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大白猪睾丸组织let-7c和miR-23a表达模式研究及表达载体构建

发表时间:2014-06-15  浏览量:1907  下载量:556
全部作者: 周佳伟,叶莲芝,罗立凡,官凯锋,马昌萍,夏宣炎,雷彬,李凤娥
作者单位: 华中农业大学农业部猪遗传育种重点开放实验室;华中农业大学理学院
摘 要: 研究扩增克隆let-7c与miR-23a,并获得其前体及成熟微小RNA(micro RNA, miRNA)序列信息。利用RT-PCR方法检测let-7c,miR-23a在大白猪35,60,90,180日龄不同发育时期睾丸组织的表达情况。结果发现:pri-let-7c在180日龄表达水平最高,而在60日龄几乎不表达,但是成熟let-7c在180日龄睾丸中显著低于其他日龄(P<0.05);pri-miR-23a在35日龄和90日龄表达量较高,而成熟miR-23a在90日龄表达量增加显著高于35日龄和180日龄(P<0.05)。通过RNAhybrid和RNA22预测到let-7c可能的靶基因精子粘附蛋白分子1(sperm adhesion molecule 1,SPAM-1)和miR-23a可能的靶基因TNF受体超家族6基因(TNF receptor superfamily member 6, FAS);构建插入pri-miRNA片段的plet-7c,pmiR-23a表达载体进行猪睾丸(swine testicular,ST)细胞转染实验。应用RT-PCR方法进行表达验证,获得相应的表达量信息。
关 键 词: 畜牧学;miRNAs;RT-PCR;猪;精子发生
Title: Expression patterns of let-7c and miR-23a in large white pig testes and miRNA expression vector construction
Author: ZHOU Jiawei, YE Lianzhi, LUO Lifan, GUAN Kaifeng, MA Changping, XIA Xuanyan, LEI Bin, LI Feng’e
Organization: Key Laboratory of Pig Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University; College of Science, Huazhong Agricultural University
Abstract: This study amplified and cloned let-7c and miR-23a, and obtained their precursor and mature sequences of miRNA. RT-PCR was used to analyze the expression pattern of let-7c and miR-23a in the porcine testes at age of 35, 60, 90, 180 d. The results showed that pri-let-7c had the highest expression at 180 d, but scarcely expressed at 60 d, while mature let-7c had a lower expression at 180 d than other age groups (P<0.05). pri-miR-23a highly expressed at 35 d and 90 d, while the mature miR-23a had a high expression at 90 d than at 35 d and 180 d (P<0.05). Using online RNAhybrid and RNA22, sperm adhesion molecule 1 (SPAM-1) and TNF receptor superfamily member 6 (FAS) genes were predicted to be the target genes of let7c and miR-23a, respectively. plet-7c and pmiR-23a expression vectors were constructed by inserting pri-miRNA fragment in, and then transfected pig swine testicular (ST) cells. RT-PCR was used for expression confirmation and obtained the corresponding expression information.
Key words: animal husbandry; miRNAs; RT-PCR; pig; spermatogenesis
发表期数: 2014年6月第11期
引用格式: 周佳伟,叶莲芝,罗立凡,等. 大白猪睾丸组织let-7c和miR-23a表达模式研究及表达载体构建[J]. 中国科技论文在线精品论文,2014,7(11):1102-1107.
 
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