斜纹夜蛾SlAtg1的克隆表达与功能初步分析

发布时间：2018-11-02 08:59

【摘要】：自噬是指膜(大部分表现为双层膜,有时多层或单层)包裹部分胞质和细胞内需降解的细胞器、蛋白质等形成自噬体(autophagosome),最后与溶酶体融合形成自噬溶酶体(autophagolysosome),降解其所包裹的内容物,以实现细胞稳态和细胞器的更新。目前普遍认为自噬是一种防御和应激调控机制,它为细胞的重建、再生和修复提供必须原料,实现细胞物质的再循环和再利用。自噬基因(autophagy-related gene,atg)的克隆始于酵母(yeast)。Atgl 蛋白(autophagy-related-gene-1)属于丝氨酸/苏氨酸蛋白激酶,是细胞自噬过程中非常关键的自噬相关蛋白之一。在自噬起始阶段时,Atg1-Atg13复合物可募集其他Atg蛋白,诱导细胞自噬的发生。鳞翅目昆虫Atg1对细胞自噬的调控作用尚不清楚,Atg1互作蛋白也研究较少。自噬相关蛋白Atg8是一种类泛素化蛋白,对自噬囊膜的延伸扩展起到重要作用,既参与底物的募集,又介导自噬小体与溶酶体的融合。它常被作为是研究细胞自噬的标记物。斜纹夜蛾Spodoptera litura(Fabricius)属于夜蛾科海灰翅夜蛾属,是一种多食性害虫,对许多农作物造成了严重的危害。本研究对已知的鳞翅目Atg1基因序列进行同源比对,根据基因片段的保守序列设计兼并引物,通过半巢式PCR方法扩增得到了 Atg1基因开放阅读框(ORF),并对此基因进行了生物信息学分析。将Atg1基因截短,分别构建Atg1-N末端600bp和Atg1-C末端500bp原核表达载体,将两种表达载体转化大肠杆菌BL21表达菌株,诱导表达并纯化截短的Atg1蛋白,用获得的纯化蛋白制备抗Atg1鼠抗和兔抗。然后,构建 Atg1 真核表达载体 pEGFP-N1-ie2-Atg1 和 pEGFP-N1-ie2-Atg1-Flag,转染S1-HP昆虫细胞,探究Atg1蛋白的表达与定位。接着将Atg1与其他自噬基因Atg8、Atg6、Atg5真核表达载体共转染S1-HP细胞,分别检测两者之间是否共定位。分别用饥饿和自噬抑制剂处理斜纹夜蛾S1-HP细胞,研究了二者对Atg1蛋白降解的影响。接着研究了 S1-HP细胞中Atg1的超表达和基因表达沉默对细胞自噬的影响。最后采用Co-IP技术探究了 Atg1与其它自噬相关蛋白的相互作用。实验结果表明,斜纹夜蛾Atg1开放阅读框全长2286bp,编码761个氨基酸,预测的分子量大小为82.6 kDa,等电点约为9.5。具有三个功能结构域,分别是激酶结构域、LIR结构域和Atg13结合结构域。在大肠杆菌中表达的全长蛋白容易降解,但是截短的蛋白比较稳定,可用于制备抗体。GFP-Atg1(或Atg1-GFP)融合蛋白分布于细胞质中,在细胞核内没有分布。它与Atg8,Atg6和Atg5部分共定位。饥饿诱导自噬导致细胞中Atg1因为降解而减少,抑制细胞自噬,可以导致Atg1积累,表明Atg1可以通过自噬溶酶体而降解。Atg1的超表达,加快了 Atg8-PE的降解;Atg1的表达沉默使Atg8-PE积累,表明Atg1促进细胞的自噬活动。免疫共沉淀技术表明Atg1可能与其它Atg蛋白存在相互作用。这些结果为揭示SlAtg1对自噬的多重调节及其分子机理提供了重要的基础。
[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.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4;Q78

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