优化的反向PCR结合TAIL-PCR法克隆棉花线粒体atpA双拷贝基因及其侧翼序列  

High efficiency genome walking method for flanking sequences of cotton mitochondrial double-copy atpA gene based on optimized inverse PCR and TAIL-PCR

作　　者：张晓[1,2] 张锐[1] 孙国清[1] 史计[1] 孟志刚[1] 周焘[1] 侯思宇[1] 梁成真[1] 于源华[2] 郭三堆[1] 

机构地区：[1]中国农业科学院生物技术研究所国家农作物基因资源与遗传改良重大科学工程,北京100081 [2]长春理工大学生命科学技术学院,吉林长春130022

出　　处：《生物工程学报》2012年第1期104-115,共12页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30771371);国家转基因重大专项(No.2008ZX08005-004)资助~~

摘　　要：双拷贝基因及其侧翼序列的克隆是分子生物学中的一个难点。将优化的反向PCR(Inverse PCR,iPCR)与TAIL-PCR相结合,有效地克隆双拷贝基因及其侧翼序列。先用Southern blotting方法确定一种能获得合适长度片段的限制性内切酶,然后用优化的iPCR方法对该酶切产物进行自连和扩增,将2个拷贝的侧翼序列区分开。根据iPCR结果,进一步用TAIL-PCR扩增更远侧翼的序列。利用这套方法,获得了棉花可育胞质和不育胞质线粒体双拷贝atpA基因的所有EcoR I限制片段(2.2～5.1 kb)和HindⅢ限制片段(8.5～11.7 kb),克隆到2个拷贝各自的侧翼序列。研究结果说明,优化的iPCR与TAIL-PCR相结合是克隆双拷贝基因及其侧翼序列的一种高效方法。Cloning of flanking sequences of double-copy gene is a challenge in molecular biology.We developed a method to solve this problem by combining an optimized inverse PCR（iPCR） with TAIL-PCR.First,Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene.Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene.Based on the obtained sequences,TAIL-PCR was performed to amplify further flanking regions of the gene.With this method,we obtained all of the EcoR I restriction fragments（2.2–5.1 kb） and Hind Ⅲ restriction fragments（8.5–11.7 kb） of mitochondrial atpA gene in cytoplasmic male sterile（CMS） line and maintainer line of Upland cotton.The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.

关 键 词：反向PCR TAIL-PCR 细胞质雄性不育 棉花 ATPA 双拷贝基因 侧翼序列 

分 类 号：S562[农业科学—作物学] Q943.2[生物学—植物学]