两种泥鳅DMRT1基因不同选择性剪接体的结构、功能及调控机制研究

DMRT1 gene is the only male development regulating factor discovered in vetebrates so far, conservative on both structure and function. In our previous studies we have isolated 6 different forms of DMRT1 from the mature testis of Misgurnus anguillicaudatus and 5 different forms of DMRT1 from Paramisgurnus dabryanus. We have determined that the diversity is resulted from alternative splicing, however, the function of different spliceosomes and the transcriptional regulation of the splicing are not clear yet, requiring further investigation. This project intends to perform gene cloning, structure and expression pattern analysis on DMRT1 genes of two loaches testis from different critical developmental period (period-specificity) and from various adult tissues (tissue specificity). Combined with RNAi technology by designing siRNA against DMRT1, we can block or reduce its expression. Following work is to track the changes in the two loaches gonadal development, and to determine the specific relationship of DMRT1 gene with gonadal development. In addition, we will also perform analyses on DMRT1 gene 5 ′flanking region and the 3′UTR regulatory region, which aims to elucidate the molecular mechanism of the DMRT1 gene. This project can help us get a more comprehensive view of DMRT1 gene function and its role in the regulatory network of sex determination.

DMRT1基因是迄今为止脊椎动物中唯一一个在结构和功能上最为保守的雄性发育调控因子。本室前期研究已分别在泥鳅（六种DMRT1s）和大鳞副泥鳅（五种DMRT1s）成熟精巢中分离得到了DMRT1基因不同形式的选择性剪接体，确定DMRT1基因在两种泥鳅中均存在选择性剪接，但是各剪接体的功能和转录调控机制目前仍不清楚，后续试验亟待进行。本项目拟进一步对两种泥鳅精巢各关键发育时期（时期特异性）和成体各种组织（组织特异性）的DMRT1基因进行克隆、结构和表达模式分析，并结合RNAi技术设计针对DMRT1的siRNA，阻断或降低其表达，追踪两种泥鳅性腺发育变化，确定DMRT1基因与性腺发育的具体关系；此外，课题还对两物种DMRT1基因的5′侧翼区和3′UTR调控区进行分析，旨在阐明DMRT1基因的分子调控机制。通过本课题实施，有助于全面解析DMRT1基因的具体功能和在性别决定调控网络中的作用。

DMRT1基因是目前发现的第一个在种属间具有进化保守性的性别分化相关基因，在动物性别决定过程中发挥重要作用。本项目首先对两种泥鳅精巢各关键发育时期（时期特异性）和成体各种组织（组织特异性）的DMRT1基因进行克隆，分离得到了DMRT1基因不同形式的选择性剪接体，并对得到的不同剪接体进行了结构分析。在此基础上利用半定量RT-PCR、荧光定量RT-PCR和原位杂交技术检测了DMRT1基因的表达模式，RNAi技术阻断或降低DMRT1的表达，追踪胚胎发育变化，推测DMRT1基因的功能；最后对DMRT1基因的5′侧翼区和3′UTR调控区进行分析。本项目实施确定了DMRT1基因的剪接方式、具体功能等，有助于阐明DMRT1在脊椎动物的性别决定和分化调控网络中的作用。