基于阻断甘蔗花叶病病原胞间移动的抗病甘蔗种质创新研究

Sugarcane is the most important sugar crop around world, especially in China. Sugarcane mosaic disease, caused by Sugarcane mosaic virus (SCMV) or Sorghum mosaic virus (SrMV), is one of the most serious viral diseases in sugarcane. The stains of SCMV or SrMV are complicate and flexible. It is time consuming and laborious to breed cultivar resistant to SMD by traditional cross breeding. So far the SCMV-CP has been extensively used to create germplasm resistant to SMD in transgenic sugarcane research. However, the resistance mediated by the PDR (pathogen-derived resistance) is not durable because it is not broad-spectrum. The mutant is susceptible to other strains of the same species virus, no mention of other species virus. Recent studies show that the general interaction mechanism in plant for viral infection was established during the long period of co-evolution between virus and its host plant. There are some specific proteins are essential for viral infection in plant host. When these host genes are silenced, the mutants, which show normal growth and development as wild types, have broad-spectrum and durable resistance to viral infection. This type of host genes acted as recessive resistance genes. In this project, we plan to construct cDNA libray of sugarcane, and then screen and identify sugarcane endogenous factors which specifically interacted with the movement protein P3N-PIPO of SCMV by yeast two-hybrid system and other protein-protein interaction techniques. The results would reveal the molecular mechanism for viral cell-to-cell movement of SCMV in sugarcane. We further plan to develop RNA interference strategy to silence the host factors which are involved in viral cell-to-cell movement through transgenic sugarcane, preventing efficient viral cell-to-cell movement in transgenic sugarcane. Thus, it is expectable to create new sugarcane germplasm with broad-spectrum sugarcane mosaic resistance. This project will elucidate the molecular mechanism of sugarcane mosaic disease casual agent cell-to-cell movement and develop a new strategy for the research of transgenic sugarcane resistant to pathogen. The objective of this project is to create new sugarcane germplasm charactering as broad-spectrum and durable resistance to sugarcane mosaic disease. It will also benefit the development of cross discipline between plant breeding and plant protection. Generally, this project is important for its scientific significance and application value.

甘蔗是重要的糖料作物，甘蔗花叶病是严重危害甘蔗生产的病毒性病害之一，其病原主要是SCMV和SrMV。甘蔗花叶病病原株系复杂多变，通过传统杂交选育抗性品种困难。目前甘蔗抗花叶病转基因研究以转CP基因为主，但这种由PDR介导的抗性不具有广谱性。研究表明，病毒与植物寄主长期协同进化并选择性利用寄主因子，沉默病毒完成侵染必须的寄主因子基因，植物可以获得对病毒广谱抗性，并且生长发育正常。鉴于此，本项目拟从病毒与寄主互作分子机制入手，利用酵母双杂交技术，筛选鉴定与SCMV运动蛋白P3N-PIPO互做的甘蔗因子；利用RNAi技术沉默参与病毒胞间移动的甘蔗基因，阻断病毒在甘蔗体内的胞间移动，获得具有花叶病广谱抗性的甘蔗新种质。本研究对于阐明甘蔗花叶病病原胞间移动的分子机制，创制对花叶病有广谱抗性的甘蔗新种质，发展甘蔗转基因抗病育种新策略，促进作物育种和植物保护交叉学科的发展，具有重要的科学意义和应用价值。

甘蔗花叶病是严重危害甘蔗生产的病毒性病害之一，其病原主要为甘蔗花叶病毒（SCMV），高粱花叶病毒（SrMV）和甘蔗条纹花叶病毒（SCSMV），由蚜虫以非持续性方式传播，常规手段难以防止。抗花叶病是甘蔗育种重要目标之一，但是传统杂交方法选育抗花叶病甘蔗品种常因花期不遇、周期长、优良基因型难以聚合等原因，难以获得高产高糖等农艺性状与抗花叶病聚合的基因型。转基因技术的发展促进了甘蔗转基因抗病育种研究。但是，通过病毒自身基因介导的抗性仅能对基因来源病毒株系或序列相近的株系产生抗性，没有广谱抗性，并因为导入外源核酸片段，尤其是导入病毒的核酸片段，使转基因植株面临复杂的安全评价问题。病毒结构简单，必须利用寄主因子才能建立系统性侵染。胞间移动是植物病毒在寄主体内建立系统性侵染的关键环节，干扰或阻断病毒胞间移动将使寄主获得对病原的抗性。为此，我们开展了以下研究工作：.克隆了SCMV、SrMV和SCSMV的11个功能蛋白的编码序列，其中P3N-PIPO的编码序列是通过融合PCR技术获得的。通过同源克隆技术，从甘蔗中分离到了阳离子结合蛋白基因ScPCaP1，利用酵母双杂交和双分子荧光互补实验证明了ScPCaP1与3种病毒P3N-PIPO的互作。以SCMV-P3N-PIPO为代表，详细解析了其与ScPCaP1互作的分子机制。构建了甘蔗栽培种ROC22的cDNA膜系统酵母文库，以SCMV-P3N-PIPO为诱饵，筛选并通过酵母双杂交验证获得6个与之互作的基因。首次在模式植物本氏烟和二穗短柄草上建立了SCMV的系统侵染体系，本氏烟无明显症状，二穗短柄草有典型的花叶病症状，为分离并验证与SCMV互作的寄主因子搭建了实验体系（可在其上进行基因敲除、基因沉默、过表达等）。利用RANi技术，沉默了ScPCaP1基因，获得12株转基因甘蔗，其中4株表现出良好的花叶病抗性。.本项目的成果发表了本研究共发表了7篇论文，其中SCI论文3篇，中文论文4篇，申请并获得授权国家发明专利4项。培养博士研究生1名，硕士研究生7名。