线粒体型蛋白frataxin在家蚕微孢子虫中的鉴定与分析

Characterization and analysis of mitochondrial protein frataxin in Nosema bombycis

摘要:

【目的】Frataxin 是铁硫簇相关蛋白，在线粒体代谢中起着重要作用。分析家蚕微孢子中该蛋白的结构特征，系统进化关系以及在孢子体内的转录翻译活性。【方法】基于家蚕微孢子全基因组序列，同源序列搜索获得该基因序列。进行蛋白二级结构比较，近缘物种共线性特征分析及系统进化树构建。此外构建pGEX-4T-1-Nbfra原核重组表达载体,转化E. coli BL21(DE3)进行目的蛋白表达纯化,以此为抗原免疫小鼠，制备抗体，并与家蚕微孢子总蛋白进行Western免疫杂交。【结果】Nbfra蛋白缺乏进入线粒体的信号序列，功能区缺乏部分a-螺旋；frataxin基因在不同微孢子基因组中的分布具有共线性特征，表明其在基因组进化中非常保守。系统进化分析显示微孢子形成独立进化支，并且与高等真核生物近缘，说明微孢子在进化地位上比其它原虫更加高等，支持了微孢子虫是真菌的姊妹枝的进化地位假说。【结论】免疫杂交结果表明Nbfra基因家蚕微孢子虫中能正常表达与翻译。本研究为微孢子的分类地位及线体假说提供了重要的补充依据。

关键词:Frataxin;共线性;家蚕微孢子虫;微孢子

Abstract:

[Aim] Frataxin protein is a component of Fe-S clusters and closely related to metabolism of mitochondria. We identified an integrity mitochondrial protein frataxin gene (Nbfra), analyzed its phylogenetic relationship, and confirmed the transcriptase activity of Nbfra in N. bombycis. [Methods] We analyzed the sequence of the second structure, gene location in genome and construction of NJ phylogenetic tree through various bioinformatics software. We constructed recombinant vector pGEX-4T-1-Nbfra, expressed the 36.5kDa recombinant protein in E. coli BL21 (DE3), and then used the protein as antigen to produce its polyantibody in mice. [Results] Nbfra was lack of targeting signal into mitochondria and part of alpha helices in functional domain, and had a synteny character between N. bombycis and E. cuniculi. Phylogenetic trees of Nbfra suggested that the evolutionary position of microsporidia was closely related to that of higher eukaryote, rather than that of other protozoa. The result of western blot suggested the expression and transcription of Nbfra gene in N.bombycis. [Conclusions] Our results offered the new evidence to analysis the conservation of Nbfra and evolutionary position of N.bombycis, and would support the hypothesis of mitosome in microsporidia.

Key words:frataxin; synteny; Nosema bombycis; microsporidia