臺灣博碩士論文加值系統

植物的生長和新陳代謝會受各種生物性和非生物性刺激的影響，包括微生物和昆蟲的攻擊以及溫度和生長素的影響。不同於動物，無法移動的植物需要更複雜和有效率的防禦系統來對抗不同的刺激。本論文著重於三個綠豆熱休克蛋白質的鑑定和高氏柴胡組培根中受甲基茉莉酸誘導基因的分析。
生物常合成分子保護子來應對高溫、低溫、傷害、重金屬以及缺水等逆境。分子量是70 kDa的熱休克蛋白Hsp70以及同源的Hsc70在各種生物之間的相似度很高。此外，Hsp/Hsc70也可以在非逆境狀態下表現於特定的發育時期。本研究之第一部分，分離三個綠豆熱休克蛋白基因並鑑定其在發育期的基因和蛋白質層次的表現。我們的結果顯示這三個綠豆熱休克蛋白質可能是參與正常的生長與發育，在種子成熟或耐受乾燥較不重要。
茉莉酸和甲基茉莉酸是廣泛存在的植物訊號分子，在調控新陳代謝的過程、繁殖、和防禦病蟲害與昆蟲中，扮演關鍵性的角色。本研究之第二部分，將甲基茉莉酸添加到高氏柴胡組培根，並研究受誘導基因間表現的資訊。我們的結果顯示茉莉酸有一種複雜的訊號網路，會牽涉大規模的轉錄重整。有些受甲基茉莉酸誘導的基因，具有農業、醫藥和食品工程應用的潛力。

Plant growth and metabolism are affected by various biotic and abiotic stimuli including microorganisms and insects attack as well as temperatural and hormonal effects. Unlike animals, plants cannot move independently and need a more complicated and efficient defense system against different stimuli. This thesis focused on the identification of three VrHsc70 proteins in Vigna radiata and the analyses of methyl jasmonate (MeJA)-induced genes in Bupleurum kaoi adventitious roots.
Organisms often synthesize molecular chaperons to cope with stress such as heat, cold, wounding, heavy metals, and water deficit. The 70 kDa heat shock protein (Hsp70) and the constitutively expressed heat shock cognate (Hsc70) are conserved among all organisms. In addition, Hsp/Hsc70s have been found to be upregulated at specific developmental stages in the absence of stress. In the first part of this study, three mungbean Hsc70 cDNAs were isolated and their developmental expressions were characterized at both transcriptional and protein levels. Our results indicate that these three VrHsc70s may not be important in seed maturation or desiccation tolerance, but probably involved in normal growth and development.
Jasmonic acid (JA) and MeJA are ubiquitous plant signaling compounds that play a key role in the regulation of metabolic processes, reproduction, and defense against pathogens and insects. In the second part of this study, MeJA was applied to adventitious root culture of B. kaoi and the expression profiles of induced genes were investigated. Our results indicate that MeJA has a complex signaling network involving a large-scale transcriptional reprogramming. Some of the MeJA-induced genes may have applications in agricultural, medicinal and food industries.

摘要---------------------------------------------------------------------------------------------------i
Abstract----------------------------------------------------------------------------------------------ii
謝誌--------------------------------------------------------------------------------------------------iv
Table of Contents-----------------------------------------------------------------------------------v
List of Tables-------------------------------------------------------------------------------------viii
List of Figures--------------------------------------------------------------------------------------ix
Abbreviations---------------------------------------------------------------------------------------x

Part I: Developmental Expression and Sequence Identification of Mungbean Hsc70s--------------------------------------------------------------------------------------1
1. Abstract-----------------------------------------------------------------------------------------2
2. Introduction------------------------------------------------------------------------------------3
3. Materials and methods
3.1 Plant materials and growth conditions-----------------------------------------------5
3.2 Isolation and sequencing of Hsc70 cDNAs from a cDNA library of mungbean seedlings---------------------------------------------------------------------------------5
3.3 RNase protection assays---------------------------------------------------------------6
3.4 Purification and peptide analysis of Hsc70 isoforms in mungbean seedlings--7
3.5 Western blotting analysis of cytosolic Hsc70s in mungbean---------------------8
4. Results and discussion
4.1 Isolation of three mungbean VrHsc70 cDNAs------------------------------------10
4.2 VrHsc70 transcript profiles in mungbean at different developmental stages------------------------------------------------------------------------------------11
4.3 Identification of the three VrHsc70 proteins using peptide mass fingerprinting--------------------------------------------------------------------------12
4.4 Cytosolic VrHsc70 proteins at different developmental stages-----------------12
5. References------------------------------------------------------------------------------------15
6. Tables and figures----------------------------------------------------------------------------18

Part II: MeJA-Induced Transcriptional Changes in Adventitious Roots of Bupleurum kaoi-------------------------------------------------------------------------29
1. Abstract---------------------------------------------------------------------------------------30
2. Introduction-----------------------------------------------------------------------------------31
3. Materials and methods
3.1 Plant growth and treatment----------------------------------------------------------33
3.2 Total cellular RNA extraction-------------------------------------------------------33
3.3 Preparation of PCR-select cDNA subtraction library and DNA sequencing--34
3.4 DNA analysis--------------------------------------------------------------------------35
3.5 DNA microarray fabrication and hybridization-----------------------------------36
3.6 Analysis of microarray data---------------------------------------------------------37
3.7 Relative quantification real-time PCR (qRT-PCR)-------------------------------37
4. Results and discussion
4.1 MeJA retarded adventitious root growth-------------------------------------------39
4.2 PCR-select cDNA subtraction library construction, sequencing and analysis---------------------------------------------------------------------------------39
4.3 Gene expression profiling of MeJA-treated B. kaoi adventitious roots--------39
4.4 Verification of microarray data with qRT-PCR-----------------------------------42
5. References-------------------------------------------------------------------------------------48
6. Tables and figures----------------------------------------------------------------------------53

Appendix------------------------------------------------------------------------------------------75
(1). Protein samples of equal amount from floral bud, flower, pod and dry seed were loaded on 10% SDS-PAGE with Coomassie staining.
(2). BkActin was used as an endogenous control gene in qRT-PCR.
(3). Paper published: Developmental Expression of Three Mungbean Hsc70s and Substrate-binding Specificity of the Encoded Proteins
(4). Paper published: Temperature effects on systemic endoreduplication in orchid during floral development
(5). Paper in press: MeJA-Induced Transcriptional Changes in Adventitious Roots of Bupleurum kaoi

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