牙鲆性腺分化相关 microRNA 的筛选与鉴定

The paralichthys olivaceus is the major marine species cultured in China,the female grow faster than the male.The sex of the paralichthys olivaceus is mainly determined by genetic and environmental factors. It has male heterogametic sex-determination. However, sex-differentiation of genetic females XX seems to be so unstable that spontaneous sex-reversal to physiological males can be caused by exogenous factors, especially high water temperature. So the genetic mechanism of gonad differentiation is an important and interesting subject for paralichthys olivaceus. The microRNAs play an key role in the gonad differentiation of animals through the mechanism of post-transcriptional regulation. In this project, next generation high-throughput DNA sequencing and Biochip technique were used to search high or unique expression microRNAs in gonad of female and male paralichthys olivaceus. Then the function of those microRNAs will be predicted using bioinformatic methods.The expression patterns of thoese microRNAs during gonad differentiation at different temperatures will be analyzed. The molecular and cell bioloby techniques will be used to verify the biological function of above selected microRNAs in vitro and in vivo.So an gene regulation pathway of gonad differentiation was take shape initially.The results will facilitate more in-depth understanding of flounder sex differentiation and to provide basic data for the the all female flounder reproductive manipulation.

牙鲆是我国重要的海水养殖品种，雌性比雄性生长更快，其性别主要由遗传和环境因子特别是温度共同决定。环境温度提高后，遗传性别为雌性的仔鱼会发生性逆转成为生理性雄鱼。因此，研究牙鲆性腺分化的遗传学机制具有重要意义。microRNA（miRNA）对靶基因进行转录后调控，在动物性腺分化中起着极其重要的作用，本项目以牙鲆精巢和卵巢特异性表达的miRNA为研究主要对象，探究其在牙鲆性腺分化中的表达规律及功能。首先通过高通量测序和生物芯片筛选得到精、卵巢特异表达的miRNA以及精、卵巢显著差异表达的miRNA基因；然后通过生物信息学和分子生物学方法预测和验证若干候选miRNA的功能，并比较不同温度组miRNA的表达规律。该项目将miRNA作为纽带联系环境因子与其它性别决定基因，研究结果将促进人们更深入理解牙鲆性腺分化的分子机制以及温度作用于仔鱼性腺的生物学效应，为牙鲆全雌化生产提供基础。

牙鲆是我国重要的海水养殖品种，研究牙鲆性腺分化和发育的遗传学机制具有重要意义。microRNA（miRNA）对靶基因进行转录后调控，在动物性腺分化和发育中起着极其重要的作用，本项目建立了牙鲆雌雄性腺miRNA文库，通过small RNA 深度测序和生物信息学分析筛选到一批雌雄性腺差异表达的miRNAs分子；通过茎环引物的定量PCR和原位杂交技术分析了5个关键miRNA在牙鲆雌雄性腺中的表达，其中miR-26a、miR-26b、miR-125 和miR-143在牙鲆性腺中表达丰富，且在精巢中的表达高于卵巢，而miR-200b在卵巢中的表达显著高于精巢；克隆和鉴定了6个miRNA的与性腺发育相关的靶基因，并分析了这些靶基因在不同组织不同发育时期的表达谱；分析和比较了这些miRNA 及靶基因在牙鲆性腺分化过程中高温诱导、雌激素和雄激素处理不同条件下的表达变化；采用双荧光素酶报告实验验证了5个miRNA的靶基因dmrt1以及miR-26a、miR-26b的靶基因emx2，初步进行了miRNA agomir 和 antagomir的显微注射，为后续miRNA的功能研究提供了基础。该项目以miRNA为联系纽带，初步探讨了miRNA及其靶基因对牙鲆性腺分化及发育的调控。