稀有[鱼句]鲫Foxo3a、Foxo3b基因克隆和表达分析  

Molecular Cloning and Expression Analyses of Foxo3a and Foxo3b Genes in Gobiocypris rarus

作　　者：钱佳铭 闵倩雯 董然然[1] 曾圣 张显波 QIAN Jiaming;MIN Qianwen;DONG Ranran;ZENG Sheng;ZHANG Xianbo(College of Animal Science,Guizhou University,Guiyang 550025,China;Guizhou Fisheries Research Institute,Guizhou Academy of Agriculture Sciences,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省农业科学院水产研究所,贵州贵阳550025

出　　处：《水产科学》2023年第2期222-232,共11页Fisheries Science

基　　金：贵州省科技厅联合基金资助项目[黔科合平台人才(2018)5768-07];淡水鱼类资源与生殖发育教育部重点实验室和水产科学重庆市重点实验室开放课题基金资助项目(2019-01);贵州省农业科学院2021年省财政科研专项资金资助项目[黔农科院种质资源(2021)06];贵州省农业委员会项目(CZ-CYTX2018-011)。

摘　　要：利用RACE技术自稀有[鱼句]鲫的卵巢中克隆叉头蛋白O3a(Foxo3a)、叉头蛋白O3b(Foxo3b)基因的全长cDNA序列,采用RT-PCR、qRT-PCR和原位杂交法检测其组织分布、时序表达、细胞定位和雷帕霉素处理后的表达。结果显示,Foxo3a基因全长cDNA序列为2562bp,包括242bp的5′端非编码区、367bp的3′端非编码区、1953bp的开放阅读框,编码650个氨基酸;Foxo3b基因全长cDNA序列为2804bp,包括470bp的5′端非编码区、375bp的3′端非编码区、1959bp的开放阅读框,编码652个氨基酸。Foxo3a基因只在脑和卵巢中表达,在其他组织中不表达;Foxo3b基因在所检测的组织中均有表达,在脑和卵巢中的表达量较高。随着卵巢的发育,Foxo3a基因表达量呈逐渐升高的趋势,Foxo3b基因表达量未出现显著变化。原位杂交结果显示,Foxo3a、Foxo3b基因定位于卵巢Ⅱ、Ⅲ、Ⅳ时相的生殖细胞和滤泡细胞中。雷帕霉素处理影响了卵巢中Foxo3a基因的表达,对Foxo3b基因的表达无显著影响。试验结果表明,Foxo3a和Foxo3b基因可能在稀有[鱼句]鲫卵子发生中发挥一定的作用。The full-length cDNA of forkhead protein O3a(Foxo3a)and forkhead protein O3b(Foxo3b)genes were cloned from the ovary of Gobiocypris rarus by using RACE.RT-PCR,qRT-PCR and in situ hybridization were employed to detect the expression of the pair of paralogous genes.A 2562 bp full-length cDNA of Foxo3a gene was abtained,containing a 1953 bp open reading frame(ORF)that encoded a protein of 650 amino acids,242 bp 5′untranslated terminal region(UTR),367 bp 3′UTR;a 2804 bp full-length cDNA of Foxo3b gene,containing a 1959 bp ORF that encoded a protein of 652 amino acids,470 bp 5′UTR,375 bp 3′UTR.Foxo3a gene was only expressed in brains and ovaries with a high level among all detected tissues.Foxo3b gene was highly expressed in brains and ovaries,and also found to be expressed in all other detected tissues.Foxo3a gene mRNA was detected in the ovaries with high levels from 2 month onward and slightly increased from 4 to 5 months.There was no significance in Foxo3b gene expression level in ovaries at all developing stages.In situ hybridization results showed that both Foxo3a and Foxo3b genes were expressed in the germ cell and follicular cell ofⅡ,Ⅲ,andⅣstage oocytes.Foxo3a gene expression was affected,but Foxo3b gene not,after the treatment by rapamycin.These findings indicate that Foxo3a and Foxo3b genes may play some roles in the oogenesis of G.rarus.

关 键 词：稀有[鱼句]鲫 Foxo3a基因 Foxo3b基因 基因克隆 基因表达 

分 类 号：Q786[生物学—分子生物学]