第三群LEA蛋白質表現與功能分析

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陳賢明(Hieng-Ming Ting) 查詢紙本館藏

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論文名稱

第三群LEA蛋白質表現與功能分析
(Functional analysis of group 3 LEA proteins)

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摘要(中)

LEA (Late embryogenesis abundant) 蛋白質在種子發育的後期被大量合成，他亦可以受乾燥逆境和離層酸 (ABA) 處理誘導產生。LEA蛋白質廣泛存在植物和動物中，依據其胺基酸組成的特性及保守性序列可分成為5大類。其中第三大類LEA蛋白質的特徵是含有一段高度重覆的11個胺基酸的結構。受ABA誘導的HVA1屬第三大類LEA蛋白質，將其轉殖並大量表現在水稻，小麥和燕麥時，可使轉殖株對逆境具有較高的忍受度。本研究欲分析HVA1在雙子葉植物的生理功能，並且找出HVA1的功能性區域。大量表現HVA1及HVA1片段的阿拉伯芥 (Arabidopsis) 轉殖株，在正常生長條件及逆境(乾旱及冷凍)條件下，發現沒有造成性狀和形態上的差異。但轉殖HVA1的N端區域 (第1至第102胺基酸)的阿拉伯芥，在種子萌芽期間，顯示高鹽和高滲透壓的忍受度。根據研究結果推論， HVA1的N端區域 (第1至第102胺基酸)足以承受逆境的忍受度。除此之外，Ose730-GFP融合蛋白質在細胞的暫時性表現研究中，證實水稻的第三大類LEA蛋白質表現在細胞核和細胞質。

摘要(英)

Late embryogenesis abundant (LEA) proteins are synthesized abundantly during late stage of seed development and can be induced by desiccation stress and/or ABA treatment. LEA proteins are widely found in plant and animal systems and classified into 5 groups according to amino acid sequences and conserved motifs. Interestingly, a tandem repeat motif of 11 amino acids is found in the group 3 LEA proteins. An ABA-responsive barley (Hordeum vulgare L.) gene HVA1, encodes a group 3 LEA protein and can improve stress tolerance when transformed into rice, wheat and oat. Here we try to analyze the physiological function of HVA1 in dicot and delineate the functional domain of HVA1. Overexpressing HVA1 variants in Arabidopsis did not change the phenotype and morphology of plants under normal or stress (drought and freezing) conditions. However, transgenic Arabidopsis plants overexpressing N-terminal region of HVA1 (amino acid residues 1 through 102 in the N-terminal region of HVA1), showed higher salt and hyperosmotic stress tolerance than that of wild-type and other transgenic lines during seed germination. These results suggest that N-terminal region of HVA1 (N1~102) is sufficient to confer stress tolerance. Furthermore, transient expression of Ose730-GFP fusion protein in cells demonstrated that rice LEA 3 protein was localized in both nuclei and cytoplasm.

關鍵字(中)

★ 阿拉伯芥
★ 離層酸

關鍵字(英)

★ Arabidopsis
★ ABA

論文目次

Chinese abstract…………………………………i
Abstract…………………………………………………………ii
Contents………………………………………………………iii
Table contents…………………………………………………………vi
Figure contents……………………………………………………………vii
Appendix contents………………………………………………viii
List of abbreviations………………………………………………ix
Chapter 1. Introduction………………………………………………1
1.1 The environmental stresses………………………………………………1
1.2 The properties of LEA proteins………………………………2
1.3 The classification of LEA proteins……………………3
1.4 Physiological functions of Group 3 LEA proteins…………5
1.5 History of HVA1……………………………………………6
1.6 History of Ose730…………………………………………6
1.7 Specific aim of this thesis……………………………7
Chapter 2. Materials and Methods…………………………………8
2.1 Materials…………………………………………………8
(A) Plant material…………………………………8
(B) Microbial material……………………………8
(C) Plasmid consturctions…………………………8
35S promoter::HVA1 and HVA1 variants ……………………8
2.2 Methods………………………………………………………9
2.2.1 Gene cloning………………………………9
(A) Preparation of E.coli competent cell……………………………………9
(B) Polymerase Chain Reaction (PCR)…………………………………………10
(C) Ligation and transformation……………………………………………11
(D) Preparation of plasmid DNA…………………………………………12
(E) Gel elution………………………………………………………………12
2.2.2 Plant transformation and stress assay…………………………………………13
(A) Agrobacterium competent cell preparation……………………………………13
(B) Agrobacterium competent cell transformation………………………………14
(C) Floral dipping……………………………………………………………14
(D) Selection of transgenic plants………………………………………………15
(E) Histochemical staining………………………………………………………16
(F) Germination and growth conditions of transgenic plants………………16
(G) Plant genomic DNA extraction……………………………………………17
(H) Plant genomic PCR…….…………………………………………………17
(I) Total protein extraction.………………………………………………………18
(J) Western blotting………………………………………………………………18
(K) Germination assay…………………………………………………………20
(L) Growth measurements……………………………………………………20
(M) Drought test………………………………………………………………21
(N) Freezing test……………………………………………………………21
(O) Electrolyte leakage test………………………………………………21
2.2.3 Hydropathy profile analysis…………………………………………22
2.2.4 Localization analysis of rice Ose730………………………………22
2.2.4.1 Transient expression assay in Arabidopsis mesophyll protoplast……22
(A) Isolation Arabidopsis mesophyll protoplast……………………………………22
(B) Plasmid transfection by the PEG/Ca2+.……………………………………24
(C) Confocal Microscope analysis………………………………………………25
2.2.4.2 Transient expression assay in onion epidermal cells……………………25
(A) Preparation of onion epidermal layer……………………………………25
(B) Preparation of gold particles……………………………………………25
(C) Coating DNA to gold particles…………………………………………26
(D) Bombardment and analysis………………………………………………26
2.2.4.3 Rice stable transformation………………………………………26
(A) Induction of rice embryogenic callus……………………………………29
(B) Transformation of immature callus…..…………………………30
(C) Resistant callus selection and plant regeneration………………………30
Chapter 3. Results………………………………………………………………31
3.1 Sequence analysis of HVA1………………………………………………………31
3.2 Identification of transgenic Arabidopsis plants………………………………32
3.3 Protein expression of transgenic Arabidopsis…………………………………33
3.4 Characterization of transgenic Arabidopsis overexpressing HVA1 variants…33
3.5 Stress tolerance assay………………………………………………………34
(1) Seed germination assay…………………………………………………………34
(2) Root growth assay……………………………………………………………35
(3) Membrane leakage assay………………………………………………………36
(4) Drought test……………………………………………………………………36
(5) Freezing test…………………………………………………………………37
3.6 Cellular localization analysis of rice Ose730……………………………………37
(1)Transient expression analysis in Arabidopsis mesophyll cells………………37
(2)Transient expression analysis in onion epidermal cells………………………37
(3)Transient expression analysis in transgenic rice callus………………………38
(4)Stable transgenic rice ………………………………………………………………38
Chapter 4. Discussion……………………………………………………………39
4.1 Potential functional domain of HVA1………………………………………………39
4.2 The expressed HVA1 protein is unstable in T3 plants……………………40
4.3 Transgenic plants develop normally…………………………………………41
4.4 Functional redundancy of Arabidopsis LEA 3 proteins……………………41
4.5 Rice Ose730 may play a function in cytoplasm…………………………45
References…………………………………………………………………………46

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指導教授

葉靖輝、賀端華
(Ching-Hui Yeh、Tuan-Hua David Ho)

審核日期

2006-7-1

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