水牛催乳素受体基因克隆及序列分析

Cloning and sequence analysis of swamp buffalo prolactin receptor partial cDNA

扩增出广西本地沼泽型水牛(Bubalus bubalis)催乳素受体(PRLR)基因部分保守序列,用于下一步实验检测体外培养的水牛乳腺上皮细胞中催乳素受体基因的表达水平。根据已报道的奶牛催乳素受体基因cDNA序列的保守区,设计一对特异性引物。提取广西本地水牛的乳腺组织总RNA,并以其为模板,以下游特异性引物为反转录引物,在反转录酶(AMV)作用下合成cDNA;用Ex-Taq酶进行PCR扩增,得到催乳素受体基因的保守序列,长度为571 bp。该扩增产物经纯化后,连接到pMD18-T载体上扩增。经序列分析结果表明:序列较保守,与GenBank上发表的奶牛催乳素受体基因的同源性达到98.9%,与绵羊的同源性达到96.5%,与猪、小鼠、人的同源性分别为86.5%、78.9%、77.1%。催乳素受体基因与奶牛相比,共有6个碱基发生了突变,其中1个为同义突变,其他5个均为错义突变,形成4个氨基酸发生变异。

Total mRNA isolated from the mammary gland tissue of swamp buffalo was used as templates to synthesize cDNA by reverse transcription polymerase. The partial sequence of prolactin receptor cDNA with length of 571 bp was obtained by PCR. By comparing with published prolactin receptor gene sequence with Basic Local Alignment Search Tool （BLAST）, the sequence showed high identity with cattle prolactin receptor （PRLR） gene. The multiple sequence homology of buffalo to cattle was 98.9%, to sheep 96.5%, to pig 86.5%, to mice 78.9%, to human 77.1%. There were four nucleotides mutations occurred in the signing peptide of PRLR, and one was nonsense mutation. These proved that the sequence is swamp buffalo prolactin receptor gene.