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標題: | 香蕉 ACC 氧化酶基因默化轉殖株分析 Silencing Analysis of 1- Aminocyclopropane-1-Carboxylate Oxidase Genes in Transgenic Banana |
作者: | Mei-Lun Yeh 葉美倫 |
指導教授: | 黃鵬林(Pung-Ling Huang) |
關鍵字: | ACC氧化酶,香蕉,RNA 干擾,T-DNA,siRNA, ACC oxidase,Banana,RNA inference,T-DNA,siRNA, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22402 |
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顯示於系所單位: | 園藝暨景觀學系 |
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