转录因子OsbHLH010介导茉莉酸途径调控水稻抗二化螟的分子机制

The plant transcription factors (TFs) were proved to be involved in regulation of many biological pathways. Previous study showed that a few TFs regulated phytohormone-mediated rice defense against herbivorous insects, but the molecular mechanism is still unknown. In our previous work, we confirmed that Chilo suppressalis- or Cnaphalocrocis medinalis-induced rice resistance to C. suppressalis was mediated by jasmonic acid (JA) by bioassay tests, chemical analyses, and RNA sequencing (RNA-seq) experiments. Based on above results, we screened a bHLH transcription factor OsbHLH010, and speculated it might regulate the JA-mediated herbivore-induced defense of rice against C. suppressalis. To confirm this hypothesis, we will first investigate the biochemical characteristics of OsbHLH010 protein including subcellular localization and transcriptional activation. Second, transgenic lines with overexpression and CRISPR-mediated knockout of OsbHLH010 gene will be developed followed by assessing their resistance to C. suppressalis and detecting the contents of JA. Third, to understand the regulatory mechanism of the OsbHLH010 functioning in JA-mediated defense response of rice plants to C.suppresalis, we will perform RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) on transgenic rice lines to determine the binding sequence and target JA pathway genes of the OsbHLH010. The interactions between OsbHLH010 and the target genes will be validated by various approaches such as ChIP-qPCR, yeast one-hybrid assay, electrophoretic mobility shift assay. The results of this project will lead to better understanding of the regulation of TFs involved in rice plant induced defense against herbivory and may provide a promising candidate gene for crop breeding.

转录因子参与调控植物的众多生理过程。前人研究显示一些转录因子参与激素途径调控水稻的诱导抗虫防御反应，但具体分子机制仍不清楚。前期，我们通过生物测定、化学分析和多组学实验，证实茉莉酸(JA)途径调控二化螟和稻纵卷叶螟为害诱导水稻对二化螟的抗性。在此基础上，筛选了一个转录因子OsbHLH010，推测其介导JA途径调控水稻的诱导抗虫性。为此，本项目拟以该基因为研究对象，研究其编码蛋白的亚细胞定位和转录活性，验证其转录因子特性；创制基因超量表达和敲除水稻材料，检测其对二化螟的抗性，并分析JA含量和JA途径基因表达，明确OsbHLH010与JA途径基因的关系；通过染色质免疫共沉淀测序技术鉴定OsbHLH010直接调控的JA途径基因，并验证二者的结合关系，以阐明其介导JA途径调控水稻抗二化螟的分子机制。研究结果将加深理解转录因子调控水稻诱导抗虫反应的作用机理，并为作物抗虫育种提供候选基因。

转录因子在调控植物对植食性昆虫的防御过程中发挥重要作用。本项目综合运用分子生物学、生物化学、昆虫生物测定、植物化学分析和转录组学等技术手段，系统研究了OsbHLH010在水稻调控二化螟抗性中的作用与机制。主要结果如下：1）成功克隆水稻OsbHLH010转录因子，其开放阅读框1422 bp，编码473个氨基酸，生物信息学分析显示，OsbHLH010蛋白与玉米ZmMYC5亲缘关系最为接近，通过酵母系统证实其没有自激活活性；二化螟（Chilo suppressalis）为害可迅速激活并诱导OsbHLH010持续表达，外源茉莉酸（Jasmonic acid, JA）处理和机械损伤也能迅速诱导其表达，但外源水杨酸（Salicylic acid, SA）处理仅诱导其微弱表达；2）室内生测和田间试验结果表明敲除OsbHLH010显著降低水稻对二化螟抗性，但也会影响水稻其他农艺性状。3）化学分析表明敲除OsbHLH010不影响水稻组成型JA信号途径激素含量，但显著降低二化螟为害后JA-Ile、JA-Val和MeJA等含量；转录组分析结果显示，OsbHLH010通过调控JA合成和降解途径关键酶基因表达而调控JAs的合成，进而影响JA介导的苯丙烷途径中抗虫防御相关的次生代谢物质合成，最终调控水稻对二化螟的抗性。本项目研究结果有助于加深理解转录因子调控水稻诱导抗虫防御反应的作用机理，为培育抗虫水稻品种提供基因资源。