香蕉ACC氧化酶基因默化轉殖株中果實後熟相關基因表現之分析

1-氨基環丙烷-1-羧酸 (1-aminocyclopropane-1-carboxylate acid, ACC) 氧化酶為參與植物荷爾蒙乙烯 (ethylene) 生合成的重要酵素。香蕉具有兩種ACC氧化酶，分別為 Mh-ACO1與Mh-ACO2，其酵素活性不同，為了瞭解此二種ACC氧化酶與香蕉果實後熟之相關性，本研究運用即時定量反轉錄聚合酶連鎖反應 (real-time reverse transcription polymerase chain reaction, real-time RT-PCR)，分別以北蕉Mh-ACO1默化轉殖株與Mh-ACO2默化轉殖株之果實為材料，進行乙烯生合成、乙烯訊息傳導、葉綠體代謝等相關基因之表現分析，結果顯示Mh-ACO1默化轉殖株中，Mh-ACO1基因之默化效果於果皮第五期尤為顯著，Mh-ACO2之基因表現也受到抑制，表明了Mh-ACO1與Mh-ACO2間存在互相調控的關係；於Mh-ACO2默化轉殖株中，Mh-ACO2最佳默化效果則出現在果皮第五至第六期。Mh-ACO1默化轉殖株果肉內，Mh-ACO1表現趨勢與未轉殖株完全相異，Mh-ACO1基因僅於第一期表現高於未轉殖株，之後呈現穩定表現，且未出現如未轉殖株之第三與第七期的表現高峰。Mh-ACO2基因於Mh-ACO2默化轉殖株果肉內第五期，具有良好之默化效果，但在第七期表現高於未轉殖株。於Mh-ACO1默化轉殖株果皮中，乙烯訊息傳導相關基因Mh-CTR1 (constitutive triple response 1) 具有不同於未轉殖株的表現趨勢，轉殖株於第五期具有顯著之基因表現高峰，而未轉殖株於第一至五期，維持低量表現。乙烯受體基因Mh-ERS1(ethylene response 1 )於Mh-ACO2默化轉殖株果肉內第四期，具有表現高峰，而未轉殖株之高峰於第五期才出現。葉綠素代謝基因CS2 (chlase 2) 於Mh-ACO2默化轉殖株與未轉殖株果皮七個時期，趨勢完全一致，但轉殖株之表現顯著低於未轉殖株。花青素生合成基因CHI (chalcone isomerase)，於Mh-ACO2默化轉殖株內之表現高峰，由未轉殖株的第三期延後至第六期。類胡蘿蔔素生合成基因ZDS1 (zeta-carotene 1) 於Mh-ACO1、Mh-ACO2默化轉殖株及未轉殖株表現相異，Mh-ACO1默化轉殖株內，ZDS1基因於第五期表現顯著高於未轉殖株，惟至第七期降為未轉殖株之一半表現量；於Mh-ACO2默化轉殖株內，ZDS1基因僅在果皮第三期出現略高於未轉殖株之表現高峰。

關鍵字

ACC氧化酶； RNA干擾；果實後熟

並列摘要

ACC oxidase is a crucial enzyme which participates in the ethylene biosynthesis of plants. Mh-ACO1 and Mh-ACO2 are two distinct ACC oxidase genes in bananas. To better understand the physiological functions of these two genes and the gene expression patterns that relate to ethylene biosynthesis and signal transduction, and chlorophyll metabolisms in ACC oxidase silenced transgenic banana fruits, the real-time RT-PCR was employed for gene expression analysis. The results showed that Mh-ACO1 gene had a significant silenced effect in the stage 5 of peels from Mh-ACO1 silenced transgenic banana, and the Mh-ACO2 gene was also repressed, indicating there existed an interaction between these two ACC oxidase genes. For the Mh-ACO2 silenced transgenic banana, the best silencing effect was shown in the stages 5 and 6 of peels. In addition, the gene expression pattern in pulps was quite different from untransformed wild type for the Mh-ACO1 gene in Mh-ACO1 silenced transgenic banana, and there was no gene expression peaks in stages 3 and 7 such as those in untransformed wild type, the Mh-ACO1 gene expressions in Mh-ACO1 silenced banana were than wild type in stages 3 and 7. The Mh-ACO2 gene expression level in pulps from Mh-ACO2 silenced transgenic banana showed the stage 5 had the most obvious silenced effect but the expression was higher than wild type in stage 7. Mh-CTR1, which is involved in ethylene signal transduction, showed a different gene expression tendency when in both Mh-ACO1 and Mh-ACO2 silenced transgenic bananas compared with wild type. The Mh-ACO1 silenced transgenic banana, expressed strongly Mh-CTR1 gene in stage 5 while the wild type retained a constant expression level. In the pulps of banana fruits, the expression of ethylene receptor gene Mh-ERS1 showed highest level in stage 4 in Mh-ACO2 silenced transgenic banana while highest level was found in stage 5 from untransformed wild type. The expression tendency of chlorophyll metabolism related gene CS2 was almost the same between Mh-ACO2 silenced transgenic banana and wild type, but the expression level for Mh-ACO2 silenced transgenic banana was quite low. The expression of CHI gene, involved in the anthocyanin biosynthesis pathway, was delayed from stages 3 to 6 in Mh-ACO2 silenced transgenic banana. As to the Carotenoid biosynthesis involved gene ZDS1, the expression patterns were totally different in Mh-ACO1, Mh-ACO2 silenced transgenic bananas, and the wild type. In the peels of Mh-ACO1 silenced transgenic banana, the ZDS1 expressed relatively high, but decreased in stage 7 where the expression level was half of the wild type. However, for ZDS1 gene in Mh-ACO2 silenced transgenic banana, the expression level was higher in stage 3.