关键词: 斜纹夜蛾, 铁硫亚基蛋白, 原核表达, 蛋白功能, SL-1细胞

Abstract: The Rieske iron-sulfur protein (RISP) is a key protein subunit of mitochondrial complex Ⅲ, which plays an important role in the respiratory electron transport chain. The opening reading-frame (ORF) of SlitRISP was cloned by RT-PCR from Spodoptera litura for the construction of prokaryotic expression vector pET32a-SlitRISP. SDS-PAGE and Western blot analysis showed that the prokaryotic protein SlitRISP was mainly present as inclusion body in the bacteria precipitate, and the antibody of RISP could be applied to the immunoblot analysis of SlitRISP successfully. In order to identify the function of SlitRISP in the cultured cell line SL-1 of S. litura, RNAi was used to silence SlitRISP by transfecting siRNA into SL-1 cells. The qRT-PCR result showed that at 48 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA, respectively, the expression levels of SlitRISP mRNA were all inhibited effectively compared with the control. The Western blotting result showed that the content of SlitRISP in SL-1 cells was obviously lower than the control. The mitochondial membrane potential (MMP), ATP content and inhibition rate of cell proliferation were detected based on the obvious silence of SlitRISP in SL-1 cells to identify the important roles of RISP in the electron-transport chain of mitochondria. The changes of MMP in SL-1 cells were monitored by flow cytometry (FCM), which decreased by 23.52% and 11.32% at 24 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA, respectively, compared with control. However, at 48 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA, the MMP increased by 5.58% and 27.66%, respectively. The ATP content in SL-1 cells at 48 h after treatment with 50 nmol/L and 100 nmol/L siRNA was decreased by 82.71% and 84.50%, respectively, measured by luminometer. Owning to the suppression of ATP synthesis by siRNA in SL-1 cells, the inhibition rates of cell multiplication reached to 53.64% and 67.94%, respectively, at 48 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA. These results demonstrate that the RISP plays an important role in the MMP formation and ATP synthesis in SL-1 cells. As RISP plays important roles in the electron-transport chain of mitochondria, it could become a new target for pest control, and this may provide reference for developing new respiration inhibitors.

Key words: Spodoptera litura, Rieske iron-sulfur protein, prokaryotic expression, protein function; Spodoptera litura cultured cell line (SL-1)