玉米抗甘蔗花叶病毒病基因Scmv1的功能和抗病机理

Based on our previous research results and the fine-mapped maize SCMV disease resistance gene Scmv1 and the obtained transgenic maize plants that contained Scmv1 complementary expression construct, this research is going to evaluate the biological function of Scmv1. First, we will screen a few BAC libraries constructed from different resistant/susceptible maize elite inbred lines and sequence the screened positive BAC clones, to analysis the full length genomic DNA sequence of Scmv1 and to compare the DNA sequence, the expression level and resistant/susceptible among the inbred lines. We will also construct RNAi vector and over-expression vector for the transformation of maize receptor HiⅡ and later transfer the transgenes from the positive transgenic plants to other maize inbred lines by crossing. The resistance of Scmv1 to SCMV infection induced disease will be evaluated with these transgenic plants. Later we will obtain the full length Scmv1 cDNA from the resistant inbred line 1145 by RACE. With the full length cDNA, the prokaryotic and transcient expression vectors will be constructed and will be expressed in E.coli and maize protoplast respectively. Scmv1 encoding protein will be purified from the transformed E.coli cells and will be used for biological function analysis by biochemical method. Yeast two hybrid cDNA library will be constructed with RNA extracted from 1145 inbred line and Scmv1 bait vector will be constructed and used for screening the library to obtain the Scmv1 interaction protein encoding gene(s). Transcient expression in maize protoplast will be used to investigate the sub-cellular localization of Scmv1 and its interaction protein. The leaves of resistant/susceptible inbred lines and transgenic maize plants inoculated with the SCMV virus will be sampled to observe the sub-cellular pathological changes induced by the infection under transmission electron microscopy. Based on these experiment results, the possible biological function and molecular mechanism that Scmv1 may involve during its performance in maize resistance to sugarcane mosaic disease induced by SCMV infection, will be investigated.

玉米甘蔗花叶病毒（SCMV）病是全球范围内普遍发生的病害，严重危害玉米的生产。经过连续多年的研究、我们已完成了玉米抗SCMV主效基因Scmv1的精细定位，确定了抗病候选基因，获得了候选基因功能互补载体的转基因植株。在此基础上，本项目将比较各种来源抗感不同自交系Scmv1候选基因的基因组结构，搜索与抗性相关的序列位点，研究获选基因基础表达量与抗性的关系。将已获得的转基因T0代植株进行自交和杂交，后代群体接种SCMV，调查抗性，确定抗病基因Scmv1。同时将构建的候选基因RNAi干扰和过表达载体转化玉米，接种SCMV，鉴定抗性，进一步明确Scmv1的功能。从抗病自交系1145中获得Scmv1的全长cDNA，构建原核和瞬时表达载体，分析Scmv1编码蛋白的生化和生物学功能。构建酵母双杂交文库，筛选与Scmv1编码蛋白互作的蛋白，验证其互作的真实性。以玉米原生质体为载体开展Scmv1编码蛋白及其互

通过抗感不同自交系的BAC 文库筛选和BAC测序，明确抗病候选基因ZmTrxh的基因组序列及其在不同抗感自交系中基因组序列差异，ZmTrxh的抗病和感病等位基因具有完全相同的基因编码区和启动子附近区，但具有不同的上游调控区。明确ZmTrxh的表达高低差异与抗性之间有直接关联，表达量高的都表现出抗性表型。互补、过表达和RNAi 干扰的转基因植株群体等田间和实验室苗期接种实验都明确证实该候选抗病基因ZmTrxh与玉米SCMV病毒病抗性成正相关。是Scmv1 抗病位点的效应基因。ZmTrxh的原核表达、蛋白纯化以及生物化学分析，揭示抗病基因ZmTrxh编码一个具有分子伴侣功能、但不具备典型的硫氧还功能的h型硫氧还蛋白，是我们前期Scmv1定位区段的效应基因，其表达丰度玉米对SCMV病毒病紧密正相关。ZmTrxh蛋白在硫氧还活性位点基序中缺少两个经典的半胱氨酸，是玉米基因组中特殊的存在。正因如此，ZmTrxh不能还原二硫键，但具有很强的分子伴侣样活性。玉米原生质体亚细胞定位实验结果表明，ZmTrxh随机分散于细胞质来抑制SCMV病毒 RNA的积累。而且，ZmTrxh介导的玉米SCMV病毒病抗性与SA和JA相关的抗病信号途径没有明显关系。筛选酵母双杂交文库，获得抗病基因ZmTrxh编码蛋白的互作蛋白PEPC大亚基，并在烟草表皮细胞中以BiFC验证二者在胞质中的互作。这可能与SCMV病毒侵染导致叶片黄化有关。同时还发现抗病基因ZmTrxh编码蛋白还可以与SCMV病毒的P1蛋白互作。这些结果表明，抗病基因ZmTrxh可能在胞质中通过与病毒编码蛋白直接互作同时也通过与宿主玉米细胞的PEPC大亚基互作而保护其功能，从而在胞质中抑制病毒繁殖同时保护宿主细胞被破坏，使得玉米表现出对SCMV病毒的抗性。探索抗病基因ZmTrxh参与调控的这些生理过程在玉米生产中的作用，为培育优良新品种服务。