[1]张久盘,宋雅萍,姜超,等.牛LATS2基因启动子克隆及转录调控分析[J].江苏农业学报,2023,(03):753-761.[doi:doi:10.3969/j.issn.1000-4440.2023.03.016]
　ZHANG Jiu-pan,SONG Ya-ping,JIANG Chao,et al.Promoter cloning and transcriptional regulation of bovine LATS2 gene[J].,2023,(03):753-761.[doi:doi:10.3969/j.issn.1000-4440.2023.03.016]
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牛LATS2基因启动子克隆及转录调控分析()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2023年03期
页码:
753-761
栏目:
畜牧兽医·水产养殖
出版日期:
2023-06-30

文章信息/Info

Title:
Promoter cloning and transcriptional regulation of bovine LATS2 gene
作者:
张久盘1;  宋雅萍2;  姜超2;  王锦1;  魏大为2
(1.宁夏农林科学院动物科学研究所，宁夏银川750002；2.宁夏大学农学院，宁夏银川750021）
Author(s):
ZHANG Jiu-pan1; SONG Ya-ping2; JIANG Chao2; WANG Jin1; WEI Da-wei2
（1.Institute of Animal Science, Ningxia Academy of Agricultural and Forestry Sciences, Yinchuan 750002, China;2.School of Agriculture, Ningxia University, Yinchuan 750021, China）
关键词:
牛; LATS2基因; 组织表达; 启动子; 转录调控
Keywords:
cattle; LATS2 gene; tissue expression; promoter; transcriptional regulation
分类号:
S823.8+1
DOI:
doi:10.3969/j.issn.1000-4440.2023.03.016
文献标志码:
A
摘要:
本研究旨在探究牛LATS2基因组织表达规律，利用相对荧光素酶活性数值确定其启动子核心区域并初步鉴定其核心区域关键转录因子，以阐明牛LATS2基因的转录调控机制。利用RT-qPCR检测牛LATS2基因在心、脾、肝、肾、肺、背最长肌、皮下脂肪、皱胃、大肠及睾丸等中的相对表达量，构建LATS2蛋白进化树。克隆LATS2基因5′端非翻译区上游1.7 kb序列，利用逐段缺失引物，巢式扩增其7个启动子区不同截断体缺失片段（-1 792～+179、-1 475～+179、-1 098～+179、-727～+179、-515～+179、-248～+179和-56～+179），并将不同截断体构建至双荧光素酶报告载体pGL3-Basic上。重组的LATS2基因启动子双荧光素载体分别转染小鼠成肌细胞（C2C12)和小鼠脂肪细胞（3T3-L1）细胞系，鉴定其启动子核心区域。进一步借助在线软件JASPAR（http://jaspar.genereg.net/）和Genomatix（http://www.genomatix.de/cgi-bin//mat-inspector）分析启动子核心区域序列特征，并预测关键转录因子结合位点。结果显示，LATS2基因在肝和背最长肌中表达量极显著高于脾(P<0.01)；LATS2蛋白构建的进化树显示反刍动物单独聚为1支，表明LATS2基因在反刍动物进化过程中保守性较高；蛋白质互作分析筛选出的与LATS2蛋白互作紧密的前10种蛋白质均为Hippo信号通路中的关键蛋白质。LATS2基因启动子核心区域位于-248～-56，预测其启动子核心区域有与肌肉发育相关的转录因子TEAD1、MEF2A、FOSL1、MyoG和Myod1的结合位点，表明LATS2基因在牛肌肉生长发育中扮演重要角色。以上结果为探究牛LATS2基因在肌肉生长发育中转录调控机制奠定基础。
Abstract:
The purpose of this study was to explore the tissue expression of bovine LATS2 gene, and identify its core promoter region and key transcription factors, so as to clarify the transcriptional regulation mechanism of bovine LATS2 gene. The relative expression levels of bovine LATS2 were detected in heart, spleen, liver, kidney, lung, longissimus dorsi muscle, subcutaneous fat, abomasum, large intestine and testis by RT-qPCR, and the evolutionary tree of LATS2 protein was constructed. The 1.7 kb sequence upstream of the 5′-untranslated region of LATS2 gene was cloned, and the promoter sequence regions of seven segments with -1 792-+179, -1 475-+179, -1 098-+179, -727-+179, -515-+179, -248-+179 and -56-+179 missing segments were amplified, and the dual-luciferase reporter vector pGL3-Basic was constructed respectively. The recombinant LATS2 gene promoter vectors were transfected into C2C12 and 3T3-L1 cell lines, respectively, and the core promoter regions were identified. With the help of online software Genomatix and JASPAR, the sequence characteristics of core promoter were analyzed to predict the binding sites of key transcription factors. The results showed that the expression of bovine LATS2 gene in liver and longissimus dorsi muscle was significantly higher than that in spleen (P<0.01). Ruminants were clustered into one branch in the evolutionary tree constructed according to LATS2 protein, which indicated that LATS2 gene was highly conserved in the evolutionary process of ruminants. The top ten proteins closely interacting with LATS2 protein screened by protein interaction analysis were the key proteins in Hippo signaling pathway. The core region of the LATS2 gene promoter was located at -248--56. It was predicted that the core promoter region of bovine LATS2 gene had binding sites of transcription factors TEAD1, MEF2A, FOSL1, MyoG and Myod1 related to muscle development. It showed that LATS2 gene played an important role in the growth and development of bovine muscle. The above results lay a foundation for exploring the transcriptional regulation mechanism of bovine LATS2 gene in muscle growth and development.

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备注/Memo

备注/Memo:
收稿日期：2022-06-15 基金项目：宁夏自然科学基金项目(2021AAC05007)；宁夏重点研发计划项目(2020BEB04011)；宁夏青年科技人才托举工程项目（TJGC2019076）作者简介：张久盘(1985-)，女，河南商丘人，硕士，助理研究员，研究方向为动物遗传育种。（E-mail）zhangjiupan@163.com 通讯作者：魏大为，（E-mail）weidaweiwdw@163.com
更新日期/Last Update: 2023-07-11