【摘要】：自噬是指膜(大部分表現(xiàn)為雙層膜,有時多層或單層)包裹部分胞質(zhì)和細胞內(nèi)需降解的細胞器、蛋白質(zhì)等形成自噬體(autophagosome),最后與溶酶體融合形成自噬溶酶體(autophagolysosome),降解其所包裹的內(nèi)容物,以實現(xiàn)細胞穩(wěn)態(tài)和細胞器的更新。目前普遍認(rèn)為自噬是一種防御和應(yīng)激調(diào)控機制,它為細胞的重建、再生和修復(fù)提供必須原料,實現(xiàn)細胞物質(zhì)的再循環(huán)和再利用。自噬基因(autophagy-related gene,atg)的克隆始于酵母(yeast)。Atgl 蛋白(autophagy-related-gene-1)屬于絲氨酸/蘇氨酸蛋白激酶,是細胞自噬過程中非常關(guān)鍵的自噬相關(guān)蛋白之一。在自噬起始階段時,Atg1-Atg13復(fù)合物可募集其他Atg蛋白,誘導(dǎo)細胞自噬的發(fā)生。鱗翅目昆蟲Atg1對細胞自噬的調(diào)控作用尚不清楚,Atg1互作蛋白也研究較少。自噬相關(guān)蛋白Atg8是一種類泛素化蛋白,對自噬囊膜的延伸擴展起到重要作用,既參與底物的募集,又介導(dǎo)自噬小體與溶酶體的融合。它常被作為是研究細胞自噬的標(biāo)記物。斜紋夜蛾Spodoptera litura(Fabricius)屬于夜蛾科�；页嵋苟陮�,是一種多食性害蟲,對許多農(nóng)作物造成了嚴(yán)重的危害。本研究對已知的鱗翅目Atg1基因序列進行同源比對,根據(jù)基因片段的保守序列設(shè)計兼并引物,通過半巢式PCR方法擴增得到了 Atg1基因開放閱讀框(ORF),并對此基因進行了生物信息學(xué)分析。將Atg1基因截短,分別構(gòu)建Atg1-N末端600bp和Atg1-C末端500bp原核表達載體,將兩種表達載體轉(zhuǎn)化大腸桿菌BL21表達菌株,誘導(dǎo)表達并純化截短的Atg1蛋白,用獲得的純化蛋白制備抗Atg1鼠抗和兔抗。然后,構(gòu)建 Atg1 真核表達載體 pEGFP-N1-ie2-Atg1 和 pEGFP-N1-ie2-Atg1-Flag,轉(zhuǎn)染S1-HP昆蟲細胞,探究Atg1蛋白的表達與定位。接著將Atg1與其他自噬基因Atg8、Atg6、Atg5真核表達載體共轉(zhuǎn)染S1-HP細胞,分別檢測兩者之間是否共定位。分別用饑餓和自噬抑制劑處理斜紋夜蛾S1-HP細胞,研究了二者對Atg1蛋白降解的影響。接著研究了 S1-HP細胞中Atg1的超表達和基因表達沉默對細胞自噬的影響。最后采用Co-IP技術(shù)探究了 Atg1與其它自噬相關(guān)蛋白的相互作用。實驗結(jié)果表明,斜紋夜蛾Atg1開放閱讀框全長2286bp,編碼761個氨基酸,預(yù)測的分子量大小為82.6 kDa,等電點約為9.5。具有三個功能結(jié)構(gòu)域,分別是激酶結(jié)構(gòu)域、LIR結(jié)構(gòu)域和Atg13結(jié)合結(jié)構(gòu)域。在大腸桿菌中表達的全長蛋白容易降解,但是截短的蛋白比較穩(wěn)定,可用于制備抗體。GFP-Atg1(或Atg1-GFP)融合蛋白分布于細胞質(zhì)中,在細胞核內(nèi)沒有分布。它與Atg8,Atg6和Atg5部分共定位。饑餓誘導(dǎo)自噬導(dǎo)致細胞中Atg1因為降解而減少,抑制細胞自噬,可以導(dǎo)致Atg1積累,表明Atg1可以通過自噬溶酶體而降解。Atg1的超表達,加快了 Atg8-PE的降解;Atg1的表達沉默使Atg8-PE積累,表明Atg1促進細胞的自噬活動。免疫共沉淀技術(shù)表明Atg1可能與其它Atg蛋白存在相互作用。這些結(jié)果為揭示SlAtg1對自噬的多重調(diào)節(jié)及其分子機理提供了重要的基礎(chǔ)。
[Abstract]:Autophagy refers to the formation of autophagy (most of which are bilayer, sometimes multilayer or monolayer) that encapsulates the degradation of part of the cytoplasm and cell internal demand, protein, etc., forming autophagy (autophagosome), and finally fused with lysosome to form autophagy (autophagolysosome), The contents are degraded to achieve cellular homeostasis and organelle renewal. At present, autophagy is generally considered as a defense and stress regulation mechanism, which provides necessary raw materials for cell reconstruction, regeneration and repair, and realizes the recycling and reuse of cellular substances. The cloning of autophagy gene (autophagy-related gene,atg) begins with yeast (yeast). Atgl protein (autophagy-related-gene-1) which belongs to serine / threonine protein kinase and is one of the most important autophagy related proteins during autophagy. At the initial stage of autophagy, Atg1-Atg13 complex can recruit other Atg proteins and induce autophagy. The regulation of Lepidoptera Atg1 on autophagy is unclear, and the Atg1 interaction protein is less studied. Autophagy associated protein (Atg8) is a kind of ubiquitin protein, which plays an important role in the extension and expansion of autophagy capsule membrane. It not only participates in the recruitment of substrate, but also mediates the fusion of autophagy body and lysosome. It is often used as a marker for the study of autophagy. Spodoptera litura (Fabricius) belongs to the genus Noctuidae of the family Noctuidae. It is a polyeating pest and has caused serious damage to many crops. In this study, homologous alignment of known Lepidoptera Atg1 gene sequences was carried out. According to the conserved sequence of Lepidoptera gene fragments, degenerate primers were designed and amplified by semi-nested PCR method. Open reading frame (ORF), of Atg1 gene was obtained. The gene was analyzed by bioinformatics. The prokaryotic expression vectors of Atg1-N terminal 600bp and Atg1-C terminal 500bp were constructed by truncating the Atg1 gene. The two expression vectors were transformed into E. coli BL21 expression strain to induce and purify the truncated Atg1 protein. The purified protein was used to prepare anti-Atg1 anti-mouse and anti-rabbit. Then, Atg1 eukaryotic expression vectors pEGFP-N1-ie2-Atg1 and pEGFP-N1-ie2-Atg1-Flag, were constructed and transfected into S1-HP insect cells to investigate the expression and localization of Atg1 protein. Then, Atg1 and other eukaryotic expression vectors of autophagy gene Atg8,Atg6,Atg5 were co-transfected into S1-HP cells to detect whether they were co-located. The effects of starvation and autophagy inhibitor on the degradation of Atg1 protein in S1-HP cells of Spodoptera litura were studied. Then the effects of Atg1 overexpression and gene silencing on autophagy in S1-HP cells were studied. Finally, the interaction between Atg1 and other autophagy related proteins was investigated by Co-IP technique. The results showed that the Atg1 open reading frame of Spodoptera litura was 2286bp, encoding 761 amino acids, and the predicted molecular weight was 82.6 kDa, isoelectric point about 9.5. There are three functional domains, namely kinase domain, LIR domain and Atg13 binding domain. The full-length protein expressed in Escherichia coli is easy to degrade, but the truncated protein is stable and can be used to prepare antibodies. GFP-Atg1 (or Atg1-GFP) fusion protein is distributed in cytoplasm but not in nucleus. It is co-located with Atg8,Atg6 and Atg5 parts. Starvation induced autophagy resulted in the decrease of Atg1 in cells due to degradation, inhibition of autophagy, which resulted in the accumulation of Atg1, indicating that Atg1 could be degraded by autophagy, and the overexpression of Atg1 could accelerate the degradation of Atg8-PE. The silencing of Atg1 expression resulted in the accumulation of Atg8-PE, suggesting that Atg1 promoted autophagy. Immunoprecipitation technique suggested that Atg1 might interact with other Atg proteins. These results provide an important basis for revealing the multiple regulation of autophagy by SlAtg1 and its molecular mechanism.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433.4;Q78

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