禾谷镰刀菌细胞自噬基因FgATG1生物学功能分析
毕业论文关键词：禾谷镰刀菌；细胞自噬；基因敲除；基因功能分析
Functional analysis of an autophagy gene FgATG1 from Fusarium graminearum
Abstract：Fusarium graminearum is a causal agent of wheat head blight, the most important fungal diseases of wheat in China, which causes great economic losses to both yield and quality. Autophagy is one of the most important processes that is conserved from yeast to man, and plays important roles in recycling of intracellular substances. In this study, the ATG1 orthologue (FgATG1) of F. graminearum was retrieved by BLASTP search of the Fusarium genome database (http://www.broadinstitute.org/annotation /genome/fusarium _group/Multi Home.html) with the saccharomyces cerevisiae Atg1 protein as a query. Deletion of the FgATG1 impaired fungal growth, conidiation and the full virulence. All of these phenotypic defects were restored by gene complementation. These results suggest that the FgATG1 is indispensable for fungal development, pathogenesis in F. graminearum. This study will be helpful to elucidate the pathogenic mechanism of pathogens and to mine potential drug targets.
Key words: Fusarium graminearum; autophagy; gene knockout; functional analysis
目 录
摘要1
关键词1
Abstract 1
Key word1
引言1
1材料与方法2
1.1材料 2
1.1.1供试菌株 2
1.1.2供试培养基2
1.2方法2
1.2.1小麦赤霉病菌种保存2
1.2.2菌丝体获得2
1.2.3用CTAB法提取禾谷镰刀菌基因组DNA2
1.2.4 PCR技术3
1.2.5 DNA琼脂糖凝胶电泳3
1.2.6禾谷镰刀菌基因敲除载体构建和敲除转化子验证3
1.2.7禾谷镰刀菌基因互补载体的构建4
1.2.8 PEG介导的禾谷镰刀菌原生质体转化4
1.2.9生长速率测定5
1.2.10产孢量测定5
1.2.11致病力测定5
2结果与分析5
2.1禾谷镰刀菌atg1敲除突变体同源臂扩增5
2.2潮霉素磷酸转移酶基因（HPH）获取6
2.3禾谷镰刀菌atg1敲除突变体巢式扩增6
2.4 禾谷镰刀菌atg1敲除突变体验证7
2.5 禾谷镰刀菌atg1敲除突变体、回复突变体生物学功能分析7
3 讨论9
致谢 9
参考文献 10
表1 PCR反应体系3
表2 本文中所用引物4
图1 禾谷镰刀菌atg1敲除突变体同源臂扩增电泳图6
图2 潮霉素基因（HPH）扩增电泳图6
图3 禾谷镰刀菌atg1敲除突变体巢式扩增电泳图7
图4 禾谷镰刀菌atg1敲除突变体验证电泳图7
图5 禾谷镰刀菌测序菌株、敲除突变体菌株、回复突变体菌株生长速率的比较8
图6 禾谷镰刀菌测序菌株、敲除突变体菌株、回复突变体菌株产孢量的比较8
图7 禾谷镰刀菌测序菌株、敲除突变体菌株、回复突变体菌株致病力的比较9
禾谷镰刀菌细胞自噬基因 FgATG1生物学功能分析
由禾谷镰刀菌复合种引起的小麦赤霉病是世界性流行病害，在温暖潮湿麦区广泛发生[1-3]。近年来，随着全球气候的变暖以及耕作制度的变化，小麦赤霉病病害发生频率增加、发生区域不断扩大，造成的损失仅次于小麦条锈病[1-3]。在我国，小麦赤霉病的主要致病菌是亚洲镰刀菌[4]。过去小麦赤霉病发生区域主要集中在长江中下游产区，近年来逐渐向北方扩展蔓延。小麦赤霉病不仅严重影响小麦产量和品质，而且禾谷镰刀菌在侵染过程中会产生对人体和动物有害的真菌毒素及次生代谢物质引起中毒。迄今为止，还没有对赤霉病具有完全免疫的抗性小麦品种。小麦赤霉病在生产上仍依赖于化学防治，目前防治小麦赤霉病的药剂主要是以多菌灵为代表的苯并咪唑类杀菌剂，但抗药性菌群体比例正迅速上升且分布范围不断扩大，药剂防效显著下降[5]。