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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。
In this study, the green onion flower buds were used to optimize the RNA reaction time and reagent addition of the reverse transcription-polymerase chain reaction (CR-RT-PCR) of circularized RNA. The optimized mitochondrial cob gene The full length of the amplified. Under the conditions of this experiment, the optimal reaction conditions were as follows: RNA was first reacted at 65 ° C for 5 min, reaction was performed at 15 ° C for 2 h after addition of the reaction reagent, and finally the enzyme was inactivated by reaction at 70 ° C for 10 min. No addition of catalyst PEG6000 . The results showed that the cob gene was 1 522 bp in length with no poly (A) tail, and contained complete N-terminal and C-terminal, encoding a total of 397 amino acids. Phylogenetic tree analysis showed that the scutellum cob gene had the highest homology with the same onions and clustered with other monocotyledons. Studies have shown that the technology obtained mitochondrial cob gene information integrity, follow-up functional studies.