诱导细胞凋亡蛋白MAPK3和MAPKK5对白木香2-苯乙基色酮生物合成的调控机制及生物学功能研究

Agarwood is most highly valuable resinous wood used as incenses, perfumes, traditional medicines in the world market. Agarwood can not generate in the normal Aquilaria sinensis (Lour.) but may form in the tissues which was wounded or infected by fungi. However, little is known about the mechanism of agarwood formation.2-phenylethyl chromones are major constituents in agarwood, and important compounds induced Aquilaria sinensis disease resistance. We have found that the activation of MAPK3 and MAPKK5 which can induce hypersensitive response (HR)-like cell death was coordinated with production of 2-phenylethyl chromones in Aquilaria sinensis callus. To evaluate the function of MAPK3 and MAPKK5 in biosynthesis of 2-phenylethyl chromones, MAPK3 and MAPKK5 was silenced in Aquilaria sinensis plants or cells, and the production of phenylethyl chromones was detected by LC-MS. The interacting proteins with MAPK3 and MAPKK5 were searched by yeast two hybrid system or combining tandem affinity purification. To find activated MAPK cascade in biosynthesis of 2-phenylethy chromones, the project will also detect the relationship between the downstream interacting proteins and biosynthesis of 2-phenylethyl chromones. Furthermore, studies on MAPK3 and MAPKK5 overexpressing plants will elucidate the MAPK3 and MAPKK5 biological functions in plant defense responses, growth and development. This investigation will contribute with an important theoretical and practical significance to illuminate mechanism of agarwood formation, enhance the quality and yield of incense, and improve the research on plant defense response.

沉香是传统名贵中药材和香料，是白木香受到伤害或者真菌侵染产生的结香物质—含脂木材，但是白木香结香机制至今尚未清楚。2-苯乙基色酮是沉香的主要特征性成分，也是白木香特殊的防御物质。前期研究发现蛋白激酶MAPK3、MAPKK5与2-苯乙基色酮生物合成密切相关，并能够诱导植物细胞凋亡产生超敏反应。本项目拟通过沉默MAPK3、MAPKK5基因，并利用LC-MS 测定2-苯乙基色酮的变化，进一步阐明该基因参与2-苯乙基色酮的生物合成调控；通过酵母双杂或串联亲和纯化技术，寻找MAPK3、MAPKK5的级联反应蛋白，并验证互作蛋白在2-苯乙基色酮生物合成过程中的调控作用，确定调控2-苯乙基色酮生物合成的级联信号途径；利用过表达植株培养研究MAPK3、MAPKK5在植物防御反应和生长发育中的生物学功能。项目研究对于阐明白木香结香机制，提升沉香品质和产量，丰富植物防御反应机理研究，均具有重要理论和应用价值。

沉香是传统名贵中药材和香料，是白木香受到伤害或者真菌侵染产生的结香物质—含脂木材，但是白木香结香机制至今尚未清楚。2-苯乙基色酮是沉香的主要特征性成分，也是白木香的防御物质。本项目通过在愈伤组织中过表达AsMAPK3和AsMAPKK5基因，利用实时荧光定量PCR检测发现过表达AsMAPK3和AsMAPKK5能够提高2-苯乙基色酮生物合成关键酶AsPKS3的表达量,利用LC-MS方法检测发现过表达AsMAPK3和AsMAPKK5能够促进盐胁迫下2-苯乙基色酮的积累。通过酵母双杂交的方式发现AsMAPKK5能够与AsMAPK11互作，并通过双分子荧光、Far-Western、GST-Pull Down 等体内外的方法验证AsMAPKK5能够与AsMAPK11互作，同时在愈伤组织中过表达AsMAPK11能够提高愈伤组织中2-苯乙基色酮生物合成关键酶AsPKS3的表达量，确定AsMAPKK5-AsMAPK11调控2-苯乙基色酮的生物合成。烟草中过表达AsMAPK3能够使植株矮小、老叶凋亡、花凋亡并且无法结种，转录组测序结果显示AsMAPK3可能通过调控乙烯途径和生长素途径蛋白调控植株凋亡的症状。过表达AsMAPKK5的烟草植株具有抗盐、抗旱和外源ABA的功能。这些结果为进一步阐明白木香结香机制奠定基础，同时也丰富了植物防御反应机制。