罗氏沼虾谷氨酸脱氢酶基因克隆及其在MrTV感染下的组织表达分析  

Molecular cloning and tissue expression analysis of glutamate dehydrogenase gene from Macrobrachium rosenbergii under MrTV infection stress

作　　者：郑群艳 潘晓艺[2] 沈锦玉[1,2] 陈少波[1,3] 徐洋[2] 许婷[2] 

机构地区：[1]温州医科大学检验医学院-生命科学学院,浙江温州325035 [2]浙江省淡水水产研究所浙江省鱼类健康与营养重点实验室,浙江湖州313001 [3]浙江省海洋水产养殖研究所,浙江温州325005

出　　处：《浙江大学学报（农业与生命科学版）》2017年第5期639-648,共10页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金(2013skj002);浙江省自然科学基金(LY12C19007);浙江省科技厅院所扶持专项(2017F30033);浙江省湖州市公益性技术应用研究项目(2015GZ09)

摘　　要：本研究利用c DNA末端快速扩增技术克隆获得罗氏沼虾(Macrobrachium rosenbergii)谷氨酸脱氢酶(glutamate dehydrogenase,GDH)基因(Mr GDH)的c DNA全长序列,对其进行生物信息学分析,并检测该基因在罗氏沼虾不同组织中的表达差异;同时,利用罗氏沼虾太湖病毒(Macrobrachium rosenbergii Taihu virus,Mr TV)感染沼虾,研究感染前后沼虾鳃和肝胰腺Mr GDH的转录特征。结果显示,Mr GDH基因c DNA序列全长2 257 bp,拥有一个1 662 bp长的开放阅读框,合计编码氨基酸553个,合成的蛋白质分子质量约为61.37 k Da。Mr GDH氨基酸序列与中国明对虾(Fenneropenaeus chinensis)、凡纳滨对虾(Litopenaeus vannamei)的同源性较高,达到92%,其二级结构和三维结构与黑腹果蝇(Drosophila melanogaster)以及家蚕(Bombyx mori)高度相似,说明Mr GDH氨基酸序列比较保守。实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,q RT-PCR)结果显示,Mr GDH基因在与机体运动和代谢有关的组织中表达量较高,其中在肌肉中表达量最高,而在血淋巴中最低。罗氏沼虾受Mr TV病毒感染48 h,肝胰腺和鳃中Mr GDH基因的表达在统计学上均极显著高于对照组(P<0.01);感染72 h,鳃中Mr GDH基因表达显著高于对照组(P<0.05),肝胰腺中Mr GDH基因表达极显著高于对照组(P<0.01):说明Mr TV感染胁迫可以刺激罗氏沼虾Mr GDH的表达上调。Macrobrachium rosenbergii is one of the major aquaculture shrimp, whose larva is the susceptible host of Macrobrachium rosenbergii Taihu virus（Mr TV） which is one kind of novel RNA virus, and a member of Dicistroviridae owning a single positive strand with two open reading frames. Glutamate dehydrogenase（GDH） is a key enzyme in glutamate biosynthesis and plays an important role in organism＇s metabolism. But there are still few reports aboutglutamate dehydrogenase in crustaceans, and almost no reports of M. rosenbergii glutamate dehydrogenase（Mr GDH）, as well as the interaction between Mr GDH and Mr TV.In this study, the complete sequence of MrG DH gene was cloned by rapid amplification of cD NA ends（RACE） technology,and the expression of MrG DH gene in different tissues was studied by quantitative real-time polymerase chain reaction（qR TPCR）. Then the transcriptional characteristics of MrG DH gene in the gill and hepatopancreas of M. rosenbergii were analyzed under Mr TV infection（positive group）, and the negative group was injected with phosphate buffered solution.The results showed that the complete c DNA sequence of Mr GDH gene was 2 257 bp in length with a 1 662 bp open reading frame（ORF） encoding 553 amino acids, which shared 92% homology with Fenneropenaeus chinensis GDH and Litopenaeus vannamei GDH. The protein molecular mass of Mr GDH was approximately 61.37 k Da. Its secondary and threedimensional structures were extremely similar to Drosophila melanogaster GDH and Bombyx mori GDH. The Mr GDH gene was expressed in all detected tissues of M. rosenbergii. After 48 h and 72 h of injection, the expression of Mr GDH gene in hepatopancreas and gill of positive group was significantly higher than the negative group.In summary, the Mr GDH gene is conservative, and its expression distribution indicates that it＇s highly expressed in the organisms extremely related to movement and metabolism, with the highest in muscle and the lowest in haemolymph.This difference may be caused by the

关 键 词：罗氏沼虾 罗氏沼虾太湖病毒 谷氨酸脱氢酶 基因克隆 组织表达差异 

分 类 号：Q17[生物学—水生生物学]