指導教授：林長平臺灣大學：植物病理與微生物學研究所賴建閔Lai, Chien-MingChien-MingLai2014-11-292018-06-292014-11-292018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/263054植物菌質體 (phytoplasma) 為重要的植物病原菌，造成嚴重的經濟損失。目前此病害仍缺乏安全有效之防治方法，釐清植物抗植物菌質體的機轉將有助於發展對抗此類病害的策略。在感染日日春葉片黃化病植物菌質體 (PLY phytoplasma)後，我們發現在有病徵與無病徵枝條皆會有 CrPR1a 的誘導表現，此外，茉莉酸生合成上一重要基因 CrLOX2 亦會受植物菌質體感染而誘導表現。因此，我們提出以病毒誘導基因靜默 (virus-induced gene silencing, VIGS) 技術來建構 CrPR1a 與 CrLOX2 的分子調控網絡之目標。我們從日日春 NGS database 中找出 55 群，共 723 個轉錄因子，初步挑選與逆境、抗病相關之類群做為篩選目標，含 AP2/EREBP (38)、ABI3/VP1 (8)、EIL (2)、NAC (29)、MADS (22)、bHLH (38)、WRKY (18)，共 155 個轉錄因子。其中，我們發現有二 AP2/EREBP 類別轉錄因子的基因靜默可使 CrPR1a 或 CrLOX2 受 TRV 感染而誘發的表現量改變。為探討篩選出之轉錄因子與植物抗病機制、病原菌間之關係，我們檢查了這兩個轉錄因子對植物菌質體感染、水楊酸 (salicylic acid, SA)、茉莉酸 (jasmonic acid, JA) 與乙烯 (ethylene, ET) 處理的反應，藉以瞭解其受荷爾蒙誘導之狀況及上下游調控網絡。同時，CrNPR1 與 CrNPR3 在調控網絡中的角色與各篩選到轉錄因子彼此間之關係亦納入討論。本研究發現 ERF-34 能負向調控 CrPR1a 的表現，其亦被另一正向調控 CrPR1a 的轉錄因子 ARF-13 調控。ERF-37，一正向調控 CrLOX2 的轉錄因子，受植物菌質體感染後誘導表現。本研究發現了新的 CrPR1a 與 CrLOX2 調控因子並建構出一調控網絡，提供了植物抗植物菌質體所啟動分子路徑上更全面的了解，以期找出更有效之防治策略。Phytoplasmas are destructive plant pathogens that cause severe loss in agriculture worldwide. However, effective disease management against phytoplasmas have not been developed. Understanding the interactions between plants and phytoplasmas may provide new insights for disease management. CrPR1a can be induced in symptomatic and non-symptomatic shoots after periwinkle leaf yellowing (PLY) phytoplasma infection. CrLOX2, a jasmonate-responsive and biosynthesis gene, is also induced by the phytoplasma infection. Therefore, we aimed to construct transcriptional regulation networks of CrPR1a and CrLOX2 by using virus-induced gene silencing (VIGS). A total of 723 transcription factors in 55 groups were identified from a pre-established transcriptome database. Seven groups of factors, including AP2/EREBP (38), ABI3/VP1 (8), EIL (2), NAC (29), MADS (22), bHLH (38), WRKY (18), that may involve in biotic or abiotic stresses were selected for the screening. Two transcription factors in the AP2/EREBP group were identified to regulate expression of CrPR1a and CrLOX2 respectively. Their expressions under phytoplasma infection, different hormone treatments, and CrNPR1, CrNPR3 gene silencing were examined, and the expression of ERF-34, the potential negative regulator of CrPR1a, was not changed under conditions tested. However, its transcript level was downregulated when ARF-13, a potential positive regulator of CrPR1a, was silenced. ERF-37, a positive regulator of CrLOX2. ERF-37, the potential positive regulator of CrLOX2, was induced after PLY and PnWB phytoplasma infection. We constructed a regulatory network of CrPR1a and CrLOX2 with novel transcription factors. This might help to better understand of plant defense against phytoplasma.Contents 致謝 i 摘要 ii Abstract iii Introduction 1 Materials and Methods 9 Plant Material and Growth Condition 9 Chemical treatment 9 Identification of periwinkle transcription factors from a transcriptome database 9 Plasmid construction 10 Agrobacterium-mediated virus-induced gene silencing 11 RNA extraction and RT-PCR 12 Regulatory network construction 12 Results 14 Data mining of putative periwinkle transcription factors 14 Screening of CrPR1a regulators using TRV-based VIGS system 14 Screening of CrLOX2 regulators using TRV-based VIGS system 15 Construction of regulatory network involving hormonal regulation and responses to phytoplasma infection 16 Construction of regulatory network among candidate factors 17 Discussion 18 Tables and Figures 24 References 41 Supplementary Figures 542692903 bytesapplication/pdf論文公開時間：2019/08/25論文使用權限：同意有償授權(權利金給回饋本人)CrPR1aCrLOX2植物菌質體病毒誘導基因靜默轉錄因子植物抗病thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/263054/1/ntu-103-R00633012-1.pdf