内蒙古绒山羊角蛋白5基因启动子克隆及序列分析

›› 2012, Vol. 39 ›› Issue (6): 12-16.

内蒙古绒山羊角蛋白5基因启动子克隆及序列分析

勿都巴拉^1,2, 阿如汗^1,2, 吴江鸿^1,2, 张燕军^1,2, 张文广^1,2, 李金泉^1,2

1. 内蒙古农业大学动物科学学院,内蒙古呼和浩特 010018;
2. 内蒙古自治区动物遗传育种重点实验室,内蒙古呼和浩特 010018

收稿日期:2011-12-15 出版日期:2012-06-20 发布日期:2012-07-02
通讯作者: 李金泉(1957－),男,内蒙古人,教授,博士生导师,研究方向:绒山羊育种原理与方法。E-mail:lijinquan_nd@126.com;Tel:0471-4309297 E-mail:lijinquan_nd@126.com
作者简介:勿都巴拉(1987－),女,内蒙古人,硕士生,研究方向:动物遗传育种。
基金资助:
现代农业产业技术体系建设专项资金资助(CARS-40-05)。

Cloning and Sequence Analysis of the Inner Mongolia Cashmere Goat Keratin 5 Gene Promoter

WUDU Ba-la^1,2, A Ru-han^1,2, WU Jiang-hong^1,2, ZHANG Yan-jun^1,2, ZHANG Wen-guang^1,2, LI Jin-quan^1,2

1. College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Key Laboratory for Animal Genetics, Breeding in Inner Mongolia Autonomous Region, Hohhot 010018, China

Received:2011-12-15 Online:2012-06-20 Published:2012-07-02

摘要/Abstract

摘要： 试验旨在了解角蛋白5(keratin 5, K5)可能的调控序列。本研究根据UCSC公布的牛K5基因5'侧翼区设计PCR引物,扩增了内蒙古绒山羊K5基因部分启动子序列。通过产物纯化、连接、转化,并对测序结果进行了生物信息学分析。结果扩增得到内蒙古绒山羊K5基因启动子序列长度为1452 bp(GenBank登录号为:JQ277735),与牛和人相应序列的相似性分别为91.5%和74%。转录起始位点位于翻译起始密码子ATG上游－101 bp位置;含有两个TATA 盒,分别位于翻译起始位点上游－129—－124 bp(ATAAAA)和－178—－174 bp(TTAAT)位置;通过在线分析软件预测发现(按5'→3')SRY,MZF1,v-Myb,SRY,AP-1,CDP CR,HNF-4,AML-1a,HSF2,AP-4,AP2,AP2,Sp1,Nkx-2,Sp1和GATA-1转录因子结合位点。其中,转录因子SRY(TGTGTTT),和CDP CR(GATTGATGGC)是绒山羊特有的;转录因子HNF-4,AML-1a,HSF2,AP-4,AP2,Sp1,Nkx-2和GATA-1(AGCCATCATG)在绒山羊、牛和人K5启动子上的结合位点高度保守。两个最小增强子分别位于翻译起始位点ATG上游－140—－91 bp和－114—－67 bp位置,含有24 bp(GCGGCTCCCAGGTAACAGAGCCGC)重叠区,预测其与绒山羊K5基因的转录调控有关。试验确定了内蒙古绒山羊K5基因启动子的转录起始位置、转录因子结合位点及最小增强子序列,为进一步研究绒山羊K5基因的表达调控机制奠定了理论基础。

关键词: 绒山羊; K5基因启动子; 克隆; 转录因子

Abstract: This experiment was conducted to study the potential regulatory sequence of Keratin 5(K5). This study according to the UCSC bovine K5 gene 5' flank regions designed the PCR primers, and Inner Mongolia Cashmere goat K5 gene promoter was amplified. Analysis the K5 gene promoter sequence after product purification,ligation,transformation and sequence. It was found that 1452 bp Inner Mongolia Cashmere goat K5 promoter sequence was confirmed, which showed 91.5% and 74% homology with that of bovine and human respectively. The transcription start site was mapped to －101 bp of translation initiation site and two TATA boxes located in －129 to －124 bp (ATAAAA) and －178 to －174 bp (TTAAT) of translation initiation site respectively. The potential transcription factor binding motifs were predicted after analysis of promoter online software, including (5' to 3')SRY, MZF1, v-Myb, SRY, AP-1, CDP CR, HNF-4, AML-1a, HSF2, AP-4, AP2, AP2, Sp1, Nkx-2, Sp1 and GATA-1, in which SRY(TGTGTTT) and CDP CR(GATTGATGGC) were specific for Cashmere goat, and HNF-4, AML-1a, HSF2, AP-4, AP2, Sp1, Nkx-2 and GATA-1(AGCCATCATG) were conserved in Cashmere goat, bovine and human. Moreover, the two minimal enhancer reside from －140 to －91 bp and －114 to －67 bp of translation initiation site respectively, and contained 24 bp (GCGGCTCCCAGGTAACAGAGCCGC) overlap, which might be related with the Cashmere goat K5 gene transcriptional regulation. In this experiment, we inferred the transcription start site, transcription factor binding motifs and minimal enhancer elements in Inner Mongolia Cashmere goat K5 gene promoter, which may be helpful for exploring the mechanism of gene expression of cashmere goat K5 gene.

Key words: Cashmere goat; K5 gene promoter; cloning; transcription factor

中图分类号:

Q785

勿都巴拉, 阿如汗, 吴江鸿, 张燕军, 张文广, 李金泉. 内蒙古绒山羊角蛋白5基因启动子克隆及序列分析[J]. , 2012, 39(6): 12-16.

WUDU Ba-la, A Ru-han, WU Jiang-hong, ZHANG Yan-jun, ZHANG Wen-guang, LI Jin-quan. Cloning and Sequence Analysis of the Inner Mongolia Cashmere Goat Keratin 5 Gene Promoter[J]. , 2012, 39(6): 12-16.

参考文献

1 于永生,王晓阳,朴庆林,等. 绵羊毛角蛋白Ⅱ型中间丝9基因的扩增及表达[J].中国畜牧兽医,2011,38(6):64~67.
2 刘永峰,李林强, 等.牛GDF5 基因启动子的克隆与序列分析[J].畜牧兽医学报,2010,41(4):392~397.
3 汤华阳, 杜卫东. 单纯型大疱性表皮松解症的K5和K14基因突变分析.安徽:安徽医科大学,2008.
4 彭振辉. LacZ转基因鼠体内角蛋白5及10的分布[J].中华医学杂志, 1998,78(8):633~635.
5 Betz R C, Planko L, et al. Loss of function mutations in the keratin 5 gene Lead to dowling-degos disease [J].The American Journal of Human Genetics, 2006,78(3):510~519.
6 Byrne C, Fuchs E. Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice [J]. Molecular and Cellular Biology, 1993, 13(6):3176~3190.
7 Casatorres J, Navarro J M, Blessing M, et al. Analysis of the control of expression and tissue specificity of the keratin 5 gene, characteristic of basal keratinocytes [J].The of Biological Chemistry, 1994, 269(32):20489~20496.
8 Lersch R, Fuchs E. Sequence and expression of a type Ⅱ keratin, K5, in human epidermal cells [J]. Molecular and Cellular Biology, 1988, 8(1):486~493.
9 Lersch R, Atellmach V, Stocks C, et al. Isolation, sequence, and expression of a kuman keratin K5 gene: transcriptional regulation of keratins and insights into pairwise control [J].Molecular and Cellular Biology, 1989, 9(9):3685~3697.
10 Liovic M, D’Alessandro M, Tomic-Canic M, et al. Severe keratin 5 and 14 mutations induce down-regulation of junction proteins in keratinocytes [J]. Experimental Cell Research, 2009, 315(17):2995~3003.
11 Martha L B. Computational prediction of transcription factor binding site locations [J]. Genome Biology, 2003, 5 (1):201~212.
12 Opitz O G, Jenkins T D, Rustgi A K. Transcriptional regulation of the differentiation-linked human K4 promoter is dependent upon esophageal-specific nuclear factors[J]. The Journal of Biological Chemistry, 1998,273(37):23912~23921.
13 Wang Y N, Chang W C. Induction of disease-associated keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun [J]. The Journal of Biological Chemistry, 2003,278(46):45848~45857.
14 Woodcock-Mitchell J, Eichner R, et al. Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies [J]. The Journal of Cell Biology, 1982,95: 580~588.

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.
[3]	刘佳隆, 张译文, 张悦然, 孙奇娟, 陈建, 何雷, 贾艳艳, 丁轲, 余祖华. 鸡ELAVL1基因克隆、原核表达及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(5): 1807-1817.
[4]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[5]	范冰峰, 李文, 刘理想, 赵向远, 邵静, 林乐丰, 孙慧敏, 张旭林, 许保增. 水貂AANAT基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(4): 1307-1318.
[6]	刘红润, 朱思燃, 奉玲丽, 张坤, 颜港, 王钰滨, 张帅, 江山, 许迪, 兰干球, 梁晶. 大白猪CDO1基因克隆、生物信息学分析及组织表达定位[J]. 中国畜牧兽医, 2023, 50(4): 1352-1363.
[7]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[8]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[9]	宋兴超, 孟金柱, 赵园园, 吴震洋, 安清明. 贵州白山羊MyoG基因启动子区序列克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(3): 893-903.
[10]	萨初拉, 吴铁成, 马跃军, 何云梅, 朱莉仙, 何托雅, 武玉平, 刘斌. 绒山羊4个多胎性状候选基因多态性及其与产羔数的关联分析[J]. 中国畜牧兽医, 2023, 50(3): 1037-1047.
[11]	张永杰, 隋志远, 张志帅, 王晨光, 李孝君, 邢凤. 多浪羊NPTX1基因克隆、生物信息学分析及在初情期的表达研究[J]. 中国畜牧兽医, 2023, 50(3): 1048-1058.
[12]	牛小杰, 徐明国, 肖莉, 刘文豪, 陈创夫, 王勇. 鸡白痢沙门氏菌IPAJ蛋白的表达纯化、多克隆抗体制备及ELISA方法的建立[J]. 中国畜牧兽医, 2023, 50(3): 1218-1228.
[13]	侯浩宇, 郑南南, 吴宏举, 陈寅龙, 李潮, 张昂克, 姬鹏超, 万博, 杜永坤, 张改平. 非洲猪瘟病毒p30蛋白单克隆抗体制备和鉴定[J]. 中国畜牧兽医, 2023, 50(3): 1241-1249.
[14]	韦义荣, 郑自华, 张叁保, 宋颖, 蒋海玉, 孙雯玥, 程鹏飞, 刘雨帆, 邹剑伟, 黄艳娜, 潘艳, 蒋钦杨. 努比亚山羊UCP1基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2023, 50(2): 440-450.
[15]	吕双飞, 马勋, 王静, 孔翠莲, 寇丽君, 刘彩霞, 史唯地, 康立超, 任慧杰. 炭疽芽孢杆菌PA-LF1融合蛋白的原核表达及其反应原性研究[J]. 中国畜牧兽医, 2023, 50(2): 674-683.