臺灣博碩士論文加值系統

Late embryogenesis abundant (LEA) 蛋白質會大量表現與累積於植物胚胎發育後期。根據這一類蛋白質的胺基酸序列相似性及收斂序列特徵，可將此類蛋白質至少區分成五群 (group)。根據前人的研究，LEA 基因會因離層酸 (ABA)、高鹽、乾旱、或是低溫等逆境誘導而表現； LEA蛋白質已被證明可在植物遭受逆境時，參與調節生理反應的角色: 如在乾燥的環境下與水分子結合維持細胞中的水含量、調節離子的種類、保護其他細胞內蛋白質及膜狀胞器的穩定性，亦有研究顯示它們可調節植物的正常發育。
第三群LEA蛋白質具有由11個胺基酸(TEAAKQKAAET)所組成的收斂(consensus)區域且重複出現數次。由於離層酸、鹽分及乾燥或是滲透壓引起的環境變化，皆能誘導此群LEA基因表現，因此第三群LEA蛋白質在植物遭遇逆境時，扮演著不可或缺的角色。大麥HVA1已被歸類於第三群LEA蛋白質，研究顯示將HVA1基因轉殖到水稻，可增加水稻對於乾旱的耐受性；其他的研究也指出含有第三群LEA基因的轉殖作物對鹽分、溫度亦具有較佳的適應能力；即使有這些實驗所得，我們對於第三群LEA蛋白質的結構與生理功能及其在細胞中所扮演的角色，卻還有許多不明瞭的地方。
水稻是一個重要的作物、亦被視為單子葉植物研究中的模式生物。本篇研究利用大麥HVA1蛋白質序列為模板，利用序列相似性比對 (BLAST)，由水稻基因體資料庫中篩選出五個水稻第三群LEA蛋白質，在資料庫中的基因/蛋白質序號分別為: AP004018/BAD19162, AP003381/BAB86507, AC073556/AAL84288, AC098833/AAU43988, AP000836/BAD81113. 分析這些基因序列特性、在發育時期及環境逆境下之基因表現狀況。結果顯示: 在生殖生長期，這五個基因在水稻植株授粉前即可測得表現量，持續到開始脫水的糊熟期時，基因表現量達高峰並延續至種子成熟；在營養生長期，可調解生長發育與遭遇環境逆境時維持正常生理功能。由基因表現數據得知，這些基因具有組織專一性，特定發育時期或逆境時，只能在特定部位偵測到基因表現訊號。利用洋蔥表皮細胞短暫表現系統研究AP004018-GFP, AP003381-GFP, AC073556-GFP, AC098833-GFP, 與AP000836-GFP綠螢光融合蛋白質在細胞內表現位置，結果顯示這些基因在不同逆境下，在洋蔥表皮細胞內會有不同的分布位置：如在離層酸或低溫處理時， AP003381、AC073556會往細胞核處集中；AC073556、AC098833處理離層酸，AP004018、 AC073556、AC098833處理氯化鈉，AP004018、AC098833在4℃低溫時在細胞質有聚集現象；AC073556在離層酸及氯化鈉，AP004018、AP003381、AC073556及AC098833 低溫處理下，集中在細胞邊緣，可能跟細胞膜的穩定性有關，此外，在正常的情況下，可以發現這些綠螢光融合蛋白質會表現在細胞核、細胞質及細胞膜。

Late embryogenesis abundant (LEA) proteins were initially found in the stage of embryo maturation. According to amino acid sequence similarities and conserved motifs, the LEA proteins can be separated into more than five groups. It is known that abscisic acid (ABA), salt, drought or cold stress can induce expression of the lea genes, conferring stress tolerance to plant cells. LEA proteins have been shown to involve in binding and replacement of water molecule, ion sequestration, maintaining of protein or cell membrane structure, and development regulation. Group 3 LEA proteins are characterized by 11-mer amino acid conserved tandem repeats (TEAAKQKAAET) and considered as the stress proteins. Barley HVA1, a member of group 3 LEA proteins, has been reported that it can improve stress tolerance in transgenic plants. However, its exact physiological functions still remain unclear. In this study we used the sequence BLAST to search Group 3 LEA protein orthologues in rice genome database and identify five lea3-like genes. They are AP004018/BAD19162, AP003381/BAB86507, AC073556/AAL84288, AC098833/AAU43988, AP000836/BAD81113 (gene/protein accession number). Here we were trying to characterize their gene sequence organization and expression patterns during developmental stages and environmental stresses. The results showed that these LEA genes played important roles on regulation of plant growth and response for varied stresses, and tissue-specific expression in root, stem, leaf, and seed. Furthermore, cellular localization analysis of the AP004018-GFP, AP003381-GFP, AC073556-GFP, AC098833-GFP, and AP000836-GFP fusion proteins in onion epidermal cells indicated that rice group 3 LEA proteins could be in nucleus (AP003381, AC073556 under ABA or 4℃ treatment), cytoplasm (AC073556, AC098833 under ABA; AP004018, AC073556, AC098833 under NaCl; AP004018, AC098833 under 4℃ treatment), or membrane structure (AC073556 under ABA and NaCl; AP004018, AP003381, AC073556, and AC098833 under 4℃ treatment ), whereas these fusion proteins localize in either nucleus or cytoplasm under non-stress conditions.

Table of contents………………………………………………………………………………I
Chinese abstract……………………………………………….….…………..………..…III
English abstract ……………………………………………….……………...………….V
List of abbreviations…………………………………………………………………..…....VII
Chapter 1. Introduction…………………………………..……….……..………………...1
Environment and stresses………………….………….………..………….……….1
Properties of LEA proteins…………………..….……….……..……………….…...2
The classification of LEA proteins………………………...…………..…………….2
Physiological functions of LEA proteins……………………………………………5
Specific aim of this thesis……………………………………………………………8
Chapter 2. Materials and Methods……………………………………………………10
2.1 Bioinformatics analysis and data mining………………………………………….….10
2.2 Plant materials………………………………………...………………………………..11
2.3 Total RNA extraction……………………………………...…………………………....11
2.4 Reverse transcriptional polymerase chain reaction………………………………...13
2.5 Polymerase chain reaction and transformation……………………………………..14
2.6 Preparation of plasmid DNA………………….…………………………………….…18
2.7 Gel elution…………………………….………………………………………………...19
2.8 Transient expression of promoter activities to different stresses………….……...20
2.9 Functional localization analysis……………………………………………………….24
Chapter 3. Results……………………………………………..……….…………………28
3.1 Orthologues of HVA1 in rice share same motif sequence and are ABA inducible
…………...……………………………………………………………………………28
3.2 Characterization for gene expression of rice group 3 lea genes during
development stages and stresses………………………………………………......29
3.3 Analysis of the promoters of rice group 3 lea genes under stresses……...31
3.4 LEA-GFP fusion proteins showed different subcellular localization under stress
treatments……………………………………………………………………………...32
Chapter 4. Discussion………………….………………………………………………33
Conserved amino acids were identified in the motif of rice group 3 LEA proteins
………………………………………………………………………………………..33
Analysis of phylogenic tree and evolution of rice group 3 lea genes………….33
Rice CBF2 gene (RCBF2) as a marker.…………………………………………..34
Physiological role of LEAs………………………………………………………….34
Prospects of LEAs…………………………………………………………………..35
Chapter 5. Tables and Figures…………………………………………………………36
Table 1. Selected genes in this study……………………………………………………36
Table 2. List of primers used in this study………………………………………………38
Table 3. Analysis of cis-acting regulatory elements on promoter regions of rice lea 3
genes………………………………………………………………………….......40
Table 4. Profiles of gene expression pattern……………………………………………42
Figure 1. Responsiveness of rice lea 3 genes to ABA treatment………………………46
Figure 2. Multiple sequence alignment of group 3 LEA proteins……………………….47
Figure 3. Analysis of hydropathicity and intrinsic disorder tendency structure………49
Figure 4. Overview of rice Tainung 67 (TNG67) growth and development stages…53
Figure 5. Expression patterns of rice lea 3 genes during developmental stages and
stress treatments…………………………..…………………………………55
Figure 6. Promoter activities of rice lea 3 genes analyze by Arabidopsis protoplast
cells………………………………………………………………………………66
Figure 7. Subcellular localization of rice LEA- GFP fusion proteins analyze by onion
epidermal cells…………………………………………………………………69
Chapter 6. References……………………………………………………………………75
Chapter 7. Appendix………………………………………………………………………80
I. Gene expression profile: electrophoresis images and normalized data…80
(A) Rice grains imbibitions in water with different time course………………………80
(B) 20-day-old rice seedling in different stress with time course……………………81
(C) Rice tillering stage in different stress and time course…………………………88
(D) Rice panicle stage in different stress and time course………………………95
(E) Seed maturation stage expression data…………………………………….…102
II. The promoter region sequences of five lea genes………………………………….104
III. Description of Cis-acting elements…………………………………………………..105

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