黄姑鱼耐寒相关基因和分子标记的筛选、精细定位及其作用机制研究

In recent years, the winter cold wave hit fish farming industry in China and caused serious damage. Nibea albiflora is an important farmed fish species in Zhejiang and Fujian Province. However, aquaculture of this kind of fish is particularly harmed by the cold wave. Therefore, identifying the cold tolerance-related genes and functional molecular markers and carrying out molecular design breeding is urgent in N. albiflora. However, genes controlling cold tolerance traits and their regulatory mechanism are still in infancy. Meanwhile, the molecular markers that can be used for molecular breeding is quite scare. This project aims to identify and characterize cold tolerance-related genes and molecular markers using transcriptomic, genomic and functional genomic analysis technologies, to elucidate the molecular mechanisms underlying the differences for cold tolerance traits in N. albiflora. Using transcriptome sequencing, gene expression profiles responding to cold stress are established to screen out and clarify the candidate genes which are regulated by cold acclimation. Using RAD sequencing technology, the high density genetic linkage map is constructed to map quantitative trait loci (QTL) for cold tolerance, and herein to determine the important cold tolerance-related genes and molecular markers. Furthermore, functional genomic analysis is used to understand the functional mechanisms of these genes and genetic markers that regulate and control cold tolerance traits. The results of current study not only provide a theoretical basis and technical support for genetic breeding in N. albiflora, but also enhance molecular breeding technology of marine fishes and hereby promote the process of breeding new varieties with high yield and quality in marine fish.

近年来，冬季寒潮侵袭给我国鱼类养殖业造成了严重的损失。黄姑鱼是浙、闽沿海重要的养殖鱼类，养殖业受寒潮危害尤为严重；因此，筛选鉴定耐寒（或耐低温）的重要基因和功能标记，进行分子设计育种显得十分迫切。但是关于养殖鱼类控制耐寒的基因及其调控机制尚不明确，可用于育种的分子标记十分匮乏。本项目旨在通过转录组学、基因组学和功能基因学的研究手段逐级筛选控制耐寒性状的重要基因和分子标记，逐步揭示黄姑鱼耐寒差异的分子机制。利用转录组测序技术构建黄姑鱼低温应答的差异基因表达谱，找出并明确与寒冷适应相关的重要候选基因；利用RAD测序技术构建高密度遗传连锁图谱并进行耐寒相关的QTL精细定位，确定耐寒的重要功能基因和分子标记，进一步利用功能基因组学分析探明其调控黄姑鱼耐寒的作用机理。本研究成果不仅为黄姑鱼耐寒优良品种的培育提供理论依据和技术手段，也将提升海水鱼类的分子育种技术水平，促进我国海水鱼类的良种化进程。

本项目以黄姑鱼为研究对象，针对养殖鱼类控制耐寒的基因及其调控机制尚不明确，可用于育种的分子标记十分匮乏等问题，利用转录组学、基因组学和功能基因组学筛选了黄姑鱼耐寒相关的基因和分子标记，定位了耐寒相关标记的功能区域，阐述了耐寒相关基因的低温应答模式，而且初步研究了其在低温适应中的调控作用。首先，发现黄姑鱼低温死亡温度与低温胁迫时间以及初始温度存在相关性，确定6.5℃为评估黄姑鱼耐低温能力的适宜温度；明确低温胁迫对黄姑鱼生长抑制、肝脏损伤以及Na+/-K+-ATP酶以及免疫相关酶的抑制作用；其次，绘制了高质量高精度的参考基因组，构建了黄姑鱼高密度遗传连锁图谱，覆盖全基因组15,686个SNP，通过耐低温试验筛分了温度敏感/耐受个体，分析了低温耐受/敏感与SNP的相关性，筛选了耐寒相关的SNP位点，并将相关的SNP位点定位到连锁群LG19；同时，通过转录组和代谢组学筛选了出黄姑鱼耐寒相关的候选基因和信号通路，系统分析了重要的候选基因如nefm、CRIP、hsp70以及APO-AⅠ的表达模式，明确其在低温应答中的作用。本项目从基因组、转录组、代谢组以及功能基因组水平，筛选了黄姑鱼耐寒相关的基因和分子标记，阐述相关基因和标记在低温适应的调控作用，初步建立了黄姑鱼耐寒的分子选育技术，相关研究工作有些在黄姑鱼中甚至鱼类中首次开展，将有助于鱼类耐低温的分子机制的全面解析，并可以指导黄姑鱼等石首鱼类的分子育种研究和生产。