三疣梭子蟹(Portunus trituberculatus)几丁质酶PtCht3基因克隆鉴定及表达分析

为初步研究三疣梭子蟹(Portunus trituberculatus)几丁质酶PtCht3的生物学功能,利用特异性引物扩增和SMARTTM RACE技术克隆获得三疣梭子蟹PtCht3基因全长cDNA序列,并对该序列进行分析.结果显示,三疣梭子蟹PtCht3基因全长为1409 bp,对PtCht3基因序列推导出的氨基酸序列进行分析可知,该基因编码由394个氨基酸组成的蛋白质,预测分子量为43.67 kDa,理论等电点为4.80.PtCht3蛋白亲水性总平均数为-0.097,属于稳定蛋白.同源性和系统进化分析发现,PtCht3与日本仿长额虾(Pandalopsis japonica)和中华绒螯蟹(Eriocheir sinensis)几丁质酶3的同源性分别为54％和53％,与其他甲壳动物几丁质酶3聚为一支.RT-PCR显示,PtCht3基因具有较强的组织表达特异性,在肝胰腺中的相对表达量最高,在三疣梭子蟹蜕皮前期表达上调,低盐胁迫后在鳃和肝胰腺中表达量出现了波动,总体呈先上升后下降的表达趋势.推测PtCht3可能在三疣梭子蟹消化和蜕皮过程中发挥重要作用,参与三疣梭子蟹渗透压调节进程.本研究为三疣梭子蟹几丁质酶的功能研究提供了重要信息.

Cloning and Expression Analysis of the cDNA of PtCht3 in Portunus trituberculatus

Portunus trituberculatus,aka the swimming crab,is an important fishery species with high commercial value in China.They mainly inhabit the sandy and muddy bottom underneath the seawater in the coast of countries such as China,Japan and Korea.The growth and development of P.trituberculatus was characterized by periodic molting activities in which the new ectoskeleton grows while the old one molting.Previous studies reported that chitinase was an essential enzyme involved in the molting of crustaceans,and that it was required in chitin digestion and the immunity to the pathogen infection.However,little has been known about the effect of chitinase on molting activities,and the expression of the enzyme during rapid salinity stress is also obscure.To understand the biological function of PtCht3 gene in P.trituberculatus,we cloned and analyzed the full-length eDNA sequence of PtCht3 gene using SMARTTMRACE amplification kit.The eDNA sequence had 1409 bp encoding 394 amino acid residues.The isoelectric point (pl) of the putative peptide was 4.80,and the predicted molecular mass was 43.67 kDa.PtCht3 could be a stable protein with the hydrophilic coefficient total average of-0.097.The homology and systematic evolution analysis revealed that PtCht3 of P.trituberculatus was highly homologous with the protein in other species;for example,it shared 54％ similarity with Pandalopsis japonica and 53％ similarity with Eriocheir sinensis.The phylogenetic analysis showed that P.trituberculatus PtCht3 was in the same class as other arthropods' PtCht3.The expression of PtCht3 in different tissues was analyzed with quantitative real-time PCR.We found that the highest expression level appeared in hepatopancreas,and that the expression was significantly up-regulated in the prophase of the molting cycle.In response to the low salinity stress the expression level fluctuated in gills and hepatopancreas,showing a general up-and-down pattern.Our results suggested that PtCht3 might play an important role in digestion and molting activities,and it could be involved in the regulation of osmotic pressure in P.trituberculatus.