三疣梭子蟹心激肽基因克隆及在低盐适应中的功能验证

Cloning of crustacean cardioactive peptide and its functional verification under low-salt adaptation in swimming crab(Portunus trituberculatus)

采用RACE技术克隆获得三疣梭子蟹(Portunus trituberculatus)心激肽(CCAP)基因。该基因全长606 bp, 5￠端非编码区72bp,3￠端非编码区108bp,开放阅读框426bp,编码141个氨基酸,预测分子量15.6kD,理论等电点9.55。同源性分析表明,三疣梭子蟹CCAP与拟穴青蟹(Scylla paramamosain)和蓝蟹(Callinectes sapidus)的CCAP同源性较高,分别为85%和82%。系统进化树分析显示,三疣梭子蟹与蓝蟹首先聚为一支,之后再与拟穴青蟹相聚。组织表达分析发现, CCAP基因在胸神经节中的相对表达量最高,其次是脑和眼柄组织。通过分析CCAP基因在低盐胁迫过程中的表达规律发现,低盐可显著改变CCAP在胸神经节中的表达模式,在24 h, 48 h和72 h实验组的表达量显著高于对照组(P<0.05),分别为对照组的1.73, 2.16和2.19倍。体外注射CCAP多肽可降低三疣梭子蟹在低盐条件下的死亡率,并诱发钠钾ATP酶(Na^+/K^+-ATPase)和V型ATP酶(V-ATPase)酶活力显著提高。本实验结果暗示CCAP通过调控Na^+/K^+-ATPase和V-ATPase活力以达到盐度适应的作用。

The swimming crab Portunus trituberculatus, an important marine economic animal widely distributed in China, belongs to Crustacea, Decapoda, Decapoda, Portunidae, P. trituberculatus is an aquatic crustacean with wide tolerance to salinity(13.7–47.7). Salinity is one of the most important environmental factors affecting the growth and development of P. trituberculatus. Ingestion, molting, growth, metabolism, immunity, and other processes in P. trituberculatus are greatly affected by salinity. Therefore, it is important to study the mechanism of salinity adaptation to breed improved salt-tolerant varieties of P. trituberculatus. In this study, random amplification of cDNA ends technology was used to clone the crustacean cardioactive peptide(CCAP) gene. The full-length CCAP gene was 606 base pairs(bp) long, including an open reading frame of 426 bp, 5′ untranslated region of72 bp, and 3′ untranslated region of 108 bp, with a poly A structure. Amino acid sequence analysis showed that the CCAP gene encodes 141 amino acids with a predicted molecular weight of 15.6 kD and isoelectric point of 9.55.Homology analysis showed that the homology between CCAP of P. trituberculatus and Scylla paramamosain and Callinectes sapidus was higher at 85% and 82%, respectively. Phylogenetic analysis showed that P. trituberculatus and C. sapidus were clustered first, followed by S. paramamosain. Tissue expression analysis revealed that the relative expression of the CCAP gene was highest in the in thoracic ganglia, followed by in the brain and eye stalk.Analysis of the expression pattern of the CCAP gene during low-salinity stress showed that low salinity significantly altered the expression pattern of CCAP in the thoracic ganglia;CCAP gene expression in the experimental group was significantly higher than that in the control group at 24, 48, and 72 h(P < 0.05) with values 1.73-, 2.16-,and 2.19-fold higher than that in the control group, respectively. In vitro injection of CCAP polypeptide reduced the mortality of P. trituberculatus under low-salinity conditions and the activities of Na^+/K^+-ATPase and V-ATPase were significantly increased. The results demonstrate that CCAP plays a protective role in P. trituberculatus under low-salinity conditions by regulating osmotic pressure and may be associated with increased Na^+/K^+-ATPase and V-ATPase activities to modulate the osmotic pressure balance in P. trituberculatus.