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Biochemical and Physiological Characterization of Transgenic Oncidium Cut Flowers Engineered to down Regulate Ethylene Biosynthesis
|作者:||林金和||關鍵字:||基礎研究;ACC deaminase;生物技術, 園藝;ACC 去胺?;切花;乙烯;文心蘭;擬胚原球體;老化;瓶插壽命;Cut flower;Ethylene;Oncidium;Protocorm-like bodies;Senescence;Vase-life||摘要:||
利用基因工程降低文心蘭乙烯生合成之生理特性中文摘要本研究自第一年開始 (2008-2009) 至接續一年 (2009-2010) 之努力，分別成功獲得1以開花專一表現起動子AtAP1與2持續表現啟動子CaMV，表現ACC去胺酶 (ACCD) 之文心蘭｀Gower Ramsey＇轉殖株 (Fig. 9-12)。期望可藉由表現ACCD 以降低文心蘭小花之乙烯生合成量，以達到延長切花瓶插壽命之效果。延續此實驗擬於新計畫年度 (2010-2011) 進一步對文心蘭轉殖株分析其生化與生理特性。包括(1) 植株ACC含量與乙烯生合成量 (2) ACC 合成酶與ACC 氧化酶活性 (3) ACCD基因持續表現之轉殖株於不同營養發育期外部型態特徵之定性。本研究並將對ACCD轉殖株於開花後，切花之瓶插壽命與植株對外加乙烯之反應進行分析。本研究目前已完成之成果摘要如下：文心蘭為一台灣重要蘭科切花作物，但其對乙烯敏感性高，導致採收後小花易提早老化凋萎，縮短切花瓶插之壽命。故此計劃擬經由基因工程操作來延長切花瓶插壽命。ACCD之表現可分解ACC以降低植物體內乙烯之生合成量。為達此目的，本研究室自促進植物生長根圈菌Pseudomonas sp.分離得ACCD基因並登錄於NCBI GenBank資料庫；代號為 #DQ830987。再構築於兩轉殖載體：pCAMBIA1301+CaMV35S-ACCD 及pCAMBIA1301+ AtAP1-ACCD，利用基因槍或農桿菌轉殖法，分別將兩載體轉殖至文心蘭｀Gower Ramsey＇擬胚原球體 (PLBs)，並以hygromycin 進行篩選，得成功存活之擬胚原球體。取其分化之葉片藉由GUS染色、PCR 及 Southern 分析，證實成功獲得開花專一表現起動子pCAMBIA1301+ AtAP1-ACCD轉殖株與持續表現啟動子pCAMBIA1301+ CaMV35S-ACCD轉殖株各兩株。目前將持續培養文心蘭轉殖株待其發育出穩定根系出瓶後，將進一步進行植株生化及生理之分析。
Biochemical and physiological characterization of transgenic Oncidium cut flowers engineered to down regulate ethylene biosynthesisSummaryOur research efforts during the first (2008-2009) and on-going second (2009-2010) year of the project have yielded a few transgenic Oncidium cv. ‘Gower Ramsey' lines containing ACC deaminase gene, under the control of either a constitutive CaMV 35S promoter or flower specific AtAP1 promoter (refer to Figures 9 to 12). We envisage that the expression of ACC deaminase in flowers would or could enhance the vase-life of Oncidium cut flowers through the down regulation of ethylene production. In continuation of our earlier work, in the next project year (2010-2011), we are intended to carry out the biochemical and physiological characterization of transgenic Oncidium plants for, (i) ACC content and ethylene production, (ii) ACC synthase and ACC oxidase enzyme activities, and (iii) morphological characteristics of Oncidium plants constitutively expressing ACC deaminase gene during different phases of vegetative developments. The vase-life of cut flowers expressing ACC deaminase and their response to external ethylene application will be studied when the transgenic plants attain the reproductive stage. The research works completed thus far are summarized below.Oncidium is an important orchid grown for cut flower production in Taiwan. Its high sensitivity to ethylene leads to early senescence of flower petals and shortens the vase-life of cut flowers. The objective of this project is to improve the vase-life of Oncidium cut flowers through genetic engineering. Ethylene synthesis can be retarded by expressing the ACC (1-aminocyclopropane-1-carboxylic acid) degrading enzyme ACC deaminase in plants. To achieve the goal, the gene encoding ACC deaminase was cloned from a plant growth-promoting rhizobacterial strain of Pseudomonas sp. The sequence data was published in the NCBI GenBank database under the accession number DQ830987. Two transformation vectors, (i) pCAMBIA1301 + CaMV 35S-ACCD, controlled by a constitutive promoter and, (ii) pCAMBIA1301 + AtAP1-ACCD, controlled by a flower-specific promoter of Arabidopsis thaliana, were constructed. The gene constructs were used to transform Protocorm-like bodies of Oncidium cultivar ‘Gower Ramsey' either through particle bombardment or Agrobacterium-mediation. The transformed PLBs were subsequently grown on the hygromycin antibiotic for the selection of transgenic plants. Histochemical GUS, PCR-Southern, RT-PCR and western analyses identified two transgenic shoots to be integrated with ACC deaminase under CaMV 35 constitutive promoter and two shoots with ACC deaminase under flower-specific AtAP1 promoter. The transformed shoots were cultured on the nutrient medium for elongation and rooting. After rooting, shoots will be transferred to greenhouse for physiological and biochemical studies.
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