(chǎn)值大大降低,其中尤以家蠶核型多角體病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)所引發(fā)的血液型膿病危害最為嚴(yán)重。細(xì)胞凋亡指細(xì)胞受基因調(diào)控的自主的細(xì)胞死亡過程,是細(xì)胞為維持內(nèi)環(huán)境穩(wěn)態(tài)的一種手段。細(xì)胞凋亡在機體的生長發(fā)育、細(xì)胞免疫、器官形成中具有重要的意義,同時在宿主免疫反應(yīng)中也發(fā)揮著重要作用。家蠶細(xì)胞凋亡在其先天性免疫反應(yīng)中具有重要作用,對家蠶細(xì)胞凋亡機理的深入解析,可為家蠶先天性免疫反應(yīng)機制的進(jìn)一步完善及家蠶抗病毒品種培育提供理論依據(jù)。本研究對家蠶細(xì)胞凋亡相關(guān)基因Bmiap進(jìn)行了克隆鑒定及功能分析,并初步解析了Bmiap基因在桿狀病毒BmNPV增殖過程中可能存在的作用機制。主要研究結(jié)果如下:1.家蠶Bmiap基因的克隆鑒定及功能分析我們成功克隆了Bmiap基因,獲得包括其完整ORF的cDNA序列,該基因CDS長度為1041bp,編碼346aa,蛋白預(yù)測分子量大小為38.86kDa,等電點為6.49。Bmiap具有保守的C-端BIR結(jié)構(gòu)域和N-端RING結(jié)構(gòu)域;構(gòu)建了Bmiap的真核表達(dá)載體和CRISPR/Cas9敲除載體,Bmiap真核表達(dá)載體轉(zhuǎn)染家蠶BmN-SWU1細(xì)胞后,免疫熒光結(jié)果顯示該基因定位于細(xì)胞質(zhì)中,western blot結(jié)果在40kDa附近檢測到特異性條帶;經(jīng)CRISPR/Cas9基因編輯技術(shù)敲除內(nèi)源性Bmiap后,細(xì)胞核皺縮并且有凋亡小體形成,Tunel染色結(jié)果綠色熒光增多,caspase酶活上升,細(xì)胞發(fā)生凋亡,說明Bmiap具有抑制細(xì)胞凋亡的作用。2.家蠶Bmiap與BmNPViaps的相互作用研究家蠶核型多角體病毒BmNPV中鑒定到兩個IAP家族同源蛋白BmNPViap1和BmNPViap2。通過免疫熒光和免疫共沉淀分析發(fā)現(xiàn)Bmiap與BmNPViap1、BmNPViap2均存在相互作用;qRT-PCR分析發(fā)現(xiàn),在BmNPV侵染細(xì)胞72h后Bmiap和BmNPViap2表達(dá)下調(diào),而BmNPViap1表達(dá)上調(diào);過表達(dá)Bmiap后,BmNPV侵染宿主細(xì)胞,BmNPViap1表達(dá)上調(diào),而BmNPViap2表達(dá)下調(diào),Vp39表達(dá)上調(diào),敲除Bmiap后,BmNPViap1表達(dá)下調(diào),BmNPViap2表達(dá)上調(diào),VP39表達(dá)下調(diào);分別過表達(dá)Bm NPViap1和BmNPViap2后以BmNPV侵染宿主細(xì)胞,結(jié)果發(fā)現(xiàn),Bmiap表達(dá)上調(diào),同時,過表達(dá)BmNPViap2會促進(jìn)Vp39的上調(diào)表達(dá),而過表達(dá)BmNPViap1后侵染BmNPV,發(fā)現(xiàn)Vp39僅在BmNPV侵染后48h表達(dá)發(fā)生輕微上調(diào),72h后無顯著差異。以上結(jié)果說明,在BmNPV增殖早期,Bmiap及BmNPViaps表達(dá)都呈上調(diào)趨勢,而在BmNPV侵染晚期Bmiap及BmNPViap2表達(dá)發(fā)上下調(diào),BmNPViap1表達(dá)進(jìn)一步上調(diào);Bmiap能促進(jìn)BmNPViap1表達(dá),抑制BmNPViap2的表達(dá),對Bm NPV的增殖具有促進(jìn)作用,而BmNPViap1和BmNPViap2都能促進(jìn)Bmiap的上調(diào)表達(dá),但是僅BmNPViap2對BmNPV的增殖具有促進(jìn)作用,而BmNPViap1對病毒的增殖無顯著作用。3.Bmiap基因在BmNPV侵染過程中作用機制研究本部分以Bmiap為誘餌蛋白通過免疫共沉淀的方法對Bmiap的相互作用蛋白進(jìn)行了篩選,鑒定到一個熱激蛋白家族成員Bmhsc70-4和一個蛋白磷酸酶Bmpp5,通過免疫熒光和免疫共沉淀驗證了Bmiap與Bmhsc70-4、Bmpp5存在相互作用關(guān)系;進(jìn)一步的研究發(fā)現(xiàn),Bmhsc70-4與Bmpp5都存在抑凋亡作用;本部分的RT-PCR數(shù)據(jù)分析發(fā)現(xiàn):BmNPV侵染過程中,Bmhsc70-4和Bmpp5的mRNA水平呈上調(diào)趨勢;過表達(dá)Bmhsc70-4和Bmpp5會促進(jìn)Bmiap和Vp39上調(diào)表達(dá),敲除Bmhsc70-4、Bmpp5,Bmiap和Vp39表達(dá)下調(diào);過表達(dá)Bmiap,Bmhsc70-4和Bmpp5的表達(dá)上調(diào),敲除Bmiap,Bmhsc70-4和Bmpp5的表達(dá)下調(diào)。以上結(jié)果說明:Bmiap與Bmhsc70-4、Bmpp5之間具有相互促進(jìn)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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S884.51

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