肥城桃果实2-Cys Prx基因克隆、生物信息学分析及原核表达

Cloning,Bioinformatics Analysis and Protein Induced Expression of 2-Cys Prx Gene in Feicheng Peach Fruits

为了深入研究2-Cys Prx基因调控桃果实氧化还原信号分子作用机制,结合了PCR技术和RT-PCR技术通过体外克隆的方法得到桃果实2-Cys Prx基因,通过对序列分析表明,2-Cys Prx基因全长1 424 bp,其中,编码区813 bp,共编码270个氨基酸。对其分子结构进行分析,2-Cys Prx蛋白分子量为29.7 ku,为疏水型蛋白,且是膜外蛋白。在线软件预测2-Cys Prx蛋白分子式为C1342H2110N348O403S4,分子量29.696 ku,由4 207个原子组成,理论等电点为6.23。使用Prot Scale分析该蛋白疏水性平均值为-0.126,说明2-Cys Prx为亲水性蛋白。TMHMM对跨膜结构进行预测,发现,2-Cys Prx氨基酸序列中不存在跨膜结构域,为膜外蛋白。此外,对该蛋白二级结构进行预测,发现2-Cys Prx有4个α螺旋结构、9个β折叠结构及无规则卷曲构成。系统进化树分析表明,肥城桃果实2-Cys Prx与其他植物氨基酸序列有较高同源性,其中与樱桃的亲缘关系最近,相似度达到了98.15%,表明植物体内2-Cys Prx具有较高的保守性。体外构建重组蛋白p ET-30a-2-Cys Prx成功在大肠杆菌BL21中表达,为下一步研究其功能与结构,从而探讨2-Cys Prx基因调控桃果实氧化还原信号作用机理奠定了基础。

In order to study the regulation mechanism of 2-Cys Prx gene to redox signal molecules. 2-Cys Prx of Feicheng peach was studied by gene cloning and prokaryotic expression including PCR technique and RT-PCR technique. The experimental results showed that the full length of 2-Cys Prx gene was 1 424 bp induced 813 bp of coding sequence encoding a polypeptide of 270 amino acids,with predicted molecular weight of 29.7 ku. The predicted protein molecular formula of 2-Cys Prx was C 1342 H 2110 N 348 O 403 S 4 with a molecular weight of 29.696 ku,which contained 4 207 atoms. For 2-Cys Prx,the theoretical isoelectric point was about 6.23,and the total average hydrophobicity index was -0.126,which indicated that 2-Cys Prx protein was a hydrophilic protein. The two level prediction of 2-Cys Prx protein composed of 9 segment of fold and 4 segment of alpha helix and the irregular. Phylogenetic tree analysis showed that the 2-Cys Prx was most closely related to Prunus avium ,with 98.15% amino acid homology rate. The high homology of amino acids of 2-Cys Prx in peach fruit and other plants,indicating that 2-Cys Prx was more conservative. These results would lay a foundation for researching the structure and function of 2-Cys Prx protein to explore the mechanism of 2-Cys Prx regulation of redox signal molecules in the further study.