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中国农学通报

中国农学通报 ›› 2008, Vol. 24 ›› Issue (12): 25-29.

所属专题： 生物技术； 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

猪骨形态发生蛋白4成熟肽cDNA的克隆及序列分析

李 明,,孙桂荣,陈其新,刘孟洲,石晓卫,赵巧辉,杨跃霞   

  • 收稿日期:2008-10-14 修回日期:2008-11-12 出版日期:2008-12-05 发布日期:2008-12-05

Cloning and Sequencing of the mature peptide cDNA of bone morphogenetic protein-4 gene in pig

Li Ming,, Sun Guirong, Chen Qixin, Liu mengzhou, Shi Xiaowei, Zhao Qiaohui, Yang yuexia   

  • Received:2008-10-14 Revised:2008-11-12 Online:2008-12-05 Published:2008-12-05

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摘要： 目的：克隆猪骨形态发生蛋白4（BMP-4）成熟肽基因。方法：提取猪肾的总RNA，通过RT-PCR技术扩增出目的基因，并将其回收后与pMD18-T载体连接，转化大肠杆菌TOP10感受态细胞，进行阳性克隆的筛选与鉴定。结果：琼脂糖凝胶电泳检测RT-PCR产物，显示结果与预期片段大小一致，质粒PCR检测、酶切鉴定证实重组克隆中插入了PCR产物，测序结果揭示BMP-4成熟肽cDNA长为351bp，编码116个氨基酸。序列对比表明，猪与人、小鼠、牛、绵羊BMP-4成熟肽cDNA序列的同源性分别为94.87%、90.60%、94.87%、94.59%,而相应的氨基酸同源性分别为99.12 %，99.12 %，100 %、100%。 结论：首次成功地克隆了猪BMP-4成熟肽编码基因，对于进一步研究猪BMP-4全基因结构、功能及其与繁殖性能的关系有重要的意义。

关键词: 耐碱, 耐碱, 根瘤菌, 16SrDNA序列, DNA－DNA杂交

Abstract: Objective: To clone the cDNA of sus scrafa mature peptide of bone morphogenetic protein-4. Methed: Totle RNA was extracted from kidney of sus scrafa and the desired segment was obtained by RT-PCR with two specific primers.The PCR products was cloned into pMD18-T vector and transformed into Top-10 competent cells. After PCR and double enzyme screening, the sequence of insert in the recombinant plasmid was assayed by DNA sequence machine. Results: DNA agarose electrophoresis showed that the products of RT-PCR was consistent with desired BMP-4. PCR, double enzyme digestion revealed that the PCR product was inserted pMD18-T vector and sequencing demonstrated that the mature protein cds of pig BMP-4 was consisted of 351 nucleotides and encoded 116 amino acids. Sequence alignments showed that the mature protein cds of pig BMP-4 had 94.87%, 90.60%, 94.87% and 94.59% homologies with that of human, mouse, cow, and sheep respectively, while the homologies for the deduced amino acids were 99.12%, 99.12%,100% and100% respectively. Conclusion: It is concluded that the mature protein cds of pig BMP-4 have been successively cloned, and this will promote the further studies on the full gene structure and function of this gene as well as its relationship with pig reproduction.

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