楊寧蓀臺灣大學：植物科學研究所謝恩蓉Hsieh, En-JungEn-JungHsieh2007-11-272018-07-062007-11-272018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/57943植物在遇到環境改變時會有不同的反應以抵抗逆境。為了找出植物在逆境下能有反應的基因群，我們由蕃茄的互補DNA微陣列 (cDNA microarray) 分析中找出了一系列植物在逆境下確實有所反應之基因。我們將研究焦點放在缺水、高鹽及低溫三種逆境下可以高度表現之基因。在這三種逆境下有反應之基因群中，我們發現其中一個基因，命名為cLEX4I22，是有C2H2之鋅指蛋白(Cys2/His2 zinc finger protein)之結構。又因該基因為一個可能的轉錄因子(putative transcription factor)，所以就選擇此基因做更進一步的研究。在動物的系統中，C2H2鋅指蛋白基因家族是最大的調節蛋白(regulatory protein)家族，且多在動物生長發育中扮演重要的角色。因此我們推理在植物中之鋅指蛋白轉錄因子在植物生長發育上也可能扮演重要的角色。此論文研究的目的即為研究蕃茄cLEX4I22之功能。在南方墨點分析(Southern blot analysis)實驗中發現在蕃茄的基因組中cLEX4I22顯然像是為一個單一基因；北方墨點分析知道cLEX4I22基因在蕃茄花部的表現程度比較高。接著，我們利用基因轉殖的方法我進一步研究了cLEX4I22的功能，分別以持續表現之啟動子(constitutive promoter) CaMV 35S及一個在逆境下可被誘導表現之阿拉伯芥啟動子cor15a來啟動cLEX4I22基因之表現。接著藉由農桿菌轉殖系統(Agrobacterium-mediated transformation)將這個基因轉入模式植物阿拉伯芥。目前得到了數個可能過度表現cLEX4I22成功之阿拉伯芥之轉殖株。我們發現大部分的轉殖植物具有極不正常之外表型，並由此推測cLEX4I22基因可能在逆境誘導下會影響植物之生長發育，或也可能與植物之節省能量機制有關。Plants are known to respond well and adapt to various environmental stresses. To identify the plant stress-responsive genes, we have obtained a large number of tomato cDNA clones that responsed to various environmental stresses, by using our homemade cDNA microarray systems. It was interesting to observe that some genes were highly expressed as a response to water deficit, salinity and low temperature stresses. One putative transcription factor, cLEX4I22, apparently belongs to a C2H2 zinc finger protein group, was chosen for this study. In animal systems, the C2H2 zinc finger protein gene family is known as the largest group of regulatory proteins and plays similar roles an important role in growth and development of a number of tested systems. It is thus hypothesized that cLEX4I22 and its homologues transcription factors may also play similar roles in plants. My study goal is to characterize this novel putative transcription factor of tomato, cLEX4I22, for functional analysis and potential application to crop plant development. Southern blot analysis showed that the endogenous cLEX4I22 apparently exist as a single copy gene in the tomato genome; Northern blot analysis of different organs revealed that the cLEX4I22 gene was expressed higher in tomato flowers then other tested organs. In addition, transgenic approach was used for functional study. We have obtained a full length cLEX4I22 cDNA, and cloned the open reading frame of cLEX4I22 into a pCAMBIA1390 binary vector, driven by a constitutive promoter CaMV35S or an Arabidopsis stress-inducible promoter cor15a. Using Agrobacterium-mediated transformation, we have obtained several putative Arabidopsis transgenic lines overexpressing cLEX4I22, which exhibited abnormal phenotypes. These results suggest that cLEX4I22 might play a role in plant growth and development.Index Abbreviation……………………………..…………………………....…….…….……i English abstract…………………………………..……………………..…….………iii Chinese abstract……………………………………………...………..…..….………iv Introduction.……………………………………………...…………..…….………1 The Cys2/His2-type zinc finger proteins……………………..………….….…….……2 The Cys2/His2-type zinc finger proteins in human……………………….…………....3 The Cys2/His2-type zinc finger proteins in plants……………….…………..…….......3 C2H2 zinc finger proteins in Arabidopsis…………………….….…………….............4 C2H2 zinc finger proteins in petunia………………….………….…………….............6 Objectives…………………………………………………………………….………..7 Materials and Methods………………………….……………………….………8 Plant materials……………….……………………………..…..…………..….………8 Stress conditions……………….………………………………..…..…………………8 Molecular Biology Analyses…………………………………….…..………...………8 Extraction of tomato DNA……………………………………..…..………….…..8 Extraction of Arabidopsis DNA………..………..………………………....……...8 Southern blot analysis…..………………………….……….................................10 Transfer of DNA or RNA from agarose gel to solid supports................................10 Northern blot analysis………………….………....................................................11 Isolation of total RNA……………….……….................................................11 RNA electrophoresis……………….………....................................................11 Preparation of probe…………….………..............................................................11 Hybridization…………….……….........................................................................12 Washing membrane.…………...............................................................................12 Stripping membrane…...........................................................................................14 Molecular cloning of cLEX4I22 gene..........................................................................14 RACE (Rapid Amplification of cDNA Ends) .......................................................14 Preparation of Agrobacterium-mediated transformation of Arabidopsis.....................15 Plasmid ..................................................................................................................15 constructs................................................................................................................15 Electroporation of E.coli........................................................................................15 Preparation of XL1-Blue competent cells..............................................................15 Transformation of E.coli........................................................................................16 Alkaline lysis of plasmid minipreparation.............................................................16 Electroporation of Agrobacterium.........................................................................16 Preparation of electro-competent Agrobacterium..........................................16 Transformation of Agrobacterium..................................................................17 Transformation of Arabidopsis......................................................................17 Plant growth conditions.................................................................................17 Agrobacterium-mediated dip transformation of Arabidopsis........................17 Hygromycin resistance test............................................................................18 Results…………………………………………………………………...…………19 Northern blot analysis of cLEX4I22 expression…………………………………...…19 Molecular cloning of the full length cLEX4I22 gene……………………………...…20 Multiple amino acid sequence alignments………………………………………...…21 Southern blot analysis of cLEX4I22 sequence in tomato genome….……………..…22 Analysis of possible organ-preference gene expression…………………………...…22 Cloning of the cLEX4I22 open reading frame for studies using transgenic approach.23 Transgenic Arabidopsis plants expressing cLEX4I22 ……………………….………24 Phenotype comparison……………………………………………………….………25 Discussion…………………………………………………………………………26 References …………………………………………………………..……………33 Index of Tables Table 1. TFⅢA-type zinc finger protein in petunia………………………..……...…39 Table 2. Comparison of the peptide sequence identity between similar genes in different species and cLEX4I22.………………..…………40 Table 3. Frequency for transforming Arabidopsis with cLEX4I22 cDNA or related genes, via hygromycin selection.……..……………..……41 Index of Figures Figure 1. Schematic diagram of a zinc finger………………………..……...……….42 Figure 2. Zinc finger motif..………………………………………...…………….….43 Figure 3. TFⅢA-type zinc finger consensus sequence. ……………….……….……44 Figure 4. Schematic representations of protein structure..…………………...………45 Figure 5. Northern blot analysis of cLEX4I22 gene in leaf and root tissue of wild type tomato plants.…………………………………...……………46 Figure 6. Rapid amplification cDNA end of Tomato cLEX4I22 cDNA………..……47 Figure 7. Full length cLEX4I22 cDNA nucleotide sequence…………….………..…48 Figure 8. Characteristic amino acid sequences of cLEX4I22.…………………...…..49 Figure 9. Comparison of cLEX4I22 amino acid sequence and related gene products from different species……………………………………………50 Figure 10. Southern blot analysis of cLEX4I22 gene in wild type tomato plants (Ⅰ)……………………………………………………….…51 Figure 11. Southern blot analysis of cLEX4I22 gene in wild type tomato plants (Ⅱ).…………………………………………………………………52 Figure 12. Northern blot analysis cLEX4I22 gene expression in different organs of wild type tomato plants.……………………………………..….53 Figure 13. PCR cloning of open reading frame from cLEX4I22 cDNA…………..…54 Figure 14. Confirmation of cloning of the cLEX4I22 open reading frame.…………55 Figure 15. Molecular construction of pCAMBIA1390-35S-cLEX4I22………..…….56 Figure 16. Molecular construction of pCAMBIA1390-cor15a-cLEX4I22.………….57 Figure 17. Phenotypes of putative transgenic plants……………….…………...……582421887 bytesapplication/pdfen-US轉錄因子transcription factorotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57943/1/ntu-93-R91226020-1.pdf