【作者】 王健；

【导师】 史建荣；

【作者基本信息】 南京农业大学 ， 植物保护， 2014， 硕士

【摘要】 麦类赤霉病(Fusarium head blight, FHB)是麦类作物(黑麦、青稞、大麦、小麦等)上的一种流行性病害,其病原菌为禾谷镰刀菌复合物(Fusarium graminearum complex)在全国各麦区都有发生,长江中下游为主要发生区。近年来由于秸秆还田等耕作方式的改变和全球变暖等气候因素的影响,赤霉病还有蔓延到黄淮麦区的趋势。小麦赤霉病不仅给麦类生产造成巨大的经济损失,还会降低籽粒的质量。由于病原菌在侵染过程中形成的多种真菌毒素如脱氧雪腐镰刀菌烯醇(DON)和玉蜀黍赤霉烯酮(ZEN),不但危害人畜健康,对食品安全也构成极大的威胁。目前生产上由于缺乏抗赤霉病的小麦品种,化学防治仍是控制小麦赤霉病流行的最有效的方法。我国自上世纪70年代,主要依赖苯并咪唑类(多菌灵)杀菌剂防治小麦赤霉病,由于常年单一用药,田间多菌灵的抗药性问题严峻。因此,新型杀菌剂的研发对赤霉病的持续防控起着至关重要的作用。从农产品的安全角度出发,某些仅存在于真菌、细菌、藻类和高等植物中,哺乳动物中没有的生物合成途径成为新型安全药剂靶标研发的热点,例如支链氨基酸(Branched-chain amino acids, BCAAs)生物合成途径,BCAAs是一类人体必需氨基酸,人体只能通过外源摄取,缺乏该类氨基酸合成相关的酶。因此,BCAAs途径仅存在于细菌、真菌以及高等植物中,在哺乳动物中不存在,这一事实使得BCAAs合成酶成为研发对哺乳动物无毒或低毒药剂的潜在药靶。本论文以小麦赤霉病的病原菌禾谷镰刀菌为研究对象,通过靶向基因敲除与互补技术对禾谷镰刀菌中编码第二步反应的酮酸-还原异构酶(keto-acid reductoisomerase, KARI) FgILV5基因的生物学功能进行了研究,论文结果将为研发靶向于BCAAs合成酶的新型安全药剂提供理论依据。通过同源比对,禾谷镰刀菌FgIlv5与啤酒酵母菌和白色念珠菌中Ilv5的氨基酸同源性分别为72.24%和71.60%。构建FgILV5基因的敲除载体,并通过原生质体转化获得敲除转化子,RT-PCR和Southern杂交均显示标号为△FgIlv5-4的转化子为单拷贝原位插入。FgILV5基因敲除突变体在营养匮乏的MM和FGA培养基上不能生长,在完全培养基PDA上气生菌丝稀疏、菌落变黄、不产红色素。Realtime-PCR测定敲除突变体中的红色素合成相关基因的表达水平仅为野生型菌株的10%。外源添加两种氨基酸(缬氨酸+异亮氨酸),且最低浓度需达到1 mM能够恢复FgILV5基因敲除突变体的营养缺乏。FgILV5基因的敲除突变体产分生孢子的能力也受到阻滞,外源添加缬氨酸(Valine)和异亮氨酸(Isoleucine)能恢复分生孢子形成,但其不能在2%蔗糖水中正常萌发。FgILV5基因的缺失还会引起突变体对渗透压力和氧化压力的敏感性。在寄主麦穗和非寄主番茄上,敲除突变体的致病力显著减弱,病斑不能扩展；另外一个重要的表型是FgILV5基因敲除突变体的DON毒素产生量与野生型菌株相比显著降低,菌体内的多个毒素合成基因TRI的表达量也明显下降。以上实验结果表明,FgILV5基因对禾谷镰刀菌生长发育、毒素合成和致病起着重要作用,是新型安全药剂研发的潜在药靶。更多还原

【Abstract】 Wheat scab (Fusarium head blight, FHB) is an epidemic disease of wheat crops (rye, barley, barley, wheat, etc.), which had occurred on all the wheat growing areas of entire country, especiallyYangtze River area is the most main area. In recent years, wheat scab has trended to Huang-Huai wheat area owing to the impact of changes in farming practices and other climatic factors (such as straw and global warming). Wheat Scab not only caused huge economic losses, but also reduced the quality of grain susceptible. It not only endangers human and animal health, but also threatens enormously food security mainly due to a variety of mycotoxins in susceptible grains as deoxynivalenol (DON) and zearalenone (ZEN). Currently due to lack of anti-scab of wheat varieties, the primary method for management of FHB is through fungicide application,because it only relies on benzimidazoles (carbendazim) fungicide, carbendazim resistant isolates have been frequently detected, Therefore, the development of new fungicide play a vital role in continued prevention and control of FHB.From the security point of agricultural products, certain biosynthetic pathways which are only present in a variety of organisms from bacteria to eukaryotes and higher plants, but not in animals become a hospot for developing a new safety drug target. E.g., BCAAs biosynthetic pathway, the body can only obtain by exogenous food, BCAAs are a class of essential amino acids, BCAAs biosynthesis enzyme is absent in the body.Thereby BCAAs synthase are only present in a variety of organisms from bacteria to eukaryotes and higher plants, but not in animals, the fact that make enzymes involved in these amino acids biosynthesis desirable targets for new herbicide and antimicrobial agents discovery. BCAAs biosynthesis enzyme becom potential drug target for developing non-toxic or low toxicity to mammals.In this study we characterized Fgllv5, a homologue of the Saccharomyces cerevisiae keto-acid reductoisomerase (KARI) from the important wheat head scab fungus Fusarium graminearum. KARI is a key enzyme in the branched-chain amino acids (BCAAs, including leucine, isoleucine and valine) biosynthetic pathway which exists in a variety of organisms from bacteria to fungi and high plants, but not mammals. The FgILV5 deletion mutant △ FgIlv5-4 failed to grow when the culture medium was nutritionally limited for BCAAs.When grown on PDA plates, △FgIlv5-4 exhibited a significant decrease in aerial hyphae formation and red pigmentation. Conidia formation was also blocked in △FgIlv5-4. Exogenous addition of 1 mM isoleucine and valine could rescue the defects of mycelial growth and conidial morphogenesis. Cellular stress assays showed that △FgIlv5-4 was more sensitive to osmotic and oxidative stresses. In addition, virulence of △FgIlv5-4 was dramatically reduced on wheat heads, and a low level of deoxynivalenol (DON) production was detected in △FgIlv5-4 in wheat kernels. The expression level of TRI4, TRI5 and TRI6 in the mutant △FgIlv5-4 was decreased by 92%,68% and 62%, respectively, as compared to those in wild type strain and complementary strain. Taken together, results of this study indicate that FgILV5 is involved in valine and isoleucine biosynthesis and is required for full virulence in F.graminearum.更多还原