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Pigments and Molecular Analysis of Transgenic Chrysanthemum Carrying Pigment Genes and Genetic Transformation of Pigment Genes to Poinsettia
|摘要:||本論文利用陳彥銘（2006）以菊花花蕾藉由農桿菌轉殖大豆種皮花青素合成基因（ANS）之菊花 ‘Linker’ 和‘Margenta Linker’ 的疑似轉殖株為材料。以PCR方法檢定菊花 ‘Linker’ 或 ‘Margenta Linker’ 二品種之疑似轉殖株，各獲得16株與9株含有大豆ANS基因之轉殖株。將轉殖株種植至隔離溫室，作為基因表現和色素分析之樣品。經南方雜交分析結果，‘Linker’ 轉殖株含有1~2個或至少5個大豆ANS基因的嵌入；而 ‘Margenta Linker’ 轉殖株亦含有至少5個ANS基因的嵌入，證實黑色種皮大豆ANS基因已嵌入菊花基因組中。從RT-PCR分析結果，白花的‘Linker’ 轉殖株花瓣因有大豆ANS基因表現，而呈現粉色；‘Linker’ 和粉色花的‘Margenta Linker’ 轉殖株花瓣有大豆ANS基因過量表現，則呈現黃色。HPLC分析結果顯示，‘Linker’轉殖株的花瓣含有cyanidin則呈現淡粉色，而含有新產物β-carotene者則呈現黃色；而‘Margenta Linker’ 轉殖株的花瓣含有新產物β-carotene亦呈現黃色。此結果證明，大豆ANS基因的嵌入和表現，使白色‘Linker’和粉色 ‘Margenta Linker’ 的花色改變成淡粉或黃色的花朵。
另以聖誕紅 ‘Nobel Star’ 和 ‘Peter Star-White’ 為材料，以農桿菌EHA105進行大豆種皮基因F3H、F3’,5’H和ANS的基因轉殖。以5 mg/l hygromycin作為篩選培養基之抗生素，以timentin抑制農桿菌對莖段培殖體的毒害最低，癒傷組織形成的比率較高並可繼續增殖，芽體篩選再生率最高為20.7%，再生篩選時間約24週。而利用胚性懸浮細胞為材料進行農桿菌基因轉殖，芽體篩選再生率平均為35.3%，再生過程大約16週，顯著較莖段培養之轉殖系統之效率高出甚多。‘Nobel Star’ 轉殖株以PCR檢測後，轉殖F3H和ANS基因各獲得5和7株轉殖株，其轉殖效率分別為1.3%和2.3%。‘Peter Star-White’，轉殖F3H 和F3’,5’H基因各獲得19和23株轉殖株，轉殖效率分別為6.3%和7.7%。以HPLC分析苞片之色素含量，‘Nobel Star’ 轉殖株F3H-4之苞片顏色與對照株無異，但花青素含量比對照株提高約2.5倍，另轉殖株ANS-7苞片顏色變為粉紅色，其花青素含量僅為對照株之35%。‘Peter Star-White’ 轉殖株F3H-16苞片顏色呈現乳黃色，轉殖株F35H-30苞片顏色呈現黃色，其β-carotene含量分別提高約3.8倍和11倍。本論文之研究，已成功建立聖誕紅基因轉殖系統並獲得花色改變之轉殖株。|
In this study putative transgenic chrysanthemum plants of ‘Linker' and ‘Margenta Linker' transformed with soybean anthocyanidin synthase (ANS) gene by Y. M. Chen (2006) using flower buds and Agrobacterium tumefaciens mediated transformation were applied as materials. According to genomic-PCR screening 16 and 9 transgenic plants of ‘Linker' and ‘Margenta Linker' were confirmed to harbor with soybean ANS gene, respectively. Plants with floral color changed were vegetatively propagated and cultivated in GM greenhouse to provide the samples for gene expression and pigment analysis. Southern analysis suggested that the transgenic plants of ‘Linker' showed 1~2 copies or more than 5 copies and of the ‘Margenta Linker' also contained at least 5 copies of soybean ANS gene. The petals of transgenic ‘Linker' plants showed light pink color however, the transgenic ‘Linker' and ‘Margenta Linker' plants with over-expressed soybean ANS gene showed yellow petals. HPLC analysis suggested that the light pink petals of ‘Linker' contained cyanidin but the yellow petals of transgenic ‘Linker' and ‘Margenta Linker' were β-carotene, a new pigment to chrysanthemum. The results demonstrated that soybean ANS gene was successfully transferred, expressed and changed the flower color in chrysanthemum. To establish a system for flower color engineering in poinsettia, explants of two varieties, ‘Nobel Star' and ‘Peter Star-White', were co-cultivated with Agrobacterium carrying soybean pigment biosynthetic genes, flavonoid 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3',5'H), and anthocyanidin synthase (ANS). Screening of transgenic plant was conducted by using 5 mg/l hygromycin containing medium. Timentin was applied to inhibit Agrobacterium tumefaciens because of its lowest toxicity to callus formation and proliferation. In the stem-explant system, about 20.7% shoots were regenerated on selection medium within 24 weeks. Using embryonic cells from suspensions for transformation 35.3% shoots were regenerated within 16 weeks and showed high transformation efficiency. Putative transgenic poinsettia of ‘Nobel Star' were verified by PCR, 5 and 7 plants transformed with soybean F3H and ANS gene and accompanied with 1.3% and 2.3% transformation efficiency, respectively. In ‘Peter Star-White' variety 5 and 7 transgenic plants with soybean F3H and F3',5'H gene, and 6.3% and 7.7% transformation efficiency were also observed, respectively. The HPLC analysis of bracts pigments showed that in ‘Nobel Star', the transgenic line F3H-4 looked similar with wild-type but accumulated 2.5 folds anthocyanidins; line ANS-7 contained only 35% anthocyanidins and turned to pink color. In ‘Peter Star-White' the bracts of transgenic line F3H-16 and F35H-30 changed to cream yellow and yellow with 3.8 and 11 folds of β-carotene increased, respectively. In this study an efficient transformation system for flower color engineering has been established and transgenic plants with altered colors were obtained in poinsettia.
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