凡纳滨对虾Crustin-like基因的克隆及在副溶血弧菌感染条件下的表达分析  

Molecular cloning of Crustin-like gene in the white shrimp (Litopenaeus vannamei) and its mRNA expression with Vibrio parahaemolyticus challenge

作　　者：张艳艳[1] 刘小林[1] 黄海洪[1] 钱昭英[1] 王宪宗[1] 相建海[2] 李义军[3] 

机构地区：[1]西北农林科技大学动物科技学院,陕西省农业分子生物学重点实验室,陕西杨凌712100 [2]中国科学院海洋研究所实验海洋生物学开放实验室,山东青岛266071 [3]海南南疆生物技术有限公司,海南三亚572000

出　　处：《西北农林科技大学学报（自然科学版）》2012年第12期41-48,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家"863"高新技术研究与发展计划项目(2006AA10A406);中国科学院实验海洋生物学重点实验室开放课题(Kf201002);科技部农业科技成果转化基金项目(2012GB2E200361)

摘　　要：【目的】研究凡纳滨对虾Crustin-like基因(即CL基因)的结构和功能,探明CL基因是否参与副溶血弧菌侵染条件下的免疫应答,为进一步研究凡纳滨对虾的免疫机制和抗病育种奠定基础。【方法】采用RT-PCR技术克隆凡纳滨对虾CL基因;用DNAstar、Clustalx、MEGA 5.0、PROSITE、TMpred和SignalP软件,分析CL基因序列及其蛋白结构,并预测蛋白功能;利用副溶血弧菌进行攻毒试验和实时定量PCR,检测CL基因在病原侵染条件下的表达情况。【结果】克隆获得了凡纳滨对虾CL基因(登录号:JQ824114)的cDNA,全长为501bp,含有33bp的5′非翻译区(1～33bp)和24bp的3′非翻译区(478～501bp),编码区为444bp,共编码147个氨基酸;对CL蛋白结构及功能的分析表明,该蛋白编码蛋白质含有1个信号肽、1个跨膜螺旋和1个WAP结构域;攻毒试验表明,副溶血弧菌刺激可导致CL基因表达量的明显变化,总体呈现出先降低后升高的变化趋势。【结论】克隆了凡纳滨对虾CL基因的全长,发现其与副溶血弧菌侵入后引发的免疫反应密切相关,可以作为副溶血弧菌感染前期诊断的参考指标。【Objective】 The research studied the structures and functions of Crustin-like（CL） gene and detected whether CL gene participated in immune response or not.It improved the understanding on the function of CL gene and rearing new resistant varieties.【Method】 The technique of RT-PCR were used to clone the coding region sequence of CL gene in Litopenaeus vannamei.The structures and functions of CL gene and CL protein were predicted by DNAstar,Clustalx,MEGA 5.0,PROSITE,TMpred and SignalP.Finally,the mRNA expression pattern of CL gene in hepatopancreas was studied by challenge assays and Real-Time quantitative PCR（qRT-PCR） at different times.【Result】 The results showed that the sequence of CL gene in L.vannamei（Accession number： JQ824114） was 501 bp including a 33 bp 5′UTR（1-33 bp）,24 bp 3′UTR（478-501 bp） and a 444 bp CDS and encoded 147 amino acids with the initial code ATG and the terminal code TGA.Sequence of amino acids was consisted of a signal peptide,a single transmembrane helix,and a WAP domain.Results of challenge assays and qRT-PCR showed that the CL gene significantly changed.It increased initially and decreased after Vibrio parahaemolyticus challenge.【Conclusion】 This research cloned full-length of CL gene and found it was closely related to immune response with V.parahaemolyticus challenge.It can be used as a preliminary indicator for pathogen infection.

关 键 词：凡纳滨对虾 Crustin-like基因 序列分析 副溶血弧菌 实时定量PCR 

分 类 号：S945.19[农业科学—水产养殖]