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目的克隆珍稀濒危药用植物铁皮石斛丙酮酸激酶(pyruvate kinase,PK)基因(DoPK),并进行生物信息学分析以及检测基因在不同器官中的表达情况。方法采用RACE技术获得基因的全长cDNA;利用生物信息学软件预测蛋白的理化性质、结构域及亚细胞定位等分子特性;用DNASTAR 6.0和MEGA 4.0分别进行氨基酸多序列比对和进化关系分析;借助r实时荧光(real-time quantitative)PCR(RT-qPCR)分析基因在不同器官中的表达情况。结果克隆获得DoPK(GenBank注册号KC178572)的cDNA全长1 895 bp,编码一条由511个氨基酸组成的多肽,相对分子质量为55 040,等电点7.00;DoPK基因与江南卷柏、拟南芥、马铃薯和葡萄等植物的PK基因有71%、86%、89%和91%的相似性;DoPK蛋白包含保守的丙酮酸激酶结构域(21~497)和活性位点(235~247);DoPK属于胞质型的CYTOSOLIC-1亚类,与单子叶植物亲缘关系较近;DoPK基因为组成型表达,其转录本在石斛茎中的相对表达量较高,为叶中的2.29倍,根中次之,为叶中的1.28倍。结论克隆了胞质型的铁皮石斛DoPK基因的全长cDNA序列,为进一步研究其在铁皮石斛生长发育中的作用奠定基础。
Objective To clone the pyruvate kinase (PK) gene (DoPK), a rare and endangered medicinal plant, and analyze its bioinformatics and detect the expression of different genes in different organs. Methods The full-length cDNA was obtained by RACE. The physical and chemical properties, domains and subcellular localization of proteins were predicted by bioinformatics software. The amino acid sequence alignment and evolutionary relationships were analyzed by DNASTAR 6.0 and MEGA 4.0. The gene expression in different organs was analyzed by real-time quantitative PCR (RT-qPCR). Results The full-length cDNA of DoPK (GenBank accession number KC178572) was cloned into GenBank with the length of 1 895 bp, encoding a polypeptide consisting of 511 amino acids with a molecular weight of 55 040 and an isoelectric point of 7.00. DoPK gene was expressed in Arabidopsis thaliana , PK of potato and grape are 71%, 86%, 89% and 91% respectively; DoPK protein contains conserved pyruvate kinase domain (21-497) and active site (235-247) DoPK belongs to the cytoplasmic CYTOSOLIC-1 subclass and has a close genetic relationship with monocotyledons. The DoPK gene is constitutively expressed and its transcript is relatively high in stems of Dendrobium, accounting for 2.29 folds In the second, 1.28 times the leaf. Conclusion Cloning the full-length cDNA sequence of the DoPK gene in the cytoplasm of Dendrobium candidum has laid the foundation for further study on its role in the growth and development of Dendrobium candidum.