基于CODEHOP克隆杉木种子14-3-3基因片段

Design degenerate primer using CODEHOP to amplify 14-3-3 gene of Chinese fir

对于未测序的物种,利用常规的引物设计很难扩增出蛋白质组获得的蛋白质基因。利用CODEHOP设计杉木种子14-3-3蛋白质的简并引物来进行反转录PCR,从杉木未成熟的种子中扩增出1 000 bp左右的产物。将PCR产物构建p MD-19Vector载体上并转化JM109菌感受态细胞中,菌落PCR扩增测序。结果显示,利用CODEHOP方法设计简并引物扩增的目标片段的大小及序列与预期的一致。这将为杉木其他蛋白质基因的克隆和研究提供参考。

It＇s difficult to amplify the gene sequence based on the protein sequence obtained from proteome using the conventional primer designed for non-sequencing species.In this study,CODEHOP software was used to design degenerate primers of Chinese fir 14-3-3 proteins,which were used for reverse transcription PCR.The results showed that 1 000 bp product was amplified from immature Chinese fir.The PCR products were constructed on pMD-19 vector,and then transformed into competent JM109 bacterial cells.Colony PCR was used to amplify the 14-3-3 fragment then 14-3-3 fragments were sequenced.The results showed that target fragment size,sequence consistent with the expected sequence by the degenerate CODEHOP method.This results would provide an new insight for cloning and studying on other proteins.