千层金叶片中丁香酚-O-甲基转移酶高效催化甲基丁香酚合成的作用机制

The essential oil from Melaleuca bracteata leaves had inhibitory activity of bacterial quorum sensing, that effective constituent was methyleugenol which contents as high as 86.5%. In addition, eugenol, which was the substrate of methyleugenol biosynthesis, was not detected in the leaves. It showed clearly that eugenol-O-methyl transferase (EOMT) had a strong catalytic activity in Melaleuca bracteata leaves, but the mechanism was unknown. In the previous research, transcriptome sequencing was performed and the full-length cDNA of 5 candidate EOMT genes were obtained. On this basis, this project intends to screen out the main effect EOMT genes that can effectively catalyze the synthesis of methyleugenol in Melaleuca bracteata leaves by using qRT-PCR and VIGS technology. Functional verification of the main effect EOMT genes was conducted through prokaryotic expression system and genetic transformation of petunia. The relationship between structure and function of EOMT was revealed through homologous modeling and site-directed mutagenesis. The aim of the project is to determine the mechanism of EOMT high-effective catalyzed the biosynthesis of methyleugenol in Melaleuca bracteata leaves, and provides theory gist and basis for further studies on the regulatory mechanisms of methyleugenol biosynthesis in Melaleuca bracteata. The results can provide new ideas and methods for efficient biosynthesis and transformation of plant secondary metabolites.

千层金叶片精油具有明显的细菌群体感应抑制活性，其有效成分甲基丁香酚含量高达86.5%，而且，叶片中未检测到甲基丁香酚合成的底物——丁香酚，说明千层金叶片中丁香酚-O-甲基转移酶（EOMT）具有很强的催化丁香酚生成甲基丁香酚的能力，但其作用机制并不清楚。课题组在前期研究中对千层金叶片进行转录组测序，获得了5条候选EOMT基因的全长cDNA。在此基础上，本项目拟通过qRT-PCR和VIGS技术，筛选出千层金叶片中高效催化甲基丁香酚合成的主效EOMT基因；通过原核表达和矮牵牛的遗传转化，对主效EOMT基因进行功能验证；通过同源建模及定点突变技术，揭示主效EOMT结构与功能的关系。旨在明确EOMT在千层金叶片中高效催化甲基丁香酚合成的作用机制，为进一步研究千层金中甲基丁香酚生物合成途径的调控机制提供理论依据和工作基础，研究结果可为植物次生代谢产物的高效合成与转化研究提供新的思路和方法上的借鉴。

千层金叶片精油具有明显的细菌群体感应抑制活性，其有效成分甲基丁香酚含量高达86.5%，而且，叶片中未检测到甲基丁香酚生物合成的底物——丁香酚，说明千层金叶片中EOMT具有很强的催化丁香酚生成甲基丁香酚的能力，但其作用机制并不清楚。本项目通过转录组和代谢组的联合分析，筛选出千层金叶片中高效催化甲基丁香酚生物合成的MbEOMT1和MbEOMT2基因，并对其进行表达谱分析，发现MbEOMT1和MbEOMT2基因的转录水平与甲基丁香酚的含量呈正相关。通过遗传转化分析发现，在瞬时过表达MbEOMT1和MbEOMT2基因的草莓果实和千层金叶片中，相应基因的表达量和甲基丁香酚的含量均显著升高；而在MbEOMT1和MbEOMT2基因沉默的千层金叶片中，甲基丁香酚的含量显著下降，相应基因的转录水平也受到抑制。构建原核表达载体pET-32a(+)-MbEOMT1，获得分子质量约为57kD的可溶性蛋白，并将纯化后的可溶性蛋白进行体外酶性分析，发现该蛋白能以SAM为甲基供体，以丁香酚为底物，催化形成甲基丁香酚，而对底物异丁香酚却没有活性。以上结果表明，MbEOMT1和MbEOMT2基因是千层金叶片中甲基丁香酚生物合成途径的关键酶基因。此外，分别对MbEOMT1和MbEOMT2进行同源建模，结合分子对接技术确定底物结合口袋附近的氨基酸残基，分析可能影响千层金MbEOMT1和MbEOMT2催化活性和底物特异性的关键位点：在MbEOMT1中，关键的氨基酸残基主要包括ALA-23、ASP-26、SER-27、ASP-127、ASN-172、ILE-207、HIS-265、ASP-266和GLU-324；在MbEOMT2中，关键的氨基酸残基主要包括LYS-19、ASN-20、ALA-23、ASP-26、LYS-79、ASN-172、LEU-207和THR-209。本项目明确了MbEOMT1和MbEOMT2基因在千层金叶片中高效催化甲基丁香酚生物合成的作用机制，为进一步研究千层金中甲基丁香酚生物合成途径的调控机制提供理论依据和工作基础，研究结果可为植物次生代谢产物的高效合成与转化研究提供新的思路和方法上的借鉴。