拟穴青蟹14-3-3基因全长cDNA的克隆及组织表达分析

The full length cDNA cloning and expression profile of 14-3-3 gene from the mud crab( Scylla paramamosain)

采用RT-PCR及RACE技术,从拟穴青蟹眼柄组织中克隆获得14-3-3基因cDNA全序列。序列分析结果表明:拟穴青蟹14-3-3基因全长1 112 bp,开放阅读框长744 bp,编码由247个氨基酸组成的蛋白,分子量为28.086 ku,等电点为4.675。与其他物种14-3-3基因氨基酸序列进行同源性比较分析显示,拟穴青蟹14-3-3基因与斑节对虾14-3-3基因同源性最高(95%),依次为墨吉对虾(93%)、苜蓿切叶蜂(92%)。聚类分析表明,拟穴青蟹14-3-3基因氨基酸序列与斑节对虾、墨吉对虾紧密聚为一支。经荧光定量检测,拟穴青蟹14-3-3基因在肝胰腺和肌肉中的表达量较高,其次为鳃、眼柄、心脏和肠,在胃中表达最少。盐度骤变实验结果表明:盐度胁迫24 h后,盐度的下降(5)或者上升(15、20、25、30)都引起了14-3-3基因在鳃中的表达量极显著上升(P<0.01),盐度变化的幅度越大,14-3-3基因的表达量越多。实验结果为进一步深入研究14-3-3基因的功能及调控机理奠定基础。

Full-length cDNA sequence of 14-3-3 gene was isolated from the eyestalk of mud crab Scylla paramamosain through RT-PCR and RACE. Sequence analysis indicated that 14-3-3 gene had an open reading frame of 744 bp encoding 247aa of 28.086 ku and pI at 4.675. The amino acid sequences of 14-3-3 gene possessed 95%, 93%, 92% identity with the 14-3-3 genes of Penaeus monodon, Fenneropenaeus merguiensis, Megachile rotundata respectively. 14-3-3 protein firstly clustered with 14-3-3 proteins of Penaeus monodon and Fenneropenaeus merguiensis in the phylogenetic analysis. The expression of 14-3-3 gene in tissues was analyzed by Real-Time PCR, the result showed that 14-3-3 gene was most expressed in hepatopancreas and muscle, then in eyestalk, intestinal, heart and gill, the lest in stomach. After 24h＇s stress in salinity, the expression of 14-3-3 gene increased great significantly （P〈0.01） whatever the salinity reduced （5） or raise （15, 20, 25, 30）. The more the salinity changed, the more the 14-3-3 gene expressed. These results will serve further studies on functions and regulation mechanism of 14-3-3 genes.