卵胎生许氏平鲉Sox9基因的克隆、启动子分析、表达及其细胞定位研究  

Cloning,promoter analysis,expression and cell localization of Sox9 gene in an ovoviviparous teleost,black rockfish Sebastes schlegeli

作　　者：马丽曼 张全启[2] 齐洁[2] 卢梦萱 王文基 MA Liman;ZHANG Quanqi;QI Jie;LU Mengxuan;WANG Wenji(School of Medicine, Taizhou University, Taizhou 318000, China;School of Life Science, Ocean University of China, Qingdao 266003, China;School of Life Science, Taizhou University, Taizhou 318000, China)

机构地区：[1]台州学院医学院,浙江台州318000 [2]中国海洋大学生命科学学院,山东青岛266003 [3]台州学院生命科学学院,浙江台州318000

出　　处：《大连海洋大学学报》2020年第5期671-679,共9页Journal of Dalian Ocean University

基　　金：浙江省科技计划项目(2017C37154);台州市科技计划项目(15ny11)。

摘　　要：为研究卵胎生许氏平鲉Sebastes schlegeli性腺分化及性别决定分子机制,采用同源克隆和RACE技术从许氏平鲉(体质量758 g±15 g)性腺组织中获得了许氏平鲉Sox9基因全长cDNA,运用生物学软件对Sox9启动子转录因子结合位点进行预测,并利用实时定量PCR和原位杂交对Sox9基因的表达进行了研究。结果表明:许氏平鲉Sox9基因cDNA全长2039 bp,包括1461 bp的ORF,336 bp的5′UTR和242 bp的3′UTR,其编码产物(486 aa)含有高度保守的HMG结构域;许氏平鲉Sox9基因启动子区存在多个转录因子结合位点,包括AP-1、GATA-3、Oct-1、FOXD3,以及性别相关SRY、Sox5、Sox9等蛋白结合位点;实时定量PCR显示,许氏平鲉Sox9基因在仔鱼发育阶段(出生后1~35日龄)均有表达,在性腺分化的敏感时期(25日龄)表达量达到较高水平;在成体性腺中显示了性别两相性差异,即在精巢中的表达高于卵巢;原位杂交显示,许氏平鲉Sox9基因在精巢的生殖细胞、Sertoli细胞,以及卵巢的卵母细胞和滤泡细胞中均有表达。研究表明,Sox9基因在许氏平鲉性腺分化及性腺发育过程中可能发挥重要作用。Sox9 gene was isolated from gonads of black rockfish Sebastes schlegeli(SsSox9)with body weight of(758±15)g by homology cloning and RACE technology to investigate the mechanisms of sex determination and gonadal differentiation in this ovoviviparous fish.Transcriptional factor binding sites of the Sox9 promoter were predicted,and the expression of Sox9 was analyzed by Real-time quantitative PCR and in situ hybridization(ISH).The results showed that the full-length of SsSox9 cDNA was 2039 bp,comprising of a 1461 bp open reading frame(ORF),a 336 bp 5′UTR and a 242 bp 3′UTR.The predicted SsSox9 protein of 486 aa contained a highly conserved HMG domain.Many common binding sites for transcription factor were found in Sox9 promoter,including AP-1,GATA-3,Oct-1,FOXD3,and the sex-related protein SRY,Sox5,and Sox9.qRT-PCR analysis indicated that the expression of SsSox9 was detected in all stages of 1,5,10,15,25 and 35 days after birth(DAB),with higher expression level of SsSox9 during the sensitive period of gonadal differentiation(25 DAB).The expression levels of the SsSox9 gene were sexually dimorphic in adult gonads,with higher level in testis than that in ovary.In situ hybridization revealed that SsSox9 mRNA was detected in the germ cells and Sertoli cells in the testis,and oocytes and follicular cells in the ovary,suggesting that SsSox9 may have an important role in gonadal differentiation and development of black rockfish.

关 键 词：许氏平鲉 启动子分析 性腺分化 性别决定 SOX9基因 

分 类 号：Q958.8[生物学—动物学]