西农萨能奶山羊β-酪蛋白基因启动子区的克隆、生物信息学分析及活性研究  

Cloning, Bioinformatic Analysis and Activity Study of Promoter Region of Xinong Saanen Dairy Goat β-Casein Gene

作　　者：梁克明[1] 朱婧[2] 童德文[2] 陈南春[1] 陈苏民[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《生物技术通讯》2005年第4期363-366,共4页Letters in Biotechnology

摘　　要：根据已发表的β-酪蛋白基因全序列设计合成引物,以西农萨能奶山羊乳腺组织基因组DNA为模板,通过长链PCR扩增β-酪蛋白基因组基因5'侧序列。将该序列克隆入pMD18-T载体,并对其进行序列测定。结果克隆得到西农萨能奶山羊β-酪蛋白基因-4359至+2106共6465bp序列。该序列保留β-酪蛋白第一内含子、第一外显子和第二外显子及其信号肽部分。将该序列与已公布的其他山羊β-酪蛋白基因序列进行比较,其同源性为96%～98%。生物信息学分析显示,β-酪蛋白基因启动子区包含有TATA启动子序列和HOXF、Oct-1、AP1、CEBP、STAT、NFKB、GR、ER、PR和ETS等转录因子识别位点。将此6465bp片段插入荧光素酶报道基因载体pGL3-Basic中,构建重组质粒pGL3/B65,瞬时转染萨能奶山羊原代培养的乳腺上皮细胞48h后检测显示,该假设的启动子区确有启动子功能,对泌乳激素发生反应。本研究为进一步构建有效的乳腺生物反应器载体奠定了基础。Special primers for goat β-casein gene promoter had been designed and synthesized according to the published nucleic acid sequence of goat complete β-casein gene. Cloning of Xinong Saanen dairy goat β-casein gene promoter region was performed by long and accurate PCR analyses. The PCR product was cloned into pMD18-T vector and then was sequenced. The cloned fragment covered the 5＇-regulative sequence, intron 1, exon 1 and exon 2 of the goat β-casein gene from -4 359 to ＋2 106 bp. The results of comparative sequence analysis indicated that putative promoter sequence of the cloned Xinong Saanen dairy goat β-casein gene promoter shared 96%-98% homology with other goat β-casein gene promoters. Bioinformatic analysis showed that Xinong Saanen dairy goat β-casein gene promoter region contained promoter sequences and putative recognition sites for several transcriptional factors, including HOXF, Oct1, AP1, CEBP, STAT, NFKB, GR, ER, PR and ETS. Reconstructed reporter plasmid pGL3/B65 was established by inserting 6 465 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. The pGL3/B65 was transfected in vitro via liposomes into the primary goat mammary epithelial cell. Transient transfection assay showed the Xinong Saanen dairy goat β-casein gene promoter region had promoter function, and it responded to hormonal treatment （14.98±2.18, P〈0.01）. This study have paved the way for further studying and application of Xinong Saanen dairy goat β-casein gene promoter.

关 键 词：Β-酪蛋白基因 西农萨能奶山羊 启动子区域 转录调控 

分 类 号：Q786[生物学—分子生物学]