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研究生:謝若微
研究生(外文):Jo-Wei Hsieh
論文名稱:新穎miRNA-tag292在甘藷傷害反應之探討
論文名稱(外文):The involvement of novel miRNA-tag292 in wounding response of sweet potato
指導教授:鄭石通鄭石通引用關係
指導教授(外文):Shih-Tong Jeng
口試日期:2017-07-26
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:109
中文關鍵詞:甘藷傷害茉莉酸新穎miRNAtag292RAP2.7PR1a
外文關鍵詞:sweet potatowoundingjasmonatenovel miRNAtag292RAP2.7PR1a
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由於植物無法移動,當面對逆境時無法藉由逃跑躲避,因此演化出許多應對的機制。在這些逆境中,傷害是最常見的逆境之一。近年研究發現microRNA (miRNAs) 參與受傷後的防禦機制。先前,實驗室前人利用次世代定序技術結合生物資訊分析找到許多在甘藷中受到傷害影響的新穎miRNA。本研究選定tag292,對其在甘藷體內的功能及目標基因IbRAP2.7進行探討。首先,tag292的前驅物可摺疊成miRNA特有的莖環結構,並於菸草短暫表現與甘藷大量表現tag292前驅物的植株中得知tag292可成功被剪切產生。於甘藷傷害反應下,tag292會受到抑制,其目標基因IbRAP2.7表現量會上升,在茉莉酸處理下也可獲得同樣的趨勢,顯示傷害對tag292與IbRAP2.7的調控可能是藉由茉莉酸來進行。tag292與目標基因IbRAP2.7的關係進一步藉由菸草短暫表現、甘藷轉殖系統以及剪切點分析證實在傷害處理下tag292確實對IbRAP2.7進行剪切並抑制其基因表現量。此外利用菸草原生質體證實IbRAP2.7主要會座落細胞核中。最後,藉由即時定量發現IbPR1a表現量在甘藷中大量表現IbRAP2.7會被誘導,反之大量表現tag292時被抑制,顯示IbPR1a在傷害下可能會被tag292和IbRAP2.7共同調控。總結以上的結論,甘藷遭受傷害會抑制tag292使其無法抑制目標基因IbRAP2.7,導致IbPR1a表現量上升。
Plants, anchored to the ground, are sessile organisms and thus have evolved complex survival mechanisms responding to stresses. Among these stresses, wounding is one of the most common ones. To date, it has been demonstrated that miRNAs participate in wounding defense mechanisms. Previously, we predicted several wound-responsible miRNA candidates based on the next generation sequencing and bioinformatics analysis. Here, we characterized the function of tag292 in planta and further identified its direct target Related to AP2.7 (IbRAP2.7). First, tag292 could be excised from putative precursor sequence (pre-tag292) proved by transient assay of tobacco and stable expression of transgenic sweet potato. Moreover, IbRAP2.7 mRNA cleavage induced by tag292 was confirmed by cleavage site mapping analysis, agro-infiltration assay and transgenic plant studies. The induction of IbRAP2.7 expression coincided with the reduction of tag292, demonstrating IbRAP2.7 was tag292 target. IbRAP2.7, a transcription factor, localized in the nucleus and endoplasmic reticulum of protoplasts. Finally, pathogenesis-related PR-1 family gene (IbPR1a) might be regulated via tag292/IbRAP2.7-mediated pathway in wounding response. These results concluded that IbRAP2.7 was induced following a decrease of tag292, leading to an increase of IbPR1a in sweet potato upon wounding.
Thesis Oral Defense Committee Report I
Acknowledgments II
摘要 III
Abstract IV
Introduction 1
1. The wounding response in plants 1
2. MicroRNAs in plants 2
2.1 The biosynthesis of miRNAs 3
2.2 The regulatory mechanisms of miRNAs 4
2.3 The roles of miRNAs in plant development 5
2.4 The roles of miRNAs in plant stress response 6
3. Prediction of novel woinding-responsive miRNAs 7
4. Prediction of miRNA targets 9
5. APETALA2/Ethylene Responsive Factors (AP2/ERF) 9
6. Role of Pathogenesis-Related (PR) proteins in plants 12
7. Research motivations and objectives 13
Materials and methods 15
1. Plant materials growth conditions and treatments 15
1.1 Sweet potato 15
1.2 Tobacco 16
2. Experimental methods 16
2.1 RNA extraction 16
2.2 RNA Electrophoresis 17
2.3 DNase treatment 18
2.4 Reverse transcription-PCR (RT-PCR) 18
2.5 PCR and qPCR 19
2.5.1 PCR 19
2.5.2 qPCR 21
2.6 Plasmid construction 22
2.6.1 Gel elution 22
2.6.2 DNA ligation 22
2.6.3 E. coli cell transformation 23
2.6.4 Plasmid DNA mini-preparation 24
2.6.4 DNA sequencing 25
2.6.4 DNA construction 25
3. Preparation of conpetent Agrobacterium cell and transformation 25
3.1 Preparation 25
3.1.1 Prepatation of Agorbacterium LBA4404 25
3.1.2 Prepatation of Agorbacterium 15834 26
3.2 Transformation 26
3.1.1 Transformation of Agorbacterium LBA4404 26
3.1.2 Transformation of Agorbacterium 15834 27
4. Agrobacterium-mediated transient expression in tobacco 27
5. Plant transformation 28
5.1 Sweet potato 28
5.2 Nicotiana tabacum 30
6. Mapping of tag292-guided cleavage sites 32
7. Subcellular licalization in tobacco 33
7.1 Target genes construction 33
7.2 Organelle markers 34
7.3 Plasmid DNA midi-preparation 34
7.4 Tissue preparation and protoplast isolation 35
7.5 Protoplast transfection 37
7.6 Protoplasts collection and GFP assay 41
8. Genomic DNA extraction 41

Results 42
1. Characterization of wounding-related miRNAs and their precursors 42
2. Production of the novel miRNA tag292 from its precursor form by transient expression of tobacco and by stable transformation of sweet potato 42
3. Expression of tag292 in sweet potato upon wounding 44
4. Validation of tag292 targets in sweet potato upon wounding 44
5. IbRAP2.7 is a target of tag292 45
5.1 Agrobacterium-mediated transient expression 45
5.2 Expression of IbRAP2.7 in transgenic sweet potato overexpression pre-tag292 47
5.3 Cleavage sites 48
6. The species-specificity of relationship between tag292 and IbRAP2.7 48
7. Effects of MeJA and ethylene on the expression of pre-tag292, tag292 and IbRAP2.7 in sweet potato 49
8. Spatiotemporal expression of pre-tag292, tag292 and IbRAP2.7 in sweet potato 50
9. Localzation of IbRAP2.7 50
10. IbPR1a was regulated by tag292 and IbRAP2.7 in transgenic sweet potato upon wounding 51
11. Putative cis-element of RAP2.7 on IbPR1a promoter region 53
Discussion 54
1. Identification of wounding-responsive novel miRNAs 54
2. Novel miRNA tag292 55
3. The prediction criteria of tag292 targets 56
4. Interaction between tag292 and its targets IbRAP2.7 transcript 58
5. IbPR1a was regulated by tag292-IbRA2.7 module 59
6. The potential role of tag292-IbRAP2.7 module 59
Conclusion 61
Tables 63
Figures 66
Supplementary Tables 94
Supplementary Figures 98
References 99
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