独脚金内酯通过木质素调控菊花抗蚜性的分子机制研究

Chrysanthemum, one of ten traditional Chinese famous flowers and one of four cut flowers over the world, of high ornamental and economic values, is extremely susceptive to aphid. Aphid causes direct damage to plants via draining the plant nutrition and indirect damage via spreading virus and pathogen disease. Chemical application fails to control the aphid efficiently, and aphid infestation has become a bottleneck of chrysanthemum industry. Lignin is the main composition of cell wall which serves as the first defensive barrier against aphid feeding, the biosynthesis of lignin is catalyzed by a series enzymes and regulatory transcription factors. We previously found that the new phytohormone, strigolactone (SL), inhibits the synthesis of lignin, while enhances the aphid feeding in chrysanthemum, however, how SL regulates the biosynthesis of chrysanthemum lignin, thereby regulating the resistance of chrysanthemum to aphid, remains unknown. Therefore, in the present proposal, we aim to isolate genes involved in the lignin biosynthesis by quantitative RT-PCR, RT-PCR and RACE cloning; to isolate transcription factors via yeast one-hybrid, and affinity trapping of DNA-binding proteins; and to identify gene function via overexpressing or artificial microRNA interfering transformation. The resistance of transgenic plants to aphid will be identified as well. Lignin content, lignin monolignols and the cell wall thickness will be detected via Phloroglucinol staining, UV autofluorescence, Nuclear magnetic resonance (NMR) and transmission electron microscope. Present study will illustrate the molecular regulatory network of lignin biosynthesis by strigolactone and how it regulates the chrysanthemum resistance to aphid. The findings will provide a guide for aphid control and minimize the application of chemicals in controlling aphid, thus the proposal is of important theoretical significance and practical value.

菊花是我国十大传统名花和世界四大切花之一，观赏、经济价值极高。蚜虫是严重为害菊花的最主要害虫，化学防治困难，已成为限制我国菊花产业化发展的瓶颈。木质素作为防御蚜虫取食的第一道有效防线，受其合成基因及转录因子调控。项目组先前发现新型植物激素独角金内酯抑制菊花木质素合成，促进蚜虫取食，而其调控木质素合成从而改变菊花抗蚜性的分子机制尚不清楚。为此，本申请拟借助qRT-PCR、RT-PCR、RACE技术发掘、克隆独脚金内酯调控的抗蚜性相关木质素合成关键基因；通过酵母单杂交、DNA结合蛋白亲和捕获技术发掘关键合成基因上游转录因子；借助超表达、基因沉默反向遗传学技术鉴定目标基因功能和抗蚜性；采用间苯三酚染色、紫外自发荧光、NMR、透射电镜检测木质素含量、单体成分及细胞壁厚度。揭示独脚金内酯借助木质素调控菊花抗蚜性的分子机制及新功能，为菊花蚜虫科学防治及减药栽培提供依据，具重要的科学意义与实践价值。

蚜虫是菊花的主要害虫，严重影响观赏性。项目组成功挖掘到响应独脚金内酯信号的木质素合成路径差异表达基因Cm4CL2，发现该基因受蚜虫取食诱导上调表达，其促进木质素合成而提高菊花抗蚜性。克隆Cm4CL2启动子，挖掘到其上游调控因子CmMYB15-like，通过酵母单杂、EMSA、原生质体双分子荧光素酶和ChIP-qPCR证实CmMYB15-like可与Cm4CL2基因的启动子AC-I元件结合，并激活其表达。CmMYB15-like遗传转化菊花，表明其可促进木质素合成，细胞壁的加厚，从而阻碍蚜虫取食，提高抗蚜性。在菊花中率先报道了CmMYB15-like-Cm4CL2通路促进木质素合成从而提高菊花抗蚜性的分子机制。挖掘到响应独脚金内酯信号的MYB类转录因子CmMYB19及其同源基因CmMYB15，通过酵母单杂交验证CmMYB19、CmMYB15可结合在木质素生物合成途径基因启动子区的AC元件上。明确了CmMYB19、CmMYB15促进木质素合成，从而提高菊花抗蚜性的功能。丰富了MYB家族对木质素合成路径的调控网络。