铁皮石斛蛋白磷酸酶2A基因的分子克隆研究  

Molecular cloning of protein phosphatase 2A gene in Dendrobium officinale

作　　者：刘亮亮 张娜 黑小斌 李欢 李元敏 李佳慧 孙涛 张岗 LIU Liangliang;ZHANG Na;HEI Xiaobin;LI Huan;LI Yuanmin;LI Jiahui;SUN Tao;ZHANG Gang(College of Pharmacy,Shaanxi University of Chinese Medicin;Shaanxi Provincial Key Laboratory for Chinese Medicine Basis ＆ New Drugs Research,Xianyang 712046,China)

机构地区：[1]陕西中医药大学药学院,陕西省中药基础与新药研究重点实验室,陕西咸阳712046

出　　处：《上海中医药杂志》2018年第9期61-64,68,共5页Shanghai Journal of Traditional Chinese Medicine

基　　金：陕西省高校青年杰出人才支持计划项目;陕西省咸阳市中青年科技领军人才项目

摘　　要：目的克隆铁皮石斛蛋白磷酸酶(protein phosphatase)2A基因,进行生物信息及表达特征分析。方法运用RACE克隆基因,生物信息学软件进行核酸及蛋白质理化性质、保守结构域、亚细胞定位等分析;用DNAStar 7.0、MEGA 6.0分别进行蛋白序列比对和进化分析。借助定量PCR检测基因表达模式。结果 Do PP2A(Gen Bank注册号KT957552)c DNA 1 410 bp,与其他植物PP2A同源性为89%~94%。ORF编码一条由306个氨基酸组成的肽链,分子量35.19 k D,等电点4.79。推导蛋白含有金属依赖性磷酸酶保守结构域,蛋白不含信号肽或跨膜残基,定位于细胞质。Do PP2A与其他植物的PP2A相比具有高度的保守性,聚在PP2A分子进化树的A组。基因转录本在石斛根和茎中分别为叶中的4.41倍和1.45倍。结论 Do PP2A分子克隆及特征为研究其在铁皮石斛生长发育中的分子功能提供基础。Objective To clone the protein phosphatase （PP） 2A gene in Dendrobium officinale, and analyze the bioinformatics and expression characteristics. Methods The RACE technology was used to clone the gene. A series of bioinformatics tools were used to analyze the physiochemical properties of nucleic acids and proteins, conserved domains and subeellular localization. The DNAStar 7.0 and MEGA 6.0 software were used for protein sequence comparison and phylogenetic analysis respectively. The quantitative PCR was used for the analysis of gene expression pattern. Results The homology of DoPP2A （ GenBank accession KT957552）cDNA, 1 410 bp in length, was 89%-94% with PP2A from other plants. The ORF encoded a peptide chain composed of 306 amino acid with a molecular weight of 35.19 kD and an isoelectric point of 4.79. The deduced protein contained the metallo-dependent phosphatase conserved domains. The DoPP2A did not contain signal peptides or trans-membrane domain, and located in the cytosol. The DoPP2A was highly conservative compared with PP2As from other plants, and was clustered into group A of the PP2A evolutionary tree. The gene transcripts in the roots and stems were 4.41 and 1.45 times respectively than that in the leaves. Conclusion The molecular clone and characterization of DoPP2A can provide basis for the research on molecular function during the growth and development of Dendrobium officinale.

关 键 词：铁皮石斛 蛋白磷酸酶 克隆 生物信息学 表达 

分 类 号：S567.239[农业科学—中草药栽培]