龙胆八氢番茄红素合成酶（PSY）基因的克隆与功能分析
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摘要
类胡萝卜素是一系列呈现黄色、橙色和红色的萜类色素物质,存在于所有的光合器官中并具有抗氧化的特性,但动物体内不能直接合成类胡萝卜素,必须从食物中摄取。近年研究表明,除了含有β-紫罗酮环的类胡萝卜素是维生素A的前体外,许多其他类胡萝卜素在增加动物营养、增强人体免疫力等方面起到重要作用,另外由于它能清除细胞内的活性氧自由基,所以类胡萝卜素在防癌抗癌方面的功效也越来越受到重视。八氢番茄红素合成酶是催化类胡萝卜素生物合成的一个特异性酶,它将2分子的牻牛儿牻牛儿焦磷酸(GGPP)催化合成第一个类胡萝卜素——八氢番茄红素。研究表明八氢番茄红素合成酶基因是类胡萝卜素生物合成途径中的限速酶。在番茄、油菜、拟南芥和马铃薯中过量表达八氢番茄红素合成酶,相应组织中积累类胡萝卜素的水平显著提高,在番茄果实中提高了1.9倍,拟南芥种子中β-胡萝卜素平均提高43倍,油菜种子中提高50倍,马铃薯块茎中提高了8倍。
本研究应用分子生物学技术从龙胆(Gentiana lutea)花瓣cDNA文库中克隆到八氢番茄红素合成酶2和3(GlPSY2和GlPSY3)基因(cDNA),经序列比对分析发现其与GlPSY1基因有高度同源性但又不完全相同。GlPSY2 cDNA核苷酸序列总长度为1923 bp,含有一段1275 bp的不间断开放阅读框(ORF),497 bp的5′末端和151 bp的3′末端非编码区,在位于1770 bp的终止密码子(TAA)与多聚腺苷酸之间有poly(A)添加信号(AATAA),位于1833-1837 bp。编码区起始于498 bp处起始密码子(ATG),ORF编码一个含424个氨基酸残基的蛋白质,分子量为48298.65 D,等电点为8.79。GlPSY3 cDNA核苷酸序列总长度为1765 bp,含有一段1278 bp的不间断开放阅读框(ORF),399 bp的5′末端和88 bp的3′末端非编码区,在位于1675 bp的终止密码子(TGA)与多聚腺苷酸之间有poly(A)添加信号(GATAAA),位于1745-1750 bp。编码区起始于399 bp处起始密码子(ATG),ORF编码一个含425个氨基酸残基的蛋白质,分子量为48399.75 D,等电点为8.79。
构建pUC-GlPSY2和pUC-GlPSY3表达载体,将pUC-PSY质粒与缺失八氢番茄红素合成酶(CrtB)基因的工程大肠杆菌pACCAR25ΔCrtB共转化进行互补实验,得到呈现出明显黄色的单克隆菌株,通过高效液相色谱分析,鉴定转化菌株的代谢产物为玉米黄质等类胡萝卜素。另外,我们将pUC-PSY质粒转入工程大肠杆菌pACCrtE(在大肠杆菌中可合成牻牛儿牻牛儿焦磷酸,GGPP)中。通过高效液相色谱分析,鉴定转化菌株经过量表达得到的代谢产物为八氢番茄红素,证明GlPSY2和GlPSY3 cDNA编码的蛋白都具有八氢番茄红素合成酶活性。
Carotenoids are one of most abundant classes of natural compounds, often highly colored with yellow, orange or red, derived from isoprenoids. They are synthesized in all photosynthetic organisms, as well as some bacteria, fungi and algae. They accumulate in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Carotenoids have fundamental roles in human nutrition as antioxidants and vitamin A precursors. It has been demonstrated their consumption is increasing associated with protection from a diverse of diseases. Animals and humans are unable to synthesize carotenoids de novo and must depend on dietary carotenoids. Thus they must acquire them from their diet. The first committed step in the plastid-localized carotenoid biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). It has been demonstrated PSY is the key enzyme limiting in carotenoid biosynthesis in tomato fruit, canola seeds, potato tubers, Arabidopsis thaliana seeds, Gentiana lutea flower petals and maize endosperm. The sole overexpression of phytoene synthase has a potent effect on carotenoid levels in storage organs generally, e.g. resulting in a 1.9-fold increase in tomato fruits, a 50-fold increase in canola seeds, a 43-fold increase in Arabidopsis seeds and a 8-fold increase in potato tubers.
In the present study, the phytoene synthase (PSY) 2 and 3 cDNAs were cloned from flower petal cDNA library of Gentiana lutea. The G. lutea PSY2 (GlPSY2) and PSY3 (GlPSY3) cDNA information were deposited in NCBI (National Center for Biotechnology Information) GenBank (Accession numbers: EF203259 and EF203260, respectively). The full-length of nucleotide sequence of GlPSY2 cDNA consisted of 1923 bp with a 1275 bp open reading frame (ORF), a 497 bp 5' terminal and a 151 bp 3' terminal non-coding regions, respectively. The deduced amino acid sequence of GlPSY2 cDNA was a polypeptide of 424 amino acid residues with molecular weight of 48298.65 D. The full-length of nucleotide sequence of GlPSY3 cDNA was 1765 bp with a 1278 bp open reading frame (ORF), 399 bp 5' terminal and 88 bp 3' terminal non-coding regions, respectively. The deduced amino acid sequence of GlPSY3 cDNA was a polypeptide of 424 amino acid residues with molecular weight of 48399.75 D. The isoelectric point of both of GlPSY2 and GlPSY3 was 8.79. The GlPSY2 and GlPSY3 amino acid sequences have higher identities with known functional PSY sequences from higher plants. The indicated function of G. lutea PSY2 and PSY3 cDNAs was estabilished by heterologous complementation in engineering Escherichia coli.
本研究应用分子生物学技术从龙胆(Gentiana lutea)花瓣cDNA文库中克隆到八氢番茄红素合成酶2和3(GlPSY2和GlPSY3)基因(cDNA),经序列比对分析发现其与GlPSY1基因有高度同源性但又不完全相同。GlPSY2 cDNA核苷酸序列总长度为1923 bp,含有一段1275 bp的不间断开放阅读框(ORF),497 bp的5′末端和151 bp的3′末端非编码区,在位于1770 bp的终止密码子(TAA)与多聚腺苷酸之间有poly(A)添加信号(AATAA),位于1833-1837 bp。编码区起始于498 bp处起始密码子(ATG),ORF编码一个含424个氨基酸残基的蛋白质,分子量为48298.65 D,等电点为8.79。GlPSY3 cDNA核苷酸序列总长度为1765 bp,含有一段1278 bp的不间断开放阅读框(ORF),399 bp的5′末端和88 bp的3′末端非编码区,在位于1675 bp的终止密码子(TGA)与多聚腺苷酸之间有poly(A)添加信号(GATAAA),位于1745-1750 bp。编码区起始于399 bp处起始密码子(ATG),ORF编码一个含425个氨基酸残基的蛋白质,分子量为48399.75 D,等电点为8.79。
构建pUC-GlPSY2和pUC-GlPSY3表达载体,将pUC-PSY质粒与缺失八氢番茄红素合成酶(CrtB)基因的工程大肠杆菌pACCAR25ΔCrtB共转化进行互补实验,得到呈现出明显黄色的单克隆菌株,通过高效液相色谱分析,鉴定转化菌株的代谢产物为玉米黄质等类胡萝卜素。另外,我们将pUC-PSY质粒转入工程大肠杆菌pACCrtE(在大肠杆菌中可合成牻牛儿牻牛儿焦磷酸,GGPP)中。通过高效液相色谱分析,鉴定转化菌株经过量表达得到的代谢产物为八氢番茄红素,证明GlPSY2和GlPSY3 cDNA编码的蛋白都具有八氢番茄红素合成酶活性。
Carotenoids are one of most abundant classes of natural compounds, often highly colored with yellow, orange or red, derived from isoprenoids. They are synthesized in all photosynthetic organisms, as well as some bacteria, fungi and algae. They accumulate in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Carotenoids have fundamental roles in human nutrition as antioxidants and vitamin A precursors. It has been demonstrated their consumption is increasing associated with protection from a diverse of diseases. Animals and humans are unable to synthesize carotenoids de novo and must depend on dietary carotenoids. Thus they must acquire them from their diet. The first committed step in the plastid-localized carotenoid biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). It has been demonstrated PSY is the key enzyme limiting in carotenoid biosynthesis in tomato fruit, canola seeds, potato tubers, Arabidopsis thaliana seeds, Gentiana lutea flower petals and maize endosperm. The sole overexpression of phytoene synthase has a potent effect on carotenoid levels in storage organs generally, e.g. resulting in a 1.9-fold increase in tomato fruits, a 50-fold increase in canola seeds, a 43-fold increase in Arabidopsis seeds and a 8-fold increase in potato tubers.
In the present study, the phytoene synthase (PSY) 2 and 3 cDNAs were cloned from flower petal cDNA library of Gentiana lutea. The G. lutea PSY2 (GlPSY2) and PSY3 (GlPSY3) cDNA information were deposited in NCBI (National Center for Biotechnology Information) GenBank (Accession numbers: EF203259 and EF203260, respectively). The full-length of nucleotide sequence of GlPSY2 cDNA consisted of 1923 bp with a 1275 bp open reading frame (ORF), a 497 bp 5' terminal and a 151 bp 3' terminal non-coding regions, respectively. The deduced amino acid sequence of GlPSY2 cDNA was a polypeptide of 424 amino acid residues with molecular weight of 48298.65 D. The full-length of nucleotide sequence of GlPSY3 cDNA was 1765 bp with a 1278 bp open reading frame (ORF), 399 bp 5' terminal and 88 bp 3' terminal non-coding regions, respectively. The deduced amino acid sequence of GlPSY3 cDNA was a polypeptide of 424 amino acid residues with molecular weight of 48399.75 D. The isoelectric point of both of GlPSY2 and GlPSY3 was 8.79. The GlPSY2 and GlPSY3 amino acid sequences have higher identities with known functional PSY sequences from higher plants. The indicated function of G. lutea PSY2 and PSY3 cDNAs was estabilished by heterologous complementation in engineering Escherichia coli.
引文
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[2] Fraser PD, Bramley PM. The biosynthesis and nutritional uses of carotenoids [J]. Prog in Lipid Res, 2004, 43: 228-265.
[3] DellaPenna D, Pogson B. Vitamin synthesis in plants: tocopherols and carotenoids [J]. Annu Rev Plant Biol, 2006, 57: 711-738.
[4] 潘瑞炽. 植物生理学(第四版) [M]. 北京:高等教育出版社, 2001. 66-76.
[5] Giuliano G, Tavazza R, Diretto G, et al. Metabolic engineering of carotenoid biosynthesis in plants [J]. Trends in Biotechnology, 2008, 26: 139-145.
[6] Bone R A, Landnum J T, Femandez L, et al. Analysis of the Mlacular pigment by HPLC: retinal distrabution and age study [J]. Invest Ophthalmol Vis Sci, 1988, 29: 843-849.
[7] Landrum J and Bone R. Lutein, zeaxanthin, and the macular pigment [J]. Arch Biochem Biophys, 2001, 385(1): 28-40.
[8] Scott KJ, Hart DJ. The carotenoid composition of vegetables and fruit commonly consumed in the UK [J]. Norwich: IFR. 1994.
[9] 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, 30(6): 609-618.
[10] Cunninghan F, Schiff J. Photoisomerization of delta-carotene stereoisiomers in cells of Euglena gracillis mutant W3BUL and in solution [J]. Photochem Photobiol Sci, 1985, 42: 295- 307.
[11] Beyer P, Kroncke U, Nievelstein V. On the mechanism of the lycopene isomerase cyclase reaction in narcissus-pseudonarcissus L chromoplasts [J]. J Biol Chem, 1991, 266: 17072- 17078.
[12] Isaacson T, Ronen G, Zamir D, et al. Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of betacaroteneand xanthophylls in plants[J]. Plant Cell, 2002, 14: 333–342.
[13] Park H, Kreunen SS, Cuttriss AJ, et al. Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis [J]. Plant Cell, 2002, 14: 321-332.
[14] Isaacson T, Ohad I, Beyer P, et al. Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants [J]. Plant Physiol, 2004, 136: 4246- 4255.
[15] Cunningham FX, Gantt E. Genes and enzymes of carotenoid biosynthesis in plants [J]. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49: 557-583.
[16] Armstrong, Albert M, Leach F, et al. Nuclcotide sequence, organization and nature of the protein products of the carotenoid biosyntheses gene cluster of Rhodobacter capsulatus [J]. Mol Gen Genet, 1989, 216: 254- 268.
[17] Misawa N, Nakagawa M, Kobayashi K, et al. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli [J]. J Bacteriol, 1990, 172: 6704-6712.
[18] Bartley GE, Scolnik PA, Beyer P. Two Arabidopsis thaliana carotene desaturases, phytoene desaturase and zeta-carotene desaturase, expressed in Escherichia coli, catalyze a poly-cis pathway to yield pro-lycopene. Eur J Biochem, 1999, 259: 396-402.
[19] Misawa N, Satomi Y, Kondo K, et al. Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level [J]. J Bacteriol, 1995, 177: 6575-6584.
[20] Bramley PM, et al. Biochemical characterization of tomato plants in which carotenoid syntheses has been inhibited through the expession of antisense RNA to pTOM5 [J]. Plant J, 1992, 2: 343-349.
[21] Fray R, Wallace A, Fraser P, et al. Constitutive expression of a fruit phytoene synthase gene in transgenic tamatoes causes dwarfism by redirecting metabolites from the gibberellin pathway [J]. Plant J, 1995, 8(5): 693-701.
[22] Zhu C, Yamamura S, Koiwa H, et al. cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea [J]. Plant Mol Biol, 2002, 48: 277-285.
[23] Mann V, Harker M, Pecker I, et al. Metabolic engineering of astaxanthin production in tobacco flowers[J]. Nat Biotechnol, 2000, 18: 888-892.
[24] Li F, Vallabhaneni R and Wurtzel ET. PSY, a new member of the phytoene synthase gene family conserved in the poaceae and regulator of abiotic stress-induced root carotenogenesis [J]. Plant Physiol, 2008, 146: 1333–1345.
[25] Iuchi S, Kobayashi M, Yamaguchi-Shinozaki K, et al. A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea [J]. Plant Physiol, 2000, 123: 553–562.
[26] Cornish K, Zeevart JAD. Abscisic acid accumulation by roots of Xanthium strumarium L. and Lycopersicon esculentum Mill. in relation to water stress [J]. Plant Physiol,1985, 79: 653– 658.
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