【作者】 孙颖；

【导师】 林浩然；

【作者基本信息】 中山大学 ， 海洋生物学， 2016， 博士

【摘要】 石斑鱼(Epinephelus spp.)是我国东南沿海及东南亚地区主要的养殖经济鱼类之一。近年来,随着养殖规模急剧扩大,石斑鱼产业面临种质资源退化及孵化种苗不足等问题。杂交育种是解决种质资源退化的有效手段,迄今已获得至少8种具有不同优良性状的杂交石斑鱼。虎龙斑是棕点石斑鱼(Epinephelus fuscogutatus♀,俗称老虎斑)和鞍带石斑鱼(Epinephelus lanceolatus♂,俗称龙趸)杂交获得的新型杂交斑。具有父母双方的优点(生长快、抗病能力强),与老虎斑相比表现出显著的生长优势,是一种极有产业化前景的石斑鱼养殖品种。早前的研究主要集中在其生理学及遗传学特征等方面,而杂交石斑鱼生长优势相关的分子机理方面的研究仍然缺乏。为探究杂交虎龙斑生长优势的分子机制,本课题对这虎龙斑、龙趸和老虎斑进行了转录组测序和分析。分别选取1.5龄的这三个不同物种,提取脑、肝脏和肌肉组织RNA,分别进行转录组测序。主要的研究结果总结如下:通过对虎龙斑、龙趸和老虎斑的脑和肝脏组织进行转录组测序,将测序结果与斜带石斑鱼(Epinephelus coioides)(又名青斑)基因组比对,共获得3.23亿条高质量reads。在允许5个碱基错配情况下,6种组织转录组共有45.92%-61.26%的reads比对上参考基因组,其中28.57%-40.58%的reads比对上参考基因。我们对虎龙斑与龙趸和老虎斑进行比较转录组研究,在脑和肝脏中分别发现662和5,239个差异表达基因(differentially expressed genes,DEGs);GO富集分析揭示代谢过程和催化活性为两个最丰富的GO term。进一步深入分析发现,脑中Gn RH1和Gn RH3,肝中GH/IGF轴相关基因(包括IGF-1、IGF-2b、IGFBP-1,IGFBP-2,IGFBP-4和IGFBP-5a)在虎龙斑中都明显上调,这与虎龙斑的生长优势相吻合。此外,与蛋白质和糖原合成途径相关基因(例如PI3KC、PI3KR、Raptor、EIF4E3以及PP1)的表达上调,PYG的表达下调。这些变化可能促进虎龙斑的蛋白和糖原合成。此外,在脑中还发现了EIF4E-BP1和POMC(促黑素细胞皮质素原基因)以及其它的食欲相关信号分子的差异表达,包括AMPK、TSC1、m TOR、EIF4E-BP1、Ag RP、leptin、tachykinin和PYY,这些变化可能与杂交子一代的食欲增强有关。肌肉是GH和IGF1的靶器官。为了深入分析GH-IGF系统及其靶器官肌肉对虎龙斑生长优势贡献的调控机理,我们进一步对这虎龙斑、龙趸和老虎斑进行了肌肉转录组测序。测序结果与斜带石斑鱼基因组比对发现,约60.79%~62.43%的高质量reads比对到参考基因组,其中51.05%~55.71%的reads比对到基因区域,48.59%~53.27%的reads比对到参考基因。对糖酵解、钙离子通路、细胞增殖、分化和代谢等过程相关的基因在虎龙斑、龙趸和老虎斑中的差异表达分析表明,虎龙斑肌肉组织中催化糖酵解的10个酶中8个基因的表达水平表达上调,说明糖酵解通路被激活,进而与上游的GH/IGF系统共同促进肌肉发育。同时,我们发现在虎龙斑肌肉中,钙离子信号通路相关基因表达增强,肌钙蛋白(Tn C、Tn T和Tn I)的表达升高,说明该通路也被活化,有利于增强肌肉收缩,进而通过促进肌纤维的合成而促进生长。此外,我们还对影响基因表达的可变剪切进行了初步探究。在虎龙斑、龙趸和老虎斑的脑和肝脏中中发现了四种主要的可变剪切形式,包括5’端可变剪切(A5SS)、3’端可变剪切(A3SS)、外显子跳跃(ES)和内含子保留(IR)。其中IR在虎龙斑、龙趸、老虎斑脑组织中发生频率最高,说明IR在石斑鱼脑组织的基因功能调节中有重要作用。另外,虎龙斑、龙趸、老虎斑中肝脏的AS事件多于脑,提示在石斑鱼的脑和肝脏通过不同的方式调节基因的表达。脑倾向于通过AS事件调节基因功能,肝脏倾向于调节基因的表达水平。此外,我们还发现虎龙斑肝脏中的可变剪切事件及相关基因数目与老虎斑相似却低于龙趸。综上所述,我们首次报道了虎龙斑肌肉生长优势的可能分子机制。在虎龙斑中找到大量的差异表达基因,尤其分布在GH/IGF轴及其下游涉及蛋白与糖原合成的信号通路上。此外,肌肉中糖酵解、钙离子信号相关基因以及肌钙蛋白的表达上调,可能是虎龙斑生长优势的重要贡献因素。这些发现为研究虎龙斑生长优势的分子机制提供了数据基础,也有利于探讨其它鱼类生长优势的分子机制和调节通路。更多还原

【Abstract】 As a novel hybrid offspring crossed between Epinephelus fuscogutatus♀ and Epinephelus lanceolatus♂, Hulong grouper exhibits significant growth superiority comparing to Efu, which made it a promising cultivated species in grouper aquaculture industry in China. Although some research efforts have been made on studying its physiology and genetic characteristics, the molecular mechanism of its heterosis remains poorly understood.Based on RNA-Seq and gene expression profiling, we conducted comparative transcriptomic analysis among Hulong grouper, Epinephelus fuscoguttatus and Epinephelus lanceolatus.We identified a number of differentially expressed genes involved in the growth hormone/insulin-like growth factor(GH/IGF) axis and its downstream signaling pathways for protein and glycogen synthesis. The expressions of GnRH1 and GnRH3, as well as GH/IGF axis related genes, such as IGF-1, IGF-2b, IGFBP-1, IGFBP-2, IGFBP-4 and IGFBP-5a were significantly up-regulated in the hybrid grouper; and genes related to protein and glycogen synthesis, such as PI3 KC, PI3 KR, Raptor, EIF4E3, and PP1 were up-regulated, while PYG was down-regulated. These results might contribute to increased protein and glycogen synthesis in hybrid grouper. In brains, we also identified higher expression of EIF4E-BP1 and lower expression of POMC in hybrid grouper, which might contribute to enhance appetite of the hybrid grouper. All these results are in accordance with the growth superiority of hybrid grouper.Glycolysis is a downstream pathway for the GH/IGF axis in muscle. In our study, we first found that almost all glycolytic genes were up-regulated, which provided evidence for a possible role of the upstream GH/IGF pathway in muscle development by stimulating and activating the downstream glycolysis in the target tissue of muscle. Also, we found Ca2+ signaling is activated and troponins are up-regulated in the muscle of hybrid grouper, which suggests an active state of muscle contraction, which leads to fiber growth. Our data, at the first time, demonstrated that the up-regulated genes of glycolysis, Ca2+ signaling and troponins might account for the growth superiority in the hybrid grouper.In addition, we identified four main types of alternative splicing(AS) in the brain and liver of Hyb, Ela and Efu, and found more alternative splicing in the brain than in the liver. Meanwhile both the number of genes involved in alternative splicing and the number of alternative splicing events in the brain of hybrid grouper were higher than those in the other two groupers. In addition, many appetite associated signaling genes were found to be involved in althernative splicing, including GH, EIF4E-BP1, Agouti-related peptide(AgRP), leptin, peptide YY(PYY), mTOR, AMP-activated protein kinase(AMPK), Tachykinin and tuberous sclerosis proteins 1(TSC-1).All these differences, to some extent, shed a light on the growth superiority of hybrid grouper and provided insights into understating of molecular basis underlying the heterosis of hybrid grouper.更多还原