香蕉ACC氧化酶基因啟動子活性分析與默化質體之轉殖

Abstract

本試驗運用RNA 干擾 (RNA interefence, RNAi)技術專一性默化香蕉乙烯生成相關酵素ACC氧化酶基因之表達，以探討該基因在果實後熟過程中所扮演的角色。同時分析Mh-ACO1及Mh-ACO2兩基因之啟動子活性，啟動子片段接於報導基因上游，構築成表達載體，利用農桿菌媒介法轉殖至阿拉伯芥 (Arabidopsis thaliana L. Columbia)，Mh-ACO1::GUS轉殖株之GUS活性，於簇生葉之葉緣根部有微量表現外，莖葉、花器中的苞片、花絲、花藥、柱頭、長角果與離層表現亦有表現， Mh-ACO1基因之表現隨著阿拉伯芥之生長發育階段不同而異；Mh-ACO2基因於阿拉伯芥任何生長發育階段並不表現。誘導試驗結果顯示，IAA、NAA、BA、GA3、ABA、JA、SA、NaCl、乙烯、光、淹水、低溫、機械傷害與乾旱可誘導Mh-ACO1基因之表現，但僅JA可誘導Mh-ACO2基因之表現。 此外，藉由農桿菌媒介法進行香蕉基因默化質體之轉殖，植株再生後，經GUS組織化學染色法與南方氏雜交分析結果顯示，已確定將基因默化質體穩定性轉殖至北蕉 (Musa spp. cv. Pei-Chiao, AAA group) ，此等植株可供日後小片段干擾RNA (siRNA) 之分析，以進一步了解對Mh-ACO基因之默化程度。

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way to shut down gene expression. In this study, transformation of silencing plasmids specific for banana ACC oxidase genes has been performed. We also analysed the promoter activity of banana ACC oxidase genes, Mh-ACO1 and Mh-ACO2. The GUS expression of Mh-ACO1::GUS transgenic Arabidopsis plants was found in rosette leaves, roots, cauline leaves, flower buds, stigma , style, anther, filament, sepals, stigmatic tissue, siliques and abscission zone. The expression of Mh-ACO1 gene differed in developmental stages, and was induced by JA, SA, IAA, GA3, ABA, NaCl, ethylene, wound, drought, flood, light and cold (4℃); On the other hand, the expression of Mh-ACO2 gene was only induced by JA and ethylene, and showed no difference at developmental stages. Besides, the gene silencing plasmids specific for Mh-ACO1 and Mh-ACO2 were stably transformed into banana (Musa spp. cv. Pei-Chiao, AAA group) by Agrobacteria-mediated transformation system.