三疣梭子蟹几丁质酶基因1的克隆及功能鉴定

Cloning and functional identification of chitinase gene 1 in swimming crab Portunus trituberculatus

中文关键词: 三疣梭子蟹  几丁质酶  基因克隆  蜕皮  低盐胁迫

英文关键词: Portunus trituberculatus  Chitinase  Gene cloning  Molting  Low salinity stress

中文摘要:

      为丰富三疣梭子蟹(Portunus trituberculatus)几丁质酶基因家族的组成，进行了几丁质酶基因1(PtCht1)的研究。PtCht1基因cDNA全长为2220 bp，编码583个氨基酸。结构域比对发现，PtCht1含有甲壳动物几丁质酶GH18家族基因的基本结构及保守序列，且进化树显示，三疣梭子蟹Cht1与拟穴青蟹(Scylla serrata)等物种的Cht1聚为一类，同源性最高，达92.80%。由此确定，PtCht1为甲壳动物GH18家族Group1基因，也是三疣梭子蟹该分类下的首个基因。此外，全组织表达分析结果显示，PtCht1在肝胰腺中较特异性高表达，且在蜕皮间期的肝胰腺中的表达量显著高于前期和后期(P<0.05)。低盐度胁迫处理后，在肝胰腺中，PtCht1基因的表达量在3 h快速升至峰值，为对照组的2.72倍；在鳃中，除12 h外，PtCht1基因的表达量均呈显著上调表达(P<0.05)，最高上调9.96倍。结果表明，PtCht1基因能够参与机体对几丁质类食物的消化过程，并且在渗透压的调控等方面发挥重要作用。本研究可为三疣梭子蟹几丁质酶GH18家族Group1基因的研究奠定基础，为解析盐度影响三疣梭子蟹生长提供参考。

英文摘要:

      Chitinase plays an important role in the molting, digestion, and pathogen defense of Portunus trituberculatus. However, little is known about the P. trituberculatus chitinase genes. To understand the P. trituberculatus chitinase gene family, we investigated its chitinase gene 1 (PtCht1). PtCht1 has a total cDNA of 2220 bp and encodes for 583 amino acids. We found that PtCht1 exhibited the basic structure and contained the conserved sequence of the chitinase GH18 family genes of crustaceans. The phylogenetic tree showed that Cht1 of P. trituberculatus was clustered with Cht1 of other species such as Scylla serrata, with 92.80% homology. Therefore, we concluded that PtCht1 is a Group1 gene of the crustacean GH18 family. Further, it is the first P. trituberculatus gene in this classification. Additionally, the results of whole tissue expression analysis showed that PtCht1 was highly expressed in the hepatopancreas, and the expression level in the hepatopancreas during the inter-molt was significantly higher than in the pre-molt and post-molt (P < 0.05). After low salinity stress treatment, the expression level of PtCht1 gene in hepatopancreas rapidly rose to the peak at 3 h (2.72-fold); in the gill, except for 12 h, the expression level of PtCht1 gene was significantly up-regulated (P < 0.05), up to 9.96-fold. Our results show that the PtCht1 gene can participate in the digestion of foods containing chitin and plays a significant role in the regulation of osmotic pressure. Thus, our study is a landmark study in Group1 gene research of the chitinase GH18 family of P. trituberculatus. To conclude, the gene function studies of PtCht1 provide new clues for analyzing the effect of salinity on the growth of P. trituberculatus.