miR393b在玉米对纹枯病免疫反应中的调控机制研究

BLSB (Banded leaf and sheath Blight) has gradually become the main disease in major maize production region of China and seriously influenced the yield and quality. miRNAs have been involved in pathogen-resistant pathway with negatively regulating special target mRNAs (up- or down-regulation) at the post-transcriptional level. Zma-miR393b plays an important role in the response to BLSB infection based on our preliminary researches. In this study, we aim to analyze molecular mechanism of Zma-miR393b response to R. solani by over-expression of Zma-miR393b. We knockout miR393b with the CRISPR method to investigate the effect on pathogen resistance to R. solani. Additionally, we perform the RNAseq of transgenic lines in response to pathogens stress during different periods, to analyze the interaction between miR393b and its target gene, as well as illustrate the molecular mechanism of Zma-miR393b in regulating pathogen-resistant pathway. It will provide theoretical guidance for demonstrating the molecular mechanism of BLSB-resistance and creating new disease-resistant resources for breeding.

玉米纹枯病逐渐成为我国玉米主产区的主要病害，严重影响玉米产量和品质性状的提高。miRNAs通过转录后调控上调或者下调其靶向基因，参与植物病原菌的抗病调控途径。前期研究中，我们发现miR393b在玉米纹枯病抗病途径中起关键调控作用。本项目拟通过超表达miR393b来系统研究miR393b在纹枯病免疫反应中的功能；通过CRISPR/Cas9技术敲除miR393b研究其对纹枯病免疫反应的影响；对转基因株系在病原真菌胁迫下不同时间段的降解组进行分析，挖掘和验证miR393b的靶基因；对转基因株系在病原真菌胁迫下不同时间段的转录组进行RNASeq分析，研究miR393b及其靶基因的相互作用关系，系统解析miR393b调控玉米纹枯病免疫反应的分子机理。开展miR393b在玉米纹枯病免疫反应调控机制中的作用，对解析玉米对纹枯病的抗病性和创制抗病育种新种质提供理论指导。

利用差异转录组、蛋白质组以及生物信息学分析发现miR393b在玉米纹枯病抗病途径中起关键调控作用，它通过负调控生长素受体蛋白基因TIR1的表达，从而调节生长素信号传导途径，并以此响应病原真菌的胁迫。为深入解析miR393b在玉米对纹枯病免疫反应中的调控机制，构建Zm-miRNA393b超表达、CRISPR/Cas9敲除载体，遗传转化获得阳性超表达和敲除转基因系，并对转基因系的抗病性、降解组和转录组开展研究，系统研究miR393b在玉米应对纹枯病免疫反应中的作用。因受遗传转化条件限制以及Cas9敲除系统未能工作，转基因系获得并不顺利，项目研究期限内仅获得了T2代过表达转基因系，Cas9敲除系未能实现打靶，没有获得miRNA393b的敲除材料。