高效热不对称交互式PCR技术克隆地黄基因

利用高效热不对称交互式PCR(hiTAIL-PCR)技术从地黄基因组中扩增出3个DNA片段,经过电泳检测、回收、纯化和测序获得2个片段的碱基序列.其中一个由578个bp组成,包含一个453bp的开放阅读框(ORF),编码151个氨基酸,与一些已知纤维素合酶基因在DNA水平和氨基酸水平的同源性分别高达81%~91%和83%~99%,而且推测蛋白质具有纤维素合酶的结构域;另一个由385个bp组成,没有ORF,不编码蛋白质,但是,预测其包含启动子元件.这些结果表明使用hiTAIL-PCR技术可以克隆地黄基因,克隆的基因将为地黄基因分子作用机制和分子育种研究奠定基础.

Gene Cloning of Rehmannia glutinosa by High-efficient Thermal Asymmetric Interlaced PCR

High-efficient thermal asymmetric interlaced PCR(hiTAIL-PCR)was used to clone genes from Rehmannia glutinosa genomic DNA.Three DNA amplified fragments were separated on agarose gel,extracted,purified,cloned and sequenced.One DNA fragment is composed of 578 bp,while another is 385 bp.Bioinformatics Analyses showed that the former contained a 453 bp open reading frame(ORF),encoding a deduced protein of 151 amino acid residues,which had a high homology of 81%-91% and 83%-99%to some known cellulose synthase genes at the DNA level and the amino acid level,respectively,and that its deduced protein is characteristic of the structure domain of other organisms cellulose synthases;that the latter did not have any homology to any known genes at the DNA level and the amino acid level,respectively,and did not contain any ORF but some promoters elements.These results indicated that hiTAIL-PCR is an efficient gene cloning technique from Rehmannia glutinosa,by which the genes were cloned and will lay a foundation for the further study of its molecular functional mechanism and its application in Rehmannia glutinosa molecular breeding in future.