大豆疫霉糖苷水解酶GH19基因家族的生物信息学分析与生物学功能研究
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
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