下丘脑关键LncRNA对肉牛剩余采食量的调控解析

Feed intake difference related to feed efficiency, has direct effects on production benefits. The hypothalamus is a critical center that regulates animal feed intake and maintains energy metabolism balance. Recently, an increasing number of studies have shown that LncRNA act as a regulators at different levels of gene expression including chromatin organization, (post)-transcriptional regulation control. Considering the regulation mechanism of LncRNAs to their target genes and the central regulatory effects of hypothalamus on animal’s feed intake behavior and feed intake differences, this project planned to screen and identify the key LncRNAs related to regulation of cattle residual feed intake (RFI) by using techniques of RANi, Pull-down, RIP, RACE and Western-blot etc. after we has obtained the miRNA data from different RFI cattle. And the cellular experiments will be used to confirm the regulation of LncRNAs to their target genes and miRNA. The aim of this study is to elucidate the mechanism of LncRNA from hypothalamus in the regulation of feed intake behavior and residual feed intake difference in cattle. The results of this project are of great significance for revealing the feed intake phenotype by non-coding RNA, as well as helpful for saving feed cost and manipulating feed intake in beef cattle.

采食量差异关系到肉牛饲料利用效率，并影响养殖经济效益。下丘脑是调控动物采食和维持能量代谢平衡的调控中枢。越来越多的研究表明，LncRNA作为调节因子参与基因表达调控的各个层次，在表观遗传及转录（后）调控等方面有着广泛的调控功能。鉴于LncRNA 对靶基因的介导机制以及下丘脑在动物采食行为中的中枢调控，在前期获得牛miRNA数据基础上，本项目拟从下丘脑入手，借助转录测序并结合RNAi、Pull-down、RIP、RACE、Western-blot等技术筛选和鉴定调控肉牛剩余采食量(RFI)差异的LncRNA分子，并在细胞水平靶向验证目的LncRNA对靶基因的调控作用及其与miRNA的互作关系，部分阐明LncRNA通过动物下丘脑中枢对动物采食行为及采食量差异调控的分子机制。本项目研究结果对于从非编码RNA分子角度揭示肉牛采食量差异具有重要意义，为节约饲养成本及人工调控肉牛采食量提供科学依据。

饲料效率的提高对于家畜生产盈利和可持续发展至关重要，人工选育低RFI（高效率）畜禽个体可加快饲料利用效率的遗传进展和维持重要经济性状遗传水平。为探究影响秦川牛饲料利用效率的分子遗传机理，本课题开展以下试验：（1）不同RFI水平肉牛下丘脑转录组差异基因与调控通路分析，筛选出AGRP、PMCH和MTNR1A等与RFI相关采食中枢基因及其富集通路，基因功能验证，将为进一步解释动物采食行为、RFI变异提供参考信息；（2）不同RFI水平肉牛下丘脑转录组差异LncRNA及其靶基因调控通路分析，通过联合分析，确定了TCONS_00226900及其靶基因MTNR1A作为候选分子；（3）基于RFI 水平肉牛下丘脑差异 circRNA 及其靶基因富集通路分析，差异circRNA及其靶向结合的bta-miR-103、bta-miR-33a/b、bta-miR-499等为后续进一步探索circRNAs参与牛RFI的调控机制提供数据支持；（4）基于脑肠轴的数据联合分析，获得9个脑肠轴关键基因，富集通路与采食与能量代谢相关的有 Rap1 信号传导途径、MAPK PI3K-Akt等信号传导途径，为脑肠轴调控RFI提供理论支持；（5）关键LncRNA共表达靶基因MTNR1A多态位点与剩余采食量的关联分析；（6）RFI 相关下丘脑SNP及可变剪切分析，差异 AS 基因可能通过能量代谢相关的信号通路和影响食欲的通路来调节能量代谢，进而参与肉牛采食量性状，为肉牛饲料利用效率相关的遗传学基础提供理论支撑；（7）目的LncRNA介导上下游靶标分子的功能分析，仍在进行中。本课题针对剩余采食量初步研究为后续挖掘动物采食、能量代谢、机体生理活动研究奠定了理论基础。饲料利用效率相关分子标记可在群体验证的基础上对节约家畜养殖成本，开发新饲料及培育高饲料效率的新品种（系）提供坚实基础。