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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)。
The full-length cDNA sequence of small G protein gene of Dunaliella salina was amplified by RT-PCR and RACE (GenBank Accession No.JN989548), named as DsRab, bioinformatics analysis was carried out, and salt was detected by real-time fluorescence quantitative PCR The gene expression under stress. The results showed that the full-length cDNA of DsRab gene was 1 299 bp with an open reading frame (ORF) of 612 bp encoding 203 amino acids. The 5 ’non-coding region was 78 bp and the 3’ non-coding region was 609 bp. The conserved domain analysis It can be seen that the encoded small G protein has 4 GTP / GDP conserved domains, 1 effector region, 1 carboxy-terminal cysteine domain and 5 Rab subfamilies shared domains; secondary structure prediction shows that the protein has 32.02% α-helix, 23.65% stretch fragment, 44.33% free-form and 3-D modeling were successful. Comparative analysis showed that DsRab protein had high homology with Ypt / Rab amino acid sequences of various organisms. Fluorescent quantitative PCR results showed that DsRab gene expression in Dunaliella salina increased significantly under high salt stress (3.0 mol / L), reached the maximum at 1 h, 4.9 times higher than that in control group (0 h) The difference was significant (P <0.01).