盐藻小G蛋白基因(DsRab)的克隆及在高盐胁迫下的表达分析  

Cloning and Expression Analysis of Small GTP-binding Protein Gene from Dunaliella salina (DsRab) Under Salt Stress

作　　者：余祝君[1] 柴晓杰[1] 张晓琳[1] 张婷[1] 薛飞[1] 

机构地区：[1]大连海洋大学农业部北方海水增养殖重点实验室辽宁省海洋生物资源恢复与生境修复重点实验室,大连116023

出　　处：《生物技术通报》2013年第9期77-83,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30972240);辽宁省教育厅科技研究项目(2008T023)

摘　　要：采用RT-PCR和RACE技术扩增了杜氏盐藻小G蛋白基因cDNA全长序列(GenBank Accession No.JN989548),命名为DsRab,对其进行生物信息学分析,并通过实时荧光定量PCR方法检测盐胁迫下该基因的表达情况。结果表明,DsRab基因的cDNA全长为1 299 bp,开放阅读框(ORF)为612 bp,编码203个氨基酸,5'非编码区78 bp,3'非编码区609 bp;保守性结构域分析可知编码的小G蛋白有4个GTP/GDP保守结构域,1个效应区、1个羧基端的半胱氨酸结构域和5个Rab亚家族共有的结构域;二级结构预测表明该蛋白有32.02%的α-螺旋,23.65%的伸展片段,44.33%的自由卷曲,三维建模成功;比对分析发现DsRab蛋白与多种生物的Ypt/Rab的氨基酸序列具有较高的同源性。荧光定量PCR结果表明,盐藻在高盐(3.0 mol/L)胁迫下,DsRab基因表达量显著上调,1 h后表达量达到最大值,为正常培养下对照组(0 h)的4.9倍,差异极显著(P<0.01)。Dunaliella salinasmall GTP-binding Protein Gene（GenBank Accession No.JN989548）was cloned by RT-PCR and RACE technology,namedDsRab,the bioinformatics-analysis was performed,and the DsRabgene expression pattern was studied under 3 mol/L NaCl by Real-time quantitative RT-PCR method.The results showed that the full length of cDNA for DsRabGene was 1 299 bp,which contains 78 bp 5＇UTR,609 bp 3＇UTR and 612 bp ORF.The ORF codes a 203 amino acids protein with four GTP/GDP binding conserved domains,one effector domain,a cysteine residues at the COOH-terminus,and five common domain of Rab sub-family;Secondary structure prediction indicated that the α-helix,β-strands and random coil in it were 32.02% of,23.65% and 44.33%.Protein homologue analysis showed that the DsRab shared high homology with Ypt/Rab from other species.The real-time quantitative PCR（RT-PCR）revealed that the expression of DsRabgene was increased by 4.9-fold under 3 mol/L NaCl comparising with that under normal level（including 1.5 mol/L NaCl）（P0.01）.

关 键 词：杜氏盐藻 小G蛋白基因 RACE技术 生物信息学 荧光定量PCR 

分 类 号：S917.3[农业科学—水产科学]