拟南芥TRX-h5基因的克隆和表达分析

Cloning and expression analysis of TRX-h 5 gene in Arabidopsis thaliana

硫氧还蛋白(TRX-h)是一类催化二硫键还原的小分子蛋白,在调控氧化应激响应中发挥重要作用。为了研究硫氧还蛋白的结构及其功能,文章以野生型拟南芥(Arabidopsis thaliana)Columbia(Col-0)为研究材料,通过反转录-聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)技术获得TRX-h5基因的编码序列(coding sequence,CDS),体外构建pET28a(+)-TRX-h5及pET28a(+)-TRX-h5M(第39位和第42位半胱氨酸位点突变)的原核表达载体,转化至大肠杆菌感受态细胞BL21(DE3)中,诱导后经镍柱纯化得到带有6个His标签的融合蛋白。通过SDS-PAGE电泳技术检测到14 kDa处的目的条带,与理论值预测一致。运用生物信息学分析得出,TRX-h5基因编码118个氨基酸,相对分子质量为13122.32,理论等电点为5.19,该蛋白的不稳定参数为26.74,属于稳定性蛋白。点突变分析发现,TRX-h5的第39位和第42位半胱氨酸位点决定了该蛋白的催化活性,为进一步探究TRX-h5的蛋白功能和高等植物氧化还原系统的研究提供了新思路。

Thioredoxin(TRX-h)is a small molecular protein that catalyzes the reduction of disulfide bonds and plays an important role in regulating the response to oxidative stress.In order to study the structure and function of TRX-h,wild type Arabidopsis thaliana Columbia(Col-0)was used as the research material.The coding sequence(CDS)of TRX-h 5 gene was obtained by reverse transcription-polymerase chain reaction(RT-PCR).The prokaryotic expression vectors of pET28a(+)-TRX-h5 and pET28a(+)-TRX-h5M(mutation at cysteine sites 39 and 42)were constructed in vitro and transformed into E.coli competent cell BL21(DE3).After induction,the fusion protein with six His tags was purified by nickel column.SDS-PAGE detected the target band at 14 kDa,which was consistent with the theoretical prediction.Bioinformatics analysis showed that TRX-h 5 gene encoded 118 amino acids,the relative molecular weight was 13122.32,the theoretical isoelectric point was 5.19,and the instability parameter of the protein was 26.74,which indicated that the protein was a stable protein.Point mutation analysis showed that the cysteine sites at 39 and 42 of TRX-h5 determined the catalytic activity of the protein,which provided a new idea for further exploring the protein function of TRX-h5 and the redox system of higher plants.