红光诱导的13-CmLOXs在薄皮甜瓜抗白粉病中作用的分子机制

Powdery mildew is one of the important diseases which restrict production in protected melon. In our previous studies, four 13-CmLOXs showed up-regulated expression in response to Powdery Mildew after Red Light-indeced; however, the molecular mechanism of the 13-CmLOXs response to the powdery mildew remains unclear. On the basis of this, firstly, to clarify the function of 13-CmLOXs response to defence reaction, effects of 13-CmLOXs differential expression by overexpression Arabidopsis thaliana and Crispr/Cas9 or virus induced gene silenced(VIGS) melon plants on the content of jasmonic acid (JA) in leaves and related resistant genes will be measured. Secondly, qRT-PCR will be used to screen the up-regulated Cm-MAPKs members which response to the RL and Podosphaera xanthii inoculation treatment. Furthermore, the inhibitor of MAPK pathway and VIGS MAPK plant will be used to verify the pathway is responsible for LOX activation, and the interaction between the key MAPK members and LOX will be verified. Finally, Yeast one-Hybrid cDNA library will be constructed after RL-induced Podosphaera xanthii inoculation, and the transcription factors(TFs) which interacts with the candidate 13-CmLOXs involved in RL-induced Podosphaera xanthii inoculation were screened by Yeast one-Hybrid system, EMSA and chromatin immunoprecipitation(Chip). Besides, we want to know whether the TFs regulated by the MAPK pathway. Collectively, the molecular mechanism of the candidate 13-CmLOXs response to powdery mildew will be clarify, which is important to potentially use the corresponding genes or to improve the resistance of melon through genetic engineering.

白粉病是制约设施甜瓜生产的重要病害。申请者前期发现红光诱导后，接种白粉病菌的甜瓜幼苗中4个13-CmLOXs上调表达，但是其抗病机制尚不明确。以此为切入点，首先利用超表达转基因拟南芥和Crispr/Cas9及VIGS甜瓜幼苗，验证13-CmLOXs基因差异表达对JA等下游产物和抗性基因表达的影响，明确13-CmLOXs在防御反应中的功能。其次筛选红光诱导后，接种白粉病菌幼苗中上调表达的MAPK成员，并通过喷施MAPK途径抑制剂及构建VIGS沉默植株验证该途径是激活LOX途径的必要条件，并验证关键MAPK成员与LOX是否互作；最后，通过构建红光诱导后白粉病侵染的酵母单杂交cDNA文库，利用酵母单杂交、EMSA及Chip筛选与13-CmLOXs互作的转录因子，并明确该转录因子是否受MAPK调控。明确候选基因在抗白粉病中作用的分子机制，为潜在利用相应基因或通过基因工程提高甜瓜抗逆性有重要意义。

白粉病严重影响设施甜瓜的产量和品质。本项目发现红光预处理可以诱导薄皮甜瓜对白粉病的防御反应，此过程中13-CmLOXs上调表达。在此基础上，首先利用病毒诱导基因沉默（VIGS）的体系，构建TRV-PHYB植株，发现白粉病菌接种后参与防御反应的13-CmLOXs是光敏色素B激活的机制。构建了TRV-CmLOX10和TRV-CmLOX13甜瓜植株，接菌后台盼蓝染色及病情指数分析表明CmLOX10和CmLOX13参与红光处理防御薄皮甜瓜白粉病的响应，且起到正调控作用。构建了红光诱导下，接种白粉病菌的酵母单杂交文库，筛选调控CmLOX10和CmLOX13表达的转录因子，通过亚细胞定位分析、启动子分析及DNA-蛋白互作验证表明CmWRKY41、CmABL5-2是调控CmLOX10转录的转录因子，CmHY5、CmERF26是调控CmLOX13表达的转录因子，同时获得TRV-CmWRKY41甜瓜植株并接种白粉病菌，结果表明CmWRKY41正调控红光诱导的白粉病抗性。此外，与空载对照相比，TRV-CmMAPK3与TRV-CmMAPK7沉默植株，LOX活性显著升高，病情指数显著降低，红光预处理后接种白粉病菌的各处理病情指数均低于单独接种白粉病菌处理，证明了在此防御反应中CmMAPK3和CmMAPK7作用于13-CmLOXs的上游并是激活脂氧合酶的必要条件。.发表相关论文3篇，其中SCI收录2篇，中文权威期刊1篇。培养博士生1名，硕士生3名。