铁皮石斛2个P型H^+-ATPase基因的克隆、表达及生物信息学分析

Cloning, Expression and Bioinformatics Analysis of Two P-type H^+-ATPase Genes in Dendrobium officinale Kimura et Migo

利用RACE进行铁皮石斛质膜PMA(plasmame mbrane H^+-ATPase)基因的全长克隆,采用生物信息学方法、DNASTAR7.0和MEGA7.0对其编码蛋白结构、序列和分子进化等进行分析,并借助实时荧光定量PCR技术检测基因表达模式。结果表明,从铁皮石斛叶片cDNA中分离到2个P型H^+-ATPase基因DoPMA1和DoPMA2(GenBank注册号KU160470和KU160471),各编码1条由973和951个氨基酸组成的肽链;2个基因编码蛋白均含有2个保守的P型ATPase结构域和5个H^+运输ATPase结合位点,不含信号肽,定位在质膜。2个蛋白与植物H^+-ATPases蛋白相似性大于72%,聚在拟南芥、水稻等植物H^+-ATPase分子进化树的Ⅴ和Ⅱ分支。实时荧光定量PCR分析表明,DoPMA1在根和茎中上调,分别为叶中的2.98、1.95倍,DoPMA2在根与叶中表达无显著差异,茎中下调为叶中的0.86倍。研究结果为进一步揭示DoPMA1和DoPMA2分子作用奠定基础。

RACE was used for full-length cloning of Dendrobium officinale Kimura et Migo membrane H+-ATPase gene.Bioinformatics method,DNASTAR 7.0 and MEGA 7.0 were used to analyze the structure,sequence and molecular evolution of the encoded protein,and real-time quantitative PCR technology was used to detect the expression pattern of gene organs.The results showed that two full length P-type H^+-ATPase genes,DoPMA1 and DoPMA2(GenBank accessions KU160470 and KU160471,respectively)were isolated from the leaves cDNAs of D.Officinale.DoPMA1 was deduced to a 973 aa(amino acid)protein,while DoPMA2 encoded a 951 aa protein.Both deduced DoPMA1 and DoPMA2 proteins had two conserved P-type H^+-ATPase domains and five H^+-transporting ATPase signature.The proteins did not contain any signal peptide,but located in plasma membrane.The two proteins were highly identical to the plant H^+-ATPases by more than 72%,similarity,and were grouped to theⅤandⅡclades of the evolutionary tree of Arabidopsis,rice,and other plants H^+-ATPase members.DoPMA1 and DoPMA2 showed deferentially expression profiles among the three organs.DoPMA1 transcripts were higher in root and stem,2.98 and 1.95 fold than that in leaf;DoPMA2 did not display significant difference between root and leaf,while it reduced in stem by 0.86 fold.The studies lay a foundation for further exploring the function of DoPMA1 and DoPMA2.