中国农业科学 ›› 2017, Vol. 50 ›› Issue (23): 4632-4643.doi: 10.3864/j.issn.0578-1752.2017.23.016

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

FAM213B基因启动子在猪子宫内膜细胞的转录调控

张爱玲1, 2,孙显月2,卢孝璋2,吴琦2,李加琪2,张豪2

 
  

  1. 1广东高校应用生态工程技术开发中心/广东第二师范学院生物与食品工程学院 广州 510310;2华南农业大学动物科学学院/广东省农业动物基因组学与分子育种重点实验室,广州 510642
  • 收稿日期:2017-03-10 出版日期:2017-12-01 发布日期:2017-12-01
  • 作者简介:张爱玲,Tel:020-85285159;E-mail:zhangmeixial@163.com
  • 基金资助:
    国家自然科学基金(31201771)、国家现代农业产业技术体系建设专项(CARS-35)、广州市科学研究专项一般项目(201707010001)

Regulation of the Promoter of FAM213B Gene in Porcine Endometrial Cells

ZHANG AiLing1,2, SUN XianYue2, LU XiaoZhang2, WU Qi2, LI JiaQi2, ZHANG Hao2   

  1. 1 Development Center of Applied Ecology and Ecological Engineering in Universities/Biology and Food Engineering Institute, Guangdong University of Education, Guangzhou 510310; 2 Guangdong Provincial Key Lab of Agro-animal Genomics and Molecular Breeding/College of Animal Science, South China Agricultural University, Guangzhou 510642
  • Received:2017-03-10 Online:2017-12-01 Published:2017-12-01

摘要: 【目的】分析猪FAM213B基因启动子转录活性区域,并检测转录因子NFκB对启动子活性及猪FAM213B基因表达影响,为深入了解FAM213B基因的转录调控机制影响前列腺素合成、母猪妊娠等繁殖活动奠定基础。【方法】采集卵泡期子宫,分离子宫内膜上皮组织,经胶原酶方法获得猪原代子宫内膜细胞,用于猪FAM213B基因启动子活性检测。参照笔者所在课题组前期研究获得的猪FAM213B基因mRNA全序列(GenBank登录号:KX444503)以及5¢调控区序列(GenBank登录号:100134955),通过PCR扩增猪FAM213B基因较长启动子序列,并进行测序鉴定;在此基础上,PCR扩增带有Mlu I和Xho I限制性酶切位点的FAM213B基因启动子7个5'端缺失片段,并通过双荧光素酶报告基因载体系统构建猪FAM213B基因启动子7个不同的5'端缺失载体。将7个载体经无内毒素处理后与pRL-TK质粒用阳离子脂质体法一起转染猪子宫内膜细胞,用双荧光素酶报告基因系统进行Luciferase活性检测,比较各启动子片段转录活性。生物信息学分析FAM213B基因启动子潜在转录因子结合位点,通过ChIP试验验证FAM213B基因启动子与潜在转录因子NFκB的相互作用。构建NFκB1和RelA的超表达载体,化学合成NFκB1和RelA的干扰siRNA片段,分别转染猪子宫内膜细胞,通过双荧光素酶报告基因系统进行Luciferase活性检测和荧光定量PCR分别检测超表达和干扰表达NFκB1和RelA对猪FAM213B基因启动子活性和mRNA表达影响。【结果】PCR和测序获得猪FAM213B基因启动子长度为 2 261 bp(-2178/+83)。生物信息学预测结果表明猪FAM213B基因启动子区存在潜在的CREB、CCAAT增强子结合蛋白、E-box因子的结合位点,炎性因子NFκB潜在结合位点分别位于-1143/-1132和-664/-655区间。启动子报告基因活性检测结果表明:重组载体P2(-1352/+30)荧光活性最高,极显著高于P1(-1 760/+30)(P<0.01),在-1 760/-1 352区域存在负调控元件;而P2显著高于P3(-919/+30)(P<0.05),表明在-1 352/- 919区域存在正调控元件;P3(-919/+30)活性极显著高于P4(-604/+80)(P<0.01),表明在-919/-604区域存在正调控元件;P4、P5、P6和P7活性差异不显著,-1352/-919区域为核心启动子区,-1352/-604对于维持该启动子较高转录活性起着重要的作用。ChIP结果表明启动子区-1143/-1132存在NFκB1结合位点,-664/-655存在RelA结合位点。超表达载体pcDNA3.1-NFκB1与P2(-1352/+30)启动子片段重组载体共转染猪子宫内膜细胞后,P2启动子活性极显著高于对照组(P<0.01),pcDNA3.1-NFκB1载体单独转染猪子宫内膜细胞后FAM213B基因mRNA表达量显著高于对照组(P<0.05);超表达载体pcDNA3.1-RelA与P3(-919/+30)启动子片段重组体共转染猪子宫内膜细胞,P3启动子活性极显著低于对照组(P<0.05),pcDNA3.1-RelA载体单独转染猪子宫内膜细胞后,FAM213B基因的mRNA表达量显著低于对照组。转染NFκB1和RelA的siRNA片段干扰片段,FAM213B基因启动子活性和mRNA表达量则表现与超表达相反的结果。【结论】获得了猪FAM213B基因启动子核心启动子序列为-1352/-919区域,NFκB是FAM213B基因启动子的转录因子,NFκB成员NFκB1和RelA在猪子宫内膜细胞中对FAM213B基因的表达起调控作用。

关键词: 猪;FAM213B基因;启动子;NF&kappa, B;子宫内膜细胞

Abstract: 【Objective】To interpret partially the role of FAM213B gene expression in prostaglandin synthetise and sow pregnancy through the identification of the transcription region of porcine FAM213B gene promoter and the effection of NFκB on the promoter. 【Method】 The porcine endometrium from the uterus in follicular phase was digested by collagenase and the isolated endometrial cells were cultured for the detection of the FAM213B promoter activity. Based on the mRNA and promoter sequences of FAM213B gene obtained in our former work (GenBank ID: KX444503 and 100134955), the longer 5′ regulationary sequence was amplified and sequenced. Then, seven promoter fragments with 5′ terminal deletion, containing Mlu I and Xho I sites, were linked into the Dual-luciferase Reporter vectors. Seven constructed vectors treated by endotoxin free and pRL-TK plasmid were co-transfected into endometrial cells through liposome method. The core region of transcriptional activity of the gene promoter was identified through the Dual-luciferase Reporter Assay System. The putative transcription factors binding sites were analyzed by bioinformatics, and the binding of NFκB with FAM213B promoter were detected by ChIP (Chromatin immunoprecipitation). The over-expression vectors of NFκB1and RelA and interference fragments of their own were transfected into endometrial cells. Then, the transcription activity of the promoter and mRNA expression of the gene were detected by the Dual-luciferase Reporter system and fluorescent quantitative, respectively. 【Result】 Through the PCR and sequencing, one fragment of 2 261 bp (-2178/+83) of porcine FAM213Bgene were obtained. The bioinformatics analysis showed that there were putative binding sites of CREB, CCAAT, E-box, and NFκB in the promoter. And the putative binding sites of NFκB were found in -1143/-1132 and -664/-655 regions. The result of the Dual-luciferase Reporter showed the region of P2 (-1352/+30) exhibited the strongest transcriptional activity, and it was significantly higher than that of P1 (-1760/+30) (P<0.01), which showed there were negative regulation elements in -1760/-1352 region. And the transcriptional activity of P2 (-1352/+30) was significantly higher than that of P3(-919/+30) (P<0.05), implying the existence of positive elements in -1352/- 919 region. The significant stronger activity of P3 (-919/+30) than P4(-604/+80) (P<0.01) meant the existence of positive elements in -919/-604 region. No significant differences were observed between P4, P5, P6, and P7. The region of -1352/-919 was the core element of the promoter. The results of Chromatin Immunoprecipitation (ChIP) demonstrated that NFκB1 binds to one site around -1143/-1132, and RelA site around -664/-655. The co-transfection of the over expression of pcDNA3.1-NFκB1 and P2 vector into endometrial cells increased the activity of the promoter (P<0.01), and the transfection of pcDNA3.1-NFκB1 enhanced the mRNA expression of FAM213B (P<0.05). While the co-transfection of the over expression of pcDNA3.1-RelA and P3 vector into endometrial cells decreased the activity of the promoter (P<0.05), and the transfection of pcDNA3.1-RelA weakened the mRNA expression of the gene. At the same time, the transfection of inference siRNA fragments of NFκB1 and RelA into endometrial cells led the contrary results for the activity of the promoter and the mRNA expression of the gene.【Conclusion】The core region of porcine FAM213B gene promoter was identified round -1352/-919. NFκB was the trancription factor of FAM213B gene. NFκB1 and RelA, two members of NFκB family, regulate the expression of FAM213B gene in endometrial cells.

Key words: pig, FAM213B gene, promoter, NFκB, endometrial cells