Scientia Agricultura Sinica ›› 2011, Vol. 44 ›› Issue (23): 4848-4857.doi: 10.3864/j.issn.0578-1752.2011.23.011

• HORTICULTURE • Previous Articles     Next Articles

Molecular Cloning，Sequence Analysis of Phytoene Synthase Gene from Cerasus humilis (Bge) Sok. and Its Functional Expression in E.coli

 ZHANG  Jian-Cheng, DU  Jun-Jie, LIU  He  , WANG  Peng-Fei, XUE  Xiao-Fang, MU  Xiao-Peng, CHEN  Jun-Qi   

  1. 1.山西农业大学园艺学院，山西太谷 030801
  • Received:2011-08-17 Online:2011-12-01 Published:2011-09-26

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Abstract: 【Objective】The present study aimed at cloning and analyzing the phytoene synthase (PSY) gene from Cerasus humilis (Bge) Sok. and verifying its catalytic function by heterologous expression in E. coli containing a β-carotene producing plasmid. 【Method】Using the total RNA from the fruit of C. humilis (Bge) Sok. as the template, the cDNA specific fragment of PSY gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), and then the full-length cDNA sequence was obtained through rapid amplification of cDNA ends (RACE) techniques and its sequence was analyzed. The full coding sequence of PSY cDNA was amplified using PCR and further subcloned into vector pET-28a (+). The recombinant plasmid pET-ChPSY was expressed in a prokaryotic expression system after its transformation into BL21 (DE3). The catalytic activity of the fusion protein (6×His-PSY) was investigated in E. coli strain engineered to accumulate β-carotene. 【Result】 Sequence analysis indicated that the full-length cDNA was 1 559 bp, with an open reading frame of 1 194 bp and encoded a protein of 398 amino acids. The cloned cDNA exhibited a homology of the nucleotides and amino acid sequences of over 70% and 65%, respectively, aligned with PSY genes from other plants. The deduced protein has a signal transit peptide consists of 55 amino acid residues in the N-terminal region and two predicted transmembrane domain in the sites of 37-58 and 219-240 amino acids. The analysis of SDS-PAGE demonstrated that the recombinant fusion proteins (6×His-PSY) was produced at a higher level by prokaryotic expression and migrated at a size of about 45.8 kD. The heterogenous expression in E. coli system confirmed that ChPSY could encode a functional phytoene synthase which could enhance β-carotene content of E. coli engineered to produce β-carotene. 【Conclusion】 The PSY gene from Cerasus humilis (Bge) Sok. was successfully cloned and fucntioally expressed in E. coli, which has established a basis for studying the protein characterization and biosynthetic mechanism of carotenoids in fruits of Cerasus humilis (Bge) Sok.

Key words: Cerasus humilis (Bge) Sok, phytoene synthase, cloning, functional expression

[1]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. 植物生理与分子生物学学报, 2004, 30(6): 609-618.

Zhu C F, Chen X, Wang Y D. Carotenoid biosynthesis in plants and application of its relative genes in gene engineering. Journal of Plant Physiology and Molecular Biology, 2004, 30(6): 609-618. (in Chinese)

[2]陶  俊, 张上隆. 园艺植物类胡萝卜素的代谢及其调节. 浙江大学学报: 农业与生命科学版, 2003, 29(5): 585-590.

Tao J, Zhang S L. Metabolism of carotenoid and its regulation in garden crop. Journal of Zhejiang University: Agriculture ＆ Life Science, 2003, 29(5): 585-590. (in Chinese)

[3]Fraser P D, Bramley P M. The biosynthesis and nutritional uses of carotenoids. Progress in Lipid Research, 2004, 43: 228-265.

[4]卢  奎, 李建伟, 毕红霞, 汤丽娜, 陈  玮. 欧李红色素的性质和应用. 食品科学, 2004, 25(12): 78-81.

Lu K, Li J W, Bi H X, Tang L N, Chen W. Studies on the properties and application of red pigment extracted from Cerasus humilis. Food Science, 2004, 25(12): 78-81. (in Chinese)

[5]刘  明, 杜俊杰. 欧李种质资源的分布与品种性状表现. 落叶果树, 2010(3): 18-21.

Liu M, Du J J. Germplasm resources distribution of Cerasus humilis sok. and its cultivars’ characteristics. Deciduous Fruit, 2010(3): 18-21. (in Chinese)

[6]Lichtenthaler H K, Schwender J, Disch A, Rohmer M. Biosynthesis of isoprenoids in higher plants chloroplasts proceeds via a mevalonate independent pathway. FEBS Letters, 1997, 40(3): 271-274.

[7]Giuliano G, Tavazza R, Diretto G, Beyer P, Taylor M A. Metabolic engineering of carotenoid biosynthesis in plants. Trends in Biotechnology, 2008, 26(3): 139-145.

[8]Giuliano G, Bartley G E, Scolnik P A. Regulation of carotenoid biosynthesis during tomato development. The Plant Cell, 1993, 5: 379-387

[9]Marty I, Bureau S, Sarkissian G, Gouble B, Audergon J M, Albagnac G. Ethylene regulation of carotenoid accumulation and carotenogenic gene expression in colour-contrasted apricot varieties (Prunus armeniaca). Journal of Experimental Botany, 2005, 56: 1877-1886.

[10]Kato M, Ikoma Y, Matsumoto H, Sugiura M, Hyodo H, Yano M. Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in Citrus fruit. Plant Physiology, 2004, 134: 824-837.

[11]Shewmaker C K, Sheehy J A, Daley M, Colburn S, Ke D Y. Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. The Plant Journal, 1999, 20(4): 401-412.

[12]Ye X D, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 2000, 287: 303-305.

[13]Fraser P D, R?mer S, Shipton C A, Mills P B, Kiano J W, Misawa N, Drake R G, Schuch W, Bramley P M. Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit specific manner. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99: 1092-1097.

[14]Ducreux L J M, Morris W L, Hedley P E, Shepherd T, Davies H V, Millam S, Taylor M A. Metabolic engineering of high carotenoid potato tubers containing enhanced levels of β-carotene and lutein. Journal of Experimental Botany, 2005, 409: 81-89.

[15]Zhang J C, Tao N G, Xu Q, Zhou W J, Cao H B, Xu J, Deng X X. Functional characterization of Citrus PSY gene in Hongkong kumquat (Fortunella hindsii Swingle). Plant Cell Reporter, 2009, 28: 1737-1746.

[16]Wan C Y, Wilkins T A. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical Biochemistry, 1994, 223: 7-12.

[17]Jayaraj J, Devlin R, Punja Z. Metabolic engineering of novel ketocarotenoid production in carrot plants. Transgenic Research, 2007, 17: 489-501.

[18]Liu Q, Xu J, Liu Y Z, Zhao X L, Deng X X, Guo L L, Gu J Q. A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). Journal of Experimental Botany, 2007, 58: 4161-4171.

[19]Hirschberg J. Carotenoid biosynthesis in flowering plants. Current Opinion in Plant Biology, 2001, 4: 210-218.

[20]Scolnik P A, Bartley G E. Phytoene desaturase from Arabidopsis. Plant Physiology, 1993, 103: 1475.

[21]Ray J, Moureau P, Bird C, Bird A, Grierson D, Maunders M, Truesdale M, Bramley M, Schuch W. Cloning and characterization of gene involved in phytoene synthesis from tomato. Plant Molecular Biology, 1992, 19(3): 401-404.

[22]Salvini M, Bernini A, Fambrini M, Pugliesi C. cDNA cloning and expression of the phytoene synthase gene in sunflower. Journal of Plant Physiology, 2005, 162: 479-484.

[23]Lindgren L O, Stalberg K G, Hoglund A S. Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids, chlorophyll, and abscisic acid. Plant Physiology, 2003, 132: 779-785.

[24]Fujisawa M, Watanabe M, Choi S K, Teramoto M, Ohyama K, Misawa N. Enrichment of carotenoids in flaxseed (Linum usitatissimum) by metabolic engineering with introduction of bacterial phytoene synthase gene crtB. Journal of Bioscience and Bioengineering, 2008, 105: 636-641.

[25]Zhu C, Naqvi S, Breitenbach J, Sandmann G, Christou P, Capell T. Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105: 18232-18237.

[26]Paine J A, Shipton C A, Chaggar S, Howells R M, Kennedy M J, Vernon G, Wright S Y, Hinchliffe E, Adams J L, Silverstone A L, Drake R. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology, 2005, 23(4): 482-487.

[27]Fujisawa, M, Takita E, Harada H, Sakurai N, Suzuki H, Ohyama K, Shibata D, Misawa N. Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation. Journal of Experimental Botany, 2009, 60: 1319-1332.

[28]Cunningham F X, Gantt E. Genes and enzymes of carotenoid biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 1998, 49: 557-583.

[29]朱跃辉, 严  媛, 姜建国, 宋冬林. 盐藻PSY基因cDNA的克隆、序列测定及其进化分析. 生命科学研究, 2004, 8(4): 329-332.

Zhu Y H, Yan Y, Jiang J G, Song D L. Cloning, sequencing and evaluation analysis of cDNA encoding PSY from Dunaliella salina. Life Science Research, 2004, 8(4): 329-332. (in Chinese)

[30]Misawa N, Masamoto K, Hori T. Expression of an Erwinia phytoene desature gene not only confers multiple resistance to herbicides interfering with carotenoid biosynthesis but also alters xanthophylls metabolism in transgenic plants. The Plant Journal, 1994, 6: 481-489.

[31]Lopez A B, Yang Y, Thannhauser T W, Li L. Phytoene desaturase is present in a large protein complex in the plastid membrane. Physiologia Plantarum, 2008, 133: 190-198.
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