影响槟榔江水牛乳脂性状重要功能SNP的鉴定

Water buffalo milk contains a high level of milk fat content and the characteristics of buffalo milk fat are obvious in composition. However, the genetic mechanism of buffalo milk fat trait is unclear. Binglangjiang buffalo has high value in dairy breeding, which is the first indigenous river buffalo found in Yunnan province of China in recent years. This project aims to dissect the molecular genetic basis of buffalo milk fat trait from the mammary gland which is the important organ of milk secretion by taking the purebred breeding core herds of Binglangjiang buffalo as the research object, which will be divided into two groups, namely, the higher milk fat group and the lower milk fat group according to the level of milk fat content. Firstly, we will performed a transcriptome sequencing and the corresponding data analysis on the pooled breast tissue samples from three different stages of lactation (15, 90 and 250 d relative to parturition) of the previous two groups, then screening differentially expressed genes between the two groups from which choose the important functional candidate genes related to milk fat trait. To determine the functional genes affecting milk fat yield and composition, we will further verify the function of candidate genes in vitro by RNA interference (RNAi) and gene over-expression technology. Finally, we intend to identify the functional SNPs which influence the milk fat trait of water buffalo over the SNPs screening of functional genes, EMSA experiment detection and the association analysis of large sample data between the genotypes of the SNP loci and the milk fat trait. This project will ultimately unravel the key genes and their regulatory networks related to milk fat metabolism in water buffalo, reveal its molecular genetic basis of high milk fat performance and will provide a basis for milk fat trait breeding of dairy buffalos.

水牛奶乳脂含量高，在乳脂组成上特征明显，但水牛乳脂性状的遗传机制尚不清楚。槟榔江水牛是近年在云南发现的我国第一个本土河流型水牛，具有较高的种用价值。本项目拟以该水牛繁育核心群为研究对象，按照乳中乳脂含量高低，将其分成高乳脂和低乳脂含量组，从泌乳的重要器官--乳腺入手，针对水牛乳脂性状的分子遗传基础开展研究。先采用RNA-Seq技术对上述两组水牛3个不同泌乳时期的乳腺组织样品池进行转录组测序分析，筛查两组水牛间的差异表达基因，从中选出影响乳脂性状的功能候选基因，并通过RNAi和基因过表达技术对其功能进行体外验证，确定影响水牛乳脂性状的重要功能基因；进一步通过对功能基因SNP的筛选及SNP位点与乳脂性状的大样本关联分析、EMSA实验，鉴定出影响水牛乳脂性状的功能SNPs。最终阐明影响水牛乳脂代谢的关键基因和调控网络，从分子水平上揭示水牛高乳脂特性的遗传基础，为奶用水牛乳脂性状的选育提供依据。

本项目在前期研究工作的基础上，以槟榔江水牛的纯种繁育核心群为研究对象，从泌乳的重要器官——乳腺入手，对水牛乳脂性状的分子遗传基础开展了研究。先采用RNA-Seq技术对槟榔江水牛高乳脂和低乳脂含量组3个不同泌乳时期的乳腺组织样品进行了转录组测序分析，筛查了两组水牛间的差异表达基因，从中选出影响乳脂性状的功能候选基因，并采用功能生物信息学、基因表达谱分析、Western blot技术及采用体外细胞水平的RNAi和基因过表达方法对上述基因进行了功能验证和功能研究，确定了影响水牛乳脂性状的重要功能基因及其相互作用；进一步通过对功能基因SNP的筛选及SNP位点变异与乳脂性状的大样本关联分析，鉴定出了影响水牛乳脂性状的功能SNP。通过项目的研究工作，获得水牛乳腺组织三个泌乳时期高、低脂肪组的转录组数据22份；阐明了水牛不同泌乳时期高、低乳脂组间及不同泌乳时期之间的差异表达基因，揭示了水牛乳脂性状基因调控的路径和遗传基础。筛选出影响水牛乳脂性状的关键候选基因74个；对筛选出来的槟榔江水牛32个乳脂性状功能候选基因进行了功能验证，揭示了它们的功能及它们之间的相互作用及调控机制。同时，对水牛DGAT1、GPRHBP1基因进行了选择性剪接研究，确定了它们在乳腺中表达的不同转录本，揭示了不同转录本的功能；阐明了水牛乳脂性状29个功能基因SNP位点的群体遗传特征及单倍型类型，揭示出水牛SNP位点的分布模式与其他牛科物种存在明显差异，获得了16个基因组上对水牛乳脂性状具有重要育种价值的SNP位点。本研究结果从分子水平上揭示了影响水牛乳脂性状的关键基因和调控路径，对阐明水牛乳脂性状的遗传机制具有重要理论意义。本项目研究筛选到的与乳脂性状相关的功能基因或功能SNP对水牛育种具有重要应用价值，可应用到水牛的分子育种中，将为我国奶用水牛特别是槟榔江水牛乳脂性状的选育提供依据。