台灣蝴蝶蘭在不同組織及不同逆境下蛋白質之PaSUMO修飾

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研究生：	陳昱伶 Chen, Yu-Ling
論文名稱：	台灣蝴蝶蘭在不同組織及不同逆境下蛋白質之PaSUMO修飾 The levels of PaSUMO conjugations in different tissues of Phalaenopsis aphrodite and the changes of SUMOylation responded to stresses
指導教授：	林彩雲 Lin, Tsai-Yun
口試委員:	楊長賢褚志斌林彩雲
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	65
中文關鍵詞：	蝴蝶蘭、SUMO蛋白、SUMO化修飾、低溫逆境
外文關鍵詞：	Phalaenopsis aphrodite, SUMO, SUMOylation, Cold stress
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SUMO蛋白質普遍存在於許多真核生物中，它經由一連串的酵素反應，與目標蛋白形成可逆的共價鍵結，進而改變目標蛋白的穩定性、位置及蛋白質與蛋白質間的交互作用，最後影響整體的生化反應。在植物中，SUMO修飾會參與訊息、發育、賀爾蒙反應以及生物和非生物性的逆境調控，前人研究發現SUMO化蛋白質累積在不同植物組織及不同環境逆境下。本研究目的在探討植物在不同環境逆境下的SUMO化修飾，為達成目標，我們利用大腸桿菌生產PaSUMO重組蛋白並製作成抗體。由蛋白質印跡的結果發現，在低溫、高溫、乾燥、高鹽及離層酸處理下，僅低溫逆境有大量的高分子量蛋白被SUMO化修飾。在熱帶生長的台灣蝴蝶蘭 aphrodite，SUMO化修飾的重要性可能是在低溫逆境下穩定蛋白質以免降解。在不同的組織中，半開的花有最多的SUMO化修飾蛋白，SUMO化修飾可能也參與花的發育。

SUMO protein is conserved in all eukaryotic organisms. SUMOylation involves a cascade of enzymatic reactions which alters target protein stability, sub-cellular localization, and protein-protein interactions through the reversible covalent attachment and affects the overall biochemical reaction chains. SUMOylation/ deSUMOylation may function in concert to regulate signaling, development, hormonal responses, biotic and abiotic stress responses in plants. SUMOylated proteins were found to accumulate in various plant tissues under different environmental stress. This thesis aimed to study the role of SUMO and SUMOylation in plants under abiotic stress. To fulfill our purpose, the PaSUMO recombinant protein was produced in Escherichia coli to raise a polyclonal antibody for SUMO detection. Upon subject to cold, heat, drought, salinity and ABA treatments, only cold stress induced accumulation of high molecular weight SUMOylated proteins in our protein blots. The induced SUMOylation in the tropical plant Phalaenopsis aphrodite under cold stress may lead to protein stabilization and protection from degradation. Among all the tissues examined, the half-opened flower displayed the highest level of PaSUMO conjugates. Our result also suggests that SUMOylation process may participate in floral transition.

摘要…………………………………………………………………………………i
Abstract…………………………………………………………………………ii
誌謝…………………………………………………………………………………iii
Table of contents……………………………………………………………iv
List of tables…………………………………………………………………vi
List of figures……………………………………………………………vii
List of appendixes………………………………………………………viii
Abbreviations………………………………………………………………ix
Introduction…………………………………………………………………1
The SUMO conjugation and deconjugation pathways………1
SUMOylation and biological processes in plants…………3
The effect of cold stress and cold stress signaling…6
The orchid plants used in this study…………………………8
The objective of the present study………………………………8
Materials and methods……………………………………………………10
1.    Plant materials and stress treatments………………10
2.    Total cellular RNA extraction and reverse transcription-PCR (RT-PCR)……………………………………………11
3.    pET28a construct, competent cell preparation, plasmid preparation and transformation, and DNA sequencing……14
4.    His tag protein expression, purification and thrombin cleavage…………………………………………………………………………17
5.    Total cellular protein extraction with phenol method20
6.    Protein quantification , one-dimensional electrophoresis, two-dimensional electrophoresis……………21
7.    Western bolt and LC-MS/MS analysis................24
Results…...............................................26
1.    Comparison of PaSUMO peptide sequence with other plant SUMO peptides……………………………………………………………26
2.    PaSUMO fusion protein was produced in E. coli using the PaSUMO-pET28a construct and the identity was verified with LC-MS/MS………………………………………………………………………27
3.    The PaSUMO antibody can recognize the PaSUMO protein in P. aphrodite……………………………………………………………………28
4.    Accumulation of PaSUMO conjugates in different tissues of P. aphrodit………………………………………………………29
5.    Effects of cold, heat, drought, salinity and ABA stress on the accumulation of PaSUMO conjugates………………30
6.    Time course analyses of the accumulation of PaSUMO conjugates in response to cold and wound stress………………31
7.    Expression of PaSUMO PaCBF and PaICE mRNA level induced by cold treatment…………………………………………………33
Discussion……………………………………………………………………………34
1.    The deduced PaSUMO protein ends in C-terminal di-glycine residues…………………………………………………………………34
2.    SUMO-conjugates accumulate in floral tissues of P. aphrodite……………………………………………………………………………35
3.    SUMOylation in responses to the environmental and hormonal stress……………………………………………………………………35
4.    Expression patterns of cold acclimation crucial genes ICE1 and CBF3………………………………………………………………………37
Conclusion……………………………………………………………………………39
References……………………………………………………………………………40
Table……………………………………………………………………………………48
Figures…………………………………………………………………………………49
Appendixes...………………………………………………………………………64

                                

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