甘蔗抗梢腐病氮代谢和系统获得性抗性途径关键组分γ-谷氨酰转移酶基因SoGGT1克隆及功能鉴定

Pokkah boeng disease (PBD) is becoming predominant among the foliar diseases only second to smut during the recent years in most of sugarcane area of China. The pathway of nitrogen metabolism and systemic acquired resistance play an active role during the process of sugarcane response to PBD pathogen. γ-glutamyltransferase gene SoGGT1, which is the key component of this pathway is considered to be an important R gene. Based on the gene cloning of SoGGT1 fragments from sugarcane, this research proposal would like to clone the full-length sequences of SoGGT1 genes from sugarcane (Saccharum officinarum) by homology cloning strategy and rapid-amplification of cDNA ends, and to analyze them with relative bioinformatics methods. We would use real-time fluorescence quota PCR to analyze the gene expression of SoGGT1 in different sugarcane varieties, different infection period and abiotic stress. We would construct sense expression vectors and RNAi expression vector, introduce the sense expression vectors into Arabidopsis and sugarcane, and introduce the RNAi expression Vector into sugarcane. We would analyze the agronomic character, morphologic variation, gene expression, physiological characteristics, resistance assessment, protein verification of the transgenic plants, and then provides the important foundation for using genetic engineering means to improve disease resistance in sugarcane.

甘蔗梢腐病在我国大部分蔗区已逐渐上升为仅次于黑穗病的主要叶片病害。氮代谢和系统性获得抗性途径在甘蔗响应梢腐病菌侵染过程中发挥积极作用。基于组学信息和参考文献将这部分通路的关键组分γ-谷氨酰转移酶基因SoGGT1视为抗病相关基因。为了解其基因表达特征及功能，本项目拟在分离到其基因片段的基础上，利用同源克隆结合RACE技术扩增其全长并进行生物信息学分析；定量PCR分析基因在不同抗性甘蔗品种、不同接种时期和非生物胁迫下的基因表达特征；构建SoGGT1基因过量表达和RNAi干扰载体，把过量载体导入拟南芥和甘蔗，RNAi载体导回甘蔗，并对转基因植株进行农艺性状观测、基因表达验证、生理特性、抗性评价、蛋白验证等分析，进而为利用基因工程手段提高甘蔗抗病性提供全新途径。

甘蔗梢腐病在我国大部分蔗区已逐渐上升为仅次于黑穗病的主要叶片病害。氮代谢和系统性获得抗性途径在甘蔗响应梢腐病菌侵染过程中发挥积极作用。本项目以典型甘蔗材料为研究对象，在前期研究基础上，围绕“甘蔗防御梢腐病菌侵染生理生化机制”这一主题，重点关注甘蔗氮代谢和系统抗性途径重要组分SoGGT1基因的热点问题，开展克隆SoGGT1基因全长，并对其进行生物信息学分析；实时定量PCR分析基因在不同甘蔗品种，同一品种不同接种时期和非生物胁迫下基因表达；通过构建过量表达和RNAi干扰载体，将SoGGT1基因导入拟南芥和甘蔗中，并对转基因植株进行综合性状分析，最终探明了SoGGT1基因在甘蔗防御梢腐病菌侵染所起的功能。该项目结果对于利用基因工程手段提高甘蔗抗梢腐病性具有重要意义。通过该项目资助，3位科技人员获得了职称晋升，发表6篇文章（其中3篇中文核心，3篇SCI），申报4项国家发明专利。