Wei RongBin, Liu Tao, Zhou ChaoWei, Zhang XingDong, Yuan DengYue, Wang Tao, Lin FangJun, Chen Hu, Wu HongEei, Li ZhiQiong
Department of Aquaculture, Sichuan Agricultural University, Ya'an 625014, PR China.
Regul Pept. 2013 Sep 10;186:18-25. doi: 10.1016/j.regpep.2013.07.002. Epub 2013 Jul 11.
Ghrelin is an important gastrointestinal hormone involved in the regulation of feeding in both mammals and fish. In this study, the preproghrelin cDNA sequence was cloning in the gut of Schizothorax prenanti (S. prenanti). The preproghrelin gene, encoding 103-amino acids, was strongly expressed in the gut and brain using real-time quantitative RT-PCR (qPCR). The S. prenanti preproghrelin was detected in embryonic developmental stages. Further, it was detectable in unfertilized eggs, suggesting that ghrelin could be classified as maternal mRNA. An experiment was conducted to determine the expression profile of ghrelin during post-feeding and fasting status of the brain and gut. The results revealed a significant postprandial decrease in ghrelin mRNA expression in the gut 6h post-feeding (hpf) and brain (1.5 and 9hpf) compared to an unfed control group, indicating that food intake and processing affect the regulation of expression of ghrelin in S. prenanti. The constructed recombinant plasmid pMD-19T-ghrelin was transformed to Escherichia coli BL21 and induced with IPTG, and the expressed product was identified by SDS-PAGE. The prokaryotic expression vector for ghrelin was constructed successfully, and fusion protein was expressed in E. coli BL21, which laid the foundation for the further study on the function of this protein and its mechanism. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin in S. prenanti. Further studies are required to identify the tissue specific functions of ghrelin in S. prenanti.
胃饥饿素是一种重要的胃肠激素，参与哺乳动物和鱼类的摄食调节。在本研究中，克隆了齐口裂腹鱼肠道中的前胃饥饿素cDNA序列。通过实时定量RT-PCR（qPCR）检测发现，编码103个氨基酸的前胃饥饿素基因在肠道和脑中强烈表达。在齐口裂腹鱼胚胎发育阶段检测到了前胃饥饿素。此外，在未受精卵中也可检测到，这表明胃饥饿素可归类为母体mRNA。进行了一项实验，以确定在摄食后和禁食状态下脑和肠道中胃饥饿素的表达谱。结果显示，与未喂食的对照组相比，喂食后6小时（hpf）肠道和脑（1.5和9hpf）中胃饥饿素mRNA表达在餐后显著下降，这表明摄食和食物处理会影响齐口裂腹鱼中胃饥饿素表达的调节。构建的重组质粒pMD-19T-胃饥饿素转化至大肠杆菌BL21并用IPTG诱导，表达产物通过SDS-PAGE鉴定。成功构建了胃饥饿素的原核表达载体，融合蛋白在大肠杆菌BL21中表达，为进一步研究该蛋白的功能及其机制奠定了基础。总体而言，我们的结果为胃饥饿素在齐口裂腹鱼中高度保守的结构和生物学作用提供了证据。需要进一步研究以确定胃饥饿素在齐口裂腹鱼中的组织特异性功能。
