低盐胁迫下三疣梭子蟹蝗抗利尿肽基因的表达

本研究采用RACE技术克隆了三疣梭子蟹(Portunus trituberculatus)蝗抗利尿肽(neuroparsin,PtNP)基因。该基因全长1920 bp,5￠端非编码区237 bp,3￠端非编码区1373 bp,开放阅读框309 bp,编码102个氨基酸,预测分子量10.8 k D,理论等电点7.42。PtNP含有12个半胱氨酸残基,具十足目动物蝗抗利尿肽典型的特征。同源性和系统进化分析表明,PtNP与拟穴青蟹(Scylla paramamosain)NP4的同源性最高(89%),并且三疣梭子蟹与拟穴青蟹首先聚为一支。组织表达分析发现,PtNP基因在脑组织中的相对表达量最高,其次是鳃和眼柄,在卵巢、肌肉、心脏和肝胰腺中表达量较低或不表达。通过分析PtNP基因在低盐胁迫过程中的表达规律发现,低盐胁迫可显著改变PtNP基因在脑、鳃和眼柄组织中的表达模式,整体呈现上调表达趋势,其中在脑、鳃和眼柄中表达量分别最高上调至7.7倍、2.8倍和2.6倍,且存在显著差异(P0.05)。去除眼柄后该基因在鳃中的表达呈上升趋势,且去除双侧眼柄组的表达量显著高于去除单侧眼柄组(P0.05)。本研究结果表明PtNP基因在三疣梭子蟹盐度适应中可能发挥一定作用且受神经内分泌系统的调控。

Expression analysis of neuroparsin gene under low salinity stress in swimming crab (Portunus trituberculatus)

The swimming crab (Portunus trituberculatus), which is an economically important marine crab in China, is a widespread species in coastal Japan, Korea, and China. Neuropeptides have a significant effect on osmoregulation, growth, development, and immunity of crabs. To investigate the function of neuroparsin under low salinity stress in P. trituberculatus, the neuroparsin gene was cloned by rapid amplification of cDNA ends. The PtNP gene is 1920 bp long, including a 309 bp open reading frame (ORF) that encodes a 102-aa polypeptide; its isoelectric point was 7.42 and the molecular mass was 10.8 kDa. The PtNP gene contains 12 cysteine residues, which is a typical characteristic of neuroparsin in decapods. The homology and phylogenetic systematic analyses revealed that the highest homology and similarity (reaching up to 89%) occurred between PtNP and the NP gene of Scylla paramamosain and P. trituberculatus clustered with S. paramamosain. The tissue expression analysis showed that the expression of PtNP gene was relatively high in the brain, followed by the gill and eye, with very little or no expression in the ovaries, muscles, heart, liver, and pancreas. The expression pattern analysis of the PtNP gene under low salt stress condition showed that low salt stress can significantly change the expression of PtNP gene in the brain, gill, and eyestalk, and the overall expression was upregulated. In the brain, gill, and eyestalk, the expression of PtNP was 7.7, 2.8, and 2.6 times higher than that of the control, respectively (P<0.05). The PtNP gene expression in the gills presented an increasing trend after the ablation of eyestalk. Furthermore, the expression of PtNP after the ablation of bilateral eyestalk was significantly higher than that after the ablation of unilateral eyestalk (P<0.05). The results of this study showed that the PtNP gene might play a role in the salinity adaptation of P. trituberculatus, which is regulated by its neuroendocrine system.