雌激素诱导的鸡MTTPL基因在肝脏脂质转运中的作用及表达调控机制

Microsomal triglyceride transfer protein (MTTP) is essential for the assembly and secretion of hepatic very low-density lipoproteins (VLDL) in mammals. However, this is not the case in chicken which also belong to amniotes. In previous studies of our research group, a novel gene MTTP-like was identified from chicken genome, which showed high homology with mammals MTTP and could play critical role on the transport of chicken hepatic VLDL. To systematically investigate the role of estrogen-induced chicken MTTPL gene in hepatic lipid transport and its regulatory mechanism of expression, the study was designed to 1) clarify the biological functions of chicken MTTPL by using the functional gain and loss strategy combined with in vivo experiments; 2) identify the critical transcription factor via the fragments deletion clone of promoter region combined with the dual-luciferase reporter system and CHIP technologies, which binds with the estrogen responsive element in the promoter region of MTTPL gene; and 3) determine the expressional characteristics of MTTPL modulated by miRNAs in the post-transcriptional level through bioinformatics analysis combined with the experimental validation. The study aimed to systematically investigate the biological function of estrogen-induced chicken MTTPL gene in hepatic lipid transport and its regulatory mechanism of expression, which will broaden the understanding of the theory of poultry hepatic lipid metabolism, and benefit the poultry industry including the improvement of production performance, and meat and egg quality.

微粒体甘油三酯转运蛋白（MTTP）是哺乳动物肝脏极低密度脂蛋白（VLDL）组装和分泌的必要因素，但同属羊膜动物鸡（禽类）MTTP在肝脏VLDL组装、分泌过程中未起关键作用。申请人前期研究发现鸡一个新基因类MTTP（MTTP-Like gene, MTTPL）与哺乳动物MTTP同源度高，且可能在鸡肝脏VLDL转运过程中起关键作用。为系统探究鸡MTTPL在肝脏脂质转运中的作用及表达调控机制，本研究拟：1）采用功能缺失或获得策略，通过在体实验阐释鸡MTTPL的生物学功能；2）采用启动子片段缺失克隆、双荧光素酶报告系统和CHIP技术，鉴定MTTPL应答雌激素的关键转录因子；3）采用生物信息学手段结合细胞学实验，鉴定靶向调控MTTPL基因表达的miRNA。本研究旨在阐释MTTPL在鸡肝脏VLDL转运中的作用及其表达调控机制，丰富禽类肝脏脂质代谢调控理论，为提高家禽生产性能和改善肉蛋品质提供理论依据。

课题组前期研究发现鸡类微粒体甘油三酯转运蛋白新基因（MTTP-Like, MTTPL）在肝脏脂质代谢中发挥重要作用，但其生物学功能和调控机制并不清楚。本项目首先构建了MTTPL启动子区域序列缺失表达载体，结合双荧光素酶报告系统试验，鉴定了MTTPL启动子的核心调控区域；采用过表达和干扰试验筛选出了与核心调控区域结合的雌激素应答调控元件，鉴定出调控MTTPL表达的关键转录因子PPARA和RXRA；其次，利用3´RACE技术克隆获得了MTTPL基因3´UTR全长序列，通过生物信息学分析结合双荧光素酶报告系统鉴定出与MTTPL基因3´UTR结合的miR-7439-3p，并阐明其靶向调控MTTPL表达的作用机制；然后，构建了MTTPL基因的腺相关病毒干扰载体，利用外源雌激素诱导MTTPL表达，进一步验证了鸡MTTPL在肝脏脂质代谢中的作用。综合以上研究结果，系统阐明了鸡MTTPL基因在肝脏脂质代谢中的作用及其表达调控机制，丰富了家禽肝脏脂质代谢调控理论，并为特色地方鸡选育提供了新思路。