棉花细胞色素P450基因GbCYP72A在黄萎病抗性中的功能分析

Verticillium wilt is a major disease in cotton planting, and seriously affects the yield and quality of cotton. Genetic engineering is an important method to resolve the problem. Hence, the research on the Verticillium wilt resistance-associated genes is of great significance. In our previously study, the novel Verticillium wilt resistance-related gene GbCYP72A was cloned through QTL fine mapping. The orthologs in other plants, including Arabidopsis, rice and tobacco, were reported to be induced by the infection of pathogens, but the function of them has not been revealed. As it was verified, the transcript abundance of GbCYP72A was highly induced by the infection of Verticillium dahlia, and that in resistant variant was significantly higher than susceptible variant. Moreover, the disease resistance of GbCYP72A silencing plant was significantly reduced. In this research, the function of GbCYP72A in Verticillium wilt resistance will be further confirmed by evaluating the disease resistance of transgenic Arabidopsis and cotton. The integration of transcriptomics and metabolomics will be applied to analyze the differentially expressed genes and metabolites. Together with the physiological analysis, those results will be used to reveal the molecular mechanism of GbCYP72A in Verticillium wilt resistance. The completion of this research can deepen the theoretical principle of Verticillium wilt resistance, and provide effectual gene resource for the gene engineering.

黄萎病是棉花生产中的主要病害，严重影响棉花的产量和质量，基因工程是解决这一问题的重要方法，对黄萎病抗性相关基因的研究具有重要意义。前期研究中，本项目组通过QTL精细定位克隆到一个新的黄萎病抗性相关基因GbCYP72A，其在拟南芥、水稻、烟草等植物中的同源基因受多种病原菌诱导表达，但这些基因的功能尚且未知。经验证，GbCYP72A的表达受黄萎病菌侵染的强烈诱导，在抗病品种中的表达量显著高于感病品种，且GbCYP72A基因沉默表达植株的抗病性显著降低。本研究拟通过转基因技术将GbCYP72A导入拟南芥和陆地棉中，并检测转基因株系的抗病性，进一步证实GbCYP72A基因的抗黄萎病功能；利用转录组-代谢组联合分析转基因株系中的差异表达基因和代谢物质，结合生理指标检测结果，阐述GbCYP72A基因抗黄萎病的分子机制。研究结果可深化棉花黄萎病抗性研究的理论基础，并为棉花抗黄萎病基因工程提供有效基因。

黄萎病是棉花生产中的主要病害，严重影响棉花的产量和质量。基因工程是解决这一问题的重要方式，因此对棉花黄萎病抗性相关基因的研究具有重要的理论价值和实践意义，但对棉花中细胞色素P450基因家族的抗黄萎病功能还少有报道。本研究通过病毒诱导的基因沉默和转基因技术证实GbCYP72A1和GbCYP72A2基因的抗黄萎病功能，并发现由于编码蛋白三维结构的不同GbCYP72A2具有更强的抗病功能。利用定量RT-PCR揭示GbCYP72A1和GbCYP72A2基因的表达特征，发现油菜素内酯信号途径在两基因介导的黄萎病菌响应途径起到重要作用。进一步通过转录组测序发掘GbCYP72A1和GbCYP72A2基因介导的黄萎病抗病途径的关键信号途径和下游基因，结果显示植物激素信号转导途径、植物-病原菌互作途径和MAPK信号途径在两基因介导的抗病中起到关键作用。同时转录组数据中大量差异基因富集在细胞膜上，这与两基因编码蛋白的亚细胞定位结果一致，说明两基因在植物细胞膜上发挥功能。此外，病毒诱导的基因沉默结果和转基因种子萌发实验结果显示，除抗黄萎病功能外，GbCYP72A2基因还在促进植物生长和对ABA响应中具有功能。本研究结果深化了棉花黄萎病抗性研究的理论基础，并为棉花抗黄萎病基因工程提供有效的基因资源。