玉米PPa基因调控自主免疫反应的分子机理研究

Identifing and excavating broad-spectrum disease resistance gene is the key to cultivate disease-resistant varieties. Plant lesion mimic mutants usually have the characteristics of resistance to disease. And the majority of mutant genes involved in plant disease resistance pathway in the key node of disease resistance control network. Studying on these mutants is significant to find excellent broad-spectrum resistance gene and unravel innate immune response and disease defense mechanism in plant. We obtained a new lesion mimic mutant in maize in the early stage. The mutant gene has been cloned by map-based cloning method which encode an pyrophosphatase (PPase). This study will combine the multidisciplinary means to study the function of the PPa gene to elucidate its biological function. At the same time, the molecular mechanism of PPa gene involved in the regulation of innate immune response was analyzed by transcriptome analysis, protein interaction analysis, combined with phenotypic identification of mutants, double mutants, transgenic lines, identifying and excavating important disease resistance genes. Adding new knowledge to explain the molecular mechanism of lesion formation, innate immune response and defense response in maize. It would be useful theoretical guidance for further use of genetic engineering methods to create and cultivate broad-spectrum resistant maize varieties, improving maize yield and quality.

鉴定和挖掘广谱抗病基因是玉米抗病品种培育的关键。植物类病斑突变体通常具有抗病的特点，其突变基因参与植物抗病调控通路，处于抗病调控网络的关键节点，对于发现优良的广谱抗病基因，解析植物自主免疫反应和抗病防御机制具有重要意义。我们前期获得了一个新的玉米类病斑突变体，并利用图位克隆策略确定该突变基因编码一种焦磷酸酶（PPase），本项目将结合多学科交叉的手段，详细阐明玉米类病斑突变体基因PPa的生物学功能；同时结合突变体、双突变体、转基因系等的表型鉴定，差异转录组分析、蛋白质互作分析等，挖掘和鉴定重要功能的抗病防御基因；解析PPa基因调控玉米自主免疫反应的分子机理。为阐释玉米类病斑形成、自主免疫反应和抗病防御反应的分子机理增添新的知识，为进一步利用基因工程的方法创制和培育广谱抗病性的玉米品种，进而提高玉米的产量和品质提供目标基因和理论指导。

鉴定和挖掘广谱抗病基因是玉米抗病品种培育的关键。为了解析植物自主免疫和抗病防御机制，项目组开展了玉米类病斑相关突变体创制与遗传分析、基因定位与功能研究等工作。主要结果如下：（1）利用图位克隆和精细定位技术，在5号染色体112 kb区间范围内克隆了类病斑突变体ppa-1的候选基因为PPa，其编码无机焦磷酸酶。（2）对突变体表型，农艺性状、生理、生化及细胞学指标进行了观察和测定，表明该突变体正经历着细胞程序性死亡，呈现叶绿体细胞结构破坏，类囊体膜解体等情况。（3）荧光定量分析和亚细胞定位结果表明，PPa定位于叶绿体内，并在玉米植株不同部位（根、茎、叶片、雄穗、雌穗等）均有表达，呈现组成型表达。（4）通过蛋白体外表达纯化及酶活性分析，表明PPa突变型蛋白的活性明显高于野生型，推测可能由于酶活性的改变诱导类病斑表型的出现。（5）通过突变体和野生型转录组的测序分析，鉴定到氧化还原、谷胱甘肽代谢、次生代谢物生物合成、α-亚麻酸代谢和二萜生物合成等通路基因富集。（6）通过互补载体构建，获得了阳性植株；提取35S::PPa-GFP的总蛋白并通过免疫共沉淀技术和蛋白质组学分析找到了259个与PPa共同表达的基因；对富集基因功能进行分析发现PPa在能量代谢、生物胁迫和非生物胁迫响应过程中发挥着重要的功能。本项目创制的类病斑突变体及挖掘新基因功能研究技术为深入阐释玉米类病斑形成、自主免疫反应和抗病防御反应的分子机理增添新的知识，为进一步利用基因工程的方法创制和培育广谱抗病性的玉米品种，进而提高玉米的产量和品质提供目标基因和理论指导。