甘薯转录因子IbPIF1调控茎线虫病抗性的分子机制解析

Sweetpotato stem nematode disease is a serious disease damaging sweetpotato and greatly reduces the quality and yield of this crop. Based on the analysis of stem nematode-resistant sweetpotato RNA-seq data in the previous studies, a stem nematode resistance related transcription factor gene IbPIF1 was screened and isolated. We found that expression of the IbPIF1 gene was significantly induced by stem nematodes and the expression level of IbPIF1 in the resistant sweetpotato varieties was significantly higher than in the susceptible varieties. In this study, IbPIF1 is transferred into sweetpotato varieties by Agrobacterium tumefaciens-mediated transformation system. Using overexpression, RNAi interference and CRISPR-Cas9 knock out, the transgenic plants are produced and the function of IbPIF1 is characterized. The IbPIF1 interacting proteins and downstream regulation genes of IbPIF1 are screened and identified using immunoprecipitation (IP), mass spectrometry, ChIP-seq and RNA-seq technologies, combining bioinformatics and molecular biology analysis methods. Molecular mechanisms of IbPIF1 for the resistance to stem nematodes will be illustrated based on the overall results. This study not only lays on an important foundation for further expounding molecular mechanisms and regulatory network of resistance genes for stem nematodes, but also provides important resistant genes and new resistant materials for improving disease resistance in sweetpotato.

甘薯茎线虫病是甘薯的主要病害之一，可导致甘薯品质和产量大幅下降，对我国甘薯生产已构成严重威胁。前期通过甘薯茎线虫诱导差异表达谱分析，筛选克隆了抗茎线虫病相关转录因子IbPIF1，该转录因子受茎线虫诱导显著上调表达，并且在抗病品种中的表达水平显著高于感病品种。本项目拟在此基础上，构建IbPIF1基因过表达、RNAi和CRISPR-Cas9等载体，用农杆菌介导法转化甘薯品种，鉴定IbPIF1基因的茎线虫病抗性功能；利用免疫沉淀与质谱分析、ChIP-seq以及RNA-seq等技术，结合生物信息学和分子生物学分析方法，鉴定IbPIF1蛋白的互作蛋白及其下游调控基因，解析IbPIF1的抗性分子机制，初步构建其参与的茎线虫病抗性分子调控网络。研究结果不仅在理论上将为阐明甘薯抗茎线虫病分子机制及调控网络奠定基础，同时可为甘薯抗病育种提供重要抗病基因和抗病新材料。

本项目利用农杆菌介导法将IbPIF1导入甘薯品种商薯19和栗子香中。IbPIF1基因的过表达显著提高了转基因甘薯植株的茎线虫病抗性，而RNAi干扰植株的茎线虫病抗性显著降低。茎线虫侵染后，IbPIF1过表达转基因植株中抗氧化酶活性、脯氨酸、木质素和胼胝质含量显著增加，而H2O2和MDA含量显著降低，木栓层细胞层数增加。对茎线虫侵染后的转基因甘薯植株进行了转录组和qRT-PCR分析，结果表明IbPIF1过表达显著上调了抗氧化系统、胁迫响应和倍半萜合成途径相关基因的表达，从而显著提高了转基因甘薯植株的茎线虫病抗性。通过酵母双杂交实验发现IbPIF1与茉莉酸信号通路关键调节因子JAZ蛋白互作。通过酵母单杂交、凝胶阻滞实验和荧光素酶报告实验发现，IbPIF1能够结合到抗茎线虫病基因IbMVD的启动子上，并激活该基因的表达。以上研究阐明了IbPIF1调控甘薯抗茎线虫病的分子机制，初步构建IbPIF1基因参与的茎线虫病抗性分子调控网络，同时可为甘薯抗病育种提供重要抗病基因和抗病新材料。通过本项目的研究，共发表论文7篇，其中SCI收录论文6篇。获得国家发明专利1项。培养博士2名、硕士2名。