荷斯坦奶牛乳腺LAP基因的克隆与序列分析

Cloning and Sequence Analysis of LAP Gene from Mammary Gland in Holstein Cow

为了研究奶牛乳腺防御素基因的表达调控机制,采用逆转录-聚合酶链式反应(RT-PCR)扩增出LAP基因的核心片段序列,并与NCBI数据库序列比对同源性;进一步扩增其DNA序列中间片段;其后运用反向PCR技术并结合半巢式PCR技术扩增其侧翼的序列。结果表明:序列与NCBI数据库中序列同源性为100%,确认为LAP基因;DNA序列扩增得到1 760 bp的中间序列,并预测了其酶切位点;得到5'侧翼序列107 bp,3'侧翼序列87 bp,经预测发现有转录因子GATA。为进一步研究防御素的防御功能、寻找防御素基因体外表达调控机制以及利用基因工程防治奶牛乳腺炎积累了基本资料。

To study the mechanisms of expression and regulation of defensins gene in mammary gland of cow, the core fragment of LAP gene sequence was amplified using the method of reverse transcription PCR （RT-PCR）, and its homology was compared with the sequences from NCBI database; followed, the middle DNA sequence was amplified; then, the sequence of flanking was amplified by using RT-PCR combined with semi-nested PCR. The results showed that the sequence was confirmed as LAP and had 100% homology with sequence from the NCBI database. The 1 760 bp of middle sequence was got from amplification of DNA sequence, and its restriction site had predicted. The 107 bp of 5＂ flanking sequence and the 87 bp of 3j flanking sequence were amplified, and transcription factor GATA was found through prediction. Therefore, the results accumulated some basic information for further study on the defense function of defensins, searching regulating mechanism of expression in vitro of defensins, and preventing and treating mastitis of cow by using genetic engineering.