葉開溫臺灣大學:植物科學研究所林沂Lin, IILin2010-05-112018-07-062010-05-112018-07-062009U0001-2201200914180100http://ntur.lib.ntu.edu.tw//handle/246246/181952Sporamin是甘藷塊根中含量超過60%的儲藏性蛋白質，經由其序列比對發現到它與大豆中的Kunitz-type trypsin inhibitor有30%的同源性，而Sporamin融合蛋白質經膠體活性分析證實具有胰蛋白酶抑制因子活性。利用北方墨點法得知該基因在受傷處理後，能系統性地在甘藷葉部表達。在sporamin啟動子的-1.1 kb~-1.02 kb中，NOS-like element: GTAAATACGT與農桿菌nopaline synthase基因的Z element有80%相似度。我們將此10鹼基對序列與其上下游共26鹼基對命名為sporamin wounding responsive element (SWRE)。本論文針對此片段進行yeast one-hybrid篩選，找到與此片段結合的兩個轉錄因子，IbNAC1和IbWRKY1。這兩個轉錄因子分別屬於NAC及WRKY轉錄因子基因家族，利用GFP融合蛋白質顯示這兩個轉錄因子皆存在於細胞核中。經由北方墨點實驗，我們發現IbNAC1和IbWRKY1在受傷後會快速的被誘導表現，IbNAC1會被MeJA及SA誘導表現；而IbWRKY1的表現會被MeJA抑制但會被SA誘導。同時也偵測了sporamin, IbNAC1和IbWRKY1在甘藷不同部位的表現情形。利用不同突變SWRE片段做為探針進行電泳移位分析(EMSA)，確認IbNAC1及IbWRKY1融合蛋白質確實會與SWRE結合，並確認IbNAC1的結合序列為A(N)3TT(N)13ACAATAT；而IbWRKY1的結合序列為(A)CATTT。帶有3XSWRE::GUS和35S::IbNAC載體的阿拉伯芥轉殖株，經GUS染色及活性測試結果顯示在沒有受傷的情況下，大量表現的IbNAC1具有很強的轉錄活性；帶有3XSWRE::GUS和35S::IbWRKY載體的阿拉伯芥轉殖株的轉錄活性則較弱。綜合以上結果，IbNAC1和IbWRKY1，兩個在甘藷中新發現的轉錄因子，且對於調控甘藷受傷誘導機制可能扮演互相拮抗的角色。Sporamin is a major storage protein accounting for more than 60% of the soluble proteins found in the tuberous root of sweet potato (Ipomoea batatas Lam.). The predicted protein sequence of Sporamin shares significant amino acid sequence identity with some Kunitz-type trypsin inhibitors and recombinant protein of Sporamin was shown to have strong inhibitory activity to trypsin in vitro. Northern blot analysis showed that sporamin transcripts could be systemically induced in leaf tissue of sweet potato under wounding stress. Our previous promoter assay showed that cis-element (GTAAATACGT) located at -1.1 kb~-1.02 kb of the promoter region displayed the wounding regulator. The cis-element of 10 bp--GTAAATACGT and its flanking sequences were designated as sporamin wounding responsive element, SWRE, encompassing 26 bp in length. The specific aim of this study is to further investigate the wounding mechanism of sporamin gene in sweet potato. In this work, two cDNA clones encoding SWRE-binding proteins were isolated by yeast one-hybrid screening: IbNAC1 and IbWRKY1. We demonstrated that the two SWRE-binding proteins belong to NAC and WRKY transcription family, respectively and their subcellular localizations were both in nucleus. Their transcripts were accumulated earlier than sporamin transcript once response to wounding. We analyzed the expression patterns of sporamin, IbNAC1 and IbWRKY1 genes at different tissues in sweet potato and treatment with MeJA and SA. Through EMSA, we confirmed that the two transcription factors can bind to SWRE in vitro. The binding sites of IbNAC1 and IbWRKY1 are demonstrated as A(N)3TT(N)13ACAATAT and (A)CATTT respectively. The GUS staining and GUS activity results showed that IbNAC1 had very strong transactivation ability while IbWRKY1 had less. Upon to our results, the two novel transcription factors found in sweet potato may play antagonistic function in wounding induced regulation.口試委員審定書 i 謝 ii文摘要 iiibstract ivbbreviation viontents viiontents of Tables ixontents of Figures xontents of Appendix xii. Introduction 1.1 Defense mechanisms of plants 2.2 sporamin gene in sweet potato 3.3 NAC transcription factors 5.4 WRKY transcription factors 7.5 Specific aims 9. Materials and methods 10.1 Experimental materials 10.2 Plasmids construction 10.3 RNA extraction, semiquantative-RT PCR, and cDNA synthesis 11.4 Construction and screening the one-hybrid library and screening interacting proteins 12.5 Northern-blot analysis 13.6 Alignment and phylogenetic analysis of NAC and WRKY domain proteins 13.7 Subcellular localization analysis of transiently expressed fusion proteins 13.8 Expression and purification of GST-recombinant proteins 13.9 Probe preparation for Electrophoretic mobility shift assay 14.10 Electrophoretic Mobility Shift Assay (EMSA) 15.11 Agrobactirum-transformation of Arabidopsis by floral dipping 15.12 Histochemical GUS staining of transgenic Arabidopsis plants 15.13 Fluorometirc GUS activity assay 16. Results 17.1 sporamin is induced by wounding in leaves of sweet potato 17.2 Screening of the cDNA clones encoding SWRE-interacting proteins 18.3 Molecular characterization of IbNAC1 and IbWRKY1 19.4 IbNAC1 and IbWRKY1 proteins are targeting to the nucleus 21.5 Quick response of IbNAC1 and IbWRKY1 genes to wounding in sweet potato leaves 21.6 Expression of sporamin, IbNAC1 and IbWRKY1 genes under hormone treatments 22.7 Expression of sporamin, IbNAC1 and IbWRKY1 at different tissues in sweet potato 23.8 Electrophoretic Mobility Shift Assay of IbNAC1 and IbWRKY1 recombinant proteins to SWRE sequence 23 9 The target binding sequence of IbNAC1 and IbWRKY1 24.10 Interaction of IbNAC1 and IbWRKY1 proteins through EMSA 25.11 Transactivation assay of the SWRE promoter by IbNAC1 and IbWRKY1 25.12 GUS activity assay of transgenic Arabidopsis, harboring 3XSWRE promoter region and overexpressed IbNAC1 or IbWRKY1 26. Discussion 27.1 ATAF subgroup and WRKY IId subgroup transcription factors 27.2 Function of IbNAC1 and IbWRKY1 in regulation of stress or hormone responses 28.3 The DNA binding sites for IbNAC1 and IbWRKY1 transcription factors 30.4 The phylogenetic relationship of NAC and WRKY transcription factors 31.5 Transactivation of downstream genes by IbNAC1 and IbWRKY1 32ables 34igures 40eference 73ppendixes 77application/pdf2537917 bytesapplication/pdfen-US甘藷受傷NAC基因家族WRKY基因家族轉錄因子電泳移位分析sweet potatowoundingyeast one-hybridNAC familyWRKY familytranscription factorsEMSAhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181952/1/ntu-98-R95b42010-1.pdf