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铁硫亚基蛋白(rieske iron-sulfur protein,RISP)是线粒体复合物Ⅲ的关键蛋白亚基之一,在呼吸链电子传递过程中起到重要作用。本研究通过RT-PCR克隆得到斜纹夜蛾Spodoptera litura RISP基因SlitRISP的ORF,构建了pET32a-SlitRISP原核表达载体,SDS-PAGE和Western blot检测结果显示,SlitRISP原核表达蛋白以包涵体的形式存在于菌体沉淀中,且RISP抗体可成功用于该蛋白的免疫印迹检测。为了进一步鉴定SlitRISP在斜纹夜蛾离体细胞系SL-1中的功能,通过向细胞内转染siRNA,利用RNAi技术沉默SL-1中的SlitRISP。qRT-PCR结果表明,分别经50nmol/L和100nmol/L siRNA处理48h后,SL-1中SlitRISP的表达均几乎完全被抑制;Western blot结果显示,SL-1中SlitRISP含量显著低于CK。当SL-1SlitRISP被成功沉默后,通过检测SL-1线粒体膜电位、细胞ATP含量和细胞增殖抑制率鉴定RISP在线粒体电子传递过程中的重要作用。流式细胞仪测定结果表明,经50nmol/L和100nmol/LsiRNA处理24h后,SL-1线粒体膜电位相对于CK分别降低23.52%和11.32%,而处理48h后,SL-1线粒体膜电位则分别升高5.58%和27.66%;siRNA处理24h和48h后,SL-1ATP含量相对于CK分别降低82.71%和84.50%,最终导致SL-1细胞增殖抑制率分别为53.64%和67.94%。这些结果表明SlitRISP在SL-1中参与线粒体膜电位的形成和细胞ATP的合成。介于RISP在线粒体电子传递链中的重要作用,其可能成为新型杀虫作用靶标,这可为研制新型呼吸抑制剂提供参考。
Rieske iron-sulfur protein (RISP), one of the key protein subunits of mitochondrial complex III, plays an important role in the electron transport of respiratory chain. In this study, the ORF of Splitoptera litura RISP gene SlitRISP was cloned by RT-PCR and the prokaryotic expression vector pET32a-SlitRISP was constructed. The results of SDS-PAGE and Western blot showed that the prokaryotic expression protein of SlitRISP existed in the form of inclusion body Body precipitate, and RISP antibodies can be successfully used for Western blot detection of this protein. To further identify the function of SlitRISP in the Spodoptera litura cell line SL-1, SlitRISP in SL-1 was silenced using RNAi technology by transfecting siRNA into cells. The results of qRT-PCR showed that the expression of SlitRISP in SL-1 was almost completely inhibited after treated with 50nmol / L and 100nmol / L siRNA for 48h, respectively. Western blot results showed that the content of SlitRISP in SL-1 was significantly lower than CK. After SL-1SlitRISP was successfully silenced, the important role of RISP in mitochondrial electron transport was identified by examining the mitochondrial membrane potential of SL-1, cellular ATP content and cell proliferation inhibition rate. The results of flow cytometry showed that the mitochondrial membrane potential of SL-1 decreased by 23.52% and 11.32% respectively when treated with 50nmol / L and 100nmol / L siRNA for 24 hours, while the mitochondrial membrane potential of SL-1 And 5.58% and 27.66% respectively. After treated with siRNA for 24 and 48 hours, the content of SL-1ATP decreased by 82.71% and 84.50% respectively compared with CK, which eventually led to 53.64% and 67.94% inhibition of SL-1 cell proliferation respectively. These results indicate that SlitRISP is involved in the formation of mitochondrial membrane potential and cellular ATP synthesis in SL-1. The important role of RISP in the mitochondrial electron transport chain, which may become a new target for insecticidal action, may provide a reference for the development of novel respiratory inhibitors.