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摘要：[目的]通过体外人工点突变和两步同源置换法研究小麦赤霉病菌β2-微管蛋白E198A突变基因型是否导致对多菌灵产生抗药性，进一步完善不同植物病原菌对多菌灵的抗药性机制。[方法]构建小麦赤霉病菌β2-微管蛋白E198A突变基因型人工点突变载体，以小麦赤霉病菌β2-微管蛋白基因缺失突变体Δβ2-23为出发菌株，将构建的人工点突变载体同源置换到β2-微管蛋白基因位点，通过潮霉素含药板和f2du含药板对转化子进行筛选，并对其进行PCR、Southern blot和测序验证，得到目的突变体，最后采用菌丝生长抑制法对突变体进行表型测定。[结果]经验证获得的E198A突变体对多菌灵表现高水平抗性，菌丝生长、产孢能力和致病力与野生型菌株2021没有显著差异。30384
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毕业论文关键词：小麦赤霉病菌；突变体；多菌灵；抗药性
Wheat fusarium graminearum beta 2 - tubulin E198A mutant of carbendazim resistant regulation research
Abstract：[Objectives] Research Fusarium graminearum Schw β2- tubulin E198A mutation genotype whether lead to resistant to carbendazim by in vitro point mutation and two step homologous displacement method, to further perfect the different plant pathogenic bacteria of carbendazim resistant mechanism.[Methods] Building Fusarium graminearum Schw β2- tubulin E198A artificial point mutation carriers. Based on the wheat fusarium graminearum β2-microtubule protein gene deletion mutant Δβ2-23 strains, take the artificial point mutation carrier homologous inserted into the β2- microtubule protein gene loci. Then filter transformant by hygromycin plate and f2du medicated medicine plate. Getting the objective transformant by PCR, southern blot and sequencing identification. Finally determine the drug susceptibility of mutant by mycelial growth inhibition method.[Results] Verified the E198A mutant showed high levels of resistance to carbendazim. Mutant strains and wild type strains 2021 don´t have significant difference in mycelial growth, spore production capacity and pathogenicity.
Key words: Fusarium graminearum Schw；mutant；carbendazim；drug resistance
目 录
摘要 1
关键词 1
Abstract. 1
Key words 1
引言 1
1 材料与方法 2
1.1供试菌株及试剂 2
1.1.1菌株 2
1.1.2 培养基 2
1.2 禾谷镰孢菌分子生物学操作方法 2
1.2.1禾谷镰孢菌基因组DNA提取（CTAB法） 2
1.2.2 原生质体的制备与转化 2
1.2.3 分生孢子悬浮液的制备 3
1.3 β2-微管蛋白基因体外人工点突变 3
1.3.1人工点突变载体的构建 3
1.3.2基于同源置换法点突变β2-微管蛋白基因 4
1.3.3 突变体的验证 4
1.4突变体的生物学特性测定 6
1.4.1 对多菌灵的药敏性测定 6
1.4.2 菌丝生长速率及菌落形态观察 7
1.4.3 致病力测定 7
1.4.4产孢量测定 7
2 结果与分析 7
2.1载体构建 7
2.2 突变体的验证 7
2.2.1药剂验证 7
2.2.2 PCR验证 8
2.2.3 Southern blot杂交验证 9
2.3 突变体的生物学特性 9
2.3.1 突变体对多菌灵的敏感性 9
2.3.2 突变体菌丝生长速率及菌落形态 9
2.3.3 突变体的致病力 10
2.3.4产孢能力 11