韧皮部miRNA调控木霉介导的黄瓜枯萎病抗性的分子机制研究

In plants, Trichoderma spp. can induce systemic resistance against biotic stresses; however, the mechanisms of systemic signaling and its regulation are largely unknown. Phloem miRNAs of plants may perform as systemic signals in response to biotic stresses. Our previous studies have discovered that systemic resistance against Fusarium wilt disease induced by Trichoderma spp.was closely related to the expression of miRNA in phloem. A total of 42 miRNA and 18 novel miRNA were identified with Solexa high-throughput sequencing technology and with miRBase,PMRD and miRNApath methods. Based on the achieved results, the project intends to analyze temporal and spatial change of miRNA in phloem of cucumber and to screen the different expression miRNA involved in induced resistance of Trichoderma against Fusarium wilt disease combined with miRNA chip and qRT-PCR. The project will also identify the target mRNAs of miRNA on a whole genome scale by using high throughput degradome sequencing approach, and then further validation by the RLM 5' race. The interaction between miRNA and their target mRNAs will be elucidated by Northern blotting. Finally, miRNA overexpressed plants will be constructed to verify the miRNA and their target genes involved in feedback communication of Trichoderma against Fusarium wilt disease between roots and shoots parts. Importantly, the project aims at elucidating the role of the long distance miRNA signal in systemic resistance induced by Trichoderma. Eventually this study will provide a new useful tool by using the communication mechanism to improve stress defense in agricultural production.

木霉可有效提高植物受到生物胁迫时的系统抗性，但其系统信号分子和调控机制尚不明确。韧皮部microRNA在植物抵御生物胁迫中起重要的系统调节作用。项目前期研究表明，木霉诱导黄瓜对枯萎病的系统抗性与韧皮液miRNA差异表达密切相关，并已鉴别出42种差异表达miRNA，18种为新的miRNA。在此基础上,本项目拟结合miRNA芯片和qRT-PCR进一步研究木霉诱导下韧皮液miRNA的时空变化，发掘与枯萎病抗性相关的韧皮液差异表达miRNA。利用降解组测序从全基因组水平获得miRNA靶基因，结合Northern杂交、RLM 5`-RACE等方法研究miRNA与靶基因的相关作用。同时构建miRNA及靶基因过量表达植株，验证miRNA及其靶标基因在木霉抗枯萎病根冠反馈交流中的作用。研究可望阐明韧皮部miRNA信号在木霉诱导的植物系统抗病性中的作用机制，探索一条基于植物通讯机制提高蔬菜抗病性的新途径。

本项目研究了哈茨木霉抑制黄瓜尖孢镰刀菌的生理与分子机制。木霉菌对病原菌的抑菌率达66.7%~85.8%，木霉菌可通过减少根系细胞的死亡、激活与胁迫相关的基因表达、提高次生代谢相关酶活性，激活AsA-GSH循环和磷酸戊糖途径相关酶活性、降低亚硝基谷胱甘肽还原酶活性，从而提高黄瓜根系中抗氧化胁迫和活性氮胁迫的能力，进而有效提高根系对病原菌的抗性。发现与木霉诱导的枯萎病抗性相关的韧皮部miRNA 69条。利用降解组测序鉴定了miRNA的靶基因，结果924个转录本被确定为61个miRNA家族的靶基因。利用qRT-PCR对Solexa测序有显著表达的18个miRNA进行了验证，主要涉及根系发育、程序性死亡、解毒作用、氧化还原信号、激素代谢。利用Gateway技术将与枯萎病侵染和木霉诱导显著相关的12个miRNA前体和相应靶标基因构建至载体pEarleyGate103中，并在烟草和黄瓜中进行遗传转化，初步结果表明过表达miRNA参与了枯萎病的发生和木霉的抗病性。发现与枯萎病相关新基因599个，其中442个得到功能注释和富集分析。利用Gateway技术将与枯萎病侵染和木霉诱导显著相关的novel-miR001前体和相应靶标基因E3 ubiquitin-protein ligase UPL7构建至植物表达载体pEarleyGate103中，并在黄瓜中进行遗传转化，表明过表达novel - miR001通过激活氧化还原信号、降低ROS积累，从而抑制了枯萎病的发生。沉默与枯萎病侵染和木霉诱导显著相关的ath -miR160a -5p3及过表达其靶基因auxin response factor ARF17，研究表明ath-miR160a-5p3促进了生长素信号抑制型转录因子ARFIO和ARFl6的表达，从而抑制了生长素信号和JA的合成。发表论文13篇，其中SCI论文11篇，出版专著1部。参与获得国家科技进步奖1项。项目研究结果对于利用木霉防控黄瓜等蔬菜枯萎病具有重要意义。