CAJ | 학술논문

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Gbchs 1,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1 238 bp,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TAII-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.
열불대칭교착PCR(TAIL-PCR)방법이엄범응용우종다충생물극륭이지DNA서렬적측익서렬.대전통적TAIL-PCR방법진행개량:(1)장TAIL기술응용우TAIL-PCR중제3보PCR순배.(2)이10bp적수궤간병인물즉RAPD인물대체기인측익간병인물.이은행품충가불수적협기인조DNA위모판,이용간병인물극륭도은행사이동합성매기인(CHS)편단서렬Gbchs 1,이차서렬설계3조특이인물,이용개량적열불대칭교착PCR방법극륭도CHS기인Gbchs2.결과표명,Gbchs2장1 238 bp,편마304개안기산병포함3'말단서렬.GbCHS2단백질서렬여기타식물적CHS단백질서렬고도동원,포함CHS단백질보수적배화작용활성위점,최화활성위점、향두소활성위점、급최화활성기서.개량후적TAII-PCR방법위기인전장적극륭제공료일충간단쾌속고효적신방법.
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 ex tensive 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 re sults showed that TA1L-PCR was useful, simple and powerful method for cloning full-length sequence of genes.