PmWRKY1 基因在马尾松抗虫防御中的作用机制研究

The Pinus massoniana Lamb. is an important timber tree species in China and plays a crucial role in the south forestry economy. However, in recent years more and more serious pest has caused great economic loss for forests of Masson pine. Using method of molecular biology becomes a trend to research the mechanism of insect resistance, mine the key genes, and carry out genome directed breeding for effective, economical, as well as environmental defense pest. At present there are few studies on the molecular mechanism of insect resistance in pines. In our previous study, expression level of PmWRKY1 gene rose sharply in insect-resistant material of Pinus massoniana Lamb.. At the same time, terpenoids concentration increased obviously. Based on these found and related study reports, we speculate that PmWRKY1 gene make Pinus massoniana L. show insect-resistant reactions by regulating of some kind or kinds of hormone signaling pathways involved in terpenoids synthesis. However, it is not clear of this specific regulatory mechanism. In this study, we focus on the function of PmWRKY1 gene during the process of insect resistant. Methods in the aspects of bioassay, chemical analysis and molecular biology will be performance to test insect resistance of Pinus massoniana L. key signaling molecules, levels of chemicals and changes of gene expression profiles. Fluorescence microscopy, yeast technology, such as single/double cross and BiFC for PmWRKY1 gene will be used to realize cell positioning, to reveal biochemical functions and to find out interactions factors. After treatments with insect pests and key signaling molecules, expression characterizations of PmWRKY1 gene and Insect behavior observation will be analyzed and find out the inherent relationship between PmWRKY1 gene and insect-resistant defense in Pinus massoniana L.. By this study, the final aim will be achieved to uncover the role of PmWRKY1 gene playing in insect-resistant defense mechanism of Pinus massoniana L.. The results of this study will provide important genetic resources and a theoretical foundation for insect-resistant material qualification and directional breeding of insect-resistant varieties in the future.

马尾松是我国南部重要用材树种，但日趋严重的虫害给马尾松林经营造成巨大经济损失。利用分子生物学方法，研究马尾松抗虫机理、挖掘关键基因，定向培育抗虫品种，是最经济有效且环保的方法。申请人前期研究表明，马尾松抗虫品种中萜类物质含量明显高于对照，PmWRKY1 基因表达明显增强，结合相关研究，推测PmWRKY1 基因可能通过调控某种或多种激素信号途径参与萜类物质合成，使植株产生抗虫反应，但其作用机制尚不明确。因此，本项目以PmWRKY1 基因为研究对象，检测马尾松抗虫重要信号分子、化学物质以及基因表达模式；进一步对基因进行细胞定位、生化功能、互作因子以及直接调控基因研究；分析PmWRKY1 与互作基因在虫害和相关信号分子处理后的时空表达特征，并进行昆虫行为学观察。全面剖析PmWRKY1 基因在马尾松抗虫中的作用，并阐明其机理。为定向培育马尾松抗虫品种和抗虫材料鉴定提供重要的基因资源与理论指导。

松毛虫是影响马尾松林的重要因素，能够造成严重的经济和生态损失。WRKY转录因子在应对各种环境胁迫中起着重要作用，尤其是最新表明在应答植物防御虫害方面起重要作用，但这些基因在马尾松防御中的作用机理尚不清楚。我们在前期的研究中，已经有证据显示WRKY在马尾松抗虫防御中可能起着重要作用。在这项研究中，我们通过外源喷施激素和Ca2+信号物质处理，使马尾松植株的内源激素含量、萜类合成酶含量、挥发性物质含量得到提高，并能有效的提高马尾松抗松毛虫能力。我们对马尾松WRKY家族分析后，选择了PmWRKY31基因进行了研究。通过酵母双杂技术获得了与PmWRKY31互作的LP8基因。荧光定量表明，外源激素和Ca2+处理后，PmWRKY31基因和激素信号、萜类合成途径相关基因的表达量得到了显著的提高，而LP8基因的表达量出现下降，说明PmWRKY31基因正调控，而LP8基因负调控激素信号途径，从而影响下游基因的表达调控。这一结果将为WRKY转录因子调控激素信号参与马尾松抗虫防御提供理论支持。