母源因子boule在光棘球海胆性腺中的表达与定位

Expression and cellular localization of maternal factor boule in gonad of sea urchin Mesocentrotus nudus

为鉴定光棘球海胆Mesocentrotus nudus生殖细胞标记基因,通过基因克隆、荧光定量PCR及切片原位杂交等技术分析了光棘球海胆(湿体质量为76.8 g±10.0 g)母源因子(boule)基因的分子特征及动态表达模式。结果表明:光棘球海胆boule cDNA序列全长为1788 bp,其中,3′非编码区为601 bp,5′非编码区为146 bp,开放阅读框(open reading frame,ORF)为1041 bp,共编码346个氨基酸,包含一个保守的RRM结构域;荧光定量PCR结果显示,boule为母源因子,在整个胚胎发育时期均有表达;boule基因在处于生长期(stageⅡ)光棘球海胆的肠、管足、体腔液和性腺中均有表达,其中,boule基因在精巢表达量最高(P<0.05);在卵巢中表达量随卵母细胞的成熟而逐渐升高,在成熟期(stageⅣ)卵巢中达到最高,精巢中表达量仅在生长期(stageⅡ)及成熟前期(stageⅢ)有较高表达;切片原位杂交显示,boule基因在光棘球海胆精巢的生殖细胞中特异表达,卵巢中未检测到阳性细胞信号。研究表明,光棘球海胆boule基因是雄性生殖细胞标记基因,本研究结果可为海胆雄性生殖细胞发育相关研究提供数据资料。

In order to identify the germ cell marker genes of sea urchin Mesocentrotus nudus,molecular characteristics and dynamic expression patterns of boule gene were elucidated in the sea urchin(body mass of 76.8 g±10.0 g)by means of gene cloning,Real-time quantitative PCR(RT-qPCR)PCR and in situ hybridization in sections.It was found that the full length of boule cDNA sequence was 1788 bp,with the length of 3′untranslated region of 601 bp,5′untranslated region of 146 bp and open reading frame of 1041 bp,encoding a total of 346 amino acids including a conservative RRM domain.Real-time quantitative PCR(RT-qPCR)analysis revealed that boule protein was a maternal factor being expressed throughout the embryonic development.In addition,boule gene was expressed in intestine,tube foot,coelomic fluid and the gonad(stageⅡ),with the maximum expression level in testis.In ovary with the maturation of oocytes,the expression of boule gene was increased gradually to stage IV,with the maximum level,and the expression of boule gene was higher in testis only at stageⅡand stageⅢ.Section in situ hybridization showed that boule gene was expressed specifically in male germ cells,without detection of boule in positive cells in ovary.By combining all the analysis results,boule gene of sea urchin is a marker gene in male germ cells and the findings lay a foundation for the study of male germ cell development of sea urchin.