黑腹果蝇ord基因的原核表达及生物信息学分析

Prokaryotic Expression and Bioinformatics Analysis of ord Gene in Drosophila melanogaster

黏连蛋白ORD(orientation disruptor)是维持黑腹果蝇(Drosophila melanogaster)减数分裂姐妹染色单体间黏连与卵母细胞同源重组、联会所必需的。本研究旨在对黑腹果蝇ord基因进行原核表达,并对其表达蛋白进行生物信息学分析。通过PCR扩增出黑腹果蝇ord基因,采用同源重组法构建pET-sumo-ORD重组质粒,经IPTG诱导表达,对表达蛋白进行SDS-PAGE及Western blotting验证。结果表明,诱导表达的His-ORD融合蛋白大小约为69 kD,为包涵体蛋白。系统发育树分析表明,黑腹果蝇与毛里求斯果蝇(Drosophila mauritiana)、塞舌尔果蝇(Drosophila sechellia)和拟果蝇(Drosophila simulans)的ord基因亲缘关系较近。生物信息学分析表明,ORD蛋白的预测等电点(pI)为5.69,疏水性平均值(GRAVY)为-0.309,属于亲水性蛋白质,其二级结构以α-螺旋和无规则卷曲为主。蛋白互作网络分析表明,ORD与INCENP存在相互作用,与CONA、CG13541共同定位。本研究结果为深入研究该基因的功能以及促进减数分裂黏连体研究提供了理论依据和试验基础。

In Drosophila melanogaster,the cohesion protein ORD(orientation disruptor)is required for sister chromatid cohesion,homologous recombination and synapsis in oocytes during meiosis.This study aimed to obtain the prokaryotic expression of ord from Drosophila melanogaster,and analyzed its functions by bioinformatics.The ord fragment was amplified by PCR,and the pET-sumo-ORD recombinant plasmid was constructed by homologous recombination.The His-ORD fusion protein was then induced by IPTG and identified by SDS-PAGE and Western blotting.The results show that the size of His-ORD fusion protein is about 69 kD,mainly in the form of inclusion body.Phylogenetic tree analysis suggests that the gene is closely related to the ord in Drosophila mauritiana,Drosophila sechellia and Drosophila simulans.Bioinformatics analysis shows that the predicted isoelectric point(pI)of ORD protein is 5.69,the average hydrophobicity(GRAVY)is-0.309,which is a hydrophilic protein,and its secondary structure is mainly alpha-helix and random coil.The protein interaction network analysis indicates that ORD interactes with INCENP,and colocalizes with CONA and CG13541.The results of this study will provide a theoretical and technical basis for further research on functions of this gene and facilitate the study of meiotic cohesion in Drosophila melanogaster.