拟穴青蟹一种含CTLD结构域基因的克隆及表达分析

Cloning and expression analysis of a novel gene containing CTLD domain in Scylla paramamosain

从拟穴青蟹(Scylla Paramamosain)中克隆得到一个含有CTLD(C-type lectin-like domain)结构域的C型凝集素基因,经分析未发现同源基因,将其命名为SpCTLD。该基因cDNA序列全长为1237 bp,包含一个468 bp的开放阅读框,编码155个氨基酸,预测分子量为18.32 kDa,等电点为7.56。经一代测序,该基因在第390位(A390G)和第1020位(T1020C)含有两个SNP位点。对SpCTLD基因在拟穴青蟹幼体不同时期和组织中的表达模式分析发现,SpCTLD在幼体的各个发育时期均有表达,其中在溞状幼体Ⅴ期表达量最高,仔蟹Ⅰ期次之,仔蟹Ⅱ期最低。对SpCTLD的组织分布分析表明,SpCTLD在鳃中表达量最高,其次为射精管,在血淋巴、肝胰腺等其他免疫组织中也有表达,其中在鳃和射精管的表达量远高于在其他组织中的表达量。副溶血弧菌胁迫实验表明,活菌组的肝胰腺、血淋巴、鳃中的SpCTLD表达量发生显著上调,且都在12 h达到最高。根据研究结果,推测SpCTLD是一种含CTLD结构域的新基因,它可能参与了拟穴青蟹抗细菌反应。该基因的发现丰富了拟穴青蟹C型凝集素相关基因的研究,有利于丰富甲壳动物先天免疫机制的研究。

A novel C-type lectin gene containing a C-type lectin-like domain(CTLD)was cloned from Scylla paramamosain,and no homologous gene was found,and was named SpCTLD.The gene sequence was 1237 bp in length and contained a 468bp open reading frame,encoding 155 amino acids.The predicted molecular weight was 18.32 kDa and the isoelectric point was 7.56.The gene contained two SNP sites at 390(A390G)and 1020(T1020C)by first-generation sequencing.The expression pattern of SpCTLD in different stages of larvae and tissues of Scylla paramamosain showed that SpCTLD was expressed in all developmental stages of larvae,and the highest expression was observed in the V phase of the zoea stage,followed by the early juvenile crab.The second juvenile crab was the lowest.The tissue distribution analysis of SpCTLD showed that the expression of gill was the highest,followed by the ejaculatory duct.The expression of both was much higher than other tissues,and it was also expressed in other immune related tissues such as hemolymph,hepatopancreas and so on.The stress test of Vibrio parahaemolyticus showed that the mRNA expression levels of hepatopancreas,hemolymph and gill in the live bacteria group were significantly up-regulated,and reached the highest at the 12th hour.Based on the results,it is speculated that SpCTLD is a new gene containing a CTLD domain,and it may be involved in the antibacterial response of Scylla paramamosain.The discovery of this gene enriches the study of C-type lectin-related genes in Scylla paramamosain,and is conducive to the improvement of the innate immune mechanism of crustaceans.