中国对虾Gs基因克隆及其在急性低盐胁迫下功能分析

采用RACE技术对中国对虾cAMP信号通路内鸟嘌呤核苷酸结合蛋白α亚基(Guanine nucleotide-binding protein G(s) subunit alpha)基因进行克隆并对其功能进行分析。将基因命名为FcGs, 该基因cDNA全长为1692 bp, 编码379个氨基酸。对同源性进行分析显示与凡纳滨对虾同源性为99%, 与日本囊对虾同源性为97%。FcGs基因组织表达分析显示, 鳃和肝胰腺表达量较高。急性低盐胁迫使FcGs基因整体呈现上调表达。对中国对虾cAMP信号通路内Gs含量及Gs基因上游多巴胺(DA)含量, 下游环腺苷酸(cAMP)和蛋白激酶A(PKA)含量及腺苷环化酶(AC)和钠钾ATP酶(Na+-K+-ATPase)活力进行测定, 发现急性低盐胁迫后整体均呈上升且变化趋势基本一致。对FcGs基因进行干扰后中国对虾死亡率上升。研究表明, FcGs基因及其所在的cAMP信号通路可以通过调控Na+-K+-ATPase的活力在中国对虾渗透压调节过程中发挥重要作用。

CLONING OF GS GENE IN FENNEROPENAEUS CHINENSIS AND ITS FUNCTIONAL ANALYSIS UNDER ACUTE LOW SALT STRESS

Guanine nucleotide binding protein (G protein), composed of α, β and γ subunits, is an important signal transduction molecule in cAMP signaling pathway by directly regulating ion channels and activating second messengers. To investigate the function of the Gs in Fenneropenaeus chinensis under acute low salt stress, the full-length cDNA of Gs in Fenneropenaeus chinensis, named FcGs, was cloned by rapid amplification of cDNA ends (RACE), and then examined its response under acute low salt stress. The full length of the FcGs gene cDNA sequences consists of 1692 base pairs (bp), including a 1140 bp open reading frame (ORF) which encodes a 379-amino acid peptide with four transmembrane domains. Homology and phylogenetic analysis showed that deduced FcGs amnio acid sequence is highly conservative, with the highest amnio acid sequence identity (99%) with Litopenaeus vannamei, and a high amnio acid sequence identity (97%) with Marsupenaeus japonicus. Results of RT-qPCR showed that FcGs gene expressed in all tissues of Fenneropenaeus chinensis, with the highest expression level in gill, followed by hepatopancreas. Acute low salt stress upregulated FcGs, which is consistent with increased Dopamine (DA) content, Gs content, adenosine cyclase (AC) activity, cAMP content, protein kinase A (PKA) activity and Na+-K+-ATPase activity in cAMP signaling pathway of Fenneropenaeus chinensis. RNA interference (RNAi) of FcGs increased the mortality of Fenneropenaeus chinensis by the acute low salt stress. This study shows that FcGs gene and its cAMP signaling pathway play an important role in the osmotic regulation of Fenneropenaeus chinensis by regulating the activity of Na+-K+-ATPase.