基于RNA-Seq与microRNA表达谱探讨杂色鲍杂种优势分子机理

Heterosis, also known as hybrid vigour, is complex biological phenomenon and has been widely utilized in aquatic animals. Many researches based on physiological and biochemical indices, molecular markers and QTL has been conducted in fish and shellfish species, but the molecular bases for this phenomenon remain elusive. Recent studies in heterosis using transcriptomic, proteomic, metabolomic, epigenomic and systems biology approaches have provided new insights in plant and animals. "Dongyou No.1 " small abalone (Haliotis diversicolor), which was hybrided between two selective lines, was conferred as the New Variety Certification by Ministry of Agriculture for its high survival rate. In this research, transcriptomic analysis for the "Dongyou No.1" and its parental lines challenged with bacteria will be studied. Based on the high-throughput RNA sequencing and bioinformatic methods, differentially expressed genes and allele-specific gene expression among the three groups will be analyzed, while some key immunology-related genes will be cloned and functional verified. Based on small RNA deep sequencing, microRNA for small abalone will be mined for the first time in abalone species, while differentially expressed profile of miRNA will be analyzed among different groups. Transcriptomic and epigenetic insights into the molecular bases of heterosis on small abalone will be studied in this project, and the conceptual advances will has an important significance on molecular breeding of abalone.

杂种优势是普遍存在的一种复杂生物学现象，在水产动物的生产实践中已获得广泛应用，虽然人们已从生理生化、分子标记和QTL定位等多个方面开展了相关研究，但对其形成机理的研究迄今尚未明晰。随着二代测序技术的发展，在作物和畜牧育种领域，越来越多地采用组学和表观遗传学技术来探讨杂种优势的分子遗传机理，并取得了重要进展。"东优1号"杂色鲍是采用选育系间杂交而培育的具有高存活率特性的国家级水产新品种。本研究拟通过构建"东优1号"杂色鲍及其双亲群体病菌胁迫条件下的转录组文库，利用RNA-Seq技术及生物信息学分析，分析基因表达水平和等位基因表达方式的差异，并对若干关键基因的功能进行生物学验证。同时，通过小RNA测序挖掘杂色鲍的microRNA，并对三个群体病菌胁迫下miRNA的差异表达特性进行分析。本研究分别从基因的转录水平与表观遗传调控水平对杂色鲍杂种优势分子机理进行研究，有望对鲍的分子育种奠定理论基础。

“东优1号”杂色鲍是以杂色鲍台湾群体为母本、杂色鲍日本群体为父本的杂交鲍新品种，表现出高于亲本的越冬养殖存活率。目前，该新品种已在生产上得到广泛的推广和应用，但对其高存活率杂种优势的生理基础和分子机制，尚缺乏较为深入的了解。本研究结合生理学实验和分子生物学手段，采用转录组、全基因组重硫酸盐甲基化测序和小RNA组测序等多种方法对其杂种优势形成机制进行了全面分析。本项目首先综合多个生理指标参数，并采用熵值法和隶属函数法，建立了鲍耐低温和抗病综合评价体系，以综合满意度代表鲍的综合耐低温和抗病能力。经计算，杂交种在这两个指标上都显著优于亲本群体。随后利用转录组高通量测序技术，分析和比较了杂色鲍3群体在低温胁迫和病原菌胁迫过程中的转录本变化，结果显示，低温胁迫下杂交种的整体基因表达模式更偏向父本，而与母本差异较大；对显性基因的功能进行进一步的分析，发现这类基因包括与鲍耐低温相关的脂质代谢、跨膜转运蛋白、抗氧化、低温保护分子、表观遗传修饰的相关基因。病原菌胁迫下杂交种的整体基因表达模式同样偏向父本，而异于母本。对在杂交种中呈显性表达的基因进行功能分析，发现这些基因主要负责细胞吞噬、病原清除、抗氧化和营养免疫等生理功能。全基因组重硫酸盐甲基化测序和小RNA组测序结果也表明，杂交种与亲本群体存在明显的全基因组甲基化修饰率的差异，以及明显的小RNA整体表达水平的差异。结合转录组分析的结果，推测杂交种中受表观修饰调控的基因的转录活动更为活跃；对转录组中鉴定出来的显性表达基因在杂色鲍3群体中的甲基化修饰状态进行分析，发现其中19.50%~29.58%的基因存在甲基化修饰水平的差异，说明甲基化修饰差异是造成杂色鲍三群体基因表达水平差异的重要原因。