水稻褐飞虱抗性调控因子bs1的分离及功能研究

Herbivore infestation causes serious yield loss in rice production. Brown planthopper (BPH), one of the most destructive pests, causes annual losses of 1.0-1.5 billion kg of rice production. Identification and characterization genes resistant to BPH and develop resistant rice cultivars is the most effective and environment-friendly strategy for protecting the crop from BPH. We previously identified a BPH sensitive mutant bs1 by EMS mutagenesis. The BS1 gene had been cloned by a positional cloning strategy and localized in chroloplast. The aim of this project is to further elucidate the role of BS1 gene in BPH resistance regulation in rice . Functional analysis of BS1 gene will be conducted following: 1) Antixenosis (number of planthopper settled and rice resistant score) and antibiosis (nymphal survival rate, honeydew weight and fresh weight increment of female adult) parameters will be investigated to determine the biochemical mechanisms of BS1 in resistance against BPH. Function of the target gene will be further verified by overexpression experiments. 2) The expression pattern in different organs/tissuses of BS1, and its response to BPH infestation of different time will be determined by qRT-PCR. 3) In order to reveal the physiological basis of BS1 gene, the difference in components or concentration of polar lipids, volatiles and phytohormones like SA, JA in vegetative tissues between wide type and bs1 mutant will be explored. Furthermore, RNA-seq and bioinformatics analysis will also be employed to screen the downstream candidate genes. The cloning and characterization of BS1 in this project will enhance not only our understanding of the molecular basis of herbivore defense responses in rice, but should also facilitate the development of BPH resistant rice varieties.

虫害是影响水稻生产的重要因素，我国每年由褐飞虱为害造成的损失有1.0-1.5亿公斤。分离鉴定BPH抗性基因、培育抗性种质，是最经济有效的虫害防控措施。通过EMS诱变我们获得了易感BPH的突变体bs1，并用图位克隆技术分离获得了候选基因BS1。本项目拟在此基础上开展后续研究：1）通过考察排趋性和抗生性指标明确BS1参与BPH防御的生理机制，并通过过表达转基因技术进行验证；2）利用qRT-PCR技术分析该基因的组织表达及虫害诱导响应模式；3）通过分析野生型和突变体植株叶片中脂肪酸、挥发物组分差异、叶片和茎秆表皮细胞结构差异等明确BS1参与水稻对BPH虫害防御的调控作用，并通过RNA-seq技术筛选BS1的下游调控因子，最终阐明脂肪酸合成相关基因BS1在水稻与褐飞虱互作中的作用机制。研究结果不仅对揭示水稻抗虫分子机理有重要价值，还将为我国水稻抗虫育种发展贡献力量。

褐飞虱为害是导致水稻减产的重要因素，分离鉴定与褐飞虱抗性相关的基因并研究其作用机理，可为是培育抗飞虱水稻品系提供重要支撑。通过对EMS诱变获得的易感褐飞虱突变体bs1的图位克隆，我们获得了BS1 候选基因，序列分析表明其编码脂肪酸合成相关的酶。本项目在此基础上对BS1基因在水稻与褐飞虱互作中的作用开展了后续研究，获得了以下几个方面的结果：1）bs1突变体对褐飞虱的抗性减弱主要表现为，易感性增强和耐受性减弱。褐飞虱趋向于为害bs1突变体，突变体植株受害程度和死亡率均高于野生型，但对褐飞虱的发育和繁殖并无显著促进作用；通过转基因回补BS1基因获得的bs1-re回复株系接虫后，能将突变体水稻对褐飞虱的耐受性回复到野生型水平，对易感性也有一定恢复效果。2）BS1编码一个在叶绿体中的脂肪酸合成相关酶，该基因在叶片和叶鞘中高表达，在根、茎秆、幼穗和花药组织中表达量较低；褐飞虱取食能显著诱导野生型茎叶中BS1基因的表达，但在突变体中变化并不显著；3）BS1基因参与了水稻中脂肪酸的合成，与野生型相比，突变体中C16:0, C16:1和C18:3三个组分的含量显著下降；褐飞虱取食导致突变体中C18:3的下降，但对野生型无显著影响。4）转录组分析结果显示，BS1基因参与的脂肪酸代谢确实与水稻对褐飞虱的抗性相关，苯丙烷类和氨基酸代谢可能作为脂肪酸代谢的下游参与了水稻对褐飞虱的应答反应。5）水杨酸和茉莉酸类激素水平的检测结果显示，突变体中OPDA和 JA-Ile含量较野生型显著增加，但是BS1基因回补并没有恢复这一性状。总之，通过本项目的实施，我们明确了BS1基因突变导致了亚麻酸（C18:3）等含量的下降，其可能通过苯丙烷类代谢调控了水稻对褐飞虱的抗性。项目研究结果不仅对揭示水稻抗虫分子机理有重要价值，还将为我国水稻抗虫育种发展贡献力量。