成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	許綺育 Hsu, Chi-Yu
論文名稱：	探討蝴蝶蘭GDPS啟動子上重複序列的差異與香味生成之相關性 Analysis of the concomitance between repeat variation on GDPS promoter and scent production in 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 - 生命科學系 Department of Life Sciences
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	46
中文關鍵詞：	花香、單萜類、蝴蝶蘭、啟動子、重複序列、GDPS
外文關鍵詞：	floral scent, monoterpenes, Phalaenopsis, promoter repeats, GDPS
相關次數：	點閱：173 下載：1
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

大葉蝴蝶蘭為原生種蝴蝶蘭，其花朵具有甜美香味，主要的香味成分為單萜類及其衍生物。PbGDPS (P. bellina geranyl diphosphate synthase) 已被確定為大葉蝴蝶蘭中單萜類物質生合成路徑中的關鍵酵素。先前研究發現，在PbGDPS啟動子序列中有一段11個核甘酸大小的片段，此片段被認為可能是受轉錄因子調控的cis-element，且可能與蝴蝶蘭的香味生成有關。利用氣相層析質譜儀分析八個蝴蝶蘭物種的香味成分，單萜類物質及其衍生物在P. amboinensis, P. stuartiana與P. mannii被偵測到。為了進一步確認11個核甘酸大小的片段與蝴蝶蘭香味生成之間的相關性，此實驗中分析了十種蝴蝶蘭的GDPS啟動子序列。結果發現，在具有強香味之P. bellina, P. amboinensis, P. lueddemanniana, P. stuartiana及P. mannii的GDPS啟動子中具有三組完整重複序列。進一步，我們發現GDPS的基因只表現在具有三組完整重覆序列之GDPS啟動子四種蝴蝶蘭。綜合以上結果可得知，GDPS啟動子序列上之三組完整重覆序列確實與GDPS基因表現以及產生單萜類物質具有相當的關連性。為了確定PbGDPS的最短啟動子序列，將PbGDPS啟動子的5’端進行連續刪除，以蝴蝶蘭花瓣之原生質體，利用雙螢光酵素系統分析連續刪除的啟動子之啟動基因表現之功能。結果顯示，571-bp啟動子的活性有明顯下降，推測可能有抑制子或者活化子會結合在571-bp到284-bp之間。而144-bp的PbGDPS啟動子已具有啟動報導基因表現之功能。此外，在P. aphrodite subsp. formosana的原生質體中，加入PbMYB1及PbMYB2並不會明顯增加PbGDPS啟動子活性。這些結果可能是由於P. aphrodite subsp. formosana的原生質體中已有內生性的MYB轉錄因子。另一種可能是仍需具有其他轉錄因子以加強PbMYBs激活PbGDPS的表現。

Phalaenopsis bellina is a native Phalaenopsis with fragrance. Monoterpenoids and their derivatives comprise most of the total volatiles in the P. bellina flowers. P. bellina geranyl diphosphate synthase (PbGDPS) has been identified as the key enzyme for monoterpenes biosynthesis in P. bellina. Previously, an 11-nucleotide unique sequence has been considered as a potential cis-element related to the scent production in Phalaenopsis orchids. In this study, the scent compounds from eight species of Phalaenopsis were collected and analyzed. Monoterpenes and their derivatives were detected in three of them, including P. amboinensis, P. stuartiana, and P. mannii. To further confirm the correlation between cis-element in the PbGDPS promoter and the scent production, the GDPS promoter sequences were analyzed in ten Phalaenopsis orchids. Three sets of repeats were identified in the GDPS promoter of P. bellina, P. amboinensis, P. lueddemanniana, P. stuartiana, and P. mannii. Expression pattern of GDPS transcript showed that it expressed in Phalaenopsis orchids that producing monoterpenes, including P. amboinensis, P. lueddemanniana, P. stuartiana, and P. mannii except P. lueddemanniana. These results indicate that the repeats within GDPS promoter are well correlated to its gene expression and concomitant with the monoterpene scent production. To determine the minimal promoter of PbGDPS, dual luciferase assay was carried out with serial deletion fragments of PbGDPS promoter, including 1 kb, 767 bp, 571 bp, 284 bp and 144 bp. A sharp drop of promoter activity was detected for 571-bp fragment as compared to that for the 284-bp fragment suggesting that the region between nucleotide -571 and -284 may reside binding sites for repressors/activator. Furthermore, the 144-bp promoter region of PbGDPS is the minimal promoter. In addition, the addition of transcription factors of either PbMYB1 or PbMYB2 did not significantly increase PbGDPS promoter activity in protoplasts prepared from P. aphrodite subsp. formosana floral buds. These results suggest that either there were background level of MYB transcription factors in the floral buds. Alternatively, other transcription factors may be needed for PbMYBs to be fully functional.

中文摘要 i
Abstract ii
致謝 iv
List of Table viii
List of Figures ix
1. Introduction	1
1.1 The economic value of Phalaenopsis in Taiwan 1
1.2 Fragrance compounds in plants 1 
1.2.1 The roles of fragrance compounds in plants 1
1.2.2 Types of fragrance compounds and their biosynthesis pathways 2
1.2.2.1 Biosynthesis of volatile compounds 2
1.2.2.2 The fragrance compounds in P. bellina flower 3
1.3 Transcription factors involved in regulation of fragrance compounds biosynthesis 4 
1.3.1 Identification of MYB transcription factors 4
1.3.2 MYB proteins regulating biosynthesis of fragrance compounds 4
1.3.2.1 Research on the regulation of fragrance compounds biosynthesis by MYB proteins in P. bellina 5
2. Purpose 6
3. Materials and methods 7
3.1 Plant materials and growth conditions 7
3.2 GDPS promoter sequences isolation and gene detection 7
3.2.1 Genomic DNA extraction 7
3.2.2 Promoter isolation 7
3.2.3 GDPS gene detection 7
3.3 Scent compounds collection and chromatographic analysis 8
3.4 Expression pattern of GDPS in various Phalaenopsis 8
3.4.1 RNA preparation 8
3.4.2 Reverse transcription-PCR (RT-PCR) 8
3.5 Transient transfection and dual luciferase reporter assay 9
3.5.1 Promoter-reporter plasmid construction 9
3.5.2 Protoplast preparation 9
3.5.3 Transient expression experiments and dual luciferase reporter assay 10
4. Results 11
4.1 Analysis of scent compounds in various Phalaenopsis	11
4.2 Expression pattern of GDPS 11
4.3 Confirmation of GDPS genes in genomic DNA 11
4.4 Comparison of the GDPS promoter sequences 11
4.5 Prediction of cis-regulatory elements in PbGDPS promoter 12
4.6 Protoplasts isolation 13
4.7 Dual-luciferase assay of serial deletion promoter sequence of PbGDPS 13

5. DISCUSSION 14
5.1 The repeat sequences of GDPS promoter in Phalaenopsis orchids 14
5.2 Transcription factor binding sites on PbGDPS promoter repeats sequences 14
5.3 The activity of PbGDPS promoter 15
5.4 Two different sequences of GDPS promoter in some species 16
5.5 Transient expression experiments by PEG transfection 16
6. CONCLUSION 18
7. REFERENECES 19
List of Table
Table 1. The list of primers used in this study	23
List of Figures
Figure 1. GC-MS analysis of the scent compounds of P. amboinensis, P. sturatiana and P. mannii. Arrows indicate the monoterpene and their derivates. 24
Figure 2. The scent trait and types of monoterpenes and derivatives detected in ten species of Phalaenopsis. ND: undetected. 26
Figure 3. Expression patterns of GDPS transcript in various Phalaenopsis orchids were analyzed by using RT-PCR. The expression of Actin was used as an internal control. 27
Figure 4. Detection of GDPS gene in the genomic DNA in ten species of Phalaenopsis. 28
Figure 5. Sequences alignment of GDPS promoter in ten Phalaenopsis species. A major difference was showed in the -820 to -635 bp region. There is a large deletion in P. equestris, P. schilleriana, and P. aphrodite subsp. formosana (red box). The 11-nt deletion in P. equestris was shown in black box. 29
Figure 6. Three sets of repeats in the GDPS promoter of P. bellina, P. amboinensis, P. lueddemanniana, P. stuartiana, and P. mannii. There was a small deletion in GDPS promoter sequences of P. equestris “S82-159” and P. javanica in the repeat 2 and repeat 2’ respectively. P. equestris, P. schilleriana, and P. aphrodite subsp. formosana contain a large deletion in this region, so there are no complete repeats on this region. 30
Figure 7. Monoterpenes and their derivatives were detected in the species, P. amboinensis, P. stuartiana, and P. mannii, whose GDPS promoter contained the three sets of repeats. GDPS was expressed in these species. However, the scent compounds of P. javanica, P. equestris “S82-159”, P. equestris, P. schilleriana, and P. aphrodite subsp. formosana, whose GDPS promoter with a variant deletion, do not produce monoterpenes. However, the GDPS promoter of P. lueddemanniana contained an intact three sets of repeats, there was no any monoterpenes detected. ND: undetected. 31
Figure 8. PbGDPS promoter contains consensus sequences of DNA binding sites of MYB transcription factors predicted by Plant Promoter Analysis Navigator (PlantPAN). The three sets of repeats on the PbGDPS promoter are in the region between -1063 and -767 bp (pink box). TSS: transcription start site. 32
Figure 9. Protoplasts prepared from flower buds of P. aphrodite subsp. formosana for transient expression experiment. (A) Protoplasts were isolated from petal of P. aphrodite subsp. Formosana flower bud. (B) The protoplasts were observed after PEG treatment for 18 hours. Photos were taken through the eye-piece of a microscope (Leica, DMI3000 B) (10x object lense, 10x ocular). 33
Figure 10. Analysis of PbGDPS promoter activity. (A) A series of 5'-deletion PbGDPS promoter fragments was generated to drive the firefly luciferase reporter gene, including fragments from nucleotide -1063, -767, -571, -284 and -144 upstream of transcription start site. (B) Dual luciferase assay for promoter activity of various promoter constructs in petal protoplasts of P. aphrodite subsp. formosana. TSS: transcription start site. 34
Figure 11. Analysis of PbGDPS promoter activity with PbMYBs. (A) Regulation of the PbGDPS promoter by PbMYB1. The PbMYB1 increased the luciferase activity about 2.2-fold, 1.4-fold and 1.1-fold for 767bp, 571-bp and 284-bp promoter fragments, respectively. However, the PbMYB1 did not increase the promoter activity of 1-kb and 144-bp fragments. (B) Regulation of the PbGDPS promoter by PbMYB2. The PbMYB2 increased the luciferase activity about 1.9-fold, 1.2-fold and 1.1-fold for 767bp, 571-bp and 284-bp promoter fragments, respectively. The PbMYB2 did not increase the promoter activity of 1-kb and 144-bp fragments. 35
Figure 12. Prediction of transcription factor binding sites on the three sets of repeats of PbGDPS promoter. There are nine DOF transcription factors binding sites on the repeats (red boxes). 36
List of Appendix figures
Appendix Figure 1. Temporal expression patterns of PbMYB1, PbMYB2 and PbMYB6 were showed by use of quantitative real-time PCR (Chuang, 2009). 37
Appendix Figure. 2. Interaction between PbGDPS promoter and PbMYB1, PbMYB2 and PbMYB6 in dual-luciferase transient promoter assay using cultured Oncidium protoplast cells. The results showed a significant increase in the case of PbMYB1, PbMYB2, and PbMYB6 with PbGDPS 1-Kb promoter region. –MYB indicates the control of the activity of the respective promoter transfected without a MYB factor (Chuang, 2009).	38
Appendix Figure 3. Yeast one-hybrid assay with PbMYB1, PbMYB2 and PbMYB6 and PbGDPS promoter subfragments. (A) Diagram of the four PbGDPS promoter subfragments used in yeast one-hybrid assay. (B) Transactivation activity of PbMYB1, PbMYB2 and PbMYB6 on four PbGDPS promoter subfragments was revealed by cell growth. After one day post incubation (DPI), yeast cells harboring PbMYB and No. 1 or No. 3’ subfragment grew on the selective medium. PbMYB1 and PbMYB2 showed higher affinities with No.1 subfragments, in contrast to PbMYB6. PbMYB1 also showed higher activating abilities for No. 3’ fragment. The No. 2 and No. 3 subfragments were not bound by any of the PbMYBs. (C) These results were more obvious after three days post incubation (DPI). Yeast cells harboring PbMYB and No. 3 subfragment started to grow on the selective medium, indicated PbMYB have lower affinities with No. 3 subfragment (Chuang, 2009). 39
Appendix Figure 4. Detection of GDPS transceipts in P. bellina, scent and scentless P. eauestris flowers on day 5 post-anthesis (Hsiao et al., 2008). 41
Appendix Figure 5. Comparison of amino acid sequences between PbGDPS and PeGDPS. The active motif (EAEVE) shown in both PbGDPS and PeGDPS (black box) (Chuang, 2009). 42
Appendix Figure 6. Sequences alignment of GDPS promoter in seven Phalaenopsis species. The 11-nt deletion in P. equestris is the major difference between scented and scentless species (Chuang, 2009). 43
Appendix Figure 7. Eight Phalaenopsis species were used for scent compounds analysis.44
Appendix Figure 8. Spatial expression levels of PbGDPS, PbMYB1 and PbMYB2 were confirmed by northern blot in P. bellina (Hsiao et al., 2008). 45
Appendix Figure 9. Southern blot analysis of PbGDPS in P. bellina genome (Hsiao et al., 2008). 46

                                    

Chang, W. C., Lee, T. Y., Huang, H. D., Huang, H. Y. and Pan, R. L. (2008). PlantPAN: Plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups. BMC Genomics 9, 561.
Chen, W.H., Tsai, W.T., Wu, C.C. and Hsieh, R.M. (1991). Electrofusion and cell division of Phalaenopsis protoplasts. Taiwan Sugar 2, 14-18.
Christenson E.A. (2001). Ecology and distribution. Phalaenopsis a monograph Portland: Timber Press.
Chuang, Y.-C. (2009). Regulation mechanism of fragrance biosynthesis in Phalaenopsis orchids: the role of MYB transcription factors. 國立成功大學生命科學研究所碩士論文.
Colquhoun, T. A., Kim, J. Y., Wedde, A. E., Levin, L. A., Schmitt, K. C., Schuurink, R. C. and Clark, D. G. (2010). PhMYB4 fine-tunes the floral volatile signature of Petuniaxhybrida through PhC4H. Journal of Experimental Botany 62, 1133-1143.
Croteau, R., Kutchan, T. M. and Lewis, N. G. (2000). Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, 1250-1318.
Du, H., Zhang, L., Liu, L., Tang, X.-F., Yang, W.-J., Wu, Y.-M., Huang, Y.-B. and Tang, Y.-X. (2009). Biochemical and molecular characterization of plant MYB transcription factor family. Biochemistry (Moscow) 74, 1-11.
Dudareva, N. (2002). Molecular control of floral fragrance. In Breeding for ornamentals: Classical and molecular approaches (Vainstein, A., ed.) . 295-309.
Dudareva, N. and Pichersky, E. (2000). Biochemical and Molecular Genetic Aspects of Floral Scents. Plant Physiology 122, 627-633.
Dudareva, N., Pichersky, E. and Gershenzon, J. (2004). Biochemistry of plant volatiles. Plant Physiology 135, 1893-1902.
Feussner, I. and Wasternack, C. (2002). The lipoxygenase pathway. Annual Review of Plant Biology 53, 275-297.
Frearson, E. M., Power, J. B. and Cocking, E. C. (1973). The isolation, culture and regeneration of Petunia leaf protoplasts. Developmental Biology 33, 130-137.
Lee, C. S. (2002). "An economic analysis of Phalaenopsis production in Tiawan." 農業經濟半年刊 71: 113-133.
Hsiao Y.-Y. (2008). Studies on the biosynthesis pathway and its related gene of Phalaenopsis bellina floral scent. 國立成功大學生命科學研究所博士論文.
Hsiao, Y.-Y., Tsai, W.-C., Kuoh, C.-S., Huang, T.-H., Wang, H.-C., Wu, T.-S., Leu, Y.-L., Chen, W.-H. and Chen, H.-H. (2006). Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway. BMC Plant Biology 6, 14.
Jürgens, A., Webber, A. C. and Gottsberger, G. (2000). Floral scent compounds of Amazonian Annonaceae species pollinated by small beetles and thrips. Phytochemistry 55, 551-558.
Loreto, F. and Velikova, V. (2001). Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol, December 2001, Vol. 127, pp. 1781-1787 127, 1781-1787.
McGarvey, D. J. and Croteau, R. (1995). Terpenoid Metabolism. The Plant Cell 7, 1015-1026.
Moreno-Risueno, M. A., Martínez, M., Vicente-Carbajosa, J., Carbonero P. (2007). Molecular genetics and genomics 4, 379-390.
Pare, P. W. and Tumlinson, J. H. (1999). Plant volatiles as a defense against insect herbivores. Plant Physiololgy 121, 325-331.
Rabinowicz, P. D., Braun, E. L., Wolfe, A. D., Bowen, B. and Grotewold, E. (1999). Maize R2R3 Myb genes: Sequence analysis reveals amplification in the higher plants. Genetics 153, 427-444.
Seskar, M., Shulaev, V. and Raskin, I. (1998). Endogenous methyl salicylate in pathogen-inoculated tobacco plants. Plant Physiology 116, 387-392.
Sharkey, T. D., Chen, X. and Yeh, S. (2001). Isoprene increases thermotolerance of fosmidomycin-fed leaves. Plant Physiology 125, 2001-2006.
Sharkey, T. D. and Yeh, S. (2001). Isoprene emission from plants. Annual Review of Plant Physiology and Plant Molecular Biology 52, 407-436.
Shulaev, V., Silverman, P. and Raskin, I. (1997). Airborne signalling by methyl salicylate in plant pathogen resistance. Nature 385, 718-721.
Skirycz A., Reichelt M., Burow M., Birkemeyer C., Rolcik J., Kopka J., Zanor M. I., Gershenzon J., Strnad M., Szopa J., Mueller-Roeber B., Witt I. (2006). DOF transcription factor AtDof1.1 (OBP2) is part of a regulatory network controlling glucosinolate biosynthesis in Arabidopsis. The Plant Journal 47,10-24.
Spitzer-Rimon, B., Marhevka, E., Barkai, O., Marton, I., Edelbaum, O., Masci, T., Prathapani, N. K., Shklarman, E., Ovadis, M. and Vainstein, A. (2010). EOBII, a gene encoding a flower-specific regulator of phenylpropanoid volatiles' biosynthesis in petunia. Plant Cell 22, 1961-76.
Stracke, R., Werber, M. and Weisshaar, B. (2001). The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology 4, 447-456.
Trappa, S. C. and Croteau, R. B. (2001). Genomic Organization of Plant Terpene Synthases and Molecular Evolutionary Implications. Genetics 158, 811-832.
Tsai, W. C., Kuoh, C. S., Chuang, M. H., Chen, W. H. and Chen, H. H. (2004). Four DEF-Like MADS Box genes displayed distinct floral morphogenetic roles in Phalaenopsis Orchid. Plant and Cell Physiology 45, 831-844.
Vainstein, A., Lewinsohn, E., Pichersky, E. and Weiss, D. (2001). Floral Fragrance. New Inroads into an Old Commodity. Plant Physiology 127, 1383-1389.
Verdonk, J. C., Haring, M. A., Tunen, A. J. v. and Schuurink, R. C. (2005). ODORANT1 regulates fragrance biosynthesis in petunia flowers. The Plant Cell 17, 1612-1624.
Xiang, L., Milc, J. A., Pecchioni, N. and Chen, L. Q. (2007). Genetic Aspects of Floral Fragrance in Plants. Biochemistry (Moscow) 72, 351-358.
Yanagisawa, S. (1996). Dof DNA binding proteins contain a novel zinc finger motif. Trends in Plant Science, 1, 213-214.
Yanagisawa, S., Schmidt, R. J. (1999). Diversity and similarity among recognition sequences of Dof transcription factors. The Plant journal, 17, 209-214.
Yoo, S. D., Cho, Y. H. and Yoo, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2, 1565-1572

校內：2021-12-31公開
校外：2021-12-20公開

簡易檢索 / 詳目顯示

相關論文