綠豆熱休克蛋白70 kDs在發育階段的表現｜國立清華大學博碩士論文庫

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研究生：	柯嘉昀 Chia-Yun Kao
論文名稱：	綠豆熱休克蛋白70 kDs在發育階段的表現 Expression levels during development and peptide ligands of mung bean Hsc70
指導教授：	林彩雲 Tsai-Yun Lin
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生命科學系 Department of Life Sciences
論文出版年：	2000
畢業學年度：	88
語文別：	英文
論文頁數：	42
中文關鍵詞：	熱休克蛋白質、伴護子、胚的形成、綠豆、七胺基酸序列、病毒胺基酸序列陳列的基因庫
外文關鍵詞：	Heat Shock Protein, Chaperone, Embryogenesis, Mung Bean, Heptapeptide, Phage Peptide Display Library
相關次數：	點閱：5 下載：0
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前人的研究發現在發芽的過程中，綠豆種子的熱休克蛋白質70 kＤa逐漸消失，而且會有另一群熱休克蛋白質70 kＤa逐漸產生。本實驗的目的是為了解在種子發芽及胚形成的過成中熱休克蛋白質70 kＤa 的表現變化。以質譜儀分析種子與發芽一天綠豆苗的熱休克蛋白質70 kＤa，發現在乾燥的種子有二種不同的熱休克蛋白質70 kＤa，但是在發芽一天的豆苗只有一種熱休克蛋白質70 kＤa。發芽一天豆苗所含的熱休克蛋白質70 kＤa與種子所含者是不同的，發芽一天豆苗的熱休克蛋白質70 kＤa比種子的熱休克蛋白質70 kＤa的分子量高且有較高疏水性。熱休克蛋白質70 kＤa在花苞、花和發芽一天的豆莢中有大量的表現在胚的發育過程中。隨著種子的逐漸成熟，熱休克蛋白質70 kＤa逐漸地在種子中累積，而果莢外皮的熱休克蛋白質則逐漸地減少。
前人研究顯示，老鼠、酵母菌、大腸桿菌的受質結合區域能與七胺基酸序列 FYQLALT結合形成複合物。但是，阿拉伯芥和綠豆的受質結合區域卻無法與FYQLALT結合形成複合物。本論文藉由病毒胺基酸序列陳列的基因庫 (phage peptide display library) 以純化的綠豆熱休克蛋白質70 kＤa當作目標蛋白來篩選出能與受質結合區域結合的七個胺基酸序列。經過三次的篩選後，我們得到五種不同的七胺基酸序列，在它們的第一個位置總是出現疏水性的胺基酸，在第五個位置經常出現帶正電的胺基酸。 PTHRHRT是五個不同的七胺基酸序列中，對熱休克蛋白質70 kＤa的親和性最高。 PTHRHRT的性質與用牛的熱休克蛋白質70 kＤa 自f2病毒胺基酸序列陳列的基因庫(f2 phage peptide display library)所篩選的NIVRKKK。它們都具有四個帶正電的鹼性氨基酸和很高的親水性的特性。

Previous studies revealed that a group of Hsc70 proteins disappeared gradually, which another group of Hsc70 emerged in mung bean seedling during germination. The purpose of this study is to examine the expression of Hsc70 during seed germination and embryogenesis. The Hsc70 proteins in seeds and seedling at 1 day after germination were analyzed by mass spectrometer. Dry seeds of mung bean contain two different Hsc70s, while seedlings of 1 dag contain only one Hsc70. The Hsc70 in seedling is different from the Hsc70s in seeds. The Hsc70 in seedling at 1 day after germination has higher molecular weight and hydrophobicity than Hsc70s in seeds. The Hsc70s present abundantly in flower bud, flower and younger legumes. During embryogenesis, the Hsc70 accumulate gradually in seeds, while decrease gradually in pods.
The substrate-binding domain of Hsc70s from bacteria, yeast and vertebrates are effective in forming complexes with FYQLALT. In contrast, the substrate-binding domain of Hsc70s from Arobidopsis and mung bean are ineffective for forming complexes with FYQLALT. Heptapetides were selected by affinity panning of phage peptide display libraries using purified the Hsc70 at 1 day after germination. After three rounds of heptapeptides selection, we obtained five different heptapeptide sequences. In these heptapeptides, the first residue is always hydrophobic, and the fifth residue has a high frequency to be positively charged. Among five selected heptapeptides, PTHRHRT exhibited the highest affinity for Hsc70 from seedling of 1 dag. The characteristics of PTHRHRT is similar to NIVRKKK, which is selected from f2 phage display libraries with bovine Hsc70. These two heptapeptides both possess four basic and positively charged amino acids with high hydrophilicity.

Table of Contents
Abstract (Chinese)  ----------------------------------------i

Abstract  --------------------------------------------------ii

Acknowledgments  -------------------------------------------iii

List of Tables  --------------------------------------------iv

List of Figures  -------------------------------------------v

Abbreviations  ---------------------------------------------vi

Table of Contents  -----------------------------------------vii

Introduction  ----------------------------------------------1

Materials and Methods  -------------------------------------9

Plant materials---------------------------------------------9

Protein extraction------------------------------------------9

Protein quantitation----------------------------------------10

Gel electrophoresis-----------------------------------------11

Western blotting--------------------------------------------11

Purification of Hsc70 from mung bean seeds------------------12

Purification of Hsc70 from mung bean seedling---------------14

Liquid chromatography/ mass spectrometer--------------------14

Panning heptapeptide for ligand-----------------------------15

Plaque amplification----------------------------------------17

Rapid purification of phage DNA-----------------------------18

Single-stranded DNA sequencing------------------------------18

Results  ---------------------------------------------------20

Discussion  ------------------------------------------------23

Reference---------------------------------------------------27

List of Tables

Table 1. Conserved features in the carboxyl terminus common to eukaryotic Hsp/c70  -------------------------------------- -32

Table 2. Nucleotide and amino acid sequences of isolated phage clones --------------------------------------------------- -33

List of Figures

Fig. 1. Purification of hsc70s of mung bean seeds with DEAE cellulose column.------------------------------------------ 34

Fig. 2. Purification of hsc70s of mung bean seeds with ATP agaose column.-------------------------------------------- -35

Fig. 3. The hybridization of anti-Hsp70 antibody to different concentrations of the Hsc70 of mun bean seeds.------------ -36

Fig. 4 Disappearance of Hsc70 protein in mung bean after germination.---------------------------------------------- -37

Fig. 5. A analysis of molecular weight of Hsc70 purified from seeds and seedlings at 1day after germination.------------ -38

Fig. 6. purification of seed Hsc70s from mung bean, Phaseolus vulgaris L. and P. acutifolius A. Gray with ATP Affinity column.-----------------------------------------------------40

Fig. 7. The cytosolic hsc70s were purified by ATP affinity column.-----------------------------------------------------41

Fig. 8. The expression level of Hsc70 in mung bean during embroygenesis.----------------------------------------------42

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