半滑舌鳎富含亮氨酸的G蛋白偶联受体基因(LGR8)的分子克隆和表达分析  

Molecular Cloning and Expression Analysis of Leucine Rich Repeat Containing G Protein Coupled Receptor 8 Gene(LGR8) in Half-smooth Tongue Sole(Cynoglossus semilaevis)

作　　者：吴娅红 陈松林[1,3] 刘肖峰[1] 董忠典[1] 张宁[1] 于洋[1] 修旺姗 

机构地区：[1]中国水产科学研究院黄海水产研究所,青岛266071 [2]上海海洋大学水产与生命学院,上海201306 [3]海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室,青岛266071

出　　处：《农业生物技术学报》2015年第9期1188-1195,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金重点项目(No.31130057);国家高技术研究发展计划(863)项目(No.2012AA10A403);山东省泰山学者攀登计划专项资助

摘　　要：富含亮氨酸的G蛋白偶联受体（leucine rich repeat containing G protein coupled receptor 8,LGR8）在哺乳类动物雄性精巢下降过程中起重要作用.本研究在前期完成的半滑舌鳎（Cynoglossus semilaevis）全基因组测序与转录组测序的基础上,通过RACE方法克隆得到半滑舌鳎LGR8基因（GenBank登录号：KP749830）,该基因全长2 768 bp,开放阅读框（open reading frame,ORF）为2 205 bp,编码734个氨基酸,5’-非翻译区（untranslated regions,UTR）为272 bp,3’-UTR为296 bp.氨基酸序列中含有10个LRRs区域、1个LDL-A区域和1个7tm-1结构域.实时定量PCR（Real-time quantitative PCR,qRT-PCR）结果显示,在3龄性成熟鱼体中LGR8主要在雄鱼脑和性腺中表达,且显著高于其他组织（P＜0.05）;在雌鱼各组织中均有表达,但是大多数组织（除肠和心脏）中表达量无显著差异;在雄鱼性腺和脑中的表达量要显著高于雌鱼（P＜0.05）.对雄鱼不同时期的性腺进行qRT-PCR检测结果显示,LGR8基因在鱼体发育80 d表达量开始上升,150～210 d表达量达到最大,且显著高于其他组织（P＜0.05）.研究表明LGR8可能在半滑舌鳎性别决定和性腺发育中起一定作用,为半滑舌鳎性别决定机制的研究提供了一定的理论基础.Leucine rich repeat containing G protein coupled receptor 8 （LGR8） plays an important role in the process of testicular descent in male mammals. LGR8 gene in half smooth tongue sole （Cynoglossus semilaevis） was cloned on the basis of the entire genome and transcriptome sequencing using RACE method. LGR8 gene contained 2 768 nucleotides, and the open reading frame （ORF） was 2 205 bp, which encoded 734 amino acids, 272 bp of 5＇-UTR and 296 bp of 5＇-UTR. The amino acid sequences contained 10 LRRs areas, a LDL-A regional structure and a 7tm-1 domain. The results of Real-time fluorescent quantitative PCR （qRT-PCR） showed that LGR8 mainly expressed in brains and gonads of the sexual maturity male bodies of three years old fish, which was extremely higher than that of other tissues. There was no significant difference among the most tissues in females（except the intestine and the heart）.. It was noteworthy that the expression level of LGR8 in the brain and gonad of males are significantly higher than that of females （P〈0.05）. The results of gonad in different periods by qRT-PCR showed that LGR8 gene began to rise at 80 d, and the expression was up to the maximum amount between 150 and 210 d, when the expression was significantly higher than that in other tissues （P〈0.05）. The results suggested that LGR8 might play a significant role in gender determination and development of the gonads in half smooth tongue sole which provides certain theoretical basis for the sex determination mechanism study of half smooth tongue sole.

关 键 词：半滑舌鳎 富含亮氨酸的G蛋白偶联受体基因(LGR8) 实时荧光定量PCR(qRT-PCR) 性别分化 

分 类 号：Q51[生物学—生物化学]