Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22815
標題: 利用基因槍轉殖法表現ACC去胺酶 (ACCD)─ 延長文心蘭切花瓶插壽命系統之建立
Transgenic expression of ACC deaminase gene in Oncidium- A first step toward enhancing vase-life of cut flowers
作者: 孫慶美
Sun, Ching-Mei
關鍵字: ACC deaminase;ACC去胺酶切花;cut flower;ethylene;Oncidium;protocorm-like bodies (PLBs);senescence;vase-life;乙烯;文心蘭;擬胚原球體;老化;瓶插壽命
出版社: 生命科學系所
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摘要: 
文心蘭 (Oncidium Gower Ramsey) 為台灣重要之外銷切花作物,但對乙烯敏感性高,導致採收後花朵易提早老化凋萎,減短切花瓶插壽命。為改善乙烯造成文心蘭切花瓶插壽命之短縮,本研究利用基因槍轉殖法,將ACC去胺酶基因 (ACC deaminase, NCBI登錄為#DQ830987; Maruthasalam et al., 2006) 轉入文心蘭之擬胚原球體 (PLBs),期藉由降低乙烯生成,達到延長其切花瓶插壽命之目的。ACC去胺酶為一細菌酵素,可將乙烯生合成前驅物ACC分解,產生α-丁酮酸及氨,達到降低乙烯生成之效。ACC去胺酶基因構築於兩個轉殖載體:pCAMBIA1301/ AtAP1-ACCD (由Arabidopsis thaliana 花器表現啟動子AP1調控) 及pCAMBIA1301/ CaMV 35S-ACCD (由持續表現啟動子35S調控),轉殖至文心蘭PLBs後,其PLBs以含潮黴素 (5.0 mg/l) 之G10培養基培養,將成功存活之植株,進行GUS組織染色、PCR與PCR-Southern分析。結果共得AtAP1-ACCD-1、AtAP1-ACCD-2和CaMV-ACCD-1三棵轉殖株系。再以RT-PCR和western分析轉殖株AtAP1-ACCD-1與AtAP1-ACCD-2得知ACCD基因在AtAP1-ACCD-2中可轉錄出mRNA,並成功轉譯成ACCD蛋白。轉殖株系CaMV-ACCD-1分化之葉片目前僅約0.3公分,尚未有足夠組織可供進行分析。目前已將確定穩定之轉殖株系培養於不含潮黴素之G10培養基以加速生長及發根。

Oncidium Gower Ramsey, an important orchid grown for cut flower production in Taiwan, is highly sensitive to ethylene leading to early senescence of flower petals and consequently reduction in the vase-life of cut flowers. To prolong the vase-life of Oncidium cut flowers by lowering the ethylene production through genetic engineering, ACC deaminase gene (NCBI database accession # DQ830987; Maruthasalam et al., 2006) was used to make two transformation vectors viz., pCAMBIA1301/ AtAP1-ACCD (controlled by a flower-specific promoter AP1 of Arabidopsis thaliana) and pCAMBIA1301/ CaMV 35S-ACCD (controlled by a constitutive promoter 35S). ACC deaminase is a bacterial enzyme which degrades ACC, an immediate precursor of ethylene biosynthesis, into equimolar amounts of α-ketobutyric acid and ammonia. The gene constructs were used for the transformation of protocorm-like bodies (PLBs) of Oncidium cultivar Gower Ramsey through particle bombardment. After bombardment, PLBs were cultured on G10 medium supplemented with hygromycin (5.0 mg/l) for obtaining transgenic plants. Through histochemical GUS, PCR and PCR-Southern analyses, two transgenic lines AtAP1-ACCD-1 and AtAP1-ACCD-2 transformed with pCAMBIA1301+ AtAP1-ACCD and one line CaMV-ACCD-1 transformed with pCAMBIA1301+ CaMV-ACCD were obtained. AtAP1-ACCD-1 and AtAP1-ACCD-2 were analyzed by RT-PCR and western blotting. The result showed a basal level accumulation of ACC deaminase transcript and protein in AtAP1-ACCD-2. The line CaMV-ACCD-1 is in the early stage of shoot elongation. After the confirmation of transgene integration, transgenic shoots were subcultured onto G10 medium without hygromycin for elongation and rooting.
URI: http://hdl.handle.net/11455/22815
其他識別: U0005-0302201015305600
Appears in Collections:生命科學系所

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