番茄AP2/ERF类转录因子协同调控蜡质芽胞杆菌AR156防治根结线虫病机理研究

Root knot nematode is a species of obligate parasite of plant root, which is a serious threat to agriculture worldwide. Bacillus cereus AR156 is our patented and sequenced strain that is registered as a biopesticide against root-knot nematode in tomato. Previous study showed that B. cereus AR156 can significant control tomato root knot disease. Other whiles, it can induce systemic resistance to pathogens in plants. In addition, the transcriptome study found that B. cereus AR156 can induced series of AP2/ERF family transcriptional factors coding genes expression during the process of B. cereus AR156 induced tomato resistance to root knot nematode. In the present project, we will firstly clone the selected AP2/ERF family transcriptional factors and do the subcellular localization analysis. Then Study on their transcriptional activity by yeast by using yeast single-hybrid technique. Secondly, we will construct the AP2/ERF family transcriptional factors’ overexpression and knock out transgenic tomato plants then evaluate the resistance activities to root knot nematode and ISR phenotype triggered by B. cereus AR156 of transgenic tomatoes. Finally, we will find out the interacted target genes of AP2/ERF family transcriptional factors by ChIP sequencing and clearly know the molecular function mechanisms of these AP2/ERF family transcriptional factors. This study will lead us to understand how AP2/ERF family transcriptional factors regulate the B. cereus AR156 induced tomato resistance to root knot nematode and thoroughly analyzes the mechanism of AP2/ERF family transcriptional factors on coordinated regulating the B. cereus AR156 biocontrol to root knot nematode disease in tomato, and provide the theoretical basis and method guidance for the application of this bacterium.

根结线虫为植物专性寄生物，严重威胁农业生产。蜡质芽胞杆菌AR156是本组已测序专利菌株，获农药登记用于防治番茄根结线虫病。前期研究发现此菌可诱导植物产生系统抗性；转录组学研究发现，AR156在诱导番茄产生对根结线虫抗性过程中，能激活一系列AP2/ERF类转录因子的上调表达。本项目首先拟对筛选的AP2/ERF类转录因子进行克隆、亚细胞定位分析，并利用酵母单杂技术研究其转录活性；另一方面通过构建过表达及敲除突变体转基因番茄，评估其对线虫的抗性及AR156诱抗活性，以此明确AP2/ERF类转录因子的生物学功能；利用ChIP测序技术对转录因子的互作靶标进行筛选及验证，明确AP2/ERF类转录因子的分子调控机制。最终将对AP2/ERF类转录因子在调控蜡质芽胞杆菌AR156防治番茄根结线虫过程中的作用机制进行分析，阐释其在防治根结线虫过程中协同调控植物抗性的作用机理，为产品的实际应用提供理论依据。

根结线虫为植物专性寄生物，严重威胁农业生产。蜡质芽胞杆菌AR156是本组已测序专利菌株，获农药登记用于防治番茄根结线虫病。前期研究发现此菌可诱导植物产生系统抗性；转录组学研究发现，AR156在诱导番茄产生对根结线虫抗性过程中，能激活一系列AP2/ERF类转录因子的上调表达。在番茄植株内对上述关键转录因子编码基因Le-ERF1a、Le-ERF4进行过表达和敲除分析，利用转基因植株对诱抗表型及对根结线虫抗性表型进行验证分析；另外，对筛选的转录因子Le-ERF1a、Le-ERF4的生物学功能及生化功能研究，对其靶标基因开展预测、筛选及验证，检测其靶标基因在AR156防治番茄根结线虫病过程中的表达情况；最后总结分析番茄AP2/ERF类转录因子协同调控AR156防治番茄根结线虫病的作用机理。延伸研究发现植物内源miR472 在生防菌控蜡质芽胞杆菌AR156诱导植物系统抗性过程中起着重要的调节作用。利用deep sequencing 以及生物信息学技术，筛选获得一组参与调控的miRNA，包括miR472。Northern Blot验证试验显示，蜡质芽胞杆菌AR156诱导拟南芥产生对Pst DC3000抗性过程中，与对照组相比miR472在蜡质芽胞杆菌AR156处理植株体内被显著下调。miR472缺失突变体致病性测定结果显示出了对病原细菌Pst DC3000的抗性；与之相反的，miR472过表达转基因植株表现得对病原菌更敏感。转录组测序分析发现miR472过表达后植株中存在上千个差异性表达基因。对miR472靶标预测结果显示，其靶标包括大量CC-NBS-LRR类基因，研究发现miR472靶基因的表达受到miRNA负向调控。对靶标基因转基因敲除突变体的致病性测定结果显示除了对病原细菌Pst DC3000的抗性下降。荧光定量PCR结果显示miR472靶基因在蜡质芽胞杆菌AR156诱导系统抗性期间上调表达。综上所述，蜡质芽胞杆菌AR156诱导植物产生系统抗性通过抑制miR472的表达，引起其靶标基因的上调，从而激活植物体自身的防卫反应。

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