分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

驴小肽转运载体1基因的克隆、序列分析及组织表达研究

  • 周苗苗 ,
  • 刘桂芹 ,
  • 刘文强 ,
  • 朱明霞 ,
  • 王长法
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  • 聊城大学农学院毛驴高效繁育与生态饲养研究院, 聊城 252000
周苗苗(1983-),女,山东东阿人,副教授,博士,从事草食动物营养研究。E-mail:zhoumm0329@163.com

收稿日期: 2020-12-30

  网络出版日期: 2021-07-06

基金资助

国家自然科学基金项目(31902177);山东省"泰山产业领军人才"项目(LJNY201713);聊城大学博士科研启动基金(318051816)

Cloning, Sequence Analysis and Tissue Expression of Donkey (Equus asinus) Oligopeptide Transporter 1 Gene

  • ZHOU Miaomiao ,
  • LIU Guiqin ,
  • LIU Wenqiang ,
  • ZHU Mingxia ,
  • WANG Changfa
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  • Liaocheng Research Institute of Donkey High-Efficiency Breeding and Ecological Feeding, College of Agriculture, Liaocheng University, Liaocheng 252000, China

Received date: 2020-12-30

  Online published: 2021-07-06

Supported by

 

摘要

小肽转运载体1(PepT1)在动物肠道小肽的摄取过程中发挥重要作用。本试验旨在研究驴(Equus asinusPepT1(ePepT1)基因的分子克隆、序列分析和组织表达。提取驴肠道组织总RNA,PCR克隆ePepT1基因片段,测序验证并进行序列分析;采用实时荧光定量PCR(real-time qPCR)方法分别测定驴肾脏、脾脏、心脏、肝脏、肺脏、胃、十二指肠、空肠和回肠组织中ePepT1基因的相对表达量。结果表明:ePepT1开放阅读框为2 124 bp,共编码707个氨基酸残基。蛋白质理化性质预测显示,ePepT1蛋白分子质量为78.6 ku,等电点为7.52,具有11个跨膜区(TMD),且TMD 9和TMD 10之间有1个大的细胞外环;ePepT1蛋白有5个胞外N-糖基化位点,5个胞外蛋白激酶C(PKC)作用位点和3个蛋白激酶A(PKA)作用位点。组织分布研究发现,ePepT1基因在各组织均有表达,但在肠道(十二指肠、空肠和回肠)中相对表达量最高。综上所述,本研究首次克隆了ePepT1基因,研究了其组织表达规律,为进一步研究驴肠道小肽吸收提供了基础。

本文引用格式

周苗苗 , 刘桂芹 , 刘文强 , 朱明霞 , 王长法 . 驴小肽转运载体1基因的克隆、序列分析及组织表达研究[J]. 动物营养学报, 2021 , 33(7) : 4030 -4037 . DOI: 10.3969/j.issn.1006-267x.2021.07.044

Abstract

Oligopeptide transporter 1(PepT1) plays an important role in the uptake of small peptides from animal intestine. The aim of this study was to study the molecular cloning, sequence analysis and tissue expression of PepT1 gene in donkey (Equus asinus) (ePepT1). Firstly, total RNA was extracted from donkey intestines, and ePepT1 gene fragment was cloned by PCR, sequenced and sequence analyzed. Then, total RNA was extracted from kidney, spleen, heart, liver, lung, stomach, duodenum, jejunum and ileum of donkey, and the relative expression level of ePepT1 gene was determined by real-time quantitative PCR. The results showed that the cloned ePepT1 cDNA sequence was 2 124 bp at full length encoding a 707 amino acids peptide with molecular of 78.6 ku and theoretical soelectric point (pI) of 7.52. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of ePepT1 identified. The ePepT1 had 11 potential transmembrane domains (TMD), with a large extracellular loop between TMD 9 and TMD 10. Five putative extracellular N-glycosylation sites, five putative extracellular protein kinase C (PKC) sites, and three protein kinase A (PKA) sites were identified in the ePepT1 sequence. The study of tissue distribution showed that ePepT1 gene was expressed in all tissues, but the relative expression level was the highest in intestinal tract (duodenum, jejunum and ileum). In conclusion, ePepT1 gene is cloned for the first time in this study, and its tissue expression is studied, which provides a basis for further study on the absorption of small peptide in donkey intestine.

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