臺灣博碩士論文加值系統

PEACI11.8為豌豆幼苗(台中十一號)受低溫馴化誘導之蛋白質，屬於第二群胚胎成熟晚期富含蛋白質 (Group 2 LEA protein)。前人研究指出PEACI11.8基因轉錄產物於低溫處理6小時開始累積，處理6到10天之間到達最大量。一旦將豌豆幼苗去馴化後，PEACI11.8的表現量則迅速下降。此外，PEACI11.8的表現還會受到ABA及PEG處理誘導。本實驗第一部分利用阿拉伯芥轉殖系統探討PEACI11.8於組織及細胞層次上的分佈情形。首先利用PEACI11.8啟動子融合β-glucuronidase (GUS)基因，之後轉殖於阿拉伯芥來探討啟動子受逆境因子的活化程度及組織上的分佈。阿拉伯芥未經處理逆境因子的幼苗組織染色結果顯示PEACI11.8主要表現於頂端分生組織、根部維管束組織及側根長出處。7天阿拉伯芥幼苗受逆境因子處理之組織染色結果得知，PEACI11.8 啟動子受逆境因子處理後，全株植物呈現藍色且其活化程度依序為ABA>PEG>NaCl>Cold。30天株齡未處理逆境因子的植株組織染色結果得知PEACI11.8主要表現於柱頭、果莢及莖的離層。柱頭及離層細胞兩者共同特徵在於這些細胞不具有機械性保護屏障，所以可能易遭受病菌感染。由PEACI11.8啟動子分析也得知此一啟動子序列之中具有許多W box及ELRE調控序列。推測PEACI11.8在這些細胞之中參與保護防衛功能。我們也利用PEACI11.8啟動子趨動GFP::PEACI11.8融合蛋白質，探討PEACI11.8於細胞層次上的分佈。T1植株觀察結果顯示PEACI11.8主要位於保護細胞、根成熟部及延長部細胞。在T2幼苗中 (23個lines)，只在其中一個ABA處理的line發現GFP::PEACI11.8融合蛋白存在於側根長出處。但是此一結果與GUS組織染色結果一致，也證明了PEACI11.8在側根長出處扮演相當重的的角色。本實驗第二部分利用酵母菌及阿拉伯芥表現系統，探討大量表現PEACI11.8是否可提升逆境忍受程度。在酵母菌中利用GAL1啟動子大量表現PEACI11.8的結果顯示，無論在1.2M NaCl、 1.2M KCl 或是4℃處理後，大量表現PEACI11.8的酵母菌皆無法有效提升逆境忍受度。在阿拉伯芥中利用35S 啟動子量表現PEACI11.8的結果顯示，無論在100 mM NaCl、 5% PEG 或是0℃處理後，大量表現PEACI11.8的阿拉伯芥植株皆無法有效提升逆境忍受度。我們推測PEACI11.8在細胞中必須與其它蛋白質共同作用才能行使其功能。

The pea cold acclimation induced protein PEACI11.8 is a group 2 late embryogenesis abundant protein involved in plant responses to stress. Previous study showed that the accumulation of its transcripts began within 6 hours of cold treatment and peaked between 4 and 10 days of treatment. The expression of this gene was rapidly down-regulated after the acclimated seedlings were transferred to normal ambient temperature. Otherwise, PEACI11.8 was also induced by ABA and PEG treatment. In the first part of this study, we provide evidence of the tissue and cell specificity of PEACI11.8 expression. We performed histochemical analysis of PEACI11.8 using β-glucuronidase (GUS) assays in Arabidopsis. The expression of PEACI11.8 promoter::GUS was detected at high level in shoot apical meristem, root vascular tissue and lateral root initiation zone of unstressed plant. In the 7-d-old stressed plants, whole plants appeared blue and the order of induction is ABA>PEG>NaCl>Cold. In adult plants, intense staining was detected in stigma, suggesting that PEACI11.8 has roles during pollen germination or pollen tube growth. Another intense staining site is abscission zone, these cells (stigma and abscission zone) share the characterization that they are unprotected by mechanical barriers and therefore are susceptible to microbial attack and one ELRE (elicitor response element) cis-element in PEACI11.8 promoter. We suggest that PEACI11.8 may play a secondary role of local defense response in stigma and abscission zone. We also used PEACI11.8 promoter to drive GFP-PEACI11.8 fusion protein in stable transformants to investigate subcellular localization. In T1 seedlings, we observed GFP::PEACI11.8 fusion protein expressed in guard cell, root maturation zones and elongation zones. Unfortunately, only one line treated with ABA (total 23 lines) in T2 seedlings expressed GFP-PEACI11.8 fusion protein in the lateral root initiation zone. This results is consistent to histochemical staining result, suggesting that PEACI11.8 may plays a important role in lateral root cells initiation. In the second part of this study, we used two expression systems to test the contribution of PEACI11.8 to stress tolerance in Saccharomyces cerevisiae and Arabidopsis thaliana. In the Saccharomyces cerevisiae expression system, PEACI11.8 was inserted into a multicopy plasmid under the transcriptional control of the yeast GAL1 promoter, and the expression of PEACI11.8 protein was confirmed by immunochemical methods. Yeast cells expressing PEACI11.8 did not show improved growth in 1.2M NaCl, 1.2M KCl and 4℃. In the Arabidopsis thaliana expression system, PEACI11.8 coding sequence was driven by cauliflower mosaic virus 35S promoter. Transgenic plants showed no differences from wild type in normal or stress conditions.

Chapter 1. Introduction……………………………………………………………..1
1.1 Abiotic stress …………………………………………………………………......1
1.2 Cold acclimation……………………………………………………..…….……...3
1.3 LEA proteins……………………………………………………………...…….....4
1.4 Properties of Dehydrins…………………………………………………………....5
1.5 Structure analysis of dehydrins………………………………………………...….8
1.6 Localization of dehydrins……………………………………………………….....9
1.7 Putative function of dehydrins…………………………………………………...10
1.8 History of PEACI11.8………………………………………………………….…12
Chapter 2. Materials and Methods…………………………………...……………13
2.1 Material…………………………………………………………………………..13
(A) Plant material…………………………………………………………………….13
(B) Microbial material……………………………………………………………….13
(C) Plasmid constructions……………………………………………………………13
(1) Native PEACI11.8 promoter::GUS construct……………………………….13
(2) Native PEACI11.8 promoter::GFP-PEACI11.8…………………………….14
(3) 35S promoter::PEACI11.8…………………………………………………..14
2.2 Methods……………………………...…………………………………………...15
2.2.1 Gene cloning………………………...…………………………………………15
(A) Preparation of E.coli competent cell……………………………………………..15
(B) Polymerase Chain Reaction (PCR)………………………………………………16
(C) Ligation and transformation……………………………………………………...16
(D) Preparation of plasmid DNA…………………………………………………….17
(E) Gel elution………………………………………………………………………..18
2.2.2 Yeast transformation and survival assay……………………………………….19
(A) Medium…………………………………………………………………………..19
(B) Yeast transformation……………………………………………………………..20
(C) Preparation and analysis of protein extracts……………………………………...21
(D) Cold tolerance assay……………………………………………………………...21
(E) Generation of S. cerevisiae growth curves……………………………………….22
2.2.3 Plant transformation and survival assay………………………………………..22
(A) Agrobacterium competent cell preparation………………………………………22
(B) Agrobacterium competent cell transformation…………………………………..23
v
(C) Floral dipping…………………………………………………………………….23
(D) Selection of transgenic plants……………………………………………………24
(E) Germination and growth conditions of transgenic plants………………………..25
(F) Genomic DNA extraction………………………………………………………..25
(G) Southern blotting…………………………………………………………………26
(H) Total protein extraction…………………………………………………………..29
(I) Western blotting………………………………………………………………….29
(J) Histochemical staining…………………………………………………………...31
(K) Quantification of GUS activity…………………………………………………..31
(L) Stress tolerance assay…………………………………………………………….33
(M) Electrolyte leakage test…………………………………………………………..33
(N) Cofocal microscopy……………………………………………………………...33
2.2.4 Phylogenetic tree analysis……………………………………………………...34
(A) Alignment……………………………………………………………………..….34
(B) Tree analysis…………………………………………………………………...…34
Chapter 3. Results……………………………………………………………..…....35
3.1 Phylogenetic tree analysis………………………………………………………..35
3.2 Using GUS reporter gene to investigate tissue specificity……………………….36
(1) Putative cis-elements in the PEACI11.8 promoter region…………………..36
(2) Southern blot analysis………………………………………………………37
(3) Histochemical analysis……………………………………………………...37
(4) PEACI11.8 promoter are activated by stress treatment……………………..38
3.3 Using GFP-PEACI11.8 fusion protein to observe subcellular localization………40
3.4 Overexpression of PEACI11.8 to test stress tolerance…………………………...40
3.4.1 Overexpression in Saccharomyces cerevisiae………………………………….41
(1) Analysis of PEACI11.8 in S. cerevisiae………………………………………….41
(2) Effect of PEACI11.8 expression on yeast growth………………………………..41
(3) Effect of PEACI11.8 expression on cold tolerance………………………………42
3.4.2 Overexpression in Arabidopsis thaliana……………………………………….42
(1) Identification of transgenic Arabidopsis plants………….………………….42
(2) Stress tolerance assay……………………………………………………….43
Chapter 4. Discussion……………………………………………………………...45
4.1 Promoter analysis………………………………………………………………...45
4.2 Histochemical studies of PEACI11.8 promoter::GUS transfomants……………..47
4.3 Localization of GFP-PEACI11.8 fusion protein………………………………….49
4.4 Overexpression of PEACI11.8 in yeast…………………………………………..50
4.5 Overexpression of PEACI11.8 in Arabidopsis…………………………………...51
References……………………………………………………………………………54

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