芽胞杆菌诱导玉米系统抗性关键miRNA鉴定及调控机理研究

Induced systemic resistance in recent years has become an important development direction of plant disease biocontrol. Many reports have confirmed that miRNA is close related to systemic acquired resistance, but fewer studies focus on the role of miRNA in induced systemic resistance. In our previous study, we constructed a Bacillus mutant FZB42△sfp△alsS, which was deficient in the induction of systemic resistance. And sRNA sequencing was used to study the maize miRNA changes in response to FZB42, FZB42△sfp△alsS and control, and a miRNA named novel_36 associated with induced systemic resistance was identified. In this project, stem-loop qRT-PCR combined with 5’ RLM-RACE and transiently expression system will be used to identify the target genes of novel_36. Then, Transgenic maize that overexpressing or silencing novel_36 and knocking out target mRNA will be constructed. Afterwards, the disease resistance of transgenic maize was determined. Moreover, high-throughput sequencing will be used to compare the difference between transgenic maize and wild type, which will facilitate us to identify potential key regulatory genes. This project would illustrate the role of maize miRNA in induced systemic resistance, and provide the theoretical foundation for biological control maize disease.

诱导植物产生系统抗性已成为生物防治植物病害的一个重要发展方向。目前已有不少报道证实植物miRNA与系统获得抗性（SAR）有着密切关系，但在诱导系统抗性（ISR）进程中的研究则较少。本课题组前期构建了丧失诱导玉米产生ISR的芽胞杆菌FZB42突变体，通过对野生型FZB42、突变体处理后的玉米进行sRNA测序和生物信息学分析，筛选出调控ISR的关键miRNA，并命名为novel_36。本项目拟通过northern blot、RACE法和瞬时表达体系鉴定novel_36的靶基因；同时创建novel_36过表达、沉默和靶基因敲除的转基因玉米株系，并测定转基因株系的抗病能力；在此基础上利用转录组测序技术分析系统抗性关键基因的表达水平，结合免疫共沉淀等技术筛选出靶基因调控的下游候选基因。本研究从miRNA角度解析了芽胞杆菌诱导玉米系统抗性的分子机制，为生物防治玉米病害打下理论基础。

诱导植物产生系统抗性已成为生物防治植物病害的一个重要发展方向。目前已有不少报道证实植物miRNA与系统获得抗性（SAR）有着密切关系，但在诱导系统抗性（ISR）进程中的研究则较少。本研究通过对野生型FZB42、FZB42△sfp△alss突变体处理后的玉米进行sRNA测序，筛选出调控ISR的关键zma-miR169i（原命名novel_36）。通过TargetFinder软件预测、降解组测序、stem-loop qRT-PCR和双荧光素酶实验证实Zm00001d027874是zma-miR169i的靶基因。同时，利用转基因技术创建zma-miR169i过表达转基因玉米，利用Crispr/Cas9技术创建zma-miR169i敲除玉米和靶基因Zm00001d027874敲除玉米。在此基础上，对转基因玉米的抗病表现、防卫基因表达水平和细胞坏死、活性氧等抗病指标进行测定，证实zma-miR169i负调控玉米抗病反应，而靶基因Zm00001d027874正调控玉米抗病反应。此外，通过RNA-seq技术初步筛选出靶基因Zm00001d027874调控的下游功能基因。本研究从miRNA角度解析了芽胞杆菌诱导玉米系统抗性的分子机制，为生物防治玉米病害打下理论基础。.通过3年的资助，本项目取得如下进展：.第一，通过TargetFinder软件预测、降解组测序、stem-loop qRT-PCR和双荧光素酶实验证实Zm00001d027874是zma-miR169i的靶基因。.第二，通过转基因技术、抗病表型和抗病指标测定，明晰了zma-miR169i在抗病进程中的生物学功能—负调控玉米抗病反应。.第三，通过RNA-seq技术初步筛选出靶基因Zm00001d027874调控的下游功能基因(Zm00001d011377, Zm00001d009714, Zm00001d041204, Zm00001d002029, Zm00001d041418, Zm00001d020043, Zm00001d043116, Zm00001d031445, Zm00001d027878, Zm00001d012603, Zm00001d033353），为下一步研究靶基因的调控机理打下基础。.项目执行期间，共发表研究性SCI文章2篇，申请发明专利1项。.本研究顺利完成了项目规定的各项考核。