铁皮石斛钙网蛋白基因DoCRT1的分子克隆与表达研究

摘要

该研究采用RACE克隆铁皮石斛钙网蛋白(calreticulin,CRT)基因DoCRT1,进行生物信息学分析,并借助定量PCR检测基因表达模式,为揭示该基因在铁皮石斛生长发育及逆境生理中的分子作用奠定基础.结果表明:(1)DoCRT1基因(GenBank登录号KT957551)cDNA全长1 672 bp,ORF长1 275 bp,编码一条由424个氨基酸组成的多肽,分子量49.05 kD,等电点4.42,具有CRT蛋白保守的钙网蛋白/钙联接蛋白P结构域(205～320)和类伴刀豆球蛋白凝集素/葡聚糖酶结构域A(20～222)以及多个基序;蛋白N端含有一个信号肽(1～23)和一个跨膜区域(8～24),预测结果显示主要定位于细胞液泡、胞外,与多种植物CRT蛋白一致性很高(80％～87％).(2)DoCRT1蛋白与OsCRT1/2、ZmCRT1亲缘关系近,聚在CRT进化树的CRT1/2分支.(3)qRTPCR检测结果显示,DoCRT1基因转录本在石斛根中表达量较高,为叶中的2.23倍,且茎与叶中表达量无显著差异.%This study was aimed to isolate the full length cDNA of DoCRT1 in Dendrobium officinale using RACE,followed by bioinformatics analysis.And the expression pattern of the gene was detected by quantitative PCR.The results showed that:(1) the full length cDNA of DoCRT1 (GenBank accession KT957551) was 1 672 bp in length with an ORF of 1 275 bp,encoding a 424 aa protein with a molecular weight of 49.05 kD and an isoelectric point of 4.42.The deduced DoCRT1 protein had the conserved calreticulin/calnexin P domain (205-320),the concanavalin A-like lectin/glucanase domain (18-243),and several motifs.DoCRT1 contained a signal peptide (1 23) and a transmembrane region (8-24) at the N terminus.The protein was predicted to locate predominantly in cell vacuolar and outside,and had high identities (80％-87％) with CRT proteins from various plants.(2) DoCRT1 was closely related to OsCRT1/2 and ZmCRT1,and was clustered to the CRT1/2 Group of the CRT evolutionary tree.(3) DoCRT1 transcripts were more abundant in roots with 2.23 fold over that in leaves,while the expression level of the gene had no apparent difference in stems and leaves.The study will be useful for uncovering molecular role of the gene involving in the growth,development,and physiological stress adaptation in D.officinale.