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.
作者
李成松
刘利娟
梁芳
赵炜栋
杨春琳
刘应高
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)
出处
《植物科学学报》
CAS
CSCD
北大核心
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)。
作者简介
李成松(1998−),男,硕士研究生,研究方向为森林保护学,E-mail:lcs2012514359@163.com;通讯作者:刘应高.E-mail:lyg927@263.net。
