槟榔江水牛乳蛋白性状关键基因的筛选及其功能研究

Because of its high milk protein content and outstanding characteristics of protein composition, water buffalo milk is an ideal raw material to manufacture high-grade dairy products. However, the key functional genes which affect buffalo milk protein traits and their genetic mechanism are unclear. Binglangjiang buffalo with high milk production has high value in dairy breeding, which is the first indigenous river buffalo found in Yunnan province of China in recent years. In this study, two groups of water buffalo with extremely high and low phenotypic values of milk protein content, namely, the higher milk protein group and the lower milk protein group, will be selected from the purebred breeding core herds of Binglangjiang buffalo according to the level of milk protein content. To identify candidate genes and candidate miRNA affecting the buffalo milk protein traits, a transcriptome sequencing (RNA-seq) and a high throughput miRNA sequencing (miRNA-seq) will be performed to generate the data of transcriptome and miRNA expression profiles using the mammary gland tissues sampled from the above-mentioned two groups. By a integrated data analysis of RNA-seq and miRNA-seq, we will identify the critical functional genes and construct functional gene network of influencing buffalo milk protein traits. Furthermore, the experiments of basic functions, expression and regulation of the key functional genes will be conducted to elucidate the expression regulation mechanism and interaction gene network of the key genes affecting buffalo milk protein traits by means of the gene cloning, qRT-PCR, gene over-expression, RNAi, immunofluorescence and Western blot techniques. This project will ultimately reveal the molecular genetic basis of high milk protein traits and will provide a basis for milk protein trait breeding of dairy buffalo.

水牛奶乳蛋白含量高，且其组成成分特征明显，是加工高档乳制品的理想原料，但水牛乳蛋白性状的关键功能基因和遗传机制尚不清楚。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。本项目拟按照乳中乳蛋白含量的高低，从该水牛纯种繁育核心群中选出两极端个体，组成高乳蛋白和低乳蛋白含量组，取乳腺组织进行转录组和高通量miRNA测序分析，筛选影响水牛乳蛋白性状的候选基因和候选miRNA，再整合分析转录组和高通量miRNA分析结果，鉴定出影响水牛乳蛋白性状的关键功能基因并构建该性状的功能基因调控网络，进一步通过基因克隆、qRT-PCR、基因过表达、RNAi抑制表达、免疫荧光、Western blot等技术对筛选出的关键基因进行基本功能及对乳蛋白合成调控的研究，阐明影响水牛乳蛋白性状的关键基因表达调控机理和互作基因网络，揭示水牛高乳蛋白特性的遗传基础，为奶水牛乳蛋白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，对水牛乳蛋白性状的分子遗传基础开展了研究。先采用RNA-seq、miRNA-seq及两者的联合分析技术对槟榔江水牛泌乳高峰期高乳蛋白和低乳蛋白含量组及泌乳前期、泌乳中期和泌乳后期组的乳腺组织样品进行了转录组测序分析，筛查了各组水牛间的差异表达基因及差异表达的miRNA，进一步通过GO和KEGG分析，确定了影响乳蛋白合成的重要功能候选基因和关键miRNA，再采用生物信息学、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转录调控研究，并对miRNA对靶基因的调控作用进行了实验研究，确定了影响水牛乳蛋白性状的重要功能基因、miRNA及其相互作用。通过项目的研究工作，获得槟榔江水牛泌乳高峰期高、低乳蛋白组及泌乳初期、泌乳中期和泌乳后期组乳腺体细胞的RNA-seq、miRNA-seq转录组测序数据各12份；阐明了水牛上述各组之间的差异表达基因，揭示了水牛乳蛋白性状基因调控的路径和遗传基础。筛选出影响水牛乳蛋白性状的关键候选基因22个、关键功能miRNA 20个；对筛选出来的槟榔江水牛10个乳蛋白性状功能候选基因进行了功能验证或转录调控研究，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，对筛选出的3个miRNA进行了功能研究，揭示它们可通过对共同靶基因PTHLH的调控，对水牛乳蛋白的合成发挥负调控作用。本研究结果从分子水平上揭示了影响水牛乳蛋白性状的关键基因、miRNA和调控路径，对阐明水牛乳蛋白性状的遗传机制具有重要理论价值。本项目研究筛选到的与乳蛋白性状相关的功能基因或miRNA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。