本研究利用反轉錄酶-聚合酶鏈反應（RT-PCR）技術，將類胡蘿蔔素生物合成路徑的關鍵基因〔包括phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS) 及 lycopene cyclase (LCY)〕大量增殖，並將這些基因選殖至植物表現載體pH2GW7上。此載體內含抗生素hygromycin篩選基因，而目標基因則插至CaMV 35S啟動子及CaMV 35S終止子之間。從無菌培養之蕃茄（Lycopersicon esculentum cv. CL5915) 切取子葉，並以農桿菌(Agrobacterium tumefaciens strain LBA4404) 感染子葉培植體。經過抗生素hygromycin篩選並以PCR方法鑑定後，4種表現載體各獲得10至24株轉殖株，最後並轉移至溫室栽種。這些轉殖株之外型，包括花及果實之顏色，均與野生型蕃茄無異。唯一例外是轉殖株PSY26，它的花朵呈微黃至純白色，而野生型花朵則為鮮黃色。再則，PSY26之果實呈微紅至黃色。收集8株轉殖株（每種表現載體任意選取2株植株）及PSY26之蕃茄，經乾燥後蕃茄內lycopene及β-carotene之含量以HPLC測定之。與野生型蕃茄比較，我們發現這些轉殖株內lycopene之含量均增加1至3倍，其中以PDS4增幅3.84倍最多。另外，轉殖株內β-carotene之含量亦有增加。相反地，lycopene及β-carotene在PSY26（黃色蕃茄）與野生型（紅色蕃茄）並無顯著差異。

In this study, key genes in carotenoid biosynyhetic pathway encoding phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS) and lycopene cyclase (LCY) were amplified by RT-PCR (reverse transcription-polymerase chain reaction) and then cloned into plant expression vector pH2GW7. This vector contains a hygromycin-resistant marker, and the target gene was inserted between CaMV 35S promoter and CaMV 35S terminator. Cotyledons from in vitro grown tomato (Lycopersicon esculentum cv. CL5915) were excised, and then were infected by Agrobacterium tumefaciens strain LBA4404. After antibiotic (hygromycin) selection and then verified by genomic PCR analysis, 10 to 24 transgenic plants from each construct were finally transferred into green house until maturity. All transgenic plants looked similar with the wild-type plant including color pattern in flowers and fruits, except for PSY26. Flowers of wild-type plant were bright yellow, whereas flowers of the transgenic line PSY26 were slight yellow to complete white. Fruits of wild-type plant were red at the end of fruit development, whereas fruits of PSY26 were slight red to yellow. Fruits from 8 transgenic plants (2 plants were randomly selected from each construct) as well as PSY26 were harvested and dried, and HPLC analysis was conducted to examine the contents of lycopene and β-carotene. We found that all the transgenic lines accumulated more lycopene, ranging from 1 to 3 folds, as compared to the wild-type plant, especially 3.84 folds was detected in PDS-4. Also, β-carotene has increased in all transgenic lines we examined. By contrast, both contents of lycopene and β-carotene in PSY26 (yellow fruits) were found similar to wild-type plant (red fruits).

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