中国水仙花色相关基因ZDS、LYCE的克隆
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摘要
中国水仙(Narcissus tazetta Lin.var.chinensis Roem)为石蒜科水仙属植物,多年生草本,是多花水仙的一个变种。福建漳州是中国水仙主要产地之一,漳州水仙于2008年获得国家地理标志驰名商标。目前,花色单一己成为制约中国水仙发展的主要障碍之一,因此,对中国水仙进行花色改良具有重大的意义。
本研究以漳州水仙品种‘金盏银台’为材料,采用RT-PCR和RACE技术,克隆得出了中国水仙ZDS(ζ-胡萝卜素脱氢酶基因)cDNA全长序列和LYCE(番茄红素ε-环化酶基因)片段序列,并对ZDS进行原核表达研究,为下一步利用花色基因进行遗传转化奠定基础,以期为中国水仙花色遗传改良提供一条新途径。主要研究结果如下:
1、提取中国水仙花瓣和副冠的总RNA,反转录为cDNA二链,并以此为模板克隆类胡萝卜素合成途径中的关键基因ZDS(登录号为:EU573238),ZDS全长2189 bp,其中包含69 bp5′非编码区,395 bp3′非编码区,1725 bp的编码区(编码574个氨基酸,分子量约为63.6 kDa)。通过BLAST工具对比发现,该ZDS氨基酸序列与黄水仙(CAA12062.1)、葡萄(CAO43021.1)和蜜柑(BAB68552.1)的ZDS基因cDNA的同源性都很高,分别为97%、85%和83%。
2、将分离的中国水仙ZDS基因cDNA的编码区序列插入含有EcoRI和XhoI酶切位点的pET-28a原核表达载体上,并在大肠杆菌BL21细胞中进行原核表达,结果表明,该编码区片段在大肠杆菌中表达,分子量约为63.6 kDa的蛋白质,与理论值相符。
3、采用RACE方法获得LYCE cDNA片段序列长1235 bp,编码371个氨基酸残基,其推导的氨基酸序列与胡萝卜(ABB52073.1)、柚子(AAX92679.1)、甜橙(AAS48096.1)和温州蜜桔(BAE93359.1)的氨基酸序列同源性分别为80%、76%、76%和75%。
Narcissus tazetta L. var. chinensis Roem, a kind of perennial plant with beautiful color and sweet fragrance, belongs to the family Amaryllidaceae. Zhangzhou which is in FuJian province is the most important produce area of Chinese narcissus and the Zhangzhou narcissus have gotten a National Geographical Indication of well-known trademarks in 2008. But the simplex color have become a main obstacle for Chinese narcissus’s development. So it's imperative to develop new varieties with modern breeding methods for the narcissus industry.
In this research, we isolated the full length cDNA of Zeta-carotene desaturase gene (ZDS) and a partial sequence of Lycopene epsilon cyclase gene(LYCE) from Chinese narcissus petals by RT-PCR and RACE and tried to express ZDS in E.coli. This laid a foundation for its application on subsequent genetic transformation study, which aims to provide a new way to the genetic improvement of the color of Chinese narcissus.
1、The RNA extracted from Narcissus flower petal was used for the isolation of ZDS by RT-PCR (GenBank accession number: EU573238). The results of analysis of the coding sequences of the genes demonstrated that the gene of ZDS is 2189bp long. It contains an ORF encoding 499 amino acid residues(Mw=63.6kDa), a 5’untranslated region of 69bp and a 3’untranslated region of 395bp. Compared with ZDSs of other plants, Chinese narcissus’s amino acid sequence had 97%, 85%, 83% identity with that of Narcissus pseudonarcissus, Vitis vinifera and Citrus unshiu.
2、The ZDS gene cDNA was insert into the vevtor pET-28a degisted by EcoR I and Xoh I. The expression construct was transformed into BL21 and was indued. The result displayed that the ZDS was expressed in E.coli. And the SDS-PAGE experiments show that the actual value of ZDS molecular weight agrees well with the theoretical one.
3、The partial sequence of LYCE isolated from Chinese narcissus by RACE is 1235bp long, encoding 371 amino acid residues. It’s amino acid sequence had 80%, 76%, 76%, 75% identity with that of Daucus carota, Citrus maxima, Citrus sinensis and Citrus unshiu.
本研究以漳州水仙品种‘金盏银台’为材料,采用RT-PCR和RACE技术,克隆得出了中国水仙ZDS(ζ-胡萝卜素脱氢酶基因)cDNA全长序列和LYCE(番茄红素ε-环化酶基因)片段序列,并对ZDS进行原核表达研究,为下一步利用花色基因进行遗传转化奠定基础,以期为中国水仙花色遗传改良提供一条新途径。主要研究结果如下:
1、提取中国水仙花瓣和副冠的总RNA,反转录为cDNA二链,并以此为模板克隆类胡萝卜素合成途径中的关键基因ZDS(登录号为:EU573238),ZDS全长2189 bp,其中包含69 bp5′非编码区,395 bp3′非编码区,1725 bp的编码区(编码574个氨基酸,分子量约为63.6 kDa)。通过BLAST工具对比发现,该ZDS氨基酸序列与黄水仙(CAA12062.1)、葡萄(CAO43021.1)和蜜柑(BAB68552.1)的ZDS基因cDNA的同源性都很高,分别为97%、85%和83%。
2、将分离的中国水仙ZDS基因cDNA的编码区序列插入含有EcoRI和XhoI酶切位点的pET-28a原核表达载体上,并在大肠杆菌BL21细胞中进行原核表达,结果表明,该编码区片段在大肠杆菌中表达,分子量约为63.6 kDa的蛋白质,与理论值相符。
3、采用RACE方法获得LYCE cDNA片段序列长1235 bp,编码371个氨基酸残基,其推导的氨基酸序列与胡萝卜(ABB52073.1)、柚子(AAX92679.1)、甜橙(AAS48096.1)和温州蜜桔(BAE93359.1)的氨基酸序列同源性分别为80%、76%、76%和75%。
Narcissus tazetta L. var. chinensis Roem, a kind of perennial plant with beautiful color and sweet fragrance, belongs to the family Amaryllidaceae. Zhangzhou which is in FuJian province is the most important produce area of Chinese narcissus and the Zhangzhou narcissus have gotten a National Geographical Indication of well-known trademarks in 2008. But the simplex color have become a main obstacle for Chinese narcissus’s development. So it's imperative to develop new varieties with modern breeding methods for the narcissus industry.
In this research, we isolated the full length cDNA of Zeta-carotene desaturase gene (ZDS) and a partial sequence of Lycopene epsilon cyclase gene(LYCE) from Chinese narcissus petals by RT-PCR and RACE and tried to express ZDS in E.coli. This laid a foundation for its application on subsequent genetic transformation study, which aims to provide a new way to the genetic improvement of the color of Chinese narcissus.
1、The RNA extracted from Narcissus flower petal was used for the isolation of ZDS by RT-PCR (GenBank accession number: EU573238). The results of analysis of the coding sequences of the genes demonstrated that the gene of ZDS is 2189bp long. It contains an ORF encoding 499 amino acid residues(Mw=63.6kDa), a 5’untranslated region of 69bp and a 3’untranslated region of 395bp. Compared with ZDSs of other plants, Chinese narcissus’s amino acid sequence had 97%, 85%, 83% identity with that of Narcissus pseudonarcissus, Vitis vinifera and Citrus unshiu.
2、The ZDS gene cDNA was insert into the vevtor pET-28a degisted by EcoR I and Xoh I. The expression construct was transformed into BL21 and was indued. The result displayed that the ZDS was expressed in E.coli. And the SDS-PAGE experiments show that the actual value of ZDS molecular weight agrees well with the theoretical one.
3、The partial sequence of LYCE isolated from Chinese narcissus by RACE is 1235bp long, encoding 371 amino acid residues. It’s amino acid sequence had 80%, 76%, 76%, 75% identity with that of Daucus carota, Citrus maxima, Citrus sinensis and Citrus unshiu.
引文
[1]庄晓英.中国水仙遗传转化及离体诱变体系的研究[D].浙江大学硕士学位论文, 2005, 5
[2]张石宝,胡虹,李树云.花卉基因工程研究进展I:花色[J].云南植物研究, 2001, 23(4): 479-487
[3]邹清成.中国水仙花色相关基因的分离和反义Psy基因的遗传转化[D].浙江大学硕士论文, 2006, 7
[4]陈振光,邱雪芝,林庆良,等.中国水仙试管育苗研究[J].福建农学院学报, 1986, 15(3): 204-210
[5]葛欣,赵宇,张建军.花卉的品质改良与反义RNA技术[J].北方园艺, 131(18): 35
[6]郑志亮, 1994.花卉作物的花色基因工程[J].北方园艺, 3: 37
[7] Dooner H K, Robbins T P. Genetic and developmental control of anthocyanin biosynthesis [J]. Ann Rev Genet, 1991, 25: 173-199
[8] Meyer P, Heidmann I, Forkmann G, et al. A new petunia flower color generated by transformation of a mutant with a maize gene[J]. Nature, 1987, 330: 677-687
[9] Mol J N M, Holton T A, Koes R E. Genetic engineering of commercial traits of floral crops[J]. Trends Biotechnol, 1995, 13: 350-355
[10] Tanaka Y, Tauda S, Kusuni T. Metabolic engineering to modify flower color[J]. Plant Cell Physiol. 1998, 11: 1119-1126
[11] Van der krol A R, Lenting R J, Veenstra J G, et al. An antisense chalone synthase gene in transgenetic plants inhibits flower pigmentation [J]. Nature, 1988, 333: 860-869
[12] Lu C Y, Nugent G, Terse W R, et al. Agrobacterium-medicatd tannsformation of carnation ( Dianthus carryophyllus L. )[J]. Bio Technology, 1991, 9: 864-868
[13] Aanhane T, Affrs D R, Lorigene B VF, et al. First rDNA flower to debut in Australia this summer. Biotechnology News[J]. 1995, 15 (14): 3-7
[14] Robinson KEP, Firoozabady E, Transformation of floriculture crops[J]. Sci Horticul, 1993, 55: 83-99
[15]储文华, 1996.用根癌农杆菌在丝石竹进行基因转化[J].上海师范大学学报(自然科学版), 25(4): 90-91
[16]朱长甫,陈星,王英典.植物类胡萝卜素生物合成及其相关基因在基因工程中的应用[J].植物生理与分子生物学学报, 2004, 06
[17] Bartley GE, Scolnik PA. Plant carotenoids: Pigments for photoprotection, visual attraction, and human health[J]. Plant Cell, 1995, 7: 1027-1038
[18] Goodwin TW, Britton G. Distribution and analysis of canotenoids in: Goodwin TW (Ed), Plant Pigments(Academic Press), 1988, 61-132
[19]黄彬城,季静,王罡,郑阳霞.植物类胡萝卜素的研究进展[J].天津农业科学, 2006, 12(02): 13-17
[20] Cunningham FX Jr, Gantt E. Genes and enzymes of carotenoid biosynthetic pathway in plants[J]. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49: 557-583
[21]陈段芬,彭镇华,高志民.中国水仙八氢番茄红素脱氢酶基因(PDS)的克隆及表达分析[J].分子植物育种, 2008, 6(03):574-578
[22] Hirschberg J, Cohen M, Harker M, et al. Molecular genetics of the carotenoid biosynthesis pathway in plants and algae[J]. Pure Appl Chem, 1997, 69: 2151-2158
[23]陈德欣.红肉蜜柚β-胡萝卜素合成途径相关酶基因的克隆[D].福建农林大学硕士学位论文, 2007, 6
[24] Cunningham F, Pogson B, Sun Z, et al. Functional analysis of theβandεlycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation[J]. Plant Cell, 1996, 8: 1613-1626
[25]徐昌杰,张上隆,植物类胡萝卜素的生物合成及其调控[J].植物生理学通讯, 2000, 36(1): 64-70
[26] Kumagai M H, ellerouvier F, et al. Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana [J]. Plant, 1998, 14: 305-315
[27] Sage D O, Lynn J, Hammatt N. Somatic embryogenesis in Narcissus pseudonarcissus cvs. Golden Harvest and St Keverne[J]. Plant Sci, 2000, 150: 209-216
[28]黄胤怡,沈明山,陈亮,等.中国水仙查尔酮合酶cDNA的克隆及序列分析(简报)[J].试验生物学报, 2002, 35(3): 195-197
[29]叶祖云,郑丽屏,贺静,等.中国水仙(Narcissus tazetta Var. chinensis)基因转化受体系统的建立[J].云南大学学报(自然科学版), 2001, 23(3): 235-237
[30] Bartley G. E, Ishida B. K. Zeta-carotene desaturase from tomato[J]. Plant Physiol, 1999, 121 (4): 1383
[31] Albrecht M., Klein A., Hugueney P., et al. Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating zeta-carotene desaturation[J]. FEBS Lett, 1995, 372 (2-3): 199-202
[32]陈选阳,林世强,袁照年,等.甘薯ZDS基因的克隆与植物表达载体构建[J].福建农林大学学报(自然科学版), 2005(34): 473-477
[33]季静,山村三郎,西源昌宏,等,通过转基因提高β-胡萝卜素生物合成量[J].中国生物化学与分子生物学报, 2004, 20(8):440-444
[34]任永霞,季静,王萍,等.农杆菌介导类胡萝卜素合成酶基因LycB转化菊花的研究[J].吉林农业大学学报, 2005, 27 (3): 255-258
[35]徐晋麟等.现代遗传学原理(修订版)[M],科学出版社, 2003
[36]陆颖,高晨曦,韩斌.水稻mRNA多聚腺苷化信号位点的序列分析[J].科学通报, 2006, 51(7): 819-826
[37] Proudfoot N. Poly(A) signals[J]. Cell, 1991, 64: 671-674
[38] Graber J H, Cantor C R, Mohr S C, et al. In silico detection of control signals: mRNA 3′-end-processing sequences in diverse species[J]. Proc Natl Acad Sci USA, 1999, 96: 14055-14060
[39] Lokc J C, Stahlberg E A, Strenski D G, et al. Compilation of mRNA polyadenylation signals in Arabidopsis revealed a new signal element and potential secondary structures[J]. Plant Physiol, 2005, 138: 1457-1468
[40] Li Q, Hunt A G. A near-upstream element in a plant polyadenylation signal consists of more than six nucleotides[J]. Plant Mol Biol, 1995, 28: 927-934
[41] J萨姆布鲁克, D W拉塞尔.分子克隆实验指南.黄培堂.第3版[M].北京:科学出版社, 2002: 644-651, 1217-1270
[2]张石宝,胡虹,李树云.花卉基因工程研究进展I:花色[J].云南植物研究, 2001, 23(4): 479-487
[3]邹清成.中国水仙花色相关基因的分离和反义Psy基因的遗传转化[D].浙江大学硕士论文, 2006, 7
[4]陈振光,邱雪芝,林庆良,等.中国水仙试管育苗研究[J].福建农学院学报, 1986, 15(3): 204-210
[5]葛欣,赵宇,张建军.花卉的品质改良与反义RNA技术[J].北方园艺, 131(18): 35
[6]郑志亮, 1994.花卉作物的花色基因工程[J].北方园艺, 3: 37
[7] Dooner H K, Robbins T P. Genetic and developmental control of anthocyanin biosynthesis [J]. Ann Rev Genet, 1991, 25: 173-199
[8] Meyer P, Heidmann I, Forkmann G, et al. A new petunia flower color generated by transformation of a mutant with a maize gene[J]. Nature, 1987, 330: 677-687
[9] Mol J N M, Holton T A, Koes R E. Genetic engineering of commercial traits of floral crops[J]. Trends Biotechnol, 1995, 13: 350-355
[10] Tanaka Y, Tauda S, Kusuni T. Metabolic engineering to modify flower color[J]. Plant Cell Physiol. 1998, 11: 1119-1126
[11] Van der krol A R, Lenting R J, Veenstra J G, et al. An antisense chalone synthase gene in transgenetic plants inhibits flower pigmentation [J]. Nature, 1988, 333: 860-869
[12] Lu C Y, Nugent G, Terse W R, et al. Agrobacterium-medicatd tannsformation of carnation ( Dianthus carryophyllus L. )[J]. Bio Technology, 1991, 9: 864-868
[13] Aanhane T, Affrs D R, Lorigene B VF, et al. First rDNA flower to debut in Australia this summer. Biotechnology News[J]. 1995, 15 (14): 3-7
[14] Robinson KEP, Firoozabady E, Transformation of floriculture crops[J]. Sci Horticul, 1993, 55: 83-99
[15]储文华, 1996.用根癌农杆菌在丝石竹进行基因转化[J].上海师范大学学报(自然科学版), 25(4): 90-91
[16]朱长甫,陈星,王英典.植物类胡萝卜素生物合成及其相关基因在基因工程中的应用[J].植物生理与分子生物学学报, 2004, 06
[17] Bartley GE, Scolnik PA. Plant carotenoids: Pigments for photoprotection, visual attraction, and human health[J]. Plant Cell, 1995, 7: 1027-1038
[18] Goodwin TW, Britton G. Distribution and analysis of canotenoids in: Goodwin TW (Ed), Plant Pigments(Academic Press), 1988, 61-132
[19]黄彬城,季静,王罡,郑阳霞.植物类胡萝卜素的研究进展[J].天津农业科学, 2006, 12(02): 13-17
[20] Cunningham FX Jr, Gantt E. Genes and enzymes of carotenoid biosynthetic pathway in plants[J]. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49: 557-583
[21]陈段芬,彭镇华,高志民.中国水仙八氢番茄红素脱氢酶基因(PDS)的克隆及表达分析[J].分子植物育种, 2008, 6(03):574-578
[22] Hirschberg J, Cohen M, Harker M, et al. Molecular genetics of the carotenoid biosynthesis pathway in plants and algae[J]. Pure Appl Chem, 1997, 69: 2151-2158
[23]陈德欣.红肉蜜柚β-胡萝卜素合成途径相关酶基因的克隆[D].福建农林大学硕士学位论文, 2007, 6
[24] Cunningham F, Pogson B, Sun Z, et al. Functional analysis of theβandεlycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation[J]. Plant Cell, 1996, 8: 1613-1626
[25]徐昌杰,张上隆,植物类胡萝卜素的生物合成及其调控[J].植物生理学通讯, 2000, 36(1): 64-70
[26] Kumagai M H, ellerouvier F, et al. Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana [J]. Plant, 1998, 14: 305-315
[27] Sage D O, Lynn J, Hammatt N. Somatic embryogenesis in Narcissus pseudonarcissus cvs. Golden Harvest and St Keverne[J]. Plant Sci, 2000, 150: 209-216
[28]黄胤怡,沈明山,陈亮,等.中国水仙查尔酮合酶cDNA的克隆及序列分析(简报)[J].试验生物学报, 2002, 35(3): 195-197
[29]叶祖云,郑丽屏,贺静,等.中国水仙(Narcissus tazetta Var. chinensis)基因转化受体系统的建立[J].云南大学学报(自然科学版), 2001, 23(3): 235-237
[30] Bartley G. E, Ishida B. K. Zeta-carotene desaturase from tomato[J]. Plant Physiol, 1999, 121 (4): 1383
[31] Albrecht M., Klein A., Hugueney P., et al. Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating zeta-carotene desaturation[J]. FEBS Lett, 1995, 372 (2-3): 199-202
[32]陈选阳,林世强,袁照年,等.甘薯ZDS基因的克隆与植物表达载体构建[J].福建农林大学学报(自然科学版), 2005(34): 473-477
[33]季静,山村三郎,西源昌宏,等,通过转基因提高β-胡萝卜素生物合成量[J].中国生物化学与分子生物学报, 2004, 20(8):440-444
[34]任永霞,季静,王萍,等.农杆菌介导类胡萝卜素合成酶基因LycB转化菊花的研究[J].吉林农业大学学报, 2005, 27 (3): 255-258
[35]徐晋麟等.现代遗传学原理(修订版)[M],科学出版社, 2003
[36]陆颖,高晨曦,韩斌.水稻mRNA多聚腺苷化信号位点的序列分析[J].科学通报, 2006, 51(7): 819-826
[37] Proudfoot N. Poly(A) signals[J]. Cell, 1991, 64: 671-674
[38] Graber J H, Cantor C R, Mohr S C, et al. In silico detection of control signals: mRNA 3′-end-processing sequences in diverse species[J]. Proc Natl Acad Sci USA, 1999, 96: 14055-14060
[39] Lokc J C, Stahlberg E A, Strenski D G, et al. Compilation of mRNA polyadenylation signals in Arabidopsis revealed a new signal element and potential secondary structures[J]. Plant Physiol, 2005, 138: 1457-1468
[40] Li Q, Hunt A G. A near-upstream element in a plant polyadenylation signal consists of more than six nucleotides[J]. Plant Mol Biol, 1995, 28: 927-934
[41] J萨姆布鲁克, D W拉塞尔.分子克隆实验指南.黄培堂.第3版[M].北京:科学出版社, 2002: 644-651, 1217-1270
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