JAK2-STAT3介导瘦素调控草鱼肠道PepT1转运小肽的作用及机制研究

The oligopeptide transporter (PepT1) plays a crucial role in transporting of dietary di-/tri-peptides in fish intestines. Our preliminary studies revealed that leptin was involved in PepT1-mediated di-/tri-peptides transport of grass carp which could be regulated by Janus kinase 2-signal transducer and activator of transcription 3 (JAK2-STAT3) signaling pathway. However, their related regulatory effect and mechanism remain mysterious and need further research. Therefore, this project will identify JAK2-STAT3 signaling pathway and analyze its response relation with leptin in grass carp intestine through multiple cellular and molecular techniques to clarify the function and mechanism of leptin in regulating intestinal JAK2-STAT3 signaling pathway. By studying regulatory effect of transcription factor STAT3 on PepT1 promoter activity, we will further reveal the regulatory mechanism of intestinal JAK2-STAT3 signaling pathway in regulating intestine PepT1 transporting oligopeptide in leptin-stimulated grass carp. Moreover, the response relation and molecular mechanisms among the grass carp leptin, intestinal JAK2-STAT3 signaling pathway and PepT1 transporting oligopeptide will be clarified by in vivo experiments through nutrition regulatory approches. Collectively, the results obtained from the present studies will elucidate the molecular mechanism of the leptin participated in regulation of PepT1 function in oligopeptide transport of grass carp mediated by JAK2-STAT3 signaling pathway, and it will also help for improving high-efficient utilization of fish transporting oligopeptide and dietary protein, reducing nitrogen pollution to the environment and formulating new environmental protection feed.

小肽转运载体1（PepT1）在鱼类肠道小肽转运过程中发挥着重要的作用。我们前期研究发现，JAK2-STAT3信号通路可介导瘦素影响草鱼肠道PepT1转运小肽，但其调控作用及机制尚不清楚。因此，本项目拟采用细胞和分子生物学技术，鉴定草鱼肠道JAK2-STAT3信号通路，分析其对瘦素的响应关系，阐明瘦素调控肠道JAK2-STAT3信号通路的作用及机制；研究转录因子STAT3对PepT1基因启动子活性的调控规律，揭示瘦素刺激下肠道JAK2-STAT3信号通路对PepT1转运小肽的调控机制；进而利用生长实验，采用营养调控技术，研究草鱼瘦素水平、肠道JAK2-STAT3通路活性、肠道PepT1转运小肽之间的响应关系及机制。最终阐明JAK2-STAT3介导瘦素调控草鱼肠道PepT1转运小肽的调控作用及分子机制，为提高鱼类肠道小肽转运及饲料蛋白吸收，减少氮对环境污染，配制高效环保饲料奠定理论基础。

研究表明，小肽转运载体1（PepT1）在鱼类肠道小肽转运过程中发挥着关键调控作用，然而其是否受到体内瘦素调节，其作用机制和分子调控网络是什么尚不清楚。本项目以草鱼为研究对象，1）利用现代分子技术鉴定了草鱼肠道瘦素信号通路JAK2-STAT3关键基因（LEPR、JAK2等）序列，生物信息学方法分析了基因的分子特征，利用离体实验和在体实验研究了草鱼肠道JAK2-STAT3通路基因在瘦素刺激下的表达水平变化规律，明确了草鱼肠道JAK2-STAT3信号通路与瘦素之间的响应关系，阐明了瘦素对草鱼肠道JAK2-STAT3信号通路的调控作用；2）人为干预JAK2通路活性，利用实时荧光定量PCR检测瘦素刺激下草鱼肠道PepT1表达变化规律，阐明草鱼肠道JAK2对PepT1的表达调控作用；利用染色体步移技术克隆PepT1基因上游调控序列，构建pGL3-PepT1-promoter报告基因载体，分析草鱼JAK2过表达后对PepT1启动子的激活作用，阐明JAK2对PepT1基因表达的转录调控作用机制，从而揭示肠道瘦素信号通路调控PepT1转运小肽功能的分子机制；3）利用生长试验，在草鱼基础饲料中添加不同剂量的大豆异黄酮，检测蛋白质效率、特定生长率等生理生化和生产性能指标，以及草鱼肠道PepT1、JAK2-STAT3信号通路基因表达等分子指标，研究体内瘦素变改变时，肠道JAK2-STAT3信号通路和PepT1转运小肽的变化规律及机理。配制不同蛋白水平和蛋白源饲料，利用生长试验，研究肠道PepT1转运小肽功能改变时，体内瘦素、JAK2-STAT3信号通路的变化规律及机理，阐明草鱼瘦素、肠道JAK2-STAT3信号通路、肠道PepT1转运小肽的响应互作关系及机制。本项目研究成果将为提高鱼类肠道小肽转运及饲料蛋白吸收，减少氮对环境污染，配制高效环保饲料奠定理论基础。