防御假单胞菌FD6中RpoS蛋白理化性质预测及rpoS-lacZ转录融合结构构建  

Prediction of physicochemical properties of RpoS in Pseudomonas protegens FD6 and construction of rpoS-lacZ transcriptional fusion structure

作　　者：尹悦 孔祥伟 张清霞[1] CHEN Yu;QIN Feng;ZHU Shanyuan(Jiangsu Provincial Key Laboratory of Veterinary Bio-Pharmaceutical High-Tech Research/Jiangsu Agri-Animal Husbandry Vocational College,Taizhou 225300,China)

机构地区：[1]扬州大学园艺与植物保护学院

出　　处：《扬州大学学报（农业与生命科学版）》2019年第4期100-105,112,共7页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(31772210);江苏省重点研发计划(现代农业)项目(BE2017344);扬州大学科技创新培育基金项目(2016CXJ066)

摘　　要：Sigma因子是一类依赖于DNA的RNA聚合酶亚基,在生防细菌中通常可调控抗生素的合成。防御假单胞菌(Pseudomonas protegens) FD6中rpoS基因编码的RpoS蛋白是一类非必需sigma因子。利用BPROM在线工具预测rpoS基因的启动子区,并用PCR技术将其克隆到pRG970Km质粒中无启动子的lacZ基因上游,将构建的rpoS-lacZ转录融合质粒转化至菌株FD6中,为后续抗生素合成调控机制的探究提供重要的试验材料。此外,利用生物信息学方法分析P.protegens FD6中RpoS的功能,并通过邻接法构建RpoS的系统发育树。结果表明:FD6中RpoS蛋白分子量约为38 ku,理论等电点为5.14;具有多个磷酸化位点;无跨膜结构,且N端无信号肽;二级结构以无规则卷曲和α-螺旋为主。系统发育树结果显示, FD6中RpoS蛋白的氨基酸序列与防御假单胞菌Pf-5相似性最高,二者在系统发育树中位于同一分支。Sigma factor is a DNA-dependent RNA polymerase subunit that regulates the biosynthesis of antibiotics in biocontrol bacteria. The RpoS protein encoded by the rpoS gene in Pseudomonas protegens FD6 is a non-essential sigma factor. In this study, BPROM online tool was used to predict the promoter region of rpoS gene, and then it was cloned into the upstream of promoterless lacZ gene in pRG970 Km plasmid by PCR. Finally, the rpoS-lacZ transcriptional fusion plasmid was transformed into strain FD6 by electroshock. The objective of this study was to provide an important experimental material for the further study of the regulation mechanism of antibiotics biosynthesis. In addition, the function of RpoS in P.protegens FD6 was analyzed by bioinformatics technology and the phylogenetic tree of RpoS was constructed by the adjacent method. The results showed the molecular mass of RpoS protein was about 38 ku, and the theoretical isoelectric point was 5.14. RpoS had many phosphorylation sites and the primary secondary structures were composed of α-helix and random coil. However, no transmembrane structure and N-signal peptide were found in it. Besides, the predicated amino acid sequence of RpoS shared the highest identity with RpoS in P.protegens Pf-5 located in the same clade with that of P.protegens FD6 in the evolution tree.

关 键 词：防御假单胞菌 RpoS蛋白 转录融合结构 理化性质 生物信息学分析 

分 类 号：Q785[生物学—分子生物学]