梅花鹿卵泡刺激素α-亚基cDNA的分子克隆与序列分析

摘要

从新屠宰的母梅花鹿脑垂体中提取总RNA,反转录获得cDNA,以此cDNA为模板用PCR法扩增目的片段,获得长为380 bp的梅花鹿卵泡刺激素α--亚基cDNA片段,将它克隆至pMD-18-T-Verctor.随机挑选3个阳性重组子进行测序,并将测序结果与绵羊、牛、猪等多种哺乳动物该基因的核苷酸序列及相应氨基酸序列进行比较.结果表明,梅花鹿卵泡刺激素α--亚基基因编码的氨基酸序列与绵羊、水牛的该基因同源性最高,达97%,只有4个氨基酸不同;与牛的该基因同源性达96%.与人的该基因氨基酸序列同源性较低,为75%.其编码的核苷酸序列与绵羊、水牛、牛的同源性最高,达96%,只有14～16个碱基不同;与人的该基因核苷酸同源性最低,为84%.总的来说,哺乳动物的卵泡刺激素α-亚基具有很高的同源性.%Total RNA was prepared from pituitary gland of new butchered sika.cDNA was synthesized by RT-PCRreaction and this cDNA was used as model in PCR amplification for α-subunit of sika follicle stimulating hormone.The PCR product was 380bp in 1.2% agarose gel electro-phoresis which just was the target fragment of predictedFSHα-subunit. It was cloned it to pMD-18-T vector. 3 positive recombinant was selected at random to analyze itssequence by DNA analysis apparatus. Its amino acid sequence was compared with some other mammalian. The resultshows that it has the highest homology with sheep and buffalo,which it reaches 97%. There are only 4 amino acidsdifference among sika ,sheep and buffalo. It has lower homology in amino acid with human, its homology is 75%. Ithas the highest homology among sika ,sheep, buffalo and bovine in nucleotide sequence, which it reaches 96%.There are 14-16 nucleotides difference among them. It has lower homology in nucleotide sequence with human, it isonly 84%. It was found that the nucleotide sequence of the o-subunit in these mammalian species are highly con-servative. According to our knowledge this sequence is first reported in the world .