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研究生：	李思誼 Lee, Szu-Yi
論文名稱：	姬蝴蝶蘭開花時間相關基因(PeSOC1) 之選殖與特性分析 Molecular cloning and characterization of SUPPRESSOR OF OVEREXPRESSION OF CO1 (PeSOC1) from Phalaenopsis equestris
指導教授：	吳文鑾 Wu, Wen-Luan
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：	2006
畢業學年度：	94
語文別：	中文
論文頁數：	73
中文關鍵詞：	開花時間、SOC1基因、姬蝴蝶蘭
外文關鍵詞：	SOC1, flowering time, Phalaenopsis equestris
相關次數：	點閱：269 下載：2
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開花為植物從營養期轉變為生殖期最重要的發育階段，也是植物繁衍後代成功與否的關鍵。在模式植物阿拉伯芥(Arabidopsis thaliana)研究中，開花過程由四條途徑所控制：光週期、春化作用、吉貝素及自發性調控途徑，而SUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1)為此四條開花路徑下游的整合者。蝴蝶蘭為國內重要經濟花卉之一，但其幼年期長，且業界常利用催花方式調控花期，因此研究蝴蝶蘭開花基因將有助於產業應用。本研究目的為選殖姬蝴蝶蘭SOC1基因並進行特性分析。首先從基因資料庫中搜尋其它物種之SOC1基因，於序列保守區設計簡併性引子(degenerate primer)，擴增蝴蝶蘭SOC1基因片段並選殖定序，接著利用RACE (rapid amplification of cDNA ends)方式獲得SOC1基因cDNA全長(命名為PeSOC1)，此基因全長為1227 bp，譯碼區有666 bp，可轉譯出221個氨基酸。另外，亦利用擴增PeSOC1基因全長之引子對選殖文心蘭SOC1基因cDNA片段(命名為OfSOC1)。比對分析PeSOC1氨基酸序列後，發現其氨基酸序列具有MADS-box與K-box兩個保留性高的區域。PeSOC1氨基酸序列與水稻OsSOC1相似度為57%，而與文心蘭OfSOC1氨基酸序列相似度高達97.8%。分子親源關係之分析顯示PeSOC1在演化上與水稻OsSOC1及文心蘭OfSOC1位於同一個分類枝(clade)。利用南方墨點法分析SOC1顯示其為單一拷貝的基因。另外，利用北方墨點法分析SOC1於幼苗、根、葉及花、花苞及開花後葉片等組織中表現量，發現PeSOC1於開花兩週後之葉片中表現量最強，在花部表現量最微弱。進一步利用農桿菌轉殖至野生型阿拉伯芥與soc1、ft突變株，T1及T2子代轉殖株均提早開花，推測PeSOC1功能與AtSOC1皆屬正向調控開花時間。在春化實驗中，蝴蝶蘭催花八週後SOC1表現量有些微上升之趨勢。最後，選殖SOC1基因啟動子，選擇轉錄起始密碼上游約2 kb區域進行軟體預測，啟動子序列分析顯示序列中具受上游MADS box 基因調控的CArG box及光週期相關之cis-elements，而CArG box為受MADS-box基因調控之序列。綜合以上結果，PeSOC1基因於姬蝴蝶蘭中為扮演促進開花之角色。

Proper timing of the transition from vegetative stage to flowering is critical to the reproductive success of plants. The floral transition in Arabidopsis is regulated by four flowering pathways: the photoperiod, vernalization, autonomous, and gibberellin dependent pathways and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) integrates signals from the four pathways. Phalaenopsis orchid is one of the most commercially important floral crops in Taiwan but usually has long juvenile periods. Therefore, studying the flowering time genes of orchid will be contributed to orchid industry. The objectives of this study were cloning and characterization of PeSOC1 from Phalaenopsis equestris. In order to isolate the SOC1 gene from Phalaenopsis equestris, we designed the degenerate primers based on the conserved domain of SOC1 genes from other species. The full length cDNA of SOC1 was obtained by using RACE (rapid amplification of cDNA end). The SOC1 homologous gene from Phalaenopsis equestris was designated as PeSOC1. The full-length cDNA of PeSOC1 was 1227 base pair (bp) and contained a 666 bp open reading frame (ORF) encoding for 221 amino-acid residues. In addition, using the primer which amplified the cDNA of PeSOC1, we obtained the cDNA of SOC1 (designated as OfSOC1) in Oncidium flexuosum. The amino acid sequence of PeSOC1 contained a conserved MADS-box domain and K-box domain. The amino acid sequence of the PeSOC1 protein was 57% identical with the rice OsSOC1 and 97.8% with OfSOC1 (Oncidium flexuosum). Molecular phylogenetic analysis of evolutionary relationship revealed that PeSOC1 was clustered with the monocot genes. Southern blot analysis indicated that PeSOC1 was a single-copy gene when 3′ UTR was used as a probe. Northern blot analysis of Phalaenopsis equestris root, leaf, floral bud and flower indicated that relative high amount of gene expression of PeSOC1 was detected in the leaf in comparison with that of flower. Furthermore, The functional study of PeSOC1 was performed by transforming wild-type, ft and soc1 mutant Arabidopsis plants with PeSOC1 gene. Transgenic plant of T1 and T2 seedlings were shown to rescue the late flowering phenotypes of mutants, suggesting that the PeSOC1 gene is a functional equivalent of AtSOC1. In the vernalization experiment, the transcript levels of PeSOC1 were higher after 8 weeks of cold treatment in Phalaenopsis. Finally, we cloned 2 kb promoter regions upstream of the transcription start site (TSS) of PeSOC1 and identified a CArG box sequence and some photoperiod-related cis-elements. In conclusion, PeSOC1 is the flowering time gene which plays the positive-regulator in Phalaenopsis equestris.

中文摘要................................................................................................................i
英文摘要...............................................................................................................ii
致謝......................................................................................................................iii
目錄......................................................................................................................iv
表目錄.................................................................................................................vii
圖目錄................................................................................................................viii
附錄目錄..............................................................................................................ix
縮寫字對照表......................................................................................................x
第一章 緒論........................................................................................................1
一、開花植物生長階段：營養生長期轉變至生殖期................................1
二、調控開花時間之基因…........................................................................1
三、開花途徑的整合者................................................................................4
四、阿拉伯芥開花基因之研究....................................................................4
五、單子葉植物水稻開花途徑之研究…....................................................5
六、開花基因SOC1之研究………………………..………………………6
七、蝴蝶蘭與文心蘭之生長特性................................................................8
八、研究目的................................................................................................9
第二章 材料與方法..........................................................................................11
一、實驗材料..............................................................................................11
二、實驗方法..............................................................................................11
1. 蝴蝶蘭total RNA 的萃取................................................................11
2. 蝴蝶蘭基因組DNA 的萃取............................................................12
3. 由瓊脂凝膠中回收DNA 片段........................................................13
4. 轉型實驗(transformation) ...............................................................14
5. 微量製備質體DNA..................................................................,.......15
6. 姬蝴蝶蘭SOC1基因序列之選殖.....................................................16
7. PCR擴增姬蝴蝶蘭SOC1 cDNA基因全長.......................................18
8. 核苷酸定序......................................................................................19
9. 序列分析..........................................................................................19
10. 隨機引子探針的製備......................................................................20
11. 南方雜合實驗(Southern hybridization) .........................................20
12. 北方雜合實驗(Northern hybridization)..........................................22
13. 阿拉伯芥轉基因實驗.....................................................................23
14. 反轉錄聚合酶連鎖反應.................................................................25
第三章 結果....................................................................................................27
一、姬蝴蝶蘭PeSOC1 cDNA之選殖.......................................................27
二、文心蘭(Oncidium flexuosum) SOC1 cDNA之選殖...........................28
三、姬蝴蝶蘭PeSOC1 cDNA之序列與親源演化分析...........................29
四、姬蝴蝶蘭PeSOC1基因之組成..........................................................30
五、姬蝴蝶蘭PeSOC1基因表現分析......................................................30
六、姬蝴蝶蘭PeSOC1基因轉殖阿拉伯芥性狀分析..............................31
七、35S::PeSOC1阿拉伯芥轉殖株中開花基因的表現.........................33
八、蝴蝶蘭SOC1表現量受催花處理之影響..........................................34
九、姬蝴蝶蘭PeSOC1啟動子之選殖與調控序列分析..........................34
第四章 討論....................................................................................................37
一、姬蝴蝶蘭PeSOC1 cDNA之選殖與序列分析...................................37
二、文心蘭(Oncidium flexuosum) SOC1 cDNA之選殖..........................38
三、姬蝴蝶蘭PeSOC1 cDNA之序列與親緣演化分析...........................38
四、姬蝴蝶蘭PeSOC1基因之組成...........................................................39
五、姬蝴蝶蘭PeSOC1基因表現分析.......................................................40
六、姬蝴蝶蘭PeSOC1基因轉殖阿拉伯芥性狀分析...............................41
七、35S::PeSOC1阿拉伯芥轉殖株中開花基因的表現...........................42
八、蝴蝶蘭PeSOC1表現量受催花處理之影響.......................................42
九、姬蝴蝶蘭PeSOC1啟動子之選殖與調控序列分析...........................43
第五章、未來展望...........................................................................................46
第六章 參考文獻.............................................................................................47

                                    

李哖、林菁敏，1984，溫度對白花蝴蝶蘭生長發育與開花之影響，中國園藝，34: 223-231.

陳加忠，1999，蝴蝶蘭溫室的結構與環控，中華盆花，30: 9-11.

蔡佩芬，2000，溫度、光度、栽培介質對文心蘭苗生育之影響，國立台灣大學園藝學研究所碩士論文.

Araki T. and Komeda Y. (1993) Analysis of the role of the late- flowering locus, GI, in the flowering of Arabidopsis thaliana. Plant J. 3: 231-239.

Bechtold N. and Pelletier G.. (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259-266.

Bernier G. and Perilleux C. (2005) A physiological overview of the genetics of flowering time control. Plant Biotechnol. J. 31: 3-16.

Blazquez M. (2000) Flower development pathways. J. Cell Sci. 113: 3547-3548.

Borner R., Kampmann G., Chandler J., Gleissner R., Wisman E., Apel K. and Melzer S. (2000) A MADS domain gene involved in the transition to flowering in Arabidopsis. Plant J. 24: 591-599.

Boss P.K., Bastow R.M., Mylne J.S. and Dean C. (2004) Multiple pathways in the decision to flower: Enabling, promoting, and resetting. Plant Cell 16: S18-S31.

Chen L., Cheng J.C., Castle L., Sung Z.R. (1997) EMF genes regulate Arabidopsis inflorescence development. Plant Cell 9: 2011-2024.

Chia T.F., Chan Y.S. and Chua N.H. (1994) The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation. Plant J. 6: 441-446.

Clough S.J., and Bent A.F. (1998) Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J. 16: 735-743.

David A.L. (1997) Comparative genetics of flowering time. Plant Mol. Biol. 35: 167-177.

Doyle J.J. and Doyle J.L. (1987) A rapod isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11-15.

He Y. and Amasino R.M. (2005) Role of chromatin modification in flowering-time control. Trends Plant Sci. 10:30-35.

Helliwell C.A., Wood C.C., Robertson M., James Peacock W. and Dennis E.S. (2006) The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. Plant J. 46:183-192.

Hepworth S.R., Valverde F., Ravenscroft D., Mouradov A., Coupland G. (2002) Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO J. 21: 4327–4337.

Honma T. and Goto K. (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409: 525-529.

Hsu H.F., Huang C.H., Chou L.T. and Yang C.H. (2003) Ectopic expression of an orchid (Oncidium Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana. Plant Cell Physiol. 44: 783-794.

Kaufmann K., Melzer R., Theißen G. (2005) "MICK-type MADS domain proteins: structural modularity, protein interactions and network evolution in land plants" Gene 347: 183-198

Kim K.W., Shin J.H., Moon J., Kim M., Lee J., Park M.C. and Lee I. (2003) The function of the flowering time gene AGL20 is conserved in Crucifers. Mol. Cells 16: 136-141.

Kornneef M., Hanhart C.J. and Van der Veen J.H. (1991) A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol. Gen. Genet. 229: 57-66.

Kornneef M., Alonso-Blanco, Peters A.J. and Soppe W. (1998) Genetic control of flowering time in Arabidopsis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 345-370.

Liau C.H., Lu J.C., Prasad V., Hsiao H.H., You S.J., Lee J.T., Yang N.S., Huang H.E., Feng T.Y., Chen W.H., Chan M.T. (2003) The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid. Transgenic Res. 2: 329-36.

Lee I., Aukerman M.J., Gore S.L., Lohman K.N., Michaels S.D., Weaver L.M., John M.C., Feldman K.A. and Amasino R.M. (1994) Isolation of LUMINIDEPENDENS: a gene involved in the control of flowering time in Arabidopsis. Plant Cell 6: 75-83.

Lee H., Suh S.S., Park E., Cho E., Ahn J.H., Kim S.G., Lee J.S., Kwon Y.M. and Lee I. (2000) The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev. 14: 2366-2376.

Lee J.H., Hong S.M., Yoo S.J., Park O.K., Lee J.S. and Ahn J.H. (2006) Integration of floral inductive signals by flowering locus T and suppressor of overexpression of Constans1. Physiol. Plantarum. 126: 475–483.

Levy Y.Y. and Dean C. (1998) The transition to flowering. Plant Cell 10: 1973-1998.

Li Z., Zhao L., Cui C., Kai C., Zhang L., Sun X. and Tang K. (2005) Molecular cloning and characterization of an anti-bolting related gene (BrpFLC) from Brassica rapa ssp. Pekinensis. Plant Sci. 168:407-413.

Macnight R., Bancroft I., Page T., Lister C., Schmidt R., Love K., Westphal L., Murphy G., Sherson S., Cobbett C. and Dean C. (1997) FCA, a gene controlling flowering time in Arabidopsis, encodes a protein contaning RNA-binding domain. Cell 89: 737-745.

Menzel G., Apel K. and Melzer S. (1996) Identification of two MADS box genes that are expressed in the apical meristem of the long-day plant Sinapis alba in transition to flowering. Plant J. 9: 399-408.

Michaels S.D. and Amasino R.M. (1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11: 949-956.

Moon J., Lee H., Kim M. and Lee I. (2005) Analysis of flowering pathway integrators in Arabidopsis. Plant Cell Physiol. 46: 292-299.

O´Neill S.D., Nadeau J.A., Zhang X.S., Bui A.Q. and Halevy A.H. (1993) Interorgan regulation of ethylene biosynthetic genes by pollination. Plant Cell 5: 419-432.

Onouchi H., Igeno M.I., Perilleux C., Graves K. and Coupland G. (2000) Mutagenesis of Plants Overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes. Plant Cell 12: 885-900.

Park D.H., Somer D.E., Kim Y.S., Choy Y.H., Lim H.K., Soh M.S., Kim H.J., Kay S.A. and Nam H.G. (1999) Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science 285: 1579-1582.

Piechulla B., Merforth N. and Rudolph B. (1998) Identification of tomato Lhc promoter regions necessary for circadian expression. Plant Mol. Biol. 38: 655-662.

Putterill J., Robson F., Lee K., Simon R. and Coupland G. (1995) The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell 80: 847-857.

Putterill J., Laurie L. and Macknight R. (2004) It’s time to flower: the genetic control of flowering time. Bioessays 26: 363-373.
Roter G. B. (1959) The photoperiod and temperature responses of orchids. In: C. L. Wither(ed). The orchids- A Scientific Survey. NewYork. Ronald Press. p.397-417.

Samach A., Onouchi H., Gold S.E., Ditta G.S., Schwarz Z.S., Yanofsky M.F. and Coupland G. (2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288: 1613-1616.

Schmitz J., Franzen R., Ngyuen T.H., Garcia-Maroto F., Pozzi C., Salamini F. and Rohde W. (2000) Cloning, mapping and expression analysis of barley MADS-box genes. Plant Mol. Biol.42: 899-913.

Shannon S. and Meek-Waanger D.R. (1991) A mutant in the Arabidopsis TFL1 gene affects inflorescence meristem development. Plant Cell 3: 877-892.

Sheldon C.C., Rouse D.T., Finnegan E.J., Peacock W.J. and Dennis E.S. (2000) The molecular basis of vernalization: The central role of FLOWERING LOCUS C (FLC). Proc. Natl. Acad. Sci. U.S.A. 97: 3753-3758.

Simpson G.G. and Dean C. (2002) Arabidopsis, the Rosetta stone of flowering time? Science 296: 285-289.

Sung S. and Amasino R.M. (2004) Vernalization and epigenetics: how plants remember winter. Curr. Opin. Plant Biol. 7:4-10.

Sutoh K. and Yamauchi D. (2003) Two cis-acting elements necessary and sufficient for gibberellin-upregulated proteinase expression in rice seeds. Plant J. 34: 635-645.

Tadege M., Sheldon C.C., Helliwel C.A., Upadhyaya N.M., Dennis E.S. and Peacock W. J. (2003) Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice. Plant Biotechnol. J. 1: 361-369.

Tang W. and Perry S.E. (2003) Binding site selection for the plant MADS domain protein AGL15 an in vitro and in vivo study. J. Biol. Chem. 278: 28154-28159

Theissen G., Becker A., Rosa A.D., Kanno A., Kim J.T., Münster T., Winter K.U. and Saedler H. (2000) A short history of MADS-box genes in plants. Plant Mol. Boil. 42: 115-149.

Theissen G. and Saedler H. (2001) Floral quartets. Nature 409: 469-471.

Tilly J.J., Allen D.W. and Jack T. (1998) The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects. Development 125: 1647-1657.

Wang Z.Y., Kenigsbuch D., Sun L., Harel E., Ong M.S. and Tobin E.M.(1997) A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene. Plant Cell 9: 491-507.

Yoo S.K., Chung K.S., Kim J., Lee J.H., Hong S.M., Yoo S.J., Yoo S.Y., Lee J.S. and Ahn J.H. (2005) CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis. Plant Physiol. 139: 770-778.

2009-08-31公開

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