粗枝云杉PaPR10-1基因的克隆表达及生物信息学分析  

Cloning,prokaryotic expression,and bioinformatics analysis of PaPR10-1 gene from Picea asperata Mast.

作　　者：李成松 刘利娟 梁芳 赵炜栋 杨春琳[1] 刘应高[1] Li Cheng-Song;Liu Li-Juan;Liang Fang;Zhao Wei-Dong;Yang Chun-Lin;Liu Ying-Gao(National Forestry and Grassland Administration Key Laboratory of Forest Resources Conservation and Ecological Safety on the Upper Reaches of the Yangtze River,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学,长江上游森林资源保育与生态安全国家林业和草原局重点实验室,成都611130

出　　处：《植物科学学报》2023年第2期224-233,共10页Plant Science Journal

基　　金：四川省教育厅重点项目(09ZA068)。

摘　　要：PR10是病程相关蛋白(PRs)家族的重要成员,能增强植物抵御外界胁迫侵扰的能力。为进一步研究PR10蛋白的生物学功能,在转录组测序的基础上,利用RT-PCR技术获得一个粗枝云杉(Picea asperata Mast.)基因PaPR10-1,利用生物信息学方法对其核苷酸和氨基酸序列进行结构和功能分析,进一步构建PaPR10-1融合蛋白原核表达系统,获得高纯度PaPR10-1融合蛋白,并采用底物法体外分析其核糖核酸酶活性。结果显示:PaPR10-1基因ORF长486 bp,编码161个氨基酸。PaPR10-1蛋白无跨膜结构和信号肽,为胞内蛋白,含有“P-Loop”和Bet_v1-like保守结构域。系统进化分析结果表明,PaPR10-1蛋白与海岸松(Pinus pinaster Aiton)PR10蛋白归为一个分支,亲缘关系较近。目的蛋白在30℃下以0.2 mmol/L的IPTG诱导1 h表达量最佳,且具有核糖核酸酶活性。PR10 is an important member of the pathogenesis-related protein(PR)family,which enhances the ability of plants to resist external stresses.In this study,a PR10 gene from Picea asperata Mast.PaPR10-1 was obtained by RT-PCR,with sequence characteristics then analyzed and prokaryotic expression system further constructed and optimized.Ribonuclease activity was then analyzed using the substrate method in vitro.Results showed that the open reading frame(ORF)of PaPR10-1 was 486 bp in length and encoded a protein with a 161 amino acid.Without a transmembrane structure and signal peptide,the PaPR10-1 protein was an intracellular protein with two conserved domains,“P-Loop”and Bet-v1-like,respectively.Phylogenetic analysis showed that PaPR10-1 was closely related to the PR10 protein of Pinus pinaster Aiton.Optimal expression conditions for the gene were 0.2 mmol/L IPTG at 30℃for 1 h induction,and the expressed target protein exhibited ribonuclease activity.

关 键 词：粗枝云杉 PR10基因 基因克隆 生物信息学 原核表达 

分 类 号：Q943.2[生物学—植物学]