臺灣博碩士論文加值系統

為探討綠豆低溫處理(4°C)所誘導表現基因，我們建立了1012個獨立的ESTs，含391個正常生長發芽三天的綠豆ESTs，和621個ESTs利用扣減法從發芽三天的綠豆分別來自低溫和高溫，利用cDNA 微陣列晶片技術去分析在低溫處理誘導的基因，從初步結果挑選了一些在低溫處理不同時間點被誘導的基因。本研究的重點在分析幾個有關泛素(ubiquitin)/蛋白解體(26S proteasome)途徑的基因，包括了泛素、泛素結合酶(ubiquitin conjugating enzyme)、泛素連接酶(ubiquitin ligase)、蛋白解體次單位(20S core protease、19S regulatory particle)，利用北方墨點法分析在不同溫度下處理的mRNA表現量，找到泛素結合基因在低溫處理下mRNA有明顯的表現量，目前在針對低溫下這途徑的研究相當的少，而這個結果建議只在低溫下這些泛素結合基因扮演終結蛋白質的角色。

To identify cold-inducible genes in mungbean, we prepared a mungbean cDNA set containing independent cDNAs combined with 391 cDNAs isolated from 3 day old seedlings and 621 cDNA from subtraction for cold- and heat-treatment. This study focused on some of the candidate genes obtained from our preliminary microarray data. We aimed to study the mungbean genes involved in the ubiqitin/26S proteosome pathway from our cold-subtracted library, including the ubiquitin, ubiquitin conjugating enzyme, RING type ubiquitin ligase, 20S core protease, and 19S regulatory particle. We confirmed those genes using Northern blot analysis and finally identified the cold-induced ubiquitin conjugation genes. These results suggest that the ubiquitin conjugation genes have roles during cold stress through the turnover of protein(s) via the ubiquitin/26S proteasome pathway, but not heat.

Abstract in Chinese••••••••••••••••••••••••••i
Abstract ••••••••••••••••••••••••••••••ii
Acknowledgement••••••••••••••••••••••••••iii
Abbreviations ••••••••••••••••••••••••••• iv
Table of Contents•••••••••••••••••••••••••• v
List of Figures & Tables••••••••••••••••••••••• vii

Introduction ••••••••••••••••••••••••••••1
Materials and Methods ••••••••••••••••••••••• 12
1. Plant growth and stress treatments••••••••••••••••••• 12
2. Subtraction library••••••••••••••••••••••••• 13
2.1 Subtraction cDNA•••••••••••••••••••••••• 13
2.2 Ligation•••••••••••••••••••••••••••• 13
2.3 Competent Cell Preparation•••••••••••••••••••• 13
2.4 Bacterial Transformation••••••••••••••••••••••14
2.5 Mini-preparation of Plasmid DNA••••••••••••••••••15
2.6 Large number of Mini-preparation of Plasmid DNA•••••••••••15
2.7 Restriction enzyme Digestion••••••••••••••••••••16
2.8 Double-stranded DNA Sequencing••••••••••••••••••17
2.9 EST library construction••••••••••••••••••••••18
3. Total cellular RNA extraction •••••••••••••••••••• 18
4. RT-PCR •••••••••••••••••••••••••••••19
5. Synthesis of DIG-labeled DNA Probes by PCR ••••••••••••• 20
6. Northern blotting ••••••••••••••••••••••••• 21
7. cDNA Microarry•••••••••••••••••••••••••• 23
7.1 PCR Products Reaction for DNA Amplification••••••••••••23
7.2 Preparation of Microarray•••••••••••••••••••• 24
7.3 Hybridization••••••••••••••••••••••••• 24
7.4 Scan•••••••••••••••••••••••••••••24
7.5 Analysis••••••••••••••••••••••••••• 25

Results ••••••••••••••••••••••••••••••26
Discussion •••••••••••••••••••••••••••••33
Tables and Figures•••••••••••••••••••••••••• 40
References •••••••••••••••••••••••••••• 55





List of Figures & Tables

Table 1. The subtraction condition including different temperatures, germination stages, and time intervals•••••••••••••••••••••• 40

Table 2. ESTs of different function were involved up-regulation under cold stress•••••••••••••••••••••••••••••••• 45

Fig. 1 Functional categories of the NG1C library •••••••••••••••41

Fig. 2 Functional categories of the NG3C library •••••••••••••••42

Fig. 3 Functional categories of the NG3H library •••••••••••••••43

Fig. 4 The morphology of mungbean at different temperatures for different periods•••••••••••••••••••••••••••••••44

Fig. 5 Comparison of the amino acid sequences of the NG3C120, NG3C153, NG3C195, NG1C339, and NG3C024 to their Arabidopsis orthologues in the TAIR database •••••••••••••••••••••••••••46

Fig. 6. Northern blot analysis of the NG1C120 (polyubiquitin) transcripts at different time and different temperatures •••••••••••••••48

Fig. 7 RT-PCR analysis of NG1C120 (Polyubiquitin) transcripts at different time and different temperatures••••••••••••••••••••• 49

Fig. 8 Northern blot analysis of the NG3C153 (Ubiquitin-conjugating enzyme E2) transcripts at different time and different temperatures•••••• ••50

Fig. 9 Northern blot analysis of NG3C195 (RING E3) transcripts at different time and different temperatures •••••••••••••••••••••51

Fig. 10 RT-PCR analysis of NG3C195 (RING E3) transcripts at different time and different temperatures ••••••••••••••••••••••• 52

Fig. 11 Northern blot analysis of NG1C339 (20S CP) and NG3C024 (19S RP) expression at 4°C treatments••••••••••••••••••••• 53

Fig. 12 Overview for ubiquitin/26S proteasome at cold stress•••••••••54

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