TAIL-PCR方法快速克隆银杏查尔酮合成酶基因及序列分析(英文)

• 作 者: 许锋 程水源 王燕 李琳玲 程述汉
• 机 构: 长江大学园艺园林学院,长江大学园艺园林学院,长江大学园艺园林学院,长江大学园艺园林学院,山东农业大学园艺科学与工程学院 湖北荆州434025山东农业大学园艺科学与工程学院,山东泰安271018,湖北荆
• 出 处: 果树学报
• 发表日期: 2007年02期
• 关键词: 合成酶基因 TAIL-PCR 查尔酮 银杏 蛋白质序列 活性位点 简并引物 末端序列 不对称 序列分析

热不对称交错PCR(TAIL-PCR)方法已广泛应用于从多种生物克隆已知DNA序列的侧翼序列。对传统的TAIL-PCR方法进行改良:(1)将TAIL技术应用于TAIL-PCR中第3步PCR循环。(2)以10bp的随机简并引物即RAPD引物代替基因侧翼简并引物。以银杏品种家佛手的叶基因组DNA为模板,利用简并引物克隆到银杏查尔酮合成酶基因(CHS)片段序列Gbchs1,以此序列设计3条特异引物,利用改良的热不对称交错PCR方法克隆到CHS基因Gbchs2。结果表明,Gbchs2长1238bp,编码304个氨基酸并包含3’末端序列。GbCHS2蛋白质序列与其他植物的CHS蛋白质序列高度同源,包含CHS蛋白质保守的环化作用活性位点,催化活性位点、香豆素活性位点、及催化活性基序。改良后的TAIL-PCR方法为基因全长的克隆提供了一种简单快速高效的新方法。

The thermal asymmetric interlaced PCR (TAIL-PCR) method has been widely used to clone the flanking sequences of known DNA sequences from a variety of organisms. The traditional TAIL-PCR method is improved: (1) The TAIL technology is applied to the third PCR loop in TAIL-PCR. (2) 10 bp random degenerate primers, ie RAPD primers, were used to replace the gene-degenerated primers. Using Ginkgo biloba leaf genomic DNA as template, degenerate primers were used to clone Gbchs1 gene of chalcone synthase gene (GST) gene. Three specific primers were designed according to this sequence. A modified thermal asymmetric staggered PCR Clone to CHS gene Gbchs2. The results showed that Gbchs2 is 1238 bp long and encodes 304 amino acids and contains the 3 ’end sequence. The GbCHS2 protein sequence is highly homologous to the CHS protein sequences of other plants and includes the conserved cyclization activity site, the catalytic active site, the coumarin active site, and the catalytically active motif of the CHS protein. The improved TAIL-PCR method provides a simple, rapid and efficient new method for the cloning of full-length genes.