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研究生：	施幸慧 Shih, Hsing-Hui
論文名稱：	大葉蝴蝶蘭GDPS_SSU II之鑑定及功能分析 Identification and functional analysis of geranyl diphosphate synthase small subunit II (GDPS_SSU II) in Phalaenopsis bellina
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 - 生命科學系 Department of Life Sciences
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	58
中文關鍵詞：	GDPS_SSU II 、類異戊二烯轉移酶、萜類生合成路徑
外文關鍵詞：	GDPS_SSU II, prenyltransferase, terpenoid biosynthesis pathway
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萜類化合物(terpenoid)為植物體中的二次代謝物，參與許多植物生理作用，包括植物生長發育、光合色素的生合成、吸引昆蟲幫助植物授粉、對草食動物或病原菌的防禦等。此類化合物的生合成由類異戊二烯轉移酶(prenyltransferase)催化，利用五個碳的單元IDP與DMADP產生不同碳鏈長度的產物，目前已有許多類異戊二烯轉移酶被發現。GDPS_SSU II為一群在親緣演化分群上不屬於任何一群已知GDPS的酵素，目前只有來自阿拉伯芥的geranylgeranyl reductase (AtGGR) 進行過功能鑑定，研究發現其單獨存在時不具有酵素活性，需與AtGGDSP11結合形成具有活性的GDPS。由於目前對於GDPS_SSU II功能的研究不多，因此目前仍不清楚他們在植物中所扮演的角色。
本研究在大葉蝴蝶蘭中找到一個與AtGGR有高度序列相似的基因，並將其命名為PbGDPS_SSU II。序列分析顯示PbGDPS_SSU II具有完整的FARM及不完整的SARM，且在蛋白質的N端帶有可轉運至葉綠體的訊號胜肽。在親緣演化上，PbGDPS_SSU II會與其它植物的GDPS_SSU II分在同一群，且與GDPS_SSU較為接近。功能鑑定顯示PbGDPS_SSU II可將IDP與DMADP或GDP結合，產生帶有十個碳及十五個碳的產物，利用大腸桿菌功能性互補實驗亦發現，PbGDPS_SSU II不具有調控PeGGDPS產物的能力。相反地， PbGDPS_SSU卻能使PeGGDPS產生的C20產物減少。進一步檢測PbGDPS_SSU II在時間與空間上的表現分布，發現其在不同花部發育時期皆有持續性表現，且在植物營養組織及生殖組織廣泛分布，顯示PbGDPS_SSU II可能與花香成分分子的合成無直接相關。鑑定PbGDPS_SSU II的功能將有助於我們了解GDPS_SSU II酵素在植物體中所扮演的角色，對蝴蝶蘭中萜類生合成路徑也將有更進一步的認識。

Terpenoids are a group of plant secondary metabolites that are involved in many biological processes, including plant growth and development, biosynthesis of photosynthetic pigments, attracting pollinators and plant defense against herbivores or pathogens. The biosynthesis of terpenoid is catalyzed by prenyltransferases using isopentenyl diphosphate (IDP, C5) and dimethylallyl diphosphate (DMADP, C5) as building blocks to form linear products with different carbon chain length. Many prenyltransferases involved in terpenoid biosynthesis have been identified and functional characterized. GDPS_SSU II is a recently identified clade of enzymes that is phylogenetically distinct from other GDPS. AtGGR, a member of GDPS_SSU II, is the only enzyme that has been functionally characterized in this clade. It is an inactive enzyme per se and only forms active GDPS with AtGGDPS11. Since the functional characterization of enzymes in this clade is scarce, the role of GDPS_SSU II in plants is still largely unknown.
In this study, an orchid gene with high similarity to AtGGR in Phalaenopsis bellina was identified and named as PbGDPS_SSU II. Sequence analysis revealed that PbGDPS_SSU II contains the first Asp-rich motif, but the second Asp-rich motif is incomplete. In addition, it possesses a chloroplast transit peptide at its N-terminus. Phylogenetically, it forms a distinct clade with other GDPS_SSU II proteins and is close to other GDPS_SSUs. Functional characterization of PbGDPS_SSU II indicated that it can accept DMADP and GDP as substrates to form GDP (C10) and FDP (C15). Moreover, the functional complementation assay in E. coli showed that PbGDPS_SSU, rather than PbGDPS_SSU II, has the ability to alter the product of PeGGDPS and reduce its C20 production. Temporal and spatial expression analyses showed that PbGDPS_SSU II was constitutively expressed during flower development and was highly expressed in both vegetative and reproductive organs. In contrast, the expression of PbGDPS_SSU is flower-specific and is highly correlated to the emission of floral scent. These results indicated that PbGDPS_SSU II may not directly involve in the biosynthesis of floral scent. Identification and functional characterization of PbGDPS_SSU II may lead to a better understanding of the function of plant GDPS_SSU II and their role in terpenoid biosynthesis in orchids.

中文摘要 I
Abstract II
致謝 III
List of Tables VI
List of Figures VII
List of Appendix Tables VIII
List of Appendix Figures VIII
1. Introduction 1
1.1 The Phalaenopsis orchids 1
1.2 Terpenoids 1
1.2.1 Plant secondary metabolites 1
1.2.2 The significance of terpenoid in higher plants 2
1.2.3 The biosynthesis pathway of terpenoids 2
1.3 Prenyltransferases 3
1.3.1 Different types of prenyltransferases 3
1.3.2 Conserved motifs in short-chain prenyltransferase 4
1.4 Geranyl diphosphate synthase 4
1.4.1 Phylogenetic relationship of GDPS 4
1.4.2 GDPS_SSU 5
1.4.3 GDPS_SSU II 6
1.5 Product chain length regulation of prenyltransferases 6
1.5.1 Mechanisms of product chain length regulation 6
1.5.2 Product chain length regulation by GDPS_SSU and GDPS_SSU II 7
1.6 Previously studies of prenyltransferases in Phalaenopsis orchids 8
1.6.1 Geranyl diphosphate synthases isolated from P. bellina 8
1.6.2 Other short-chain prenyltransferases from Phalaenopsis orchids 8
2. Purpose 10
3. Materials and methods 11
3.1 Plant materials 11
3.2 Molecular cloning of GDPS_SSU II from P. bellina 11
3.3 Sequence analysis 12
3.4 Phylogenetic analysis 12
3.5 Isolation of total RNA 13
3.6 RT-PCR 13
3.7 Expression and purification of PbGDPS_SSU II recombinant proteins 14
3.8 Prenyltransferase activity assay 15
3.9 Thin layer chromatography (TLC) 16
3.10 Measurement of kinetic properties of PbGDPS_SSU II 16
3.11 Functional complementation assay 17
3.12 Molecular modeling of PbGDPS_SSU II 18
3.13 Yeast two-hybrid analysis 18
4. Results 20
4.1 Isolation and sequence analysis of GDPS_SSU II from P. bellina 20
4.2 Phylogenetic analysis 20
4.3 Functional characterization of PbGDPS_SSU II 20
4.4 Functional complementation assay of PbGDPS_SSU II 22
4.5 Protein-protein interaction between PbGDPS_SSU II and PeGGDPS 23
4.6 Temporal and spatial expression patterns of PbGDPS_SSU II 23
4.7 Homology modeling structure of PbGDPS_SSU II 24
5. Discussion 25
5.1 PbGDPS_SSU II is a functional prenyltransferase that generate GDP and FDP 25
5.2 PbGDPS_SSU II could not generate products longer than C15 26
5.3 PbGDPS_SSU, rather than PbGDPS_SSU II, has the ability to regulate the product chain length specificity of PeGGDPS 27
5.4 The role of PbGDPS_SSU II in P. bellina 29
5.5 Functional redundancy of PbGDPS_LSU, PbGDPS_SSU and PbGDPS_SSU II 30
6. Conclusion 31
7. Perspectives 32
8. References 33

List of Tables 
Table 1. Apparent Km and Kcat values for recombinant PbGDPS_SSU II 37
Table 2. Templates used for constructing 3D structure of prenyltransferases 38
Table 3. All primers used in this study 39

List of Figures
Figure 1. Multiple sequence alignment of AtGGR-like proteins 40
Figure 2. Phylogenetic tree of plant prenyltransferases 41
Figure 3. Protein purification and prenyltransferase activity of PbGDPS_SSU II 42
Figure 4. TLC characterization of PbGDPS_SSU II products 43
Figure 5. in vivo functional complementation assay 44
Figure 6. Yeast two-hybrid analysis of PbGDPS_SSU II and PeGGDPS interaction 45
Figure 7. Temporal and spatial expression patterns of PbGDPS_SSU II 46
Figure 8. Molecular models of PbGDPS_SSU II, AtGGR, PbGDPS_SSU and SaGGDPS 47
Figure 9. Amino acid residues referred in discussion 48
Figure 10. Overview of terpenoid biosynthesis pathway in Phalaenopsis orchids 49

List of Appendix Table
Appendix Table. Short-chain prenyltransferases isolated from Phalaenopsis orchids 50

List of Appendix Figures
Appendix Figure 1. The terpenoid biosynthesis pathway 51
Appendix Figure 2. Phylogenetic analysis of GDPSs and GGDPSs 52
Appendix Figure 3. The chain-length determination region 53
Appendix Figure 4. Regulation of GGDPS product chain length specificity by GDPS_SSU and AtGGR 54
Appendix Figure 5. The pACCAR25ΔcrtE plasmid 55
Appendix Figure 6. Temporal expression of PbGDPS_SSU and the emission of linalool and geraniol in P. bellina 56
Appendix Figure 7. Spatial expression pattern of AtGGR 57
Appendix Figure 8. Spatial expression patterns of short-chain prenyltransferases identified in Phalaenopsis orchids 58

                                    

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