番茄OPA3-like基因的生物信息学分析及原核表达

Bioinformatic analysis and prokaryotic expression of OPA3-like gene from tomato

[目的]获得高效表达的类视神经萎缩蛋白3(OPA3-like),并分析其生物信息学功能。[方法]利用Vector NTI和ExPASy,Signal P,Plant-mPLoc等在线工具对OPA3-like蛋白基本理化性质、信号肽、跨膜区及亚细胞定位进行分析;构建原核表达载体pET22b-OPA3-like、pET28a-OPA3-like、pET32a-OPA3-like并分别转化至大肠杆菌BL21(DE3)和Rosetta(DE3)中,经IPTG诱导融合蛋白表达,SDS-PAGE对结果进行验证。[结果]OPA3-like蛋白是不稳定蛋白,含有跨膜区,可能位于叶绿体;原核表达表明,对于同一重组质粒而言,融合蛋白在两种菌中的表达量无明显差别,其中,转化pET28a-OPA3-like可表达20kDa的融合蛋白;转化pET32a-OPA3-like可表达37kDa的融合蛋白,且主要都以包涵体形式存在,转化pET22b-OPA3-like无融合蛋白的表达。[结论]转化pET32a-OPA3-like的表达菌可以高效表达融合蛋白(37 kDa),有利于进一步研究OPA3-like在植物中的亚细胞定位及功能。

[ Objective] To obtain high expression optic atrophy 3 -like protein（ OPA3 -like） and to analyze its bioinformat- ies function. [ Methods] The basic physical and chemical properties, signal peptide, transmembrane region and subceliular lo- calization of OPA3 - like protein were analyzed by the Vector NTI and online software such as ExPASy, SignalP, Plant - mPLoc, et al. Three prokaryotic expression vectors, pET22b - OPA3 - like, pET28a - OPA3 - like and pET32a - OPA3 - like, had been constructed,and then transformed each of them into E. coli BI21 （DE3） and Rosetta（ DE3 ） respectively to analyze the prokaryotic expression of OPA3 - like gene. The expression of fusion protein was induced by IPTG, then the results were ver- ified by SDS- PAGE. [ Results] The OPA3 -like is an unstable protein, which may have transmembrane regions and may lo- cate at the chloroplast. Prokaryotic expression results showed that for the same recombinant plasmid, the expression of fusion protein in two kinds of bacteria had no significant difference, in addition, pET28a- OPA3 -like transformation could express a 20 kDa fusion protein;pET32a - OPA3 - like transformation could express a 37 kDa fusion protein and both were mainly existed as inclusion bodies, but pET22b - OPA3 - like transformation had no fusion protein expression. [ Conclusion ] The expression of fusion protein （37 kDa） can be efficiently expressed by transforming pET32a - OPA3 - like ,which provides a foundation for further studies on functions and subcellular location of OPA3 - like in plants.