铁皮石斛丙酮酸激酶基因的克隆与表达分析

Cloning and expression analysis of pyruvate kinase gene in Dendrobium officinale

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 in a rare and endangered medicinal plant Dendrobium officinale, followed by bioinformatic analysis and to detect the expression in different organs. Methods RT-PCR and RACE technologies were used to clone the full length cDNA ofDoPK gene. The molecular characteristics such as physiochemical properties, conserved domain, and sub-cellular localization of the deduced DoPK protein were determined using a series of bioinformatic tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 4.0 soflwares, respectively. Real time quantitative PCR （RT-qPCR） was used for gene expression analysis in different organs. Results The full length cDNA ofDoPKwas 1 895 bp in length （GenBank accession KC178572） and encoded the protein of 511 amino acids with a molecular weight of 55 040 and an isoelectric point （PI） of 7.00. Sequence analysis demonstrated that DoPKwas similar to PK genes from Selaginella moellendorffli, Arabidopsis thaliana, Solanum tuberosum, and Fitis vinifera with the identities of 71%, 86%, 89%, and 91%, respectively. The deduced DoPK protein contained the conserved PK domain （21~97） and the active site （235--247）. Phylogenetic analysis indicated that DoPK was grouped into the CYTOSOLIC-1 subfamily and was closely related to monocots. The transcription level of DoPK in the stems ofD. o^cinale was the highest （2.29-fold higher than that in the leaves）, followed by that in the roots （1.28-fold）. Conclusion The full length cDNA sequence in a CYOTOSLIC DoPKgene is identified, facilitating further functional analysis of the gene involving in the growth and development ofD. officinale.