团头鲂HPG轴相关基因对其性早熟的分子调控机理研究

Early puberty is one of the major problems in many farmed fish species due to negative effects on growth performance. As a main aquaculture species in the Chinese freshwater polyculture system with high economic value, the Megalobrama amblycephala is gradually exhibiting early sexual maturity under a long cultured period. Many studies had revealed the molecular regulation mechanism of HPG related genes on the early puberty in mammalian vertebrates and the SNPs of related genes in HPG axis have been demonstrated to be correlated with early puberty. This study was designed to screen the SNPs correlated with the early maturation of M. amblycephala and analyze the regulation mechanism of HPG related genes on its early maturation. Based on the ESTs and SNPs of the related genes in the HPG axis from the 454 pyrosequencing in our research team, the correlations of these SNPs with early maturation of M. amblycephala will be analyzed using its artificial reproduction population in this study to identify the SNPs significantly related to its early maturation through DNA MassArray? Technology. Also, allelic expression of the correlated SNPs in the genome DNA and cDNA will be compared to evaluate whether the allelic expression imbalance is existed during the gonadal development. In the meantime, the mRNA expression level of the HPG axis related genes will be compared in the different gonadal development stages to evaluate the regulatory relationships of these genes and its effect on the gonadal development. As the KiSS/GPR54 system plays a key role in sexual precocity in mammals, the over-expression and RNA interference vectors of these two genes will be constructed and its effect on the HPG related genes mRNA and protein expression in M. amblycephala will be analysed. The results from the present study could offer certain evidence for the molecular mechanism of fish early maturation. The identified SNPs related to the early maturation of M. amblycephala would facilitate its selective breeding. This study will be found to have the important meaning on theory and practical breeding.

鱼类的性早熟是在人工养殖过程中出现并影响其生长速度的主要因素之一。团头鲂作为我国重要的草食性经济鱼类,在长期人工养殖过程中,性早熟现象明显。HPG轴相关基因的SNP已经被证实与哺乳动物的性早熟密切相关,其中KiSS/GPR54被认为是动物性成熟的关键基因。本研究拟基于课题组454测序获得的团头鲂HPG轴相关基因的EST序列以及SNPs位点,在人工繁育群体中分析基因SNPs与其性早熟的相关性,筛选与其性早熟相关联的SNPs。同时分析HPG轴相关基因在个体性腺发育不同阶段的表达水平及基因之间的调控关系,构建KiSS和GPR54基因的超表达和RNA干扰表达载体,在活体水平上检测相关基因的转录和蛋白表达变化,初步解析团头鲂性早熟的分子调控机理。本项目的研究结果可为鱼类性早熟的分子机制提供一定的理论依据,筛选到的团头鲂性早熟相关联的SNP标记可促进其非性早熟品种的选育工作,具有一定的理论和育种意义。

鱼类的性早熟是在人工养殖过程中出现并影响其生长速度的主要因素之一。团头鲂作为我国重要的草食性经济鱼类，在长期人工养殖过程中，性早熟现象明显。本研究(1)克隆获得了团头鲂Kiss2, Kiss2R,leptin,leptin-R,leprotl-1等基因的cDNA序列及部分基因的基因组DNA序列。其中Kiss2和Kiss2R 基因cDNA全长分别为623 bp和1176 b；两个基因的基因组DNA全长分别为791 bp和4775 bp，均包括2个外显子和1个内含子。Leptin,leptin-R和leprotl-1的cDNA全长分别为953 bp，3432 bp和1676 bp；三个基因的基因组DNA全长分别为1836 bp，28528 bp和5480 bp，分别包含3、15和4个外显子。(2)采用荧光定量PCR方法分析了这些基因在团头鲂雌雄性腺发育不同时期的表达量，初步解析了这些基因对团头鲂性早熟的调控作用。其中Kiss2/Kiss2R系统主要是通过增加团头鲂卵巢发育Ⅱ期时脑和垂体的表达量以及Ⅲ期精巢的表达量来发挥生殖启动作用。Leptin和leptinR基因在性腺组织中，I到IV期的表达量均较低，V期随之表达量增高后，在VI期出现峰值；而在脑组织III期的表达量显著性增高。leprotl1基因发挥着降低leptinR基因的表达，抑制leptin基因与leptinR在靶器官结合并发挥功能的作用。(3)采用候选基因SNP筛选法，筛选到与团头鲂性腺发育相关的SNP位点2个，同时对团头鲂2个亲本和198尾子代进行RAD测序，首次构建团头鲂高密度遗传连锁图谱，共包含24个连锁群，连锁群有效SNP位点达到5676个，标记间遗传距离为0.57 CM，获得了团头鲂生长、性腺发育性状以及性别相关QTL位点共12个。(4)通过构建家系分析评估了团头鲂1龄生长和性腺发育相关性状的遗传力及性状间的遗传和表型相关性。所有生长指标均有较高遗传力，而GSI遗传力较低。生长性状之间的遗传和表型相关值都较高，而性腺发育指数与净重间没有显著表型相关性，且遗传相关值也较低。项目研究结果可为鱼类性早熟的分子机制提供一定的理论依据,筛选到的团头鲂性早熟及生长相关QTL位点可促进其非性早熟品种的选育工作,具有一定的理论和育种意义。