跳到主要內容

臺灣博碩士論文加值系統

(44.192.22.242) 您好!臺灣時間:2021/07/31 11:11
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:洪承裕
研究生(外文):Cheng-yu Hung
論文名稱:美女櫻花瓣中flavonoid3’,5’-hydroxylase(F3’,5’H)基因之選殖
論文名稱(外文):Molecular cloning of flavonoid 3’,5’-hydroxylase cDNA from the petals of Verbena x hybrida
指導教授:周昌弘周昌弘引用關係
指導教授(外文):Chang-hung Chou
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:69
中文關鍵詞:RACEPCR5''HF3''
外文關鍵詞:RACE5''HPCRF3''
相關次數:
  • 被引用被引用:2
  • 點閱點閱:172
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
Flavonoid 3’,5’-hydroxylase (F3’,5’H) 在花青素生合成途徑中扮演一關鍵酵素,催化反應產生藍色或紫色花瓣。本實驗以園藝作物美女櫻為材料,選殖其F3’,5’H 基因,以供給花色改良及花色調控之研究。根據已發表物種之F3’,5’H 基因序列,設計退化性引子,利用RT-PCR 方法選殖出預期片段,再配合5’及3’RACE (rapid amplification of cDNA ends) 策略,將全長F3’,5’H cDNA 序列選殖出,命名為VhFH1 (accesion number:AY604727);另外利用IPCR (inverse PCR) 策略進行F3’,5’H 基因組DNA之選殖,同時將基因組DNA 序列與cDNA 序列經排序對比後發現,F3’,5’HDNA 序列具有兩個introns 及1542 bp 的ORF (open reading frame),可轉譯成514 個胺基酸,並包含41 bp 的5’端非轉譯區域(UTR) 及58 bp 的3’UTR,且於基因上游發現可能為TATA box 序列的啟動子區域。將基因序列經NCBI 資料庫比對後,顯示其胺基酸序列與其他已發表之物種F3’,5’H序列有50-77﹪的相似度(identity) 。在預測蛋白質分析方面,VhFH1 的分子量為57 KD,等電點(pI) 為7.69,除了在N 端及C 端序列有較大差異外,其他序列部分皆有較高的相似度,並具備屬於細胞色素P450
(cytochrome P450) 家族所擁有的三個典型motifs,故推測此VhFH1 應為美女櫻F3’,5’H 基因。將此cDNA 序列構築於植物表現載體上,經轉型至大腸桿菌後,亦有RT-PCR 產物出現,未來更可轉殖於植物體上,以提供花色創新之原料。在不同品系下的美女櫻F3’,5’H 基因表現,由RT-PCR 顯示出藍色或紫色花品系之美女櫻皆具有此基因表現,而紅色花之美女櫻則缺乏,值得注意的是,在白色花品系中也有其表現,推測可能在後轉譯時期受到調控因子的抑制,才無法累積藍色色素,此現象提供了有利於美女櫻花色調控機制之研究。
Flavonoid 3’,5’-hydroxylase (F3’,5’H) is the key enzyme that catalyzesthe anthocyanin biosynthesis pathway for the expression of blue or purple flower color. The garden crops of Verbena x hybrida were used to clone its F3’,5’H gene for the investigation of flower color engineering and regulatory program. Degenerate primers were designed from the conservative regions of other published F3’,5’H genes to amplify the expectant DNA fragment . A full-length cDNA of the F3’,5’H gene designated VhFH1 (AY604727) was
cloned by the method of 5’and 3’ RACE, and its genomic DNA sequence was isolated by the IPCR strategy. Nucleotide sequence alignment revealed that VhFH1 contains two introns and a 1542 bp open reading frame encoding a polypeptide of 514 amino acid residues, and there could be a promoter
sequence with TATA box signal in the upstream of the transcription start site. The amino acid sequence of VhFH1 was compared with the previous reported F3’,5’H and showed between 50﹪and 77﹪identity with those species. The
expectant molecular mass and isoelectric point of VhFH1 protein is 57 KD and 7.69, respectively. There are three typical motifs of the F3’,5’H that belongs to
the cytochrome P450 proteins in the VhFH1 predicted protein. According to the above-mentioned conjecture, VhFH1 is a full-length cDNA of the F3’,5’H gene in the V. hybrida. This cDNA fragment was inserted into the plant expression vector pCambia 1304 and could be detected the expression in E. coli by RT-PCR and protein electrophoresis. It is practicable to transform the
horticultural plants with these vectors to create novel flower colors in the future. Furthermore, transcripts of the F3’,5’H gene were detected in the blue, purple
and white flowers but not in the red one as revealed by RT-PCR. These results are advantageous in the further investigation of regulatory factors of the
anthocyanin biosynthesis pathway in the V. hybrida.
文摘要----------------------------------------------------------------------------------1
英文摘要----------------------------------------------------------------------------------2
前言----------------------------------------------------------------------------------------3
前人研究----------------------------------------------------------------------------------6
一. 類黃酮及花青素的生化合成途徑-------------------------------------------6
二. 影響花色呈現之因子----------------------------------------------------------8
三. 影響花色的調節基因----------------------------------------------------------9
四. Flavonoid 3’,5’-hydroxylase 之介紹--------------------------------------11
五. 利用分子育種改變花色之可行性-----------------------------------------12
材料及方法-----------------------------------------------------------------------------14
一. 材料-----------------------------------------------------------------------------14
二. 方法-----------------------------------------------------------------------------15
(一) 總量RNA (total RNA) 之萃取---------------------------------------15
(二) 總量DNA (total DNA) 之萃取---------------------------------------15
(三) F3’,5’H 基因之選殖及分析--------------------------------------------16
1. F3’,5’H 退化性引子(degenerate primers) 之設計--------------16
2. 反轉錄酵素反應(reverse transcription reaction, RT) ----------16
3. cDNA聚合酵素連鎖反應(polymerase chain reaction)---------16
4. PCR 產物之回收及純化----------------------------------------------17
5. 接合反應(ligation) --------------------------------------------------17
6. 大腸桿菌的轉型作用(transformation)----------------------------17
7. 少量質體DNA 的抽取及殖入片段(insert) 之篩選------------18
8. F3’,5’H 基因片段之定序分析---------------------------------------18
9. 5’及3’ RACE 之引子設計及產物分析----------------------------19
(四) F3’,5’H genomic DNA 之選殖及分析-------------------------------191. IPCR (inverse PCR) 模板之製備---------------------------------19
2. IPCR 選殖分析-------------------------------------------------------20
3. Genomic DNA 之exon 及intron 之選殖-------------------------20
結果--------------------------------------------------------------------------------------25
一. 美女櫻F3’,5’H 基因選殖----------------------------------------------------25
二. 美女櫻F3’,5’H 之genomic DNA 選殖------------------------------------27
三. 美女櫻F3’,5’H 基因表現----------------------------------------------------30
討論--------------------------------------------------------------------------------------41
一. 美女櫻F3’,5’H cDNA 選殖方法------------------------------------------41
二. 美女櫻F3’,5’H cDNA基因確認及分析----------------------------------42
三. 美女櫻F3’,5’H genomic DNA 及cDNA 序列之分析------------------52
四. 不同美女櫻品系中F3’,5’H 之表現---------------------------------------53
五. 植物表現載體之建構--------------------------------------------------------53
六. 未來展望-----------------------------------------------------------------------56
參考文獻--------------------------------------------------------------------------------57
參考文獻
Aida, R., Kishimoto, S., Tanaka, Y., and Shibata, M. 2000. Modification of
flower color in torenia (Torenia fournieri Lind.) by genetic transformation.
Plant Sci. 153:33-42.
Aida, R., Yoshida, K., Kondo, T., Kishimoto, S., and Shibata, M. 2000.
Copigmentation gives bluer flowers on transgenic torenia plants with the
antisense dihydroflavonol-4-reductase gene. Plant Sci. 160:49-56.
Bloomquist, B. T., Johnson, R. C., and Mains, R. E. 1992. Rapid isolation of
flanking genomic DNA using biotin-RAGE, a variation of single-sided
polymerase chain reaction. DNA Cell Biol. 11:791-797.
Bozak, K. R., Yu, H., Sirevag, R., and Christoffersen, R. E. 1990. Sequence
analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit. Proc.
Natl. Acad. Sci. 87:3904-3908.
Brugliera, F., Barri-Rewell, G., Holton, T. A., and Mason, J. G. 1999.
Isolation and characterization of a flavonoid 3''-hydroxylase cDNA clone
corresponding to the Ht1 locus of Petunia hybrida. Plant J. 19:441-451.
Chapple, C. 1998. Molecular-genetic analysis of plant cytochrome
P450-dependent monooxygenases. Annu. Rev. Plant Physiol. Plant Mol. Biol.
49:311-343.
Consonni, G., Geuna, F., Gavazzi, G., and Tonelli, C. 1993. Molecular
homology among members of the R gene family from maize. Plant J.
3:335-346.
de Vettern, N., Quattrocchio, F., Mol, J., and Koes, R. 1997. The an11 locuscontrolling flower pigmentation in petunia encodes a novel WD-repeat protein
conserved in yeast, plants and animals. Genes Dev. 11:1422-1434.
de Vetten, N., ter Horst, J., van Schaik, H. P., de Boer, A., Mol, J., and Koes,
R. 1999. A cytochrome b5 is required for full activity of flavonoid 3'',
5''-hydroxylase, a cytochrome P450 involved in the formation of blue flower
colors. Proc. Natl. Acad. Sci. USA. 96:778-783.
Doodeman, M., Tabak, A. J. H., Schram, A. W., and Bennink, G. J. H. 1982.
Hydroxylation of cinnamic acids and flavonoids during biosynthesis of
anthocyanins in Petunia hybrida. Hortic. Planta 154:546-549.
Dooner, H. K., Robbins, T., and Jorgensen, R. A. 1991. Genetic and
developmental control of anthocyanin biosynthesis. Annu. Rev. Genet.
25:173-199.
Forkmann, G. and Ruhnau, B. 1987. Distinct substrate specificity of
dihydroflavonol 4-reductase from flowers of Petunia hybrida. Z. Naturforsch
42c:1146-1148.
Forkmann, G. 1991. Flavonoidas flower pigments:The formation of the
natural spectrum and its extension by genetic engineering. Plant
Breed.106:1-26.
Forkmann, G. and Martens, S. 2001. Metabolic engineering and application
of flavonoid. Curr. Opin. Biotech.12:155-160.
Giusti, M. M. and Wrolstad, R. E. 2003. Acylated anthocyanins from edible
sources and their applications in food systems. Biochem. Eng. J. 14:217-225.
Hannon, G. J. 2002. RNA interference. Nature 418:246-251.Harborne, J. B.1989. General procedures and measurement of total phenolics.
Method in plant biochemistry. Vol. 1. Harbone, J. B. ed. Academic Press.
U.S.A. pp. 1-28.
Harborne, J. and Williams, C. 2000. Advance in flavonoid research since.
Phytochemistry 55:481-504.
Helariutta,Y., Elomaa, P., Kotilainen, M., Seppanen,P., and Teerl, T.H.
1993. Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and
characterization of dfr expression in the corollase of Gerbera hybrida war.
Regina (Composites) . Plant Mol. Biol. 22:83-193
Holton, T. A., Brugilera, F., Lester, D. R., Tanaka, Y., Hyland, C., Menting,
J. G. T., Lu, C. Y., Farcy, E., Stevenson, T. W., and Cornish, E. C. 1993.
Cloning and expression of cytochrome P450 genes controlling flower colour.
Nature 366:276-279.
Holton, T., and Cornish, E. 1995. Genetic and biochemistry of anthocyanin
biosynthesis. Plant Cell 7:1071-1083.
Kaltenbach, M., Schroder, G., Schmelzer, E., Lutz, V., and Schroder, J.
1999. Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous
expression, enzyme properties and cell-type specific expression in plants. Plant
J. 19:183-193.
Kim, G. T., Tsukaya, H., and Uchimiya, H. 1998. The ROTUNDIFOLIA3
gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450
family that is required for the regulated polar elongation of leaf cells. Genes
Dev. 12:2381-2391.
Koes, R. E., Quattrocchio, F., and Mol, J. N. M. 1999. The flavonoidbiosynthetic pathway in plant:function and evolution. BioEssaya 16:123-132.
Koornneef, M. 1990. Mutations affecting the testa color in Arabidopsis.
Arabidopsis Inf. Serv. 28:1-4.
Landry, L. G., Chapple, C. C. S., and Last, R. L. 1995. Arabidopsis mutants
lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative
damage. Plant Physiol. 109:1159-1166.
Li, J., Ou-Lee, T. M., Raba, R., Amundson, R. G., and Last, R. L. 1993.
Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant
Cell 5:171-179.
Lloyd, A. M., Walbot, V., and Davis, R. W. 1992. Arabidopsis and Nicotiana
anthocyanin production activated by maize regulators R and C1. Science
258:1773-1775.
Martin, V. J., and Mohn, W. W. 1999. An alternative inverse PCR (IPCR)
method to amplify DNA sequences flanking Tn5 transposon insertions. J.
Microbiol. Meth. 35:163-166.
Matousek, J., Novak, P., Briza, J., Patzak, J., and Niedermeierova, H. 2002.
Cloning and characterization of chs-specific DNA and cDNA sequences from
hop (Humulus lupulus L.) . Plant Sci. 162:1007-1018.
Meyer, K., Cusumano, J. C., Somerville, C., and Chapple, C. C. 1996.
Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of
cytochrome P450-dependent monooxygenases. Proc. Natl. Acad. Sci. USA.
93:6898-6874.
Meyer, P., Heidmann, I., Forkmann, G., and Saedler, H. 1987. A newpetunia flower colour generated by transformation of a mutant with a maize
gene. Nature 330:677-678.
Mol, J., Jenkins, G., Schafer, E., and Weiss, D. 1996. Signal perception,
transduction, and gene expression involved in anthocyanin biosynthesis. Crit.
Rev. Plant Sci. 15:525-557.
Mol, J., Grotewold, E., and Koes, R. 1998. How genes paint flowers and
seeds. Trends Plant Sci. 3:212-217.
Mol, J., Cornish, E., Mason, J., and Koes, R. 1999. Novel coloured flowers.
Curr. Opin. Biotechnol. 10:198-201.
Mori, S., Kobayashi, H., Hoshi, Y., Kondo, M., and Nakano, M. 2004.
Heterologous expression of the flavonoid 3'',5''-hydroxylase gene of Vinca
major alters flower color in transgenic Petunia hybrida. Plant Cell Rep.
22:415-421.
Napoli, C., Lemieux, C., and Jorgensen, R. 1990. Introduction of a Chimeric
Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of
Homologous Genes in trans. Plant Cell 2:279-289.
Noda, K., Glover, B. J., Linstead, P., and Martin, C. 1994. Flower color
intensity depends on specialized cell shape controlled by a Myb-related
transcription factor. Nature 369:661-664.
Ochman, H., Gerber, A. S., and Hartl, D. L. 1998. Genetic applications of an
inverse polymerase chain reaction. Genetics 120:621-623.
Quattrocchio, F., Wing, J. F., Leppen, H. T. C., Mol, J. N. M., and Koes, R.
1993. Regulatory genes controlling anthocyanin pigmentation are functionallyconserved among plant species and have distinct sets of target genes. Plant Cell
5:1497-1512.
Quattrocchio, F. 1994. Regulatory genes controlling flower pigmentation in
Petunia hybrida. PhD thesis (Amsterdam: Vrije Universiteit) .
Quattrocchio, F., Wing, J. F., van der Woude, K., Mol, J. N. M., and Koes,
R. 1998. Analysis of bHLH and MYB domain proteins: Species-specific
regulatory differences are caused by divergent evolution of target anthocyanin
genes. Plant J. 13:475-488.
Robert, S. C., and Imre, E. S. 1997. Rapid amplification of genomic ends
(RACE) as a simple method to clone flanking genomic DNA. Gene
194:273-276.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn G.
T., Mullis, K. B., and Erlich, H. A. 1988. Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase. Science
239:487-491.
Shibata, K., Shibata, Y., and Kasiwagi, I. 1919. Studies on anthocyanins:
color variation in anthocyanins. J. Am. Chem. Soc. 41:208-220.
Shimada, Y., Nakano-Shimada, R., Ohbayashi, M., Okinaka, Y., Kiyokawa,
S., and Kikuchi, Y. 1999. Expression of chimeric P450 genes encoding
flavonoid 3’,5’-hydroxylase in transgenic tobacco and petunia plant. FEBS Lett.
461:241-245
Shimada, Y., Ohbayashi, M., Nakano-Shimada, R., Okinaka, Y., Kiyokawa,
S., and Kikuchi, Y. 2001. Genetic engineering of the anthocyanin biosynthetic
pathway with flavonoid-3’,5’-hydroxylase: specific switching of the pathwayin petunia. Plant Cell Rep. 20:456-462.
Spelt, C., Quattrocchio, F., Mol, J., and Koes, R. 2000. anthocyanin1 of
petunia encodes a basic-helix loop helix protein that directly activates structural
anthocyanin genes. Plant Cell 12:1619-1631.
Spelt, C., Quattrocchio, F., Mol, J., and Koes, R. 2002. ANTHOCYANIN1
of petunia controls pigment synthesis, vacuolar pH, and seed coat development
by genetically distinct mechanisms. Plant Cell 14:2121-2135.
Szekeres, M., Nemeth, K., Koncz-Kalman, Z., Mathur, J., Kauschmann, A.,
Altmann, T., Redei, G. P., Nagy, F., Schell, J., and Koncz, C. 1996.
Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450,
controlling cell elongation and de-etiolation in Arabidopsis. Cell 85:171-182.
Tanaka, Y., Fukul, Y., Fukuchi-Mizutani. M., Holton, T., Higgins, E., and
Kusumi, T. 1995. Molecular cloning and characterization of Rosa hybrids
dihydroflavonol-4-reductase gene. Plant Cell Physiol. 36:1025-1031.
Tanaka, Y., Tsuda, S. and Kusumi, T. 1998. Metabolic engineering to modify
flower color. Plant Cell Physiol. 39:1119-1126
Walker, A. R., Davison, P. A., Bolognesi-Winfield, A. C., James, C. M.,
Srinivasan, N., Blundell, T. L., Esch, J. J., Marks, M. D., and Gray, J. C.
1999. The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome
differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40
repeat protein. Plant Cell 11:1337-1350.
Wang, H., Cao, G., and Prior, R. L. 1997. Oxygen radical absorbing capacity
of anthocyanins. J. Agric. Food Chem. 45 (2):1612-1615.Winkler, R. G. and Helentjaris, T. 1995. The mazie Dwarf3 gene encodes a
cytochrome P450-mediated early step in gibberellin biosynthesis. Plant Cell 7
(8):1307-1317.
Ueyama, Y., Suzuki, K., Fukuchi-Mizutani, M., Fukui, Y., Miyazaki, K.,
Ohkawa, H., Kusumi, T., and Tanaka, Y. 2002. Molecular and biochemical
characterization of torenia flavonoid 3’-hydroxylase and flavone synthase II
and modification of flower color by modulating the expression of these genes.
Plant Sci. 163:253-263.
Van Houwelingen, A., Souer, E., Spelt, K., Kloos, D., Mol, J., and Koes, R.
1998. Analysis of flower pigmentation mutants generated by random
transposon mutagenesis in Petunia hybrida. Plant J. 13:39-50.
Yoshida, K., Kondo, T., Okazaki, Y., and Katou, K. 1995. Cause of blue
petal colour. Nature 373:291.
吳宜娟2000. 大豆種皮flavonoid 3’,5’-hydroxylase (F3’,5’H) 基因之選殖
與基因表現分析。國立中興大學農業生物科技學研究所碩士論文。
謝文蘋2000. 非洲菊花色和類黃素(flavonoid) 的遺傳。國立嘉義大學農
學院園藝學系二技專題討論報告。
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top