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)

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。

Efficient amplification and sequence analysis of chalcone synthase gene from Ginkgo biloba by thermal asymmetric interlaced PCR

TAIL-PCR is a powerful tool for the recovery of DNA fragment adjacent to known sequences.With a modified TAIL-PCR technique,the genomic DNA sequence(named as Gbchs2)of chalcone synthase gene fragment was cloned from Ginkgo biloba cv.Jiafoshou.Two novel modifications in the TAIL-PCR procedures were introduced here:(1)the use of TAIL-PCR cycle in tertiary PCR,and(2)the use of a battery of random 10 bp RAPD primers as the short arbitrary primers.Gbchs2 was 1 238 bp long,containing 3'-flanking regions and encoding 304 amino acids.GbCHS2 was found to have extensive homology with those of other plant CHS based on multiple alignments,and the active site of the CoA binding,cumaroyl pocket,cyclization pocket and active site motif in CHS protein of other plants were also found in GbCHS2.The results showed that TAIL-PCR was useful,simple and powerful method for cloning full-length sequence of genes.