巴西橡胶树白粉病广谱抗性基因HbMlo的功能研究

Powdery mildew is one of main diseases in rubber tree which inhibits plant growth and dry latex yield. Mlo is an important member of plant diseases resistance genes, and mlo mutants result in durable, broad-spectrum resistance in plants. Identification of Hevea Mlo gene (HbMlo) is very important to breeding disease resistance clones in rubber tree. In our previous study, four HbMlos were identified, and they shared high homology with Mlo with known function in disease resistance from other species. Moreover, the transcripts of these four HbMlos were significantly induced by powdery mildew. These suggested that these HbMlos may play a crucial role in the pathogenesis of powdery mildew. In this study, we will focus on: 1) identification of HbMlo family members based on systematically analyzing the Hevea transcriptome database; 2) characterization of the expression profiles of HbMlos upon powdery mildew, various phytohormones and stresses treatments by Q-PCR in rubber tree, and identification of disease resistance-related HbMlos; 3) analysis of DNA sequence polymorphisms of disease resistance-related HbMlos between powdery mildew resistant and susceptible clones, and development of molecular markers associated with powdery mildew resistance; 4) functional assessment of disease resistance-related HbMlos using a transient assay system by transient over-expression and silence of HbMlos in the leaf of rubber tree, and identification of HbMlo which conferred broad-spectrum powdery mildew resistance. This study will provide a foundation for elucidating the molecular mechanism of disease resistance, and provide important genes for breeding of disease-resistant clones in rubber tree.

白粉病是橡胶树的主要病害，严重影响胶树生长和干胶产量。Mlo基因是植物重要的抗病基因，其隐性突变可赋予植物广谱持久的抗病性。橡胶树Mlo基因（HbMlo）功能鉴定对橡胶树抗病育种具有重要意义。我们在前期研究中发现4个HbMlo与其他物种已知具有抗病功能的Mlo高度同源，并受白粉菌诱导显著上调表达，推测这些基因可能参与橡胶树对白粉菌的抗性反应。本项目拟利用橡胶树转录组数据库全面地鉴定并克隆HbMlo家族成员；采用Q-PCR技术系统地分析HbMlo在白粉菌、植物激素和胁迫处理后的表达模式，确定抗病相关HbMlo；分析抗病相关HbMlo在抗、感白粉病种质中的序列多态性并开发抗白粉病相关分子标记；采用瞬间表达技术在橡胶树叶片中瞬间过表达和沉默抗病相关HbMlo，鉴定对白粉病具有广谱抗性的HbMlo并阐明其抗病功能，为阐明抗病分子机制提供理论基础，为橡胶树抗病育种提供重要基因资源。

白粉病是橡胶树主要叶部病害之一，严重影响橡胶树生长和干胶产量。植物Mlo基因是重要的抗白粉病基因，其隐性突变可赋予植物广谱持久的抗病性。橡胶树Mlo基因家族（HbMlos）功能鉴定尤其是白粉病专性抗性成员的功能鉴定对橡胶树阐明白粉病抗性机制进而在橡胶树白粉病病害监测、预测预报和防控技术研发具有重要意义。在国家基金项目资助下，我们首先采用RT-PCR技术结合橡胶树基因组和转录组数据库全面地鉴定并克隆HbMLO1(Qin et al., 2015)，HbMLO1-1(何海霞 等, 2016)，HbMLO7(何海霞等, 2016)，HbMLO8(张宇 等, 2016)，HbMlo9(张宇 等, 2018)和Hbmlo12(张宇 等, 2015)6个HbMlo家族成员；进而采用qRT-PCR技术系统地分析6个HbMlos在不同抗感种质、白粉菌侵染、植物激素和干旱等胁迫处理下表达规律，结合生物信息学分析，确定抗病相关基因HbMlo12；随后采用亚细胞定位技术证明HbMlo12定位于细胞质中；并采用过表达HbMlo12转拟南芥atmlo突变体和白粉菌侵染技术证明敲除HbMlo12提高拟南芥抗白粉病功能，证明抗病性与其C端DFSF基序相关；在调控机制研究中，发现橡胶树MYB转录因子HbMYB1(Qin et al., 2014)和PRS磷酸焦磷酸激酶HbPRS4(Yu et al., 2017)受白粉菌侵染上调表达。在应用领域，系统分析抗病相关HbMlo12在抗、感白粉病种质中的序列多态性，在外显子和内含子区域分别发现16和31位点差异，并选取960,2007,2762,2917和2922五个位点开发抗白粉病相关分子标记；结合白粉病病级等级和流行强度预测方法，修订了白粉病行业标准（NYT1089-2015）。本项目在鉴定HbMlos家族成员的基础上，证明了对白粉病具有广谱抗性的HbMlo12并阐明其抗病机制，为阐明橡胶树抗白粉病分子机制提供理论基础，为橡胶树抗病育种提供重要基因资源。本项目根据抗病基因开发的分子标记为橡胶树白粉病病害监测和预测预报系统建立和应用打下坚实的应用基础，并在5-10年可应用。