海南黑山羊MBL2基因的转录调控元件的筛选与分析  

Screening and Analysis of Transcriptional Regulatory Elements of MBL2 Gene in Hainan Black Goat

作　　者：王雪梅[1] 翟哲 陈巧玲 吴艳茹 吴昊天 黄惠娴 刘志勇 李崇瑞 满初日嘎[1] 王凤阳[1] 杜丽[1] 陈思 WANG Xuemei;ZHAI Zhe;CHEN Qiaoling;WU Yanru;WU Haotian;HUANG Huixian;LIU Zhiyong;LI Chongrui;MANCHU Riga;WANG Fengyang;DU Li;CHEN Si(Key Laboratory of Animal Genetic Engineering of Haikou, Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research of Hainan Province, College of Animal Science and Technology, Hainan University, Haikou 570228, China)

机构地区：[1]海南大学动物科技学院,海南省热带动物繁育与疫病研究重点实验室,海口市动物基因工程重点实验室,海口570228

出　　处：《畜牧兽医学报》2022年第6期1795-1806,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：海南省自然科学基金项目(2019RC123);国家现代农业产业技术体系项目(财政部和农业农村部-CARS38);海南省院士创新平台科研专项(YSPTZX202153);海南省院士团队创新平台资金项目。

摘　　要：甘露聚糖结合凝集素C(mannose binding lectin C,MBL-C)是C型(Ca^(2+)依赖型)凝集素超家族的成员,其作为一种急性期蛋白,具有抗细菌感染的功能,参与机体的天然免疫反应。为鉴定出结合在MBL2基因启动子区(1009 bp)的重要转录因子,探寻该基因的转录调控机制,本研究选取海南黑山羊MBL2基因的启动子序列1009 bp,采用DNA重组技术克隆6个转录起始位点上游1009 bp的启动子5′端侧翼缺失序列,克隆片段经双酶切后连接至pGL3-Basic载体。重组质粒转染至293T细胞中,结合双荧光素酶活性检测系统筛选MBL2基因的核心启动子区域。通过在线生物信息学软件预测山羊MBL2基因的核心启动子区域的转录因子结合位点,利用点突变技术构建转录因子结合位点缺失的载体,转染293T细胞后结合双荧光素酶活性检测系统分析其转录活性。结果表明,海南黑山羊MBL2基因的核心启动子区域位于转录起始位点上游-304~-45 bp范围内,在线软件分析该区域存在RELA、NF-κB2、MZF1等3种转录因子结合位点。双荧光素酶报告分析结果表明,RELA和NF-κB2的结合位点缺失后均使山羊MBL2基因的转录活性极显著下降(P<0.01)。结果提示,RELA和NF-κB2对山羊MBL2基因的转录活性可能具有重要的正调控作用。该研究为进一步探寻海南黑山羊MBL2基因的功能提供理论依据。Mannose binding lectin C(MBL-C)is a member of the C-type(Ca^(2+) dependent)lectin superfamily.As an acute phase protein,MBL-C plays an important role in the innate immune response.The aim of this study was to identify the key transcriptional factors in the promoter region(1009 bp)of MBL2 gene,and explore the expression regulation mechanism of MBL2 gene.The promoter sequence 1009 bp of MBL2 gene was selected from Hainan black goat,and a serial of 5′-flanking deletion regions of the goat MBL2 promoter were amplified and subcloned into pGL3-Basic vector.The recombined plasmids were transfected into 293T cells and detected with the dual-luciferase activity detection system to screen the core promoter region of MBL2 gene.The transcription factor binding site of the core promoter region of Hainan black goat MBL2 gene were predicted by online bioinformatics software.The site mutation technology and dual-luciferase activity detection system were utilized to identify the transcription factor binding sites.The results showed that the core promoter region of Hainan black goat MBL2 gene located in the range of-304 to-45 bp of the transcription initiation site.Online software analysis indicated that this region existed three transcription factors binding sites,including RELA,NF-κB2 and MZF1.Dual-luciferase report analysis results showed that the deletion of the binding sites of RELA and NF-κB2 extremely significantly reduced the transcriptional activity of goat MBL2 gene(P<0.01).These results indicated that RELA and NF-κB2 might positively activate transcriptional regulation of goat MBL2 gene.This study can provide a theoretical basis for further exploring the function of MBL2 gene in Hainan black goat.

关 键 词：海南黑山羊 MBL2基因 转录调控 双荧光素酶 核心启动子 

分 类 号：S827.2[农业科学—畜牧学]