洋葱AcFMO克隆及其在蒜氨酸生物合成过程中的功能分析

Cloning and functional analysis of AcFMO from onion during alliine biosynthesis

黄素单氧化酶(flavin-containing monooxygenase,FMO)是CSOs生物合成途径中的关键酶,具有硫氧化功能。为了探讨FMO在洋葱CSOs合成中的功能,本研究基于转录组数据库和系统发育分析获得了一个可能参与蒜氨酸合成的AcFMO基因,该基因的cDNA为1374 bp,编码457个氨基酸,在进化上与大蒜的AsFMO亲缘关系最近。实时荧光定量PCR(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)表明AcFMO在洋葱的花中表达量最高,在叶鞘中表达量最低。亚细胞定位结果显示AcFMO基因产物广泛分布于整个细胞。构建了酵母表达载体,转化酵母后进行体外酶功能分析,确定通过酵母真核表达的AcFMO蛋白在体外具有催化活性,能够催化S-烯丙基-L-半胱氨酸合成蒜氨酸。总之,AcFMO的克隆和功能鉴定对了解洋葱中CSOs的生物合成具有重要的参考价值。

Flavin-containing monooxygenase(FMO)is the key enzyme in the biosynthesis pathway of CSOs with sulfur oxidation.In order to explore the molecular regulatory mechanism of FMO in the synthesis of onion CSOs,based on transcriptome database and phylogenetic analysis,one AcFMO gene that may be involved in alliin synthesis was obtained,the AcFMO had a cDNA of 1374 bp and encoded 457 amino acids,which was evolutionarily closest to the AsFMO of garlic.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)indicated that AcFMO was the highest in the flowers and the lowest in the leaf sheaths.The results of subcellular localization showed that the AcFMO gene product was widely distributed throughout the cell A yeast expression vector was constructed,and the AcFMO gene was ecotopically overexpressed in yeast to further study the enzyme function in vitro and could catalyze the synthesis of alliin by S-allyl-L-cysteine.In summary,the cloning and functional identification of AcFMO have important reference value for understanding the biosynthesis of CSOs in onions.