波叶山蚂蝗根系分泌物中诱导寄生杂草独脚金 “自杀性萌发”化感物质的生物合成研究

Intercrop Desmodium genus is one of the most effective way to protect crops from parastic weed Striga genus due to the allelochemicals from root exudates of Desmodium induce Striga ‘suicidal germination’. On the basis of Desmodium sequax which has been screened from common Desmodium genus in China shows significant inhibition activity against Striga asiatica radicle development. Targeting the allelochemicals from Desmodium sequax root exudates contain germination stimulants strigolactones and biosynthesis of radicle inhibitor C-glucosylflavones is essential to uncover the mechanism of ‘suicidal germination’. In this study, both allelopathic strigolactones and C-glucosylflavones will be isolated from root exudate of Desmodium sequax hydroponic solution though bio-guided isolation technology before NMR and MS structure determination. The synthetic labelled substrate of C-glucosylflavones and sugar donors constitute enzymatic reaction system will be established in vitro which provides synthetic labelled products subsequently following analyzed by NMR and LCMS. As a result, the order of glycosylation reaction and biosynthesis of allelopathic C-glucosylflavones will be eventually elucidated. Within this framework, the application of intercroping Desmodium genus agaist Striga asiatica in China and anti-Striga crop culturing research will be boosted which will take a great effect on agriculture sustainable development.

间作山蚂蝗属植物作为保护农作物免受独脚金属寄生杂草侵害的新型生物防治方法，其根系分泌物中的化感物质具有诱导独脚金属寄生杂草“自杀性萌发”的功能。本项目在前期对我国山蚂蝗属植物进行筛选并确定波叶山蚂蝗具有显著诱导活性的基础上，拟对其根系分泌物中具有刺激独脚金寄生种子萌发的独脚金内酯类和抑制胚根发育的黄酮苷类化感物质的进行解析，揭示诱导“自杀性萌发”机制，并确定黄酮苷类化合物的生物合成途径。通过活性跟踪分离技术，对波叶山蚂蝗根系分泌物中的化感物质进行分离，结合NMR、MS技术确定化感物质结构；采用人工合成的稳定同位素标记底物及单糖供体，构建体外酶促反应体系；使用LCMS、NMR技术对酶促产物结构进行分析、确定糖基化反应顺序，最终阐明生物合成途径。该项研究不仅为推广间作山蚂蝗属植物防治寄生杂草独脚金提供理论依据，也为培育具有抗寄生杂草的农作物提供理论基础，对促进农业的可持续性发展具有重要意义。

间作山蚂蝗属Desmodium植物作为保护农作物免受独脚金属寄生杂草侵害的新型生物防治方法，其根系分泌物中的化感物质具有诱导独脚金属寄生杂草“自杀性萌发”的功能。在项目申请前期确定波叶山蚂蝗Desmodium sequax具有诱导活性的基础上，项目执行期内在全国范围内收集23个种山蚂蝗属植物进行水培养和诱导试验，最终确定假地豆Desmodium heterocarpon根系分泌物诱导活性最为显著，在10 ppm下抑制率达到63.8%高于波叶山蚂蝗（58.4%）。在此基础上，通过活性跟踪分离结合LCMS技术，确定假地豆中含有诱导寄生种子萌发的独脚金内酯类化合物和诱导吸器发育的异夏佛塔苷。通过对假地豆根组织进行转录组数据进行进行序列比对和聚类分析，筛选到Unigene0019190和Unigen0029705为异夏佛塔苷生物合成途径中的关键基因。通过在大肠杆菌BL21(DE3)表达菌株中进行异源表达、蛋白纯化，获得DhCGT1和DhCGT2。采用体外酶促反应验证了DhCGT1和DhCGT2具有以2-羟基柚皮素为底物、UDP-glucose和UDP-arabinose为糖基供体催化生成异夏佛塔苷、夏佛塔苷、牡荆苷和异牡荆苷功能。酶底物专一性研究表明，DhCGT1和DhCGT2以UDP-xylose和UDP-rhamnose为糖基供体生成一系列黄酮碳苷和黄酮双碳苷类化合物。本项目在确定假地豆根系分泌物中诱导独脚金吸器发育化感物质为异夏佛碳苷的基础上，进一步阐明了阐明了异夏佛碳苷、夏佛塔苷、牡荆苷和异牡荆苷的生物合成途径。为培育具有抗寄生杂草的农作物提供理论基础，对促进农业的可持续性发展具有重要意义。