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奶山羊POU1F1基因CDs区的克隆、分析及原核表达
李建华1, 罗军, 张晓, 樊睿, 王伟, 郝娟, 赵旺生
西北农林科技大学 动物科技学院,陕西省农业分子生物学重点实验室,陕西 杨凌 712100
摘要:
【目的】 克隆奶山羊垂体特异性转录因子POU1F1基因CDs区,并对其进行生物信息学分析和原核表达。【方法】 采集关中奶山羊垂体组织,提取其总RNA,根据绵羊POU1F1基因序列,利用反转录RT-PCR克隆POU1F1基因CDs区,对其进行生物信息学分析,并构建原核表达载体pET-32a-POU1F1,在大肠杆菌BL21(DE3)pLysS中进行表达。【结果】 关中奶山羊POU1F1基因(GenBank登陆号:FJ547813)开放读码框由876个碱基组成,编码291个氨基酸,基中包含1个POU-specific 结构域(从第124~198个氨基酸)和1个Homeobox 结构域(从第214~276个氨基酸);奶山羊POU1F1基因CDs区核苷酸序列与绵羊(NM_001009350)、牛(NM_174579)、人(NM_000306)和小鼠(NM_008849)的同源性分别为98%,97%,91%和86%,其氨基酸序列与绵羊、牛、人和小鼠的同源性分别为98%,98%,96%和92%;成功构建了重组质粒pET-32a-POU1F1的原核表达系统,并在体外条件下诱导表达出融合蛋白His-POU1F1。【结论】 POU1F1基因编码的功能氨基酸在奶山羊、绵羊、牛、人和小鼠上高度保守,推测奶山羊POU1F1基因与其他物种的POU1F1基因具有相似的功能。
关键词:  关中奶山羊  POU1F1  克隆  生物信息学分析  原核表达
DOI:
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基金项目:国家“863”高新技术研究与发展计划项目(2008AA10Z135);国家公益性行业(农业)科研专项经费项目(nyhyzx07-037)
Cloning and analysis of POU1F1 gene in dairy goat and its prokaryotic expression
LI Jian-hua,LUO Jun,ZHANG Xiao,FAN Rui,WANG Wei,HAO Juan,ZHAO Wang-sheng
Abstract:
【Objective】 The study cloned prokaryotic expression of the CDs of dairy goat pituitary-specific transcription factor POU1F1 gene and analyzed the biological information.【Method】 The CDs of goat pituitary-specific transcription factor POU1F1 gene was amplified and cloned from total RNA of goat pituitary by RT-PCR according to the corresponding sequence in sheep.The biological information analysis was performed with it.The prokaryotic expression vector pET-32a-POU1F1 was constructed and expressed in E.coli BL21(DE3)pLysS.【Result】 The gene POU1F1 CDs is cloned and registered in GenBank (GenBank accession No.FJ547813).The ORF of POU1F1 gene consisted of 876 nucleotides encoding 291 amino acids with a POU-specific (POUs) domain from 124-198 amino acids and a Homeobox domain from 214-276 amino acids.The nucleotide sequence homology of POU1F1 CDs of Guanzhong dairy goat was found to be 98%,97%,91% and 86% compared with that of sheep (NM_001009350),bovine (NM_174579),human (NM_000306) and mouse (NM_008849),while the amino acid sequence homology was 98%,98%,96% and 92% respectively.The prokaryotic expression system of recombined vector pET-32a-POU1F1 was constructed successfully.The fusion protein His-POU1F1 was expressed by inducing in vitro.【Conclusion】 The functional amino acids coded by the gene POU1F1 are highly conserved in goat,sheep,cattle,human and mouse.It is implied that the gene POU1F1 in dairy goat shows the same function as it does in other species.
Key words:  Guanzhong dairy goat  POU1F1  cloning  biological information analysis  prokaryotic expressiondomain gene Pit-1