玉米大斑病菌论文：玉米大斑病菌CWI-MAPK级联途径中StBCK1基因功能的研究.doc

玉米大斑病菌论文：玉米大斑病菌CWI-MAPK级联途径中StBCK1基因功能的研究 【中文摘要】玉米大斑病是由玉米大斑病菌(Setosphaeria turcica)引起的重要的玉米叶部病害,在流行年份造成巨大的经济损失。立足于分析病原菌致病性的调控机制,探讨更加有效的防治途径已成为目前研究的热点。在前期研究中克隆了玉米大斑病菌CWI-MAPK途径中的1个MAPKKK基因,命名为StBCK1。在此基础上,本研究利用基因敲除技术创制StBCK1基因敲除突变体,分析突变体,进而初步明确该基因的功能,主要试验结果如下:1.利用DNAstar、DNAMAN软件及NCBI网站中BLAST软件对玉米大斑病菌StBCK1基因的结构进行分析,发现该基因具有MAPKKK类基因的保守结构域。同源性分析表明,玉米大斑病菌StBCK1基因与酿酒酵母中BCK1基因等其它真菌有较高的同源性,属于MAPK信号转导途径中的细胞壁完整性途径。2.采用聚乙二醇(PEG)介导的转化方法,将StBCK1基因的同源重组载体转化到玉米大斑病菌野生型菌株的原生质体中,经潮霉素抗性(Hgy+)筛选、PCR及RT-PCR检测得到1株基因缺失突变体。3.玉米大斑病菌StBCK1基因调控病菌的生长速度、菌丝菌落形态、分生孢子的产量。StBCK1基因缺失突变体的生长速度明显减慢;菌落颜色呈灰白色,会发生自溶现象;菌丝形态不规则,颜色较浅,分隔不明显,细胞内部出现许多液泡状结构;分生孢子的产量明显低于野生型。4.玉米大斑病菌StBCK1基因调控病菌的细胞壁发育。显微观察发现,StBCK1基因缺失突变体的菌丝细胞壁呈透明状,扫描电镜观察发现,菌丝表面出现明显褶皱;突变体对细胞壁降解酶高度敏感,在细胞壁降解酶作用2.5 h、3 h、3.5 h时原生质体的释放量均比野生型多,其中3 h时突变体中的释放量是野生型菌株的20倍;突变体对SDS、H2O2高度敏感,且其敏感性随浓度的增加而增加。5.玉米大斑病菌StBCK1基因调控病菌的致病性。突变体产生的分生孢子在48 h内不能穿透玻璃纸;突变体对刺伤和健康的玉米叶片均不能侵入,突变体致病性丧失。6.玉米大斑病菌CWI-MAPK途径参与调控病菌的黑色素形成。 【英文摘要】Northern Leaf Blight of Corn caused by Setosphaeria turcica, is one of the most important diseases in corn production areas and often results in significant economic losses in epidemic years. Analyzing pathogenic regulatory mechanism and exploring more effective control measures have become a hot topic in the research field. A MAPKKK gene of S.turcica named StBCK1 was cloned in our previous research and target gene mutagenesis techniques was used to obtain StBCK1 gene deleted mutants. The founction of StBCK1 gene was identified by analyzing the characteristic of mutant. The main results were as follows:1. DNAstar, DNAMAN, and BLAST from NCBI website were used to analyze the gene structure of StBCK1. It was showed that StBCK1 gene had the conservative domains of MAPKKK-like gene in plant pathogenic fungi. Homology analysis showed that there was high homology between StBCK1 and BCK1genes of other filamentous fungi, suggesting that StBCK1 gene belonged to cell wall integrity pathway of S.turcica.2. StBCK1 gene homologous recombination plasmid we