更年性果實中乙烯生理作用是由系統一和系統二所構成，系統一主要於營養生長時期及果實中表現，提供一般生理用的微量乙烯；而系統二為更年性果實與花器老化所特有，系統一能產生微量乙烯誘發系統二表現。本研究應用 RNA 干擾的策略並藉由農桿菌轉殖法，取得默化 Mh-ACO1 和 Mh-ACO2 香蕉轉殖株。經 GUS 活性組織化學染 色分析與南方氏雜交，確認 T-DNA 均完全嵌入香蕉染色體中。默化 Mh-ACO1 及默化Mh-ACO2 之北蕉轉殖株與未轉殖株相比，生長速度及下位葉片萎凋速度均較慢。
進一步以北方雜交分析轉殖株，了解 Mh-ACO1 和 Mh-ACO2 於轉殖北蕉之葉片、創傷處理與果實表現情形。分析結果顯示 Mh-ACO1 於葉片、創傷處理與果實中均有表現，Mh-ACO2 僅於創傷處理與果實表現，且Mh-ACO1 和 Mh-ACO2 隨著果實後熟程度的增加，表現量也隨之增強。而默化 Mh-ACO1 或 Mh-ACO2 基因之北蕉轉殖株，均會影響另一 ACC 氧化酶基因表現。默化 Mh-ACO1 轉殖株創傷葉片，以Mh-ACO1 cDNA 為探針可偵測到介於 21-25 nt siRNA；於默化 Mh-ACO2 後熟果實中，亦可以 Mh-ACO2 cDNA為探針可偵測到介於 21-25 nt siRNA 表現，顯示默化構築默化構築 pBI121-1AnS 與 pBI121-2AnS 能有效啟動 RNAi 的機制，有效且專一的默化北蕉中 Mh-ACO1 與 Mh-ACO2 表現。
利用 IPCR (Inverse Polymerase Chain Reaction) 與 APCR (Anchored Polymerase Chain Reaction) 分析默化轉殖株邊界序列，從 Mh-ACO1 轉殖株品系 1AS-1 與1AS-25 取得二段默化 T-DNA 左邊界序列；及 Mh-ACO2 轉殖品系 2AS-1 與 2AS-79 中，取得三段 T-DNA 左邊界序列。2AS-1、1AS-25、與 2AS-79 中嵌入染色體的四段左邊界序列中，帶有全長或部分左邊界保守序列，左邊界保守序列出現 26 bp、23 bp 、19 bp 與 16 bp，而 1AS-1 不帶有左邊界保守序列。 2AS-1 與 2AS-79 所嵌入之邊緣序列都帶有非 T-DNA 載體序列，且部分左邊界序列接有右邊界非 T-DNA 載體序列，顯示農桿菌侵染植物細胞的過程中，會帶入部分非 T-DNA 載體序列，且發生重組或刪除 DNA 的現象。

Ethylene biosynthesis in climacteric plants is regulated by two systems. System 1 functions during normal vegetative growth and is responsible for producing the basal levels of ethylenedetected in all tissues including mature fingers before the onset of fruit ripening. System 2initiates during the ripening of climacteric fruit when ethylene is auto-stimulatory. In this study we examine the regulation of ACO gene family of banana. In banana, two transcripts corresponding to Mh-ACO1 and Mh-ACO2 have been identified. RNAi (RNA interference) strategy was employed to knock down either of the two ACO genes to elucidate their functions. Agrobacterium-mediated transformation was used to produce the Mh-ACO1 or Mh-ACO2 silenced transgenic plants. β-glucuronidase (GUS) activity and Southern blot analysis indicated that the T-DNA was inserted into genomic DNA. Most transgenic plants carried a multiple number of inserts. The growth rate and lower leaves wilting of ACO silenced transgenic plants were slower than Un transgenic plant.
The transcript levels of the two ACO genes of wounded leaves and fruits in the transgenic and Un transgenic plant, were examined using Northern blot analysis. The data indicated that Mh-ACO1 expresses constitutively whereas Mh-ACO2 expresses only in wounded leaves and fruits from Un transgenic plant. No matter Mh-ACO1 or Mh-ACO2 silencing, it affects the performance of the other ACC oxidase gene. We can detect the range of 21-25 nt siRNA in Mh-ACO1 silenced wounded leaves and Mh-ACO2 silenced ripening banana fruits. The results showed that silenced construct pBI121-1AnS and pBI121-2AnS can inhibit Mh-ACO1 and Mh-ACO2 in banana plant specifically by RNAi mechanism.
The integration of T-DNA border regions were determined by IPCR (Inverse Polymerase Chain Reaction) and APCR (Anchored Polymerase Chain Reaction). We got five left border region fragments from transgenic plants 1AS-1, 1AS-25, 2AS-1 and 2AS-79. Transgenic plant 2AS-1, 1AS-25 and 2AS-79 contained part of left border consensus sequence and transgenic plant 1AS-1 had no consensus sequence. The integration of T-DNA border regions of 2AS-79 and 2AS-1 had Un-T-DNA vector sequences. The results showed that T-DNA complex integrated into plant genome will carry some Un-T-DNA vector sequences. Furthermore, recombination and deletion of T-DNA may also occur during this integration process.

目錄
中文摘要 I
Abstract II
壹、 前言 1
貳、 前人研究
一、乙烯於植物生理反應與生合成途徑
(一)乙烯於植物生理影響 2
(二)植物更年性生理表現 2
(三)香蕉果實後熟相關基因與乙烯生理研究 3
二、ACC oxidase特性與基因研究 4
三、RNAi (RNA interference) 原理與應用 6
四、農桿菌 T-DNA 嵌入植物基因組之邊緣序列分析
(一)農桿菌 T-DNA 嵌入植物基因組之原理及其影響 7
(二) T-DNA 嵌入植物染色體組位置之分析策略 8
參、 材料與方法
一、植物材料 10
二、試驗方法
(一)GUS活性組織化學染色分析 10
(二)石蠟切片 10
(三)植物基因組 DNA 之抽取 10
(四)植物基因組 RNA 之抽取 10
(五)南方氏雜交分析 11
(六)北方雜交分析 13
(七)小片段 RNA 北方雜交分析 14
(八) IPCR (Inverse Polymerase Chain Reaction) 14
(九) APCR (Anchored Polymerase Chain Reaction) 15
(十)香蕉葉片創傷試驗 16
肆、 結果
一、轉殖株分子驗證 17
二、石蠟切片組織學觀察 18
三、默化 Mh-ACO1 與默化 Mh-ACO2 轉殖北蕉生長情形 18
四、Mh-ACO1 與 Mh-ACO2 於不同北蕉轉殖株之差異性基因表現
18
五、香蕉 ACC 氧化酶基因於創傷逆境表現 18
六、默化 ACC 氧化酶基因於創傷逆境之 siRNA 表現 19
七、ACC氧化酶基因於默化 Mh-ACO2轉殖株之果實不同後熟時期
表現 19
八、ACC氧化酶基因於默化 Mh-ACO2轉殖株後熟果實之siRNA表現
20
九、ACC 氧化酶默化轉殖株 T-DNA 邊界序列分析 20
伍、 討論
一、默化 Mh-ACO1 與默化 Mh-ACO2 轉殖株外表型差異 44
二、ACC氧化酶基因於不同默化 Mh-ACO1 與默化 Mh-ACO2轉殖株葉
片表現 45
三、ACC氧化酶基因於默化 Mh-ACO1 與默化 Mh-ACO2轉殖株葉片創傷逆
境表現 45
四、ACC氧化酶基因於默化 Mh-ACO2轉殖株果實不同後熟時期表現46
五、siRNA 於乙烯生理表現情形47
六、ACC 氧化酶默化轉殖株T-DNA邊界序列分析 48
陸、 結語 50
柒、 參考文獻 51


圖表目錄
圖一、香蕉轉殖默化 Mh-ACO1 表現質體 pBI121-1AnS 之香蕉(Musa ‘Pei Chiao’,
AAA group) 擬轉殖株葉片 GUS 活性組織化學染色分析。 22
圖二、北蕉默化 Mh-ACO1 轉殖株以 GUS 為探針之南方氏雜交分析。 23
圖三、北蕉默化 Mh-ACO1 轉殖株以 Mh-ACO1 為探針之南方氏雜交分析。 24
圖四、北蕉默化 Mh-ACO2 轉殖株以 GUS 為探針之南方氏雜交分析。 25圖五、北蕉默化 Mh-ACO2 轉殖株以 NPT ΙΙ 為探針之南方氏雜交分析。 26
圖六、以石蠟切片分析非轉殖株北蕉、Mh-ACO1 RNAi 2 與 Mh-ACO2 RNAi
79 葉片構造。 27
圖七、香蕉轉殖默化 Mh-ACO1 表現載體 pBI121-1AnS 擬轉殖癒傷組織再生
情形 28
圖八、香蕉 (Musa ‘Pei Chiao’, AAA group) 轉殖默化 Mh-ACO1 表現質體
pBI121-1AnS 的植株葉片形態觀察。 29
圖九、未轉殖株與默化 ACC 氧化酶基因之轉殖香蕉 (Musa ‘Pei Chiao’, AAA
group) 於精密溫室生長情形。 30
圖十、Mh-ACO1 RNAi 轉殖株葉片之 Mh-ACO1 與 Mh-ACO2 基因表現分析。 31
圖十一、Mh-ACO2 RNAi 轉殖株葉片之 Mh-ACO1 與 Mh-ACO2 基因表現分析 32
圖十二、創傷 Mh-ACO1 RNAi 2、 Mh-ACO2 RNAi 79 轉殖株與未轉殖株葉片之
Mh-ACO1 與 Mh-ACO2 基因表現分析。 33
圖十三、Mh-ACO1 RNAi 2 與 Mh-ACO2 RNAi 79 轉殖株創傷處理後 Mh-ACO1 基因之 siRNA 表現情形。 34
圖十四、Mh-ACO1 RNAi 2 與 Mh-ACO2 RNAi 79 轉殖株創傷處理後 Mh-ACO2 基因之 siRNA 表現情形。 35
圖十五、Mh-ACO2 RNAi 79 轉殖果實於不同後熟程度之 Mh-ACO1 與 Mh-ACO2 基因表現。 36
圖十六、Mh-ACO2 RNAi 79 轉殖株果實於不同後熟程度 Mh-ACO1 與 Mh-ACO2 基因表現之定量。 37
圖十七、Mh-ACO2 RNAi 79 (Musa ‘Pei Chiao’, AAA group) 轉殖株後熟香果實之 Mh-ACO1 siRNA 表現情形。 38
圖十八、Mh-ACO2 RNAi 79 (Musa ‘Pei Chiao’, AAA group) 轉殖株後熟果實之Mh-ACO2 siRNA 表現情形。 39
圖十九、默化 Mh-ACO1 與 Mh-ACO2 轉殖香蕉 (Musa ‘Pei Chiao’, AAA
group) 之T-DNA 嵌入染色體邊界序列分析 40
表一、香蕉組織培養之培養基 41
表二、分析 T-DNA 邊界所用引子與連結子序列。 42
表三、默化 Mh-ACO1、默化 Mh-ACO2 與香蕉未轉殖株之外表形態差異。 43

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