香蕉ACC氧化酶基因默化轉殖之研究

Abstract

香蕉 (Musa spp.) 為更年性果實，乙烯在果實後熟中扮演重要之角色，而ACC氧化酶 (ACC oxidase, ACO) 為乙烯生合成最終步驟酵素。由香蕉中選殖出兩個ACC氧化酶基因為Mh-ACO1及Mh-ACO2。分別將兩個香蕉ACC氧化酶基因，利用農桿菌媒介法轉殖以及RNA干擾 (RNA interference, RNAi) 技術，默化其在香蕉中的表現，以探討兩基因的功能。建立北蕉懸浮培養細胞系統後，進行農桿菌媒介法轉殖，將默化Mh-ACO1基因之表現載體轉殖進入北蕉細胞之中，並利用25 mg/L的G418抗生素進行初期篩選，並在體胚形成後再提高篩選濃度，經過轉殖後的北蕉細胞，於轉殖後5個月長出胚根，待發芽後可進行GUS活性組織化學染色分析以確認轉殖株。將Mh-ACO2默化擬轉殖株利用南方氏雜交分析，結果可將擬轉殖系分成三群，並且得知為多拷貝的穩定轉殖。Mh-ACO2默化轉殖株的Mh-ACO2表現量在各轉殖株系間具有不同程度之默化效果。而在花序的不同器官上，Mh-ACO2及siRNA的表現量，也是不盡相同的。創傷逆境的處理下， Mh-ACO1會提高表現，而Mh-ACO2則否，並且Mh-ACO2默化的轉殖香蕉，Mh-ACO1表現不會受到影響。Mh-ACO2默化之轉殖香蕉果實，在後熟過程中，其果皮轉色情形、乙烯之生合成與呼吸速率之增加，均有延遲的情形，可同時達到控制果實後熟及延緩果實後熟的產業利用目的。並且推測，在香蕉中果皮及果肉表現上ACC氧化酶的表現差異，應主要為Mh-ACO2所造成，而非Mh-ACO1。Mh-ACO1表現在轉殖株與非轉殖株的果皮與果肉上，具有相同的表現趨勢，而Mh-ACO2則不同。

Banana (Musa spp.) is a climacteric fruits. Mh-ACO1 and Mh-ACO2 genes isolated from banana (Musa spp., AAA group) encode fruit ripening related ACC oxidase (ACO), the last enzyme in the ethylene biosynthetic pathway. Agrobacterium-mediated method and RNA interference (RNAi) were used as a strategy of Mh-ACO1 and Mh-ACO2 gene inhibition to clarify their function in banana. In the Agrobacterium-mediated method, banana (Musa ‘Pei Chiao’, AAA group) suspension cells were co-cultivated with Agrobacterium and selected with 25 mg/L G418. In 5 months, the embryos developed roots. Southern blot analysis indicated that the Mh-ACO2 silenced transgenic banana lines were divided into three types and the transformed fragments were high copy insertion. The Mh-ACO2 was decreased in different lines and different organs in transgenic bananas. The siRNAs were only expressed in petal. Mh-ACO1 was increased in wounded leaves but Mh-ACO2 was not. When compared with wild type banana, the change of peel color, ethylene production and respiration rate in MAO2 silenced transgenic banana fruits was delayed. The Mh-ACO1 and Mh-ACO2 gene expression pattern in peel and pulp were different.