禾谷镰孢菌中6个同源FgAAT2基因敲除载体的构建及敲除突变体的获得与鉴定
毕业论文关键词：小麦赤霉病；禾谷镰孢菌；天冬氨酸氨基转移酶；基因敲除；FgAAT2基因
Deletion of 6 paralogous FgAAT2 gene in Fusarium graminearumum
Abstract：The wheat scab pathogen anamorph Fusarium graminearum, sexual state of g.zeae, is the world's most important disease of wheat. Wheat scab occurs mainly in the middle and lower reaches of the Yangtze River, the Huaihe River Basin and the eastern coastal areas. But in recent years, the disease in the northern and western parts of China has also increased the trend of wheat. Fusarium head blight of wheat will not only lead to loss of yield, but also cause DON toxicity to food safety because of pathogenic bacteria. Over the past 30 years, China has mainly relied on the use of chemical methods to prevent and control wheat scab. Through the comparison to the identification of 6 homologous genes encoding aspartate aminotransferase in Fusarium graminearum genome caused by wheat scab, proposed by gene knockout technology study on the biological function of these 6 AAT2 genes. The main contents of this paper include 6 AAT2 gene knockout vector constructed by PEG mediated protoplast transformation of transformants obtained the corresponding gene, and then verify the transformants obtained in situ insertion mutant analysis, the next step for the biological function of material.
Key words: Wheat scab; Fusarium graminearumum; Aspartate aminotransferase; Gene knockout; FgAAT2 gene
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法2
1.1 材料 2
1.2.1 实验菌株与质粒 2
1.2.2 常用培养基 2
1.2.3 常用试剂3
1.1.4 实验材料与仪器4
1.2 方法 4
1.2.1 小麦赤霉菌种的保存、培养、产孢4
1.2.2 DNA的提取（小抽DNA）4
1.2.3 pBlueScript-hph质粒的提取5
1.2.4 禾谷镰刀菌基因敲除技术5
1.2.5 DNA琼脂糖电泳8
1.2.6 PCR产物提纯8
1.2.7 小麦赤霉病菌的遗传转化8
2 结果与分析9
2.1 禾谷镰刀菌FgAAT2基因敲除载体的构建9
2.1.1 引物设计 9
2.1.2 构建基因敲除片段10
2.2 敲除突变体的获得与鉴定12
2.2.1 PCR鉴定 12
3 讨论 15
致谢15
参考文献16
禾谷镰孢菌中6个同源FgAAT2基因敲除载体的构建及敲除突变体的获得与鉴定
引言
禾谷镰刀复合种（Fusarium graminearumum complex）引起的赤霉病是小麦的重要病害之一[1]，主要发生在长江中下游麦区和东北春麦区，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。在我国，赤霉病年发生7000万亩以上，占全国小麦种植面积的16%；2010年和2012年赤霉病大发生，发病面积均超过1亿亩，给农业生产造成了巨大的经济损失。赤霉病除了造成减产外，感病籽粒中含有的多种真菌毒素如脱氧雪腐镰刀烯醇（DON）和玉蜀黍赤霉烯酮（ZEN），危害人畜健康，对食品安全构成严重威胁[2]。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途径，但是目前对镰刀菌有效的药剂品种很少，我国自上个世纪70年代以来长期使用苯并咪唑类杀菌剂（如多菌灵）防治小麦赤霉病。由于长期单一使用，多菌灵的抗药性问题严峻[3,4]，在长江中下游麦区多菌灵的防效已显著下降。深入研究禾谷镰刀菌的生物学以及产毒致病机制对于赤霉病和赤霉毒素的持续防控具有重要的理论指导意义。