利用CR-RT-PCR技术克隆大葱线粒体cob基因

Cloning Allium fistulisum Mitochondria cob Gene by CR-RT-PCR Technology

本研究以大葱花蕾为材料,对环化RNA反转录聚合酶链式反应(CR-RT-PCR)技术在RNA反应时间和试剂添加上进行了优化,并用优化后的技术对大葱线粒体cob基因的全长进行了扩增。在本实验条件下,最优环化反应条件为:RNA首先65℃反应5 min,加入反应试剂后15℃反应2 h,最后70℃反应10 min使酶失活,反应体系中不必添加催化剂PEG6000。实验结果表明,cob基因全长为1 522 bp,无poly(A)尾,包含完整的N端和C端,共编码397个氨基酸。系统进化树分析显示大葱cob基因与同属的洋葱同源性最高,且与其他单子叶植物聚为一类。研究表明此技术获得的线粒体cob基因信息完整,可进行后续功能研究。

We used Allium fistulisum as materials, to optimized RNA reverse transcription polymerase chain reaction(CR-RT-PCR) technique on RNA Reaction time and reagent addition. Also used optimized technology to amplified the full length of onion mitochondrial cob gene. In this research, the optimal reaction conditions were:firstly RNA 65℃ for 5 min, then added reaction reagent in 15℃ for 2 h, lastly 70℃ for 10 min to inactivate the enzyme. The reaction system without adding a catalyst PEG6000. The results showed that the length of the cob gene was 1 522 bp poly(a tail), it contains the complete N-terminal and C-terminal and the encoding 397 amino acids Phylogenetic tree analysis shows that Allium fistulisum cob gene clustered with the monocots clade, and most similar to the Allium cepa. The research showed that we can obtain the mitochondria cob gene complete information through CR-RT-PCR technology, this information was suitable for subsequent gene functional studies.