臺灣博碩士論文加值系統

本論文利用菸草屬 (Nicotiana sp.)植物設計簡併式引子對，從圓葉菸草 (Nicotiana benthamiana L.) 中分別選殖多元胺代謝相關基因片段，其分別為大小1,112 bp、1,010 bp和1,648 bp的SPDS (spermidine synthase)、SPMS (spermine synthase) 以及PAO (polyamine oxidase) 基因。從即時轉錄聚合酶連鎖反應的結果發現進行乾旱處理會提高SPDS和SPMS基因的表現，抑制PAO基因的表現，乾旱回水處理則提高SPDS和PAO基因的表現，抑制SPMS基因的表現。本論文利用帶有目標基因片段之菸草鑲嵌病毒 (TMV)，構築於Ti質體進行生體內 (in vivo) 感染，以進行目標基因之靜默。將已接種TMV 28天圓葉菸草之切離葉片進行乾旱試驗，TMV-PAO靜默植株在乾旱逆境下較控制組累積較多的游離spermine，而MDA (malondialdehyde) 和H2O2則累積較少，因此推測菸草在乾旱逆境下累積多元胺與PAO表現量降低有關，而多元胺的降解可能導致植物累積H2O2造成膜系的損壞。

The fragments of polyamine metabolism related genes, including SPDS, SPMS and PAO, of 1,112 bp, 1,010 bp and 1,648 bp respectively were obtained from Nicotiana benthamiana by using degenerate primers. The results of real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression of SPDS and SPMS gene was induced by drought, and the expression of PAO gene was inhibited. Drought rewatering treatment increased the expression of SPDS and PAO genes and inhibited the expression of SPMS gene. In this thesis, the full-length cDNA of TMV with the target gene fragment was constructed an in vivo system to silencing the target gene. Drought tests were carried out on the 28 days post infection. Results showed that TMV-PAO silenced plants accumulated more free spermiine under drought stress, while MDA and H2O2 accumulated less. Therefore, it was suggested that accumulation of polyamine in tobacco under drought stress was related to the decrease of PAO expression. Degradation of polyamines may lead to the accumulation of H2O2 and caused damage on the cell membrane.

摘要................................ i
英文摘要................................................................ ii
表次索引................................................................................................. viii
前人研究.....................................................................................................1
植物的多元胺與其合成代謝途徑.....................................................1
逆境誘導植物多元胺累積..................................................................2
利用病毒誘導基因沉寂系統研究基因功能.....................................4
研究目的..............................................................................................7
材料與方法.................................................................................................8
試驗材料..............................................................................................8
菸草總量 RNA (total RNA)之萃取....................................................8
反轉錄聚合酶鏈鎖反應(Reverse Transcription-Polymerase Chain
Reaction, RT-PCR)...............................................................................9
SPDS、SPMS、PAO 基因片段之選殖............................................10
基因片段的選殖................................................................................12
圓葉菸草 PAO、SPDS、SPMS 基因的VIGS 構築........................12
大腸桿菌的轉型作用(transformation) ...........................................13
質體 DNA 之萃取.............................................................................14
農桿菌的轉型作用(transformation) ...............................................14
農桿菌接種短暫表現系統(Agrobacteria-mediated transient
expression system) .............................................................................15
酵素免疫分析法(Enzyme-linked immunosorbent assay, ELISA) ...15
逆境試驗............................................................................................16
即時定量反轉錄聚合酶連鎖反應...................................................19
vii
結果...........................................................................................................21
多元胺生和成基因(SPDS、SPMS)和降解基因(PAO) 之選殖.21
圓葉菸草之多元胺於乾旱逆境下的表現.......................................22
農桿菌短暫表現系統........................................................................24
利用 VIGS 探討在乾旱逆境下游離多元胺與多元胺合成及降解相
關酵素的調控關係............................................................................25
討論...........................................................................................................27
乾旱處理下圓葉菸草多元胺的累積...............................................27
多元胺降解基因(PAO) 之選殖......................................................28
利用 VIGS 分析植物多元胺合成及降解基因與乾旱逆境間的關係
............................................................................................................29
參考文獻...................................................................................................31

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