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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 沈偉強 | |
| dc.contributor.author | Yi-Hsuan | en |
| dc.contributor.author | 賴羿璇 | zh_TW |
| dc.date.accessioned | 2021-05-19T18:03:30Z | - |
| dc.date.available | 2023-08-14 | |
| dc.date.available | 2021-05-19T18:03:30Z | - |
| dc.date.copyright | 2013-08-20 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8030 | - |
| dc.description.abstract | Brassica black spot disease fungus, Alternaria brassicicola is an important plant pathogenic fungus that produces plenty of secondary metabolites under both in vitro and in vivo conditions, including 3 most well-known mycotoxins: alternariol (AOH), alternariol-9-methyl ether (AME) and tenuazonic acid (TA). Thus far, very limited information is available concerning their biosynthesis and roles in pathogenesis. In this study, we aim to identify the genes involved in the biosynthesis of TA in A. brassicicola. With the knowledge of structural discovery and isotope feeding researches on TA, the activities of both polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) are speculated to be essential in the biosynthesis of TA while isoleucine and acetoacetate were identified to be its precursors. We first examined the TA production in A. brassicicola wild type strain by agar diffusion assay on honey foulbrood disease pathogen, Paenibacillus larvae subsp. larvae. Candidate genes were identified from the genome database of A. brassicicola and applied to bioinformatical analysis on evolutionary relationship and functional domain prediction with known PKS/NRPS genes. Further, AB04556.1 gene was chosen from candidate genes for constructing a gene-disrupted mutant on basis of gene expression level examined by qPCR. The transformants which constructed via biolistic transformation were purified and compared gene expression as well as TA production with wild type. Additionally, we construct a heterologous expression system on Aspergillus nidulans LO2026 strain in order to find out the role that AB04556.1 gene played in the biosynthesis of TA. In this study, we found a potentially candidate gene from the genomic database of A. brassicicola and generated a gene-disrupted mutant for analyzing of the biosynthesis of TA. Ultimately, inoculation was held to understand how TA affects to the pathogenesis during infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T18:03:30Z (GMT). No. of bitstreams: 1 ntu-102-R99633003-1.pdf: 2134144 bytes, checksum: 67343f24111d56612d9240adfcc0da6f (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | Table of Contents
中文摘要 i Abstract iii List of figures viii List of tables viii List of appendix ix Chapter I Introduction 1 1.1 Fungal secondary metabolites (polyketides, non-ribosomal peptides, terpenes, and alkaloids) 1 1.2 PKS-NRPS hybrids 4 1.3 Mycotoxin studies of Alternaria brassicicola 5 1.4 Tenuazonic acid 6 Chapter II Materials and methods 8 2.1 Fungal strain and growth conditions 8 2.2 Bioinformatics 8 2.2.1 Characterization of PKS/NRPS genes in A. brassicicola 8 2.2.2 Alignment 8 2.3 Induction of toxin production in A. brassicicola 9 2.4 RNA extraction and reverse transcription 9 2.5 Real-time PCR 10 2.6 Tenuazonic acid assay 10 2.6.1 Chemical analysis (TLC/HPLC) 10 2.6.2 Bioassay 11 2.7 Construction of gene disruption mutant 12 2.7.1 Generation of a disruption plasmid pBSAB1 12 2.7.2 Biolistic transformation 12 2.8 Southern blot 13 2.8.1 Genomic DNA preparation 13 2.8.2 Southern blot 14 2.9 Heterologous expression 17 2.9.1 Construction of expression plasmid 17 2.9.2 Protoplast transformation 18 2.9.3 Induction of heterologous expression 19 2.10 Inoculation 20 Chapter III Results 21 3.1 Bioinformatical analysis 21 3.2 TA induction and gene expression (real-time PCR) 22 3.3 Gene disruption mutants 23 3.3.1 Generation of mutants 23 3.3.2 Analysis of TA biosynthesis through down-regulation 24 3.4 Heterologous expression in Aspergillus nidulans LO2026 26 3.5 Inoculation 27 Chapter IV Discussion 29 Figures and tables 38 Appendix 54 References 57 | |
| dc.language.iso | en | |
| dc.title | 十字花科黑斑病菌生合成tenuazonic acid之PKS-NRPS基因鑑定 | zh_TW |
| dc.title | Identification of a PKS-NRPS gene involved in the biosynthesis of tenuazonic acid in Alternaria brassicicola | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉瑞芬,呂廷璋,王嘉駿,李宗徽 | |
| dc.subject.keyword | 十字花科黑斑病菌 (Alternaria brassicicola),二級代謝 (secondary metabolites),tenuazonic acid,異源表現 (heterologous expression), | zh_TW |
| dc.subject.keyword | Alternaria brassicicola,secondary metabolites,tenuazonic acid,heterologous expression, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2013-08-14 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-14 | - |
| 顯示於系所單位： | 植物病理與微生物學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-102-1.pdf | 2.08 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。