【作者】 张大军；

【导师】 蒋伶活；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 禾谷镰刀菌(Fusarium graminearum)是引发我国小麦赤霉病的优势种之一。该病菌侵染植物的过程包括孢子的产生与寄主表面的附着、孢子萌发以及侵染菌丝的形成与扩展等关键阶段。这些阶段也是许多其它植物病原真菌所共有的,缺少其中任何一步将导致病菌不能侵染寄主。因此,研究禾谷镰刀菌这些发育阶段的分子基础不仅在理论上有助于阐明病原真菌致病性的分子机理,在应用上也有助于发现筛选和设计新型农药的靶标基因。近年来全球对禾谷镰刀菌开展了大量的研究工作,并取得了一系列重大进展。但由于病菌在侵染过程中产生毒素,使得人们至今对禾谷镰刀菌的致病机理及寄主的抗病机制仍缺乏全面的了解与认识。目前,对禾谷镰刀菌的研究热点逐渐集中在弄清其致病机理和发现新的药物作用靶位点。本研究中,作者鉴定了一个控制禾谷镰刀菌分生孢子产生的新基因TEP1,并发现该基因与磷脂酰肌醇信号通路和致病性有关。具体结果如下:首先,作者通过构建pMDa+b+G418敲除质粒,原生质体转化,转化子抗药性筛选和基因型鉴定,最终得到两个Fgtep1-t敲除体。与野生型PH-1菌株相比,发现Fgtep1-t缺失株对锂盐和3-磷酸磷脂酰肌醇(PI3)激酶抑制剂—涡曼青霉素敏感性显著增加,分生孢子产量下降约56%,对寄主小麦的致病力也下降了约38.5%。为了进一步验证FgTEP1基因的生物学功能,作者构建了pUC-hph-FgTEP1回复质粒,并对Fgtep1 -t缺失株进行了回复实验。表型回复实验说明,包含该基因完整启动子和编码区的一个3.4kb的DNA片段能够使缺失株Fgtep1-t在锂盐和涡曼青霉素耐受性、分生孢子产量以及致病力等方面均恢复到野生型水平。这些结果证明:Fgtep1-t的突变表型是由于FgTEP1基因的缺失而引起的。此外,本研究还运用了比较基因组学方法,探索了FgTEP1基因在模式真菌酿酒酵母中的互补功能。作者通过RT-PCR获得了不含内含子的FgTEP1 cDNA序列,并将其克隆到带有强启动子ADH的酵母表达载体YPB1-ADH1上,转化双倍体酿酒酵母Sctep1/tep1缺失株。互补实验结果表明,FgTEP1基因能够使双倍体酿酒酵母Sctep1/tep1缺失株对涡曼青霉素的耐受性恢复到野生型水平。这说明FgTEP1与ScTEP1都是磷脂酰肌醇信号通路调控中的功能蛋白,在生物学功能上存在相似性。本研究对于进一步研究FgTEP1基因调控禾谷镰刀菌磷脂酰肌醇信号通路、分生孢子的产生以及致病机理奠定了基础,对于广谱新型杀菌剂的开发具有重要的指导意义。更多还原

【Abstract】 Fusarium graminearum is the major causal agent of Fusarium head blast (FHB) disease on wheat, The infection process of F. graminearum in host plants includes conidia production and surface attachment,conidia germination,infectious hypha differentiation and spreading.All these stages are common in many other plant pathogenic fungi.Therefore,studying on the molecular basis of these developmental stages in F. graminearum will help to elucidate the molecular mechanism of fungal pathogenicity and to discover candidate target genes for screening and designing novel fungicides.In recent years, F.graminearum was carried out a great deal of studies in the world and made a series of significant progress. However, due to toxins in the infection process, making it so far on the pathogenesis of F.graminearum and resistance mechanisms of host is still a lack of comprehensive understanding and awareness.At present,the studies on F. graminearum have been focused on the pathogenesis and the discovery of drug targets. In this study,a novel gene FgTEP1 required for conidiation was identified and linked to the phosphatidylinositol pathway and the pathogenicity of F. graminearum.The results in details are as the following:Firstly,we constructed the knock-out plasmid pMDa+b+G418 for the FgTEP1 gene and successfully deleted the ORF of FgTPE1 gene by homologous recombination. Compared with wild-type strain PH-1, the knock-out strain Fgtep1-t are sensitive to both lithium ions and phosphatidylinositol-3-phosphate(PI3)kinase inhibitor wortmannin,the conidia production of Fgtep1-t is less than 56% and its virulence is less about 38.5%.To further validate the function of FgTPE1 gene,we constructed the complemental plasmid pUC-hph-FgTEP1 for gene complement to Fgtep1-t.The gene complement results showed that all the capacities of the resistance to wortmannin and to lithium ions,the conidia production and the pathogenicity in the complementary transforments were restored to the level of those in the wild-type strain PH-1 by 3.4kb DNA fragment,including functional promoter and the entire coding region of the FgTPE1.These results proved that the mutant phenotype was caused by the detection of FgTPE1.This study also exploits the possible complementary function of the FgTEP1 gene in Saccharomyces cerevisiae. We cloned the cDNA sequence of the FgTEP1 gene into a yeast expression vector YPB1-ADH1 with a strong promoter of ADH1, the alcohol dehydrogenase gene. As a result, we have found that the FgTEP1 cDNA sequence could complement the function of ScTEP1 in wortmannin tolerance, which indicates that the FgTEP1 and ScTEP1 genes are functional homologues and linked to the phosphatidylinositol pathway.This study provides a basis for further studies on the phosphatidylinositol pathway, conidia production and virulence of Fusarium graminearum. It also provides an important guiding significance for the development of a new type of broad-spectrum fungicide.更多还原