仿刺参ghitm基因的克隆及LPS诱导后的表达分析

Cloning and LPS-induced expression analysis of ghitm gene in sea cucumber Apostichopus japonicus

根据GenBank中登录的仿刺参Apostichopus japonicus ghitm基因的EST片段,采用RACE扩增法克隆了仿刺参ghitm基因的c DNA全长序列,并利用qRT-PCR技术检测了经LPS诱导后仿刺参ghitm的表达变化情况。结果表明:仿刺参ghitm基因(Aj-ghitm)的c DNA序列全长为1325 bp,包含一个1005 bp编码334个氨基酸的开放阅读框,序列两端分别为140 bp的5'UTR和180 bp的3'UTR。序列分析结果显示,Aj-ghitm编码的蛋白含有一个8次跨膜结构域,与Gen Bank中登录的紫色球海胆GHITM蛋白的氨基酸序列相似度为65.4%,且在系统发育树中聚为一支;qRT-PCR检测结果表明,经LPS刺激可诱导仿刺参体腔细胞Aj-ghitm mRNA的表达且呈先下降后升高最后回复到接近正常水平的趋势,在24 h时上升到最高,随后下降。本研究是有关棘皮动物ghitm基因的首次报道,其结果可为进一步探讨ghitm在仿刺参抗细菌感染以及生长发育等方面的功能及作用机制提供参考。

The full-length c DNA sequence of ghitm were firstly cloned by RACE technique from coelomocytes of sea cucumber Apostichopus japonicus according to the EST sequence previously obtained from Genbank,and we named it as Aj-ghitm（ Gen Bank： KC886718）. The full-length c DNA of Aj-ghitm was 1325 bp,containing a5＇UTR of 140 bp and a 3＇ UTR of 180 bp,with an open reading frame of 1005 bp encoding 334 amino acids.SMART analysis showed that the deduced protein has an eight-transmembrane domain. Multiple alignment and phylogenic analysis showed that Aj-GHITM and the sea urchin GHITM were closely clustered in one group with the highest similarity of 65. 4%. Quantitative real-time PCR revealed that Aj-ghitm mRNA expression level was decreased first and then increased,finally recovery to the normal level after LPS-induced,with the maximum expression 24 h followed by a lower expression. The findings give a reference for further study of the role of GHITM in the development and anti-bacterial infection in sea cucumber.