植物菌質體 (phytoplasma) 為寄生在植物篩管內無細胞壁的絕對寄生性原核生物，在世界各地造成嚴重的經濟損失。從日日春的研究中發現，植物抗植物菌質體可能與 PR 基因相關之系統性抗病反應有關。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。因調控CrPR1a 和CrLOX2表現的因子極可能參與對抗植物菌質體，故在本研究中擬透過病毒誘導基因靜默 (virus-induced gene silencing, VIGS)技術，篩選調節CrPR1a和CrLOX2的轉錄因子。我們從台大生技所及植微系團隊所建立之日日春轉錄子資料庫中找出 55群，共 723個轉錄因子。從中選取抗病、抗逆境相關的類群作為篩選目標共6群，152個轉錄因子，分別為ARF (18)、Aux-IAA (8)、bZIP (27)、C2H2 (40)、MYB (45)、MYB-related (14)。其中，我們發現有三個 ARF、一個 Aux-IAA、以及一個 bZIP 類別的轉錄因子的基因靜默可使CrPR1a受 TRV 感染而誘發的表現量改變。為了探討篩選出的轉錄因子與植物抗病機制及與病原菌間之關係，我們檢查了這五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。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。由本研究的結果發現，正向調控CrPR1a的轉錄因子IAA-6會受到菌質體的感染而被誘導，同時 IAA-6同時會被另一個負向調控CrPR1a的因子，bZIP-7，所調控。此外，我們也發現了ARF-12這個正向調節因子，會受另一正向調節因子ARF-13的正向調控。透過本研究，我們發現了新的CrPR1a 與CrLOX2 調控因子並建構出一調控網絡，使得植物抗植物菌質體所啟動分子路徑上有了更全面的了解，以便日後找出更有效之防治策略。

Phytoplasmas are wall-less prokaryotic obligate plant pathogens that are restricted in phloem. They have caused serious economic loss worldwide. In periwinkle, a PR gene-related defense mechanism was found to be related with defense against phytoplasmas. In addition, CrLOX2, which is a jasmonate-responsive and biosynthesis gene, is also induced by the phytoplasma infection. Because regulators of CrPR1a and CrLOX2 may participate in defense against phytoplasmas, both genes were used as marker genes to construct a defense regulatory network using VIGS. We identified 55 groups of 723 transcription factors from periwinkle transcriptome database generated by NTU research team. Among them, we selected those potentially related to biotic or abiotic stresses, including a total of 6 groups, 152 transcription factors, which are ARF (18)、Aux-IAA (8)、bZIP (27)、C2H2 (40)、MYB (45)、MYB-related (14). Results showed that three ARF, one Aux-IAA, and one bZIP transcription factors were identified to regulate the expression of CrPR1a. Their expressions under phytoplasma infection, different hormone treatments, and CrNPR1 or CrNPR3 gene silencing were examined. Results showed that IAA-6, which is a positive regulator of CrPR1a, was induced under phytoplasma infection. IAA-6 is also positively regulated by a negative regulator of CrPR1a, bZIP-7. In addition, ARF-12, which is a positive regulator of CrPR1a, is positively regulated by another positive regulator of CrPR1a, ARF-13. Through the research, we have discovered novel transcription factors regulating CrPR1a and constructed a transcriptional regulatory network. This might help us to better understand the interaction between phytoplasma and its host plant , which would facilitate the development of more effective control strategies.

摘要 ii
Abstract ii
Introduction 1
Materials and Methods 11
Plant Materials and Growth Conditions 11
Chemical treatments 11
Identification of Putative Transcription Factors from Periwinkle Transcriptome Database 11
Plasmid construction 12
Agrobacterium-mediated virus-induced gene silencing 13
RNA extraction and semi-quantitative RT-PCR 14
Establishment of CrPR1a and CrLOX2 regulatory network 15
Results 16
Data mining of putative periwinkle transcription factors 16
CrPR1a is regulated by five transcription factors transcriptionally 16
No current transcription factors selected for screening regulates CrLOX2 transcriptionally 18
Construction of regulatory network involving hormonal regulation, responses to phytoplasma infection, and related to CrNPR1, CrNPR3 18
Construction of regulatory network among candidate factors 19
Discussion 20
Tables and Figures 25
References 43

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