臺灣博碩士論文加值系統

MicroRNA為一段21~24核甘酸長度，不轉譯蛋白質的 RNA，它透過降解目標基因mRNA及抑制轉譯來阻止基因的表現。miR160的目標基因為ARF10、ARF16和ARF17。在熱逆境下miR160會被誘導，並且阿拉伯芥miR160大量表現株 (160OE) 比起野生株具有較好的耐熱性。雖然miR160大量表現株的種子發芽率較野生型高，對於miR160及其下游基因的傳遞路徑依然尚未明朗。藉由將短時間熱處理的samples RNA進行NGS測定後，從NGS的結果中篩選了四種被熱及miR160調控的不同基因群。透過GO term 分析基因的種類後發現在熱逆境和160OE株中受誘導的基因大多與強光、熱反應及H2O2 相關，其中包含熱休克因子/蛋白相關基因HSP26.5、HSP17.6、CLPB1 和 HSFA2及離層酸 (ABA) 相關基因。另外被熱逆境誘導，但在160OE株中被抑制的基因大多與種子休眠有關，包括APUM9、MSE65、MSE66、LEA18 和LEA46，其中也有生長素 (auxin) 相關基因IAA32和對吉貝素 (GA) 反應的SWEET14基因。在熱逆境及160OE株中都被抑制的基因除了參與細菌防禦反應的基因外，還有參與吉貝素訊息傳遞路徑和細胞分裂素催化過程的基因。在熱逆境下被抑制，但在160OE株中被誘導的基因除了有參與光訊號傳遞路徑的PIL1和LAF1基因以外，還有抑制花朵發育的PIE1基因及身為轉錄因子的anac028和ARF19基因。透過NGS基因的篩選及GO term的分析，有助於了解熱逆境和miR160在植物體內的交互作用機制。

MicroRNA is the 21~24 nucleotide non-coding small RNA. It represses target genes expression by degrading mRNA or repressing translation. MiR160 targeted and regulated ARF10, ARF16 and ARF17. Also, miR160 is induced by heat stress, and miR160 over-expressing transgenic plants (160OE) have higher thermotolerance than wild-type in Arabidopsis thaliana. Although overexpression of miR160 improves the survival rate of seedlings, the signaling pathway of miR160 and its downstream genes are still unclear. After next generation sequencing of heat shock samples, the data of wild-type and 160OE with four different screening conditions were analyzed. GO term schematics were used to know the gene species in these four conditions. According to results, heat stress and 160OE induced the genes about response to heat, high light intensity and H2O2, including heat shock factor/protein genes HSP26.5, HSP17.6, CLPB1 and HSFA2. ABA-related genes were also affected. The genes induced by heat stress but repressed in 160OE were seed dormancy-related genes, including APUM9, MSE65, MSE66, LEA18, and LEA46, auxin-related gene, IAA32, and gibberellic acid responsive gene, SWEET14. The genes repressed in 160OE and heat stress were involving in defense response to bacterium, gibberellic acid signaling pathway and cytokinin catabolic process. Additionally, the genes repressed by heat stress but induced in 160OE were light signaling pathway genes PIL1 and LAF1, negative regulator of flower development PIE1, and transcription factor genes anac028 and ARF19. Therefore, the interactions of heat stress and miR160 in plants were known by gene selected and GO term schematics.

目次
致謝................................................................................................................i
中文摘要.......................................................................................................ii
Abstract.......................................................................................................iii
目次..............................................................................................................iv
表目次..........................................................................................................vi
圖目次.........................................................................................................vii
第一章 前言..................................................................................................1
一. 植物熱逆境......................................................................................1
1. 熱逆境對植物的影響…..........................................................1
2. 植物在熱逆境的應變方法…..................................................1
2.1細胞內的訊息傳遞….........................................................1
2.2熱休克蛋白 (heat shock protein, HSPs).............................2
二. 植物小分子RNA………………………………………………….2
1. 植物小分子RNA的作用機制……………………………...3
2. 植物小分子RNA對植物生理型態的影響………………...3
3. 植物小分子RNA對非生物逆境的影響…………………...4
三. 基因定序…………………………………………………………4
四. miRNA160和生長素反應因子…………………………………..5
1. miRNA160…………………………………………………...5
2. 生長素反應因子 (Auxin response factor, ARFs)...........5
3. 生長素與離層酸……………………………………………..6
4. 生長素與熱逆境的關係……………………………………..6
五. 研究方向與目的…………………………………………………..7
第二章 實驗材料與方法…………………………………………………..8
一. 實驗材料…………………………………………………………..8
1. 阿拉伯芥……………………………………………………..8
2. 無菌培養植株生長條件……………………………………..8
二. 實驗方法…………………………………………………………..8
1. 阿拉伯芥短時間熱處理……………………………………..8
2. 阿拉伯芥植物RNA萃取…………………………………...8
3. 植物RNA純化……………………………………………...8
4. 次世代定序…………………………………………………..9
5. 基因本體論 (Gene ontology term) 圖表繪製……………….9
6. 使用文氏圖的基因篩選……………………………………..9
第三章 結果………………………………………………………………11
一. RNA的QC檢測…………………………………………………11
二. 基因的篩選方式…………………………………………………11
三. 受熱逆境誘導，以及在miR160大量表現株中也被誘導的基因
……………………………………………………………………11
1. 基因數量及gene ontology (GO) term 圖………………….11
2. 篩選出的基因………………………………………………11
四. 受熱逆境誘導，但在miR160大量表現株中受抑制的基因….12
1. 基因數量及gene ontology (GO) term 圖………………….12
2. 篩選出的基因………………………………………………12
五. 受熱逆境抑制，但在miR160大量表現株中受誘導的基因….12
1. 基因數量及gene ontology (GO) term 圖……………….....12
2. 篩選出的基因………………………………………………12
六. 受熱逆境抑制，以及在miR160大量表現株中也被抑制的基因
……………………………………………………………………13
1. 基因數量及gene ontology (GO) term 圖…………………13
2. 篩選出的基因………………………………………………13
第四章 討論……………………………………………………………....14
一. MiR160的抗熱機制與植物賀爾蒙的作用…………………….14
二. 熱逆境下 160OE株中被抑制的基因與種子休眠的關係……..14
三. 熱逆境下光訊息傳遞基因及抑制開花基因對植株的調控……15
四. 植物賀爾蒙在熱逆境下對植物發育的調控……………………16
五. 總結………………………………………………………………16
第五章 參考文獻…………………………………………………………33

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