農桿菌1D1609腫瘤誘生質體上的冷休克蛋白基因簇之實驗與生物資訊分析

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研究生：	李庭儀 Ting-Yi Lee
論文名稱：	農桿菌1D1609腫瘤誘生質體上的冷休克蛋白基因簇之實驗與生物資訊分析 Experimental and Bioinformatic Characterization of a Cold Shock Protein Gene Cluster on the Tumor-inducing Plasmid (pTi) of Agrobacterium tumefaciens 1D1609
指導教授：	郭志鴻 Chih-Horng Kuo 吳少傑 Shaw-Jye Wu
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2021
畢業學年度：	109
語文別：	中文
論文頁數：	57
中文關鍵詞：	農桿菌、冷休克蛋白、腫瘤誘生質體
外文關鍵詞：	Agrobacterium tumefacien, Cold Shock Protein, tumor inducing plasmid
相關次數：	點閱：11 下載：0
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農桿菌為一種植物病原細菌，可以將其DNA嵌入受感染宿主的染色體中，是一種重要的基因轉形生物技術工具。為了提高轉殖效率和拓展植物宿主範圍，更深入解細菌與宿主之間的分子相互作用有其重要性。前人研究發現有一個位於農桿菌1D1609菌株腫瘤誘導質體上的冷休克蛋白基因簇會在毒力誘導時有較高的表現量。因為冷休克蛋白是會被植物免疫系統識別的病原體分子標記，因此推論這個基因簇可能會影響感染效率。為驗證此假說，本研究建構剔除此基因簇的突變株，用來與野生型1D1609菌株進行比較。在圓葉煙草的農桿菌滲入法實驗中，野生型菌株對6週齡植株的感染效率顯著低於對4週齡植株的感染效率。此結果符合預期，因前人研究已發現圓葉煙草在接近開花期時可產生較高的免疫反應。相較之下，兩個突變株皆對 4 週齡和6 週齡植株有相近於與野生型對4週齡植株的感染效率，而此結果可支持本研究之假說。根據比較基因體學分析，這個基因簇普遍存在於不同農桿菌屬物種的第二型腫瘤誘導質體中且具有高度相似序列，亦部分存在於第三型腫瘤誘導質體中且具有中度相似序列。根據此發現可推論，此基因簇的剔除可能可用於改善農桿菌屬其他物種及菌株在圓葉煙草接近開花期時的短暫轉形。總結而言，本研究為此基因簇參與農桿菌與植物宿主的分子相互作用提供實驗證據。但此基因簇中四個基因的個別功能仍須未來研究釐清，而所得之知識預期將可應用於進一步改善利用農桿菌進行的基因轉形。

Agrobacterium tumefaciens is a plant pathogen that could insert its DNA into the chromosome of infected hosts, and is an important biotechnology tool for genetic transformation. To improve the transformation efficiency and plant host range, it is important to understand the molecular interactions between this bacterium and its hosts. In a previous study, a cold shock protein gene cluster located on the tumor-inducing plasmid (pTi) of A. tumefaciens 1D1609 was found to be up-regulated upon virulence induction. Because cold-shock proteins were reported to be pathogen-associated molecular patterns (PAMPs) recognized by the plant immune system, we hypothesize that this gene cluster may encode antivirulence factors that affect the infection efficiency. To test this hypothesis, we generated the deletional mutants, Δcspgc-1 and Δcspgc-2 that both lack this gene cluster for comparisons with wild-type 1D1609. For agroinfiltration in leaves of Nicotiana benthamiana, the transient transformation efficiency of the wild-type in 6-week-old plants is significantly lower than in 4-week-old plants, which is consistent with the expectation based on the high immune responses in restricting genetic transformation when it reaches the flowering stage. In comparison, both deletional mutants have similar transformation efficiencies in 4-week-old and 6-week-old plants as the wild-type in 4-week-old plants, supporting our hypothesis. Further comparative genomics analysis revealed that this gene cluster is highly conserved among all type II pTi and partially conserved among type III pTi found in different Agrobacterium species. This finding suggests that deletion of this gene cluster may be applied to improve transient transformation in older N. benthamiana plants using other Agrobacterium species and strains. In conclusion, this study provided evidence for the involvement of this gene cluster in Agrobacterium-plant interactions. For future studies, functional characterization of each gene in this four-gene cluster is necessary to understand their roles. The knowledge may contribute to future improvements of Agrobacterium-mediated transformation.

中文摘要    i
Abstract    ii
誌謝    iii
Table of Contents    iv
List of Figures    vi
List of Tables    vii
Introduction    1
1    Introduction of Agrobacterium tumefaciens    1
2    Agrobacterium-plant interaction    1
3    Identification of candidate genes controlling Agrobacterium virulence    2
4    Pathogen-associated-molecular patterns (PAMP)    3
5    Hypothesis    4
Materials and methods    5
1    Cold shock protein of 1D1609    5
1.1    Cold shock protein epitope alignment    5
1.2    Promoter prediction    5
2     Generation of deletional mutant    5
2.1    Competent cell prepration    5
2.2    Homologous recombination    6
2.3    RNA extraction and quantitative PCR    7
3     Phenotyping of transformation efficiency    7
3.1    AGROBEST    7
3.2    Agroinfiltration of Nicotiana benthamiana    9
3.3    Tumor assay on Nicotiana benthamiana stems    10
4     CSP gene clusters of other pTi    10
4.1    Synteny plot    10
4.2    Cold shock protein epitope alignment and phylogeny    10
Result and Discussion    11
1     Cold shock protein of 1D1609    11
1.1    Cold shock protein epitope alignment    11
1.2    Promoter prediction    11
2     Compare wild-type and deletion mutants    12
2.1    Double crossover result    12
2.2    RNA expression    12
3     Phenotyping of transformation efficiency    13
3.1    AGROBEST    13
3.2    Agroinfiltration    14
3.3    Tumor assay    15
4   CSP gene cluster of other Agrobacterium pTi    16
4.1    Cold shock protein gene cluster    16
4.2    Cold shock protein    16
Conclusions    17
References    18
Figures    21
Tables    32


                                

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