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挖掘并利用作物优异抗病基因是进行农作物品种遗传改良的重要途径。辣椒是我国乃至世界最重要的蔬菜作物之一，黄瓜花叶病毒病（CMV）是目前影响辣椒生产的一种重要病害。基于项目组多年的前期研究，本项目针对栽培辣椒品种中缺乏抗CMV基因的现状，以高抗CMV辣椒Vc16a为材料，首先采用抗病基因同源序列法和cDNA-AFLP技术，克隆抗CMV相关候选RGAs或差异表达基因，其次通过RACE技术获得抗CMV相关候选RGAs或差异表达基因的全长，分析其结构和序列特征，再利用荧光定量PCR技术分析抗CMV候选基因在不同组织的时空表达特性，最后利用转基因技术对抗CMV候选基因进行功能验证，明确其与抗CMV的相关性，最终克隆出抗CMV基因。项目挖掘的优异基因及相关基础理论研究结果对于辣椒及其它作物的抗CMV遗传育种将具有重要的理论和实践意义。 Pepper is one of the most cultivated Solanaceae species worldwide. Several disease caused by virus, fungi, bacteria and nematodes can reduce yield and quality of pepper fruits, and cucumber mosaic virus (CMV) is the most important virus disease. Genetic improvement of a variety with a resistance to CMV is slower, most due to the absence of resistance gene of CMV. Capsicum frutescens var. Vc16a with a high resistance to CMV is used in this study. First, CMV resistance gene or differential expression gene is cloned by resistance gene analogue (RGA) and cDNA amplified fragment length polymorphism (cDNA-AFLP). Second, full-length of these candidate gene is obtained by rapid amplification of cDNA ends (RACE), gene structure and sequence features is described, spatial expression characteristics in different tissues is analyzed based on quantitative fluorescent PCR (QF-PCR). Finally, a genetic transformation procedure is obtained for functional validation of candidate genes, if the correlation between the resistance to CMV and candidate genes is significant, resistance gene then is cloned. This approach will support an intensive study of functional analysis of potential resistance genes, and this is particularly well adapted to CMV-resistance-based approaches to breed a new pepper variety. |
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