北京奶山羊β乳球蛋白基因(βlg)的克隆及其调控序列的分析  

Cloning of Beijing Milk Goat β-lactoglobulin Gene(βlg)and Analyzing Its Control Regions

作　　者：林爱星[1] 刘建喜[1] 周云[1] 胡宏宇[1] 陈永福[1] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室,北京100094

出　　处：《农业生物技术学报》2001年第3期226-229,共4页Journal of Agricultural Biotechnology

摘　　要：从北京奶山羊白细胞中提取高分子量基因组ＤＮＡ，用Ｓａｕ３Ａ部分酶切，连接到经ＢａｍＨＩ－ＥｃｏＲＩ酶切过的λＥＭＢＬ３载体上，构建了６．７×１０～６ｐｆｕｓ的基因组文库。荧光标记牛βｌｇ５’端０．５ｋｂ片段作探针，原位杂交得到４个阳性斑：Ａ、Ｂ、Ｃ和Ｄ。三轮杂交后，经ＰＣＲ产物序列分析和物理图谱分析，发现克隆Ｄ（１０．６ｋｂ）包括完整的　乳球蛋白基因，其５’侧翼序列长为３．８　ｋｂ，３’侧翼序列长为２．２　ｋｂ。将克隆Ｄ５’　和３’调控区亚克隆，发现５’侧翼序列包含βｌｇ核心启动子，３’侧翼序列具有ＭＡＲ序列。比较北京奶山羊与绵羊β乳球蛋白基因的５’侧翼序列和３’侧翼序列，发现它们的同源性分别是９０％、８９％。结果分析表明，克隆Ｄ为β乳球蛋白的编码基因。High molecular genomic DNA was prepared from a Beijing milk goat blood sample,partially digested with Sau3A and ligated to BamHI and EcoRI cleaved λEMBL3 phage arms by standard methods. The goat genomic DNA library, consisting of 6.7×10～6 pfus,was screened with 0.5 kb fragment from bovine βlg 5'flanking which had been labeled using fluorescence gene image system.Four plaques designated A,B,C and D exhibited the strongest hybridization signals under condition of high stringency and each was subjected to three rounds of plaque purification.Analyzing with PCR,PCR product sequencing and physical map revealed that the insert (10.6 kb) with recombinant phage D includes the goat βlg gene.The 3.8 kb 5'flanking contained complete core promoter and the 2.2 kb 3'flanking included complete matrix attachment region.Restriction enzymes generated DNA fragments derived from clone D were subcloned and sequenced.The homology of theβlg5'and 3'between goat and sheep was 90%,89% respectively.According to the analysis of sequences,clone D was the βlg coding gene.

关 键 词：北京奶山羊 β乳球蛋白 基因 克隆 调控序列 

分 类 号：S827.2[农业科学—畜牧学]