半滑舌鳎star基因的克隆和表达分析及性别相关基因(ftz-f1、neurl3)原位杂交分析-海洋生物学专业论文.docxVIP

志。 通过原位杂交技术，分别对 FTZ-F1 基因和 neurl3 基因进行了组织学定位。 结果显示 FTZ-F1 基因在半滑舌鳎的雌鱼和雄鱼中都有杂交信号，但在精巢中的 杂交信号明显强烈，随着精巢的成熟杂交信号明显加强，44d 的雄鱼主要在精原 细胞中信号最强，205d 的精巢中杂交信号主要集中在精子细胞中。卵巢中该基 因的杂交信号较弱，44d 的雌性原始性腺中原始生殖细胞有较弱的表达，在 205d 的雌鱼卵巢中该基因主要在卵母细胞的膜中表达。neurl3 基因原位杂交表达分 析结果显示，该基因在半滑舌鳎的精巢中表达信号强，2 龄的雄鱼中检测到了该 基因的杂交信号，杂交信号主要集中在成熟的精子细胞表达，成年的卵巢中没有 该基因的表达。对于三倍体的半滑舌鳎雄鱼也检测到了该基因的表达，但表达程 度明显比正常的雄鱼弱。伪雄鱼中成熟的精子细胞表达信号明显。由上述可以推 断 neurl3 基因与半滑舌鳎精子细胞的发生有很密切的联系，很可能是半滑舌鳎 精子发生过程中一个重要的基因。该研究结果为半滑舌鳎性别基因的研究提供了 相关的理论基础，为更好的开展半滑舌鳎选择育种工作做好相关铺垫。 关键词：半滑舌鳎；克隆；荧光定量；原位杂交；性别相关基因；StAR；FTZ-F1； neurl3 Ⅱ Abstract  PAGE 6 Abstrct Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture fish， Due to the half-smooth tongue sole have high nutritional value. A large differences in male and female adult is that adult females are generally 2-3 times the adult males. And in the development of process , Half-smooth tongue sole occur sex reversal under natural conditions. Therefore , in the study process, mechanisms for gender related decisions controlled studies should be part of a very important. Based on the StAR gene cloning of Half-smooth tongue sole, The gene was found very clear expression in the testis , suggesting that development of testes is closely related to the sperm cells.It has done the foundation work and make pathe way to Half-smooth tongue sole breeding. Partial StAR gene sequence obtained from whole genome sequencing results of half-smooth tongue sole(Cynoglossus semilaevis) and its full-length cDNA sequence was acquired by SMART-RACE.The StAR complete cDNA was 1294bp in length containing 1470bp open reading frame (ORF) and encoded 284 amino acids, 132bp 5′un-translated region(5′-UTR) and 310bp 3′un-translated region(3′-UTR). Alignment analysis with other species and Homologous analysis of the amino acids showed that StAR of Cynoglossus semilaevis was 85℅ similar to most species of StAR including Solea senegalensis, Micropterus salmoides,Lateolabrax japonicus and Sparus aurata.RT-PCR results indic