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摘要：大豆疫霉是一种大豆卵菌病害。由于其经济重要性，大豆疫霉已成为研究卵菌生理学、遗传学和病理学的模式物种。本实验前期初步发现大豆疫霉糖苷水解酶基因家族GH19的一个基因PsGH19在大豆疫霉侵染过程中显著上调，其编码的蛋白质有信号肽和跨膜结构域，为分泌蛋白。将该基因与其他物种中的同源基因进行序列比对，发现GH19基因相对保守。利用CRISPER/Cas9系统对大豆疫霉进行快速有效的基因组编辑，以PsGH19基因为靶标，在同源模板缺少的情况下，由Cas9介导DNA双链断裂进而引起了缺失，获得大豆疫霉PsGH19基因沉默的转化株。对转化子进行表型分析发现：与野生型P6497相比，敲除转化子的生长速率显著下降，卵孢子的形成与产量明显减少，说明该基因参与大豆疫霉有性生殖的过程，对大豆根腐病的防控提供理论基础。29468
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毕业论文关键词:大豆疫霉；糖苷水解酶家族19；PsGH19；生物信息学；CRISPR/Cas9
Bioinformatics Analysis and Biological Function Research of Glycoside Hydrolase Family 19 in Phytophthora sojae
Abstract:Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete physiology, genetics and pathology. We found a gene PsGH19 expression increased significantly which belongs to Glycoside Hydrolase family 19 in Phytophthora sojae, and PsGH19 encoded protein with signal peptide and transmembrane domain, which indicates that PsGH19 belongs to secreted protein. Aligned with the homologous genes in other species, we found that GH19 gene was relatively conserved. Here, we use a CRISPR/Cas9 system enabling rapid and efficient genome eding in P.sojae. Using the gene PsGH19 as a target, we observed that in the absence of a homologous template, the repair of Cas9-induced DNA double-strand breaks occurs, and result in the short indels of PsGH19. Implementing phenotypic analysis of transformants, we found that compared with wild type P6497 strain, the growth rate and oospores’ formation and production of knockout transformants decreaced significantly, suggesting that PsGH19 participates in the process of sexual reproduction, which laid the theoretical fundation for proventing the soybean root rot.
Key words: Phytophthora sojae；Glycoside Hydrolase family 19；PsGH19；Bioinformatics；CRISPR/Cas9
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 GH19基因的生物信息学分析 2
1.1.1 基因组数据来源 2
1.1.2 基因同源检索与多重序列比对 2
1.1.3 蛋白质结构预测 2
1.2 供试菌株 3
1.3 培养基的制备 3
1.4 CRISPR/Cas9技术对GH19基因的敲除 3
1.4.1 sgRNA的设计 3
1.4.2 大豆疫霉CRISPR/Cas9系统的构建 3
1.5 大豆疫霉瞬时转化 4
1.5.1 试剂和材料 4
1.5.2 大豆疫霉瞬时转化的具体步骤 5
1.6 大豆疫霉菌丝基因组DNA的提取 6
1.7 大豆疫霉敲除转化子表型分析 6
1.7.1 转化子生长速率的统计 6
1.7.2 转化子卵孢子产量测定 7
2 结果与分析 7
2.1 大豆疫霉基因组中GH19蛋白序列分析 7
2.2 大豆疫霉基因敲除转化子的获得 8
2.3 PsGH19转化子生长速率减慢 8