甘薯抗茎线虫病基因在转基因甘薯中的抗病分子机理研究

Sweetpotato stem nematode disease is a serious disease damaging sweetpotato and greatly reduces the quality and yield of this crop. In this study RNA interference vectors and overexpression vectors are constructed with IbMIPS1,IbMVD,IbChi and IbGlu genes cloned by the former project of National Natural Science Foundation of China. The four genes are transferred into sweetpotato varieties susceptible to stem nematodes by Agrobacterium tumefaciens-mediated transformation system. Large numbers of transgenic plants resistant to stem nematodes are obtained. The expression of the four genes in different tissues of transgenic plants is analyzed by real-time quantitative PCR. The expression of the four genes in the cells of transgenic plants is analyzed by immunogold labeled technique and enzyme activity analysis. Differentially expressed proteins in transgenic sweetpotato plants are screened using two-dimensional gel electrophoresis and mass spectrometry technique, and the proteins interacted with the four genes are further screened using yeast two-hybrid system to analyze the resistance mechanism of the four resistance genes. This success will lay on an important foundation for further expounding molecular mechanism of resistance of stem nematode resistance genes.

甘薯茎线虫病是甘薯的严重病害，导致甘薯品质和产量严重下降，对我国甘薯生产已构成严重威胁。本项目用上一个国家自然基金项目克隆得到的甘薯抗茎线虫病基因IbMIPS1、IbMVD、IbChi和IbGlu，构建RNAi干扰表达载体和超表达载体；用根癌农杆菌介导法，转化感茎线虫病甘薯品种，获得大量抗茎线虫病转基因植株；通过实时荧光定量PCR和酶活性分析研究这4个基因在转基因甘薯植株不同组织中的表达，用免疫金标记技术研究其在细胞内的表达；用蛋白质双向凝胶电泳及质谱技术分析抗性相关蛋白的表达，用酵母双杂交分析与目的基因互作的蛋白，明确这4个基因的抗茎线虫病分子机理。本研究的成功，对进一步阐明甘薯抗茎线虫病分子机理及抗病育种具有重要的理论意义和应用前景。

本项目按原计划完成，达到了预期的研究目标，并取得重要研究进展：用农杆菌介导法将IbMIPS1、IbMVD、IbChi和IbGlu基因分别导入感茎线虫病甘薯品种，获得抗茎线虫病转基因植株。茎线虫侵染后，过表达IbMIPS1基因植株的肌醇含量、IP3含量、PA含量、Ca2+含量、ABA含量、胼胝质含量、木质素含量和抗氧化酶活性显著增加，MDA含量显著降低，H2O2含量在侵染1～2 d迅速增加之后再降低。过表达IbMVD基因植株的倍半萜含量、胼胝质含量、木质素含量和抗氧化酶活性显著增加，MDA含量显著降低。转录组及qRT-PCR等分析表明，在茎线虫侵染下，转基因植株与野生型植株相比，IbMIPS1基因的过表达上调了肌醇合成、磷脂酰肌醇信号途径、ABA信号途径和抗病相关基因的表达；IbMVD基因的过表达上调了甲羟戊酸代谢途径、倍半萜合成途径和胁迫响应相关基因的表达；IbChi和IbGlu基因的过表达上调了胁迫响应相关基因的表达，从而显著提高转基因甘薯植株的茎线虫病抗性。研究结果对进一步阐明甘薯抗茎线虫病分子机理具有重要理论意义，这些基因对改良甘薯生物胁迫和非生物胁迫抗性具有应用价值。发表论文8篇，其中SCI收录论文5篇，国内会议论文3篇。获得国家发明专利2项。培养博士2名、硕士2名。