林秋榮臺灣大學：植物科學研究所官建洲Guan, Jiahn-ChouJiahn-ChouGuan2007-11-272018-07-062007-11-272018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/57953第一族低分子量熱休克蛋白質（class I small heat shock protein, sHSP-CI）是植物於熱逆境下累積量及種類最多的熱休克蛋白質，本研究針對水稻(Oryza sativa Tainung No.67)中sHSP-CI基因群進行詳細的基因鑑定及基因表現分析。水稻sHSP-CI基因群成員總共有九個，分別為Oshsp16.9A、Oshsp16.9B、Oshsp16.9C、Oshsp16.9D及Oshsp17.9B位於第一號染色體上，而Oshsp17.3、Oshsp17.7、Oshsp17.9A及Oshsp18.0則位於第三號染色體上，這九個基因在編譯區(coding region)具有很高的相似度(>60 %)，而歧異度較高的區域則位於其3’-UTRs (<40%)，根據α-helix 2 (α2) 氨基酸序列的變異性，我們建議進一步將單子葉植物的sHSP-CI基因群分成subclass A及subclass B兩群。利用二維膠體電泳分析各個基因的in vitro transcription/translation產物或者重組蛋白質，已經確認這九個基因產物在二維膠體上pI值及分子量的相對應位置以及構成heat shock complex (HSC)的主要基因成員。以RT-PCR分析各個水稻class I sHSP基因在熱逆境下的表現，在熱處理下，除了Oshsp17.9B之外，其它基因皆會被誘導大量表現，位於第三號染色體上的sHSP-CI基因在32℃與41℃下很迅速地被誘導表現，而相同處理下第一號染色體上的sHSP-CI基因則較慢被誘導表現。以砷處理白化幼苗，除了Oshsp16.9D和Oshsp17.9B之外，其餘七個sHSP-CI基因皆會誘導表現，但是以第三號染色體上sHSP-CI基因的表現量較多。在鎘、azetidine-2- carboxylic acid（Aze, proline的類似物）及canavanine（Can, arginine的類似物）處理之下則只觀察到第三號染色體上的sHSP-CI基因被誘導表現，相同的sHSP-CI基因表現形式也可受到酒精、NaCl、H2O2和CuCl2引發。此外，我們也觀察到在水稻種子發育成熟過程中只有Oshsp16.9A大量表現，顯示此蛋白質在發育中扮演重要角色。藉由報導基因短暫性表現分析顯示Oshsp17.3和Oshsp18.0共享一個356 bp的雙向啟動子（bidirectional promoter），由此分析亦證明Aze所造成的選擇性差異表現確實是源自於水稻sHSP-CI基因啟動子的原本特性，亦即水稻sHSP-CI基因的選擇性差異表現最有可能是受轉錄層次的調節，進一步由Oshsp17.3啟動子剔除分析確認一段對Aze有專一性反應元素（cis-responsive element, GTCCAGGACG）位在相對於轉錄啟始點-181至-171間。此外也發現一水稻OsMAPK2基因mRNA會受Aze誘導而增加約3倍的表現量，而鎘則只增加約1.5倍。綜合目前的結果顯示已經可以知道氨基酸類似物會以不同於熱休克的方式誘導調節水稻sHSP-CIs基因轉錄表現。最後我根據本研究所得的結果與前人關於訊息傳導的研究，提出一個訊息傳導的可能模式來解釋熱訊息傳導與化學誘導物如Aze或鎘訊息傳導之間的互相關聯性。The cytosolic class I small heat shock proteins (sHSP-CI) represent the most abundant sHSP in plants. Here, we report the characterization and the expression profile of 9 members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67), of which Oshsp16.9A, Oshsp16.9B, Oshsp16.9C, Oshsp16.9D and Oshsp17.9B are clustered on chromosome 1, and Oshsp17.3, Oshsp17.7, Oshsp17.9A and Oshsp18.0 are clustered on chromosome 3. The rice sHSP-CI genes share high homology in the coding regions (>60%) and low homology in the 3’-UTRs (<40%). According to the amino acid variation of theAbstract in Chinese I Abstract in English III Abbreviations V Introduction 1. Heat Shock (HS) Response and HSPs 2 2. The Biological Significance of sHSPs in Plants 3 3. Diversity and Classification of Plant sHSP Family 5 4. Physical-Chemical Properties and Structure of Plant sHSPs 9 5. Chaperone Mechanism of sHSP Action in Plants 12 6. Plant sHSP Expression during Heat Stress 16 7. Expression of Plant sHSP under Normal Growth Conditions 18 7.1 Constitutive Expressions in Vegetative Tissues 18 7.2 Constitutive Expressions in Reproductive Tissues 19 8. Expression of sHSPs in Response to Other Environmental Stresses 21 8.1 Osmotic Stress 21 8.2 Oxidative Stress 22 8.3 Chilling Stress 22 8.4 Chemical Inducers 23 8.5 Other Stresses 24 9. Structural Features Required for Regulation of Plant sHSP Genes 24 9.1 The Essential Cis-response Elements in Promoter Regions 25 9.2 Cis-responsive Motifs in the Enhancer Regions 25 9.3 The Function of 5'-Untranslated Regions of Plant sHSP Genes 26 10. Transcriptional Regulation of sHSP Genes by HSFs 27 11. Developmental Regulation of Plant sHSPs by HSFs 31 12. Signal Transduction Pathway of Plant sHSP Gene Expression 33 13. Progresses and Goals 37 13.1 Progresses of Research Project in Recent Ten Years 37 13.2 Goals of Current Research Project 38 Materials and Methods 1. Plant Materials 41 2. Chromosome Mapping 41 3. RNA Isolation and RT-PCR 41 4. Primer Extension Analysis 43 5. Coupled in Vitro Transcription/Translation and Expression of Recombinant Proteins 43 6. Two Dimensional Gel Electrophoresis (2-DE) and Western Blotting Analysis 44 7. Particle Bombardment and Transient Expression Assays 45 8. Preparations of DNA Constructs for Promoter Deletion Analysis 47 9. Bioinformatics 47 Results 1. Rice sHSP-CI Gene Family Contains Nine Members 48 2. Sequence Analysis of the Rice sHSP-CI Gene Family 48 3. Genomic Organization and Promoter Sequences of the Rice sHSP-CIs Gene Family 49 4. Seven Major sHSP-CI Are Present in the Rice HSCs 51 5. The Heat Stress Responsiveness of the Rice sHSP-CI Genes 52 6. Rice sHSP-CI Genes Are Induced by Various Chemical Inducers in a Selective Manner 53 7. ROS May Have a Role in the Selective Induction of Rice sHSP-CI Genes in Response to Chemical Inducers 54 8. Transient Expression Assays of the Promoter Activity Supported the in Vivo Selective Expression of Rice sHSP-CI Genes by Aze Treatment 54 9. Specific Members of Rice sHSP-CIs Are Induced During Seed Development 55 10. OsMAPK2 Transcripts Are Enhanced by Aze Treatment 55 11. Requirement of a Specific Cis-Responsive Element for Induction of Oshsp17.3 by Aze treatment 56 Discussions 1. Rice sHSP-CI Gene Family 58 2. Structural Features of Monocot sHSP-CIs 59 3. Genomic Organization of the Rice sHSP-CI Gene Family 60 4. Oshsp17.3 and Oshsp18.0 Share a Bidirectional Promoter 61 5. HS Response 63 6. HS-like Response Induced by Chemical Inducers 64 7. Oshsp16.9A Is Developmentally Regulated During Rice Grain Maturation 65 8. Role of H2O2 in the Selective Induction of Rice sHSP-CI Genes 66 9. OsMAPK2 May Be Involved in the Selective Induction of Rice sHSP-CI Genes 67 10. GTCCTGGACG motif (Box1) May Be an Aze-Responsive Element (AZRE) Involved in the Selective Induction of Rice sHSP-CIs by Aze 68 11. A Model of Oshsp17.3 and Oshsp18.0 Induction by Aze and Its Relationship with HS 70 Prospectus 72 Tables 74 Figures 77 References 992276347 bytesapplication/pdfen-US訊息傳導基因表現啟動子短暫性表現分析水稻熱休克蛋白質重金屬氨基酸類似物transient expression analysispromotergene expressionRiceheat shock proteinheavy metalsamino acid analoguessignal transductionotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57953/1/ntu-94-D89226004-1.pdf