广西水牛ghrelin cDNA的克隆及序列分析

目的]克隆广西水牛ghrelin的cDNA并对其进行序列分析。方法]以广西沼泽型水牛的真胃底组织总RNA为模板，用特异性引物扩增ghrelin的cDNA。扩增产物经凝胶回收纯化后与pMD 18-T连接并转化大肠杆菌DH5d，转化产物经PCR和双酶切鉴定后筛选出阳性克隆，阳性克隆经5×1ml LB液体培养基培养鉴定后测序。结果]成功获得了广西水牛ghrelin的cDNA，该片段长339bp，编码113个氨基酸。BLAST分析结果表明，该片段与黄牛前原ghrelin基因仅有6个碱基的差异，同源性为98．2％；而两者ghrelin均由27个氨基酸组成，序列同源性为1013％。结论]为进一步研究ghrelin基因的生物学功能提了供理论基础。

Clone and Sequence Analysis on cDNA of Ghrelin Gene from Guangxi Buffalo

Objective] The study was to clone cDNA of ghrelin gene from Guangxi buffalo and analyze its sequence.Method]With the total RNA from true stomach tissue of Guangxi swamp buffalo as the template,the specific primers were taken to amplify cDNA of ghrelin gene.The amplified products were connected with pMD 18-T and transformed into Escherichia coli DH5α after it was recovered and purified with gel.The positive clones were screened out from transformation products after they were identified by PCR and double digestion.The positive clones were taken to do sequence analysis after they were cultured by liquid medium of 5×1 ml LB.Result]The cDNA of ghrelin gene from Guangxi buffalo was successfuly gained in the test,and the length of cDNA fragment was 339 bp and it coded 113 amino acid.The result of BLAST analysis showed that the fragment only had 6 basyl difference with that of prepro ghrelin gene from yellow cattle and their homology was 98.2%.The ghrelin gene of Guangxi buffalo and yellow cattle were all composed by 27 amino acid,and the sequence homology of their ghrelin genes was 100%.Conclusion] The test provided the theoretical basis for further study on biological function of ghrelin gene.