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研究生：	鄭佩玲 Cheng, Pei-Ling
論文名稱：	蝴蝶蘭花色相關UDP轉醣酶之生化特性研究 Biochemical characterization of flower color related UDP-glycosyltransferases from Phalaenopsis orchids
指導教授：	陳虹樺 Chen, Hong-Hwa
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 熱帶植物科學研究所 Institute of Tropical Plant Sciences
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	61
中文關鍵詞：	蝴蝶蘭、花色、轉醣酶、花青素
外文關鍵詞：	flower color, anthocyanin, UFGT, glucosyltransferase, Phalaenopsis
相關次數：	點閱：127 下載：8
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蝴蝶蘭(Phalaenopsis)在市場上是其中一種最受歡迎的蘭花之一，對商業生產而言，其多變的花色是重要的性狀。從姬蝴蝶蘭 (Phalaenopsis equestris) 花苞的EST (expressed sequence tag) 資料庫中，得到三個可能參與花青素生合成路徑的 UFGT (UDP-glucose:flavonoid glucosyltransferase) 基因，分別命名為PeUFGT1、 PeUFGT2 和PeUFGT3。其中，只有 PeUFGT3 在紅色品種中有差異性表現但是在白色品種中沒有。UFGT負責催化醣基從UDP-glucose到黃酮上的轉移，此種醣基化可以增加黃酮類在液泡中之可溶性及穩定性。親源演化分析顯示PeUFGT3與順式玉米素(cis-zeatin) O-轉醣酶相關。利用大腸桿菌外源表現重組PeUFGT3蛋白驗證PeUFGT3的功能，並以高效液相層析法(high-performance liquid chromatography)與液相層析質譜法(liquid chromatography–mass spectrometry )分析反應產物，顯示PeUFGT3可以接受cis-zeatin，但卻不能接受kaempferol當作受質。為了探討這三個PeUFGT蛋白在生物體中的功能，通過藉由CymMV進行病毒誘導的基因沉默(virus-induced gene silencing)比較PeUFGT靜默後植株的花色改變情形。在PeUFGT1-與PeUFGT2-靜默的植株中花朵顏色比控制組要深。反之，在PeUFGT3靜默植株中花朵顏色卻變淡。花青素含量在PeUFGT3靜默植株比控制組減少50%，而在PeUFGT1-與PeUFGT2-靜默的植株增加30%含量。PeUFGT3基因表現量在PeUFGT1-與PeUFGT2-靜默植株中mRNA的含量與控制組相比分別多了兩倍與一倍。這些結果顯示PeUFGT3在蘭花中的紅色形成中扮演著關鍵性的角色。
為了進一步尋找蝴蝶蘭花青素3-O轉醣酶，我們以目前已發表且經功能確認植物花青素3-O轉醣酶的序列搜尋姬蝴蝶蘭的部分基因體序列。PeUFGT4，是一個花青素3-O轉醣酶，並藉由3'端與5'端的cDNA末端選殖技術得到其全長序列。經多序列比對和親緣演化分析顯示PeUFGT4乃歸類在花青素3-O轉醣酶中，並且具有 plant secondary product glycosyltransferase 共同保留區域。然而，PeUFGT4在白色與紅色蝴蝶蘭品種中並無差異性表現，顯然PeUFGT4並非蘭花花色成色之關鍵酵素，雖然它真正的酵素功能仍有待進一步確認。

Phalaenopsis is one of the most popular orchids in markets, and the various flower colors are important traits for commercial production. Three UFGT (UDP-glucose:flavonoid glucosyltransferase) genes involved in anthocyanin biosynthesis pathway have been identified from floral bud EST database of Phalaenopsis equestris, and named as PeUFGT1, PeUFGT2 and PeUFGT3. Among them, only PeUFGT3 differentially expressed in red but not in white cultivars. UFGT was responsible for catalyzing the transfer of glucosyl moiety from UDP-glucose to flavonoid, which increases the stability and solubility of flavonoid. Phylogenetic analysis results reveal that PeUFGT3 is related to cis-zeatin O-glucosyltransferase. To identify the function of PeUFGT3, heterologous expression of recombinant PeUFGT3 in Eschericia coli was performed. High-performance liquid chromatography and liquid chromatography–mass spectrometry analyses of PeUFGT3-catalyzed products indicated that PeUFGT3 could use cis-zeatin rather than kaempferol as a substrate.
To investigate the function of these three PeUFGT proteins in vivo, CymMV-based virus-induced gene silencing was adopted for comparing the flower color change in the PeUFGTs-silenced plants. The flower color became darker than the mock in both PeUFGT1-silenced and PeUFGT2-silenced plants. In contrast, the red flower color became bleached in the plants knockdown expression of PeUFGT3. The anthocyanin content of PeUFGT3-silenced plants was 50% lower than that of mock, while that was 30% increase in both PeUFGT1-silenced and PeUFGT2-silenced plants. The mRNA abundance of PeUFGT3 in PeUFGT1-silenced and PeUFGT2-silenced plants was 2-fold and 1-fold higher than that in mock, respectively. Together, these results suggest that PeUFGT3 plays a crucial role in red color formation of orchid flowers.
We then searched against the P. equestris partial genomic sequence with the published and function characterized anthocyanin 3-O glucosyltransferase from various plants. PeUFGT4, an anthocyanin 3-O glucosyltransferase was identified, and its full length sequence was obtained by using 3’- and 5’- rapid amplification of cDNA ends. Multiple alignment and phylogenetic analysis indicated that the PeUFGT4 is clustered into anthocyanin 3-O-glucosyltransferase with the conserved plant secondary product glycosyltransferase motif. However, there was no differential expression of PeUFGT4 between white and red cultivars in Phalaenopsis, suggesting that it is not a crucial enzyme for red color formation in orchid flowers, although its enzymatic activity remained to be confirmed.

中文摘要 III
Abstract V
致謝 VII
List of Table X
List of Figures X
List of Appendix Figures XII

1. Introduction 1
1.1 Economic importance of Phalaenopsis 1
1.2 Significance of flower color in higher plants 1
1.2.1 Main pigment components of flower color 1
1.2.2 Anthocyanin 2
1.3 Biological roles of anthocyanin 2
1.3.1 The biosynthesis pathway of anthocyanin 2
1.3.2 The modification of flavonoids 3
1.4 The glucosyltransferases of anthocyanin 3
1.4.1 The importance of plant UGTs in the anthocyanin biosynthesis pathway 3
1.4.2 PeUFGTs isolated from P. equestris 4
1.5 Glycosyltransferases and cytokinin 5
1.6 Genetic engineering of flower color 6

2. Purpose 8

3. Materials and methods 9
3.1 Plant materials and chemicals 9
3.2 RNA extraction and reverse transcription-PCR (RT-PCR) 9
3.3 Molecular cloning of recombinant PeUFGTs protein 10
3.4 Heterologous expression of PeUFGTs 11
3.5 Purification of his-tagged and GST-fusion proteins 11
3.6 Enzyme activity assay 12
3.7 High-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC-MS) 12
3.8 Virus-induced gene silencing 13
3.9 Determination of total anthocyanin content 14
3.10 Quantitative real-time PCR 14
3.11 Protoplast isolation 15
3.12 PEG-mediated transfection of protoplasts 16
3.13 5’-RACE and 3’-RACE 16
3.14 Sequence alignments and phylogenetic analysis 17

4. Results 18
4.1 The phylogenetic analysis of PeUFGT1, PeUFGT2 and PeUFGT3 18
4.2 Heterologous expression of the recombinant PeUFGT3 protein in E. coli 18
4.3 Enzyme activity and substrate specificity assays of recombinant PeUFGT3 19
4.4 Loss-of-function assay by using virus-induced gene-silencing of PeUFGT1 and PeUFGT2 20
4.5 Effects of gene silencing of PeUFGTs in Phalaenopsis flower color formation 21
4.6 Transient expression of PeUFGT3 in Phalaenopsis flowers 22
4.7 Anthocyanin UFGT candidates investigation from OrchidBase 23
4.8 Spatial expression patterns of PeUFGT genes 23

5. Discussion 25
5.1 PeUFGT3 is a glycosyltransferase which can use cis-zeatin as the substrate 25
5.2 Alter PeUFGT3 gene expression on flowers in Phalaenopsis 27
5.3 PeUFGT4, a candidate gene responsible for glycosylation of UDP-glucose to anthocyanin 3-O-position 29

References 30

List of Table
Table 1. The primers used in this study 36

List of Figures
Figure 1. Spatial expression patterns of PeUFGT1, PeUFGT2 PeUFGT1, and PeUFGT4 by RT-PCR 37
Figure 2. The expression vectors used for the construction with PeUFGTs coding sequence 38
Figure 3. Expression of recombinant PeUFGT1 and PeUFGT3 proteins 39
Figure 4. Enzyme activity of purified recombinant PeUFGT1 protein 40
Figure 5. Enzyme activity of purified recombinant PeUFGT3 protein 41
Figure 6. HPLC of PeUFGT3 and cis-zeatin reaction products 42
Figure 7. HPLC of PeUFGT3 and kaempferol reaction products 43
Figure 8. Mass spectra of standard of cis-zeatin and reaction products 44
Figure 9. PeUFGT1, PeUFGT2 and PeUFGT3 silencing fragments for VIGS 45
Figure 10. Flower morphology of normal plant, PeUFGT1-, PeUFGT2- and PeUFGT3-silenced plants 46
Figure 11. Total anthocyanin contents detected in flowers of mock plant, PeUFGT1-, PeUFGT2- and PeUFGT3-silenced plants 47
Figure 12. The PeUFGTs gene expression patterns of mock and PeUFGT1-, PeUFGT2- and PeUFGT3-silenced plants by RT-PCR 48
Figure 13. The relative PeUFGTs mRNA abundance of mock and PeUFGT1-, PeUFGT2- and PeUFGT3- silenced plants 49
Figure 14. Transient expression of PeUFGT3 in Doritaenopsis “OX1313” protoplasts treated with PEG-mediated transfection 50
Figure 15. GUS staining of PeUFGT3 transgenic P. aphrodite flowers after particle bombardment 51
Figure 16. Multiple alignment of deduced amino acid sequences of four three PeUFGTs and PeUFGT4 52
Figure 17. Neighbor-joining phylogenetic tree of deduced amino acid sequences of three PeUFGTs, PeUFGT4 and glucosyltransferases from other plants 53
Figure 18. Phylogenetic tree of PeUFGT1, PeUFGT2 and PeUFGT3 57

List of Appendix Figures
Appendix 1. The anthocyanin biosynthesis pathway of the most abundant anthocyanin pigments 58
Appendix 2. Expression pattern of anthocyanin biosynthetic genes 59
Appendix 3. The major anthocyanin and flavone derivatives synthetic pathway in Phalaenopsis 60
Appendix 4. The expression patterns of PeUFGT4 in different color cultivars of Phalaenopsis by using RT-PCR 61
                                    

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校內：2017-08-31公開
校外：2017-08-31公開

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