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研究生:吳靜芬
研究生(外文):Ching-fen Wu
論文名稱:蝴蝶蘭色素合成轉醣酶之鑑定及功能分析
論文名稱(外文):Identification and functional analysis of UDP-glucosyltransferase for pigments biosynthesis in Phalaenopsis orchids
指導教授:陳虹樺陳虹樺引用關係
指導教授(外文):Hong-Hwa Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:54
中文關鍵詞:轉醣酶色素花色蝴蝶蘭
外文關鍵詞:pigmentPhalaenopsisglucosyltransferaseflower color
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蝴蝶蘭 (Phalaenopsis) 是高經濟價值的花卉,而多樣化的花色是其具高價值的主因之一。屬於黃酮類化合物 (flavonoid) 之一的花青素 (anthocyanin) 是形成紅色、紫色、藍色等花色的主要色素,而花青素的呈色會受到黃酮 (flavone) 等共色素的影響,形成肉眼看到的花色。在蝴蝶蘭屬,主要的色素為apigenin 6-C-glucosido-7-O-glucoside (saponarin) 和cyanidin 3-O-glucoside-7,3’-di-O-[6-O- (sinapyl)-glucopyranoside],他們分別為黃酮和花青素的衍生物。另外,從姬蝴蝶蘭 (Phalaenopsis equestris) EST (expressed sequence tag) 資料庫中,得到參與花青素生合成路徑的酵素基因序列,分別為CHS (chalcone synthase), CHI (chalcone isomerase), ANS (anthocyanidin synthase),以及三個 UFGT (UDP-glucose:flavonoid glucosyltransferase) 酵素。比較這些酵素在紅花和白花間的表現差異,發現除了 PeUFGT3 在紅花有較高的基因表現,其他酵素則無顯著差異。 而 UFGT 是一種轉醣酶,將來自 UDP-glucose的醣基加到黃酮類化合物。
本論文研究三個 PeUFGT 轉醣酶在蝴蝶蘭兩個主要色素的生合成所扮演的角色。依據上述的色素分析及PeUFGT和其他轉醣酶演化樹分析結果,針對兩個色素的醣化推論出一個合成路徑假說,並依此假說證明三個PeUFGT 轉醣酶的弁遄C首先,以大腸桿菌(E. coli)分別表現三個轉醣酶的重組蛋白。經過各種誘導表現系統的嘗試,最佳的系統被用以表現轉醣酶蛋白。三個 PeUFGT 轉醣酶的弁鄔吨尷R結果顯示,apigenin 首先被PeUFGT1在7-OH位置上醣化,接著被PeUFGT2在6-C位置上被醣化,形成apigenin 6-C-glucosido-7-O-glucoside。PeUFGT3則參與花青素合成路徑最後,初花青素 (anthocyanidin) 醣化成花青素的催化。此外,三個PeUFGT轉醣酶蛋白分子結構顯示,三個轉醣酶有類似的蛋白結構,可分成N端及C端二區塊( N- and C-terminal domain)。而測量受質結合位寬度後,發現PUFGT2相較於其他二酵素有較寬的受質結合位,此結果和PUFGT2有結構較長的受質相呼應。總結以上結果,PeUFGT1及PeUFGT2酵素參與在蝴蝶蘭主要黃酮衍生物的醣化,PeUFGT3參與花青素的生合成,而蛋白質的受質接受位寬度可能是這些酵素接受不同受質的原因。
Phalaenopsis is a high-value cash crop, and their variant flower colors are an important factor for commercial orchid production. Anthocyanin belongs to a class of flavonoids that is the main pigment contributing to the red, violet, and blue color of flowers, and the visible color is modified by copigment such as flavonols and flavones. In Phalaenopsis, one flavone derivative, apigenin 6-C-glucosido-7-O-glucoside (saponarin), and an anthocyanin, cyanidin 3-O-glucoside-7,3’-di-O-[6-O-(sinapyl) -glucopyranoside], have been identified in flowers of Phalaenopsis hybrids with a substantial amount. Previously, full length cDNAs encoding putative major enzymes in the anthocyanin biosynthetic pathway have been identified from floral bud EST (expressed sequence tag) database of Phalaenopsis equestris. These include chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase (ANS), and UDP glucose:flavonoid glucosyltransferase (UFGT). Most enzymes involved in the anthocyanin biosynthesis showed similar transcriptional activity in both red and white flower of Phalaenopsis cultivars, such as CHS, CHI, ANS, PeUFGT1 and PeUFGT2. In contrast, the PeUFGT3 was highly expressed only in the red flower but not in the white flower. UFGT catalyzes the transferring of glucosyl moiety from UDP-glucose to flavonoid, which increase the solubility of flavonoid.
To investigate the function of the three PeUFGT proteins involved in the formation of pigments in Phalaenopsis, a hypothesis of the major flavonoid derivatives synthesis pathway was proposed according to the study of pigment in Phalaenopsis and phylogenetic analysis. The glucosyltransferase activity of the three PeUFGT enzymes were analyzed. First, recombinant PeUFGT proteins were ectopically expressed in E. coli respectively. The proper expression systems for the optimal induction of recombinant proteins have been monitored. Then functional analysis of three PeUFGT enzymes were carried out, and modified pathway for pigment formation was postulated. Apigenin was added first glucose to the 7-OH position by PeUFGT1 to form apigenin-7-O-glucoside, which is converted to apigenin 6-C-glucosido-7-O-glucoside by PeUFGT2 through the addition of glucose to the 6-C position. In the anthocyanin synthesis pathway, PeUFGT3 might add glucose to the 3-OH position of anthocyanidin to form anthocyanin. Besides, molecular modeling of these three enzymes was performed. The structure of PeUFGTs molecular model were similar with N- and C-terminal domain. The estimation of the width of sugar acceptor binding site showed that PeUFGT2 have a larger pocket than PeUFGT1 and PeUFGT3, which was coincident with the larger dimension of the substrate of PeUFGT2 than that of PeUFGT1 and PeUFGT3. In conclusion, PeUFGT1 and PeUFGT2 were involved in the glucosylation of the major flavone derivative, and PeUFGT3 were involved in the formation of anthocyanin in Phalaenopsis. The width of the sugar acceptor binding site was deduced to be responsible for the substrate specificity.
中文摘要 i
Abstract iii
誌謝 v
Table of contents vi
List of tables viii
List of figures ix
List of appendix figures x
1. Introduction
1.1 Phalaenopsis species 1
1.2 Pigments of flower color 1
1.3 Biosynthesis of pigments 3
1.4 Gene regulation of anthocyanin biosynthetic pathway 5
1.5 UFGT genes involved in color formation in plant 6
1.6 Specific aims 7
2. Materials and methods
2.1 Plant materials 9
2.2 RNA extraction and cDNA synthesis 9
2.3 Cloning of PeUFGT1, PeUFGT2, and PeUFGT3 gene 9
2.4 Heterologous expression of UDP-glucoside:flavonoid glucosyltransferase 10
2.5 Purification of GST-fused UFGT protein 10
2.6 Purification of his-tagged protein 10
2.7 Enzyme activity assay 11
2.8 Thin layer chromatography (TLC) analysis of enzyme assay products 11
2.9 High-performance liquid chromatography (HPLC) analysis of enzyme assay products 11
2.10 Determination of kinetic constants 12
2.11 Phylogenetic analysis 12
2.12 Protein secondary structure alignment 12
2.13 Analysis of the enzyme molecular model 12
3. Results
3.1 Phylogenetic analysis of GT proteins 14
3.2 Heterologous expression of PeUFGT proteins 14
3.3 Enzymatic activity assays of the heterologously expressed PeUFGT proteins 16
3.4 Postulated modified pathway for anthocyanin and flavonoid derivatives in Phalaenopsis 17
3.5 Molecular modeling of three PeUFGTs 17

4. Discussion
4.1 Role of PeUFGTs catalytic products in Phalaenopsis 19
4.2 Protein structures of PeUFGTs and other plant GTs 19
4.3 Loss-of-function assay of PeUFGTs 21
4.4 Heterologous expression of proteins 21
5. Conclusion and prospect 23
References 24
Tables 28
Figures 34
Appendix figures 50
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