Cloning and Expression of psy Gene from Rhododendron molle

doi:10.11924/j.issn.1000-6850.casb17070067

Abstract

Abstract: We aim to study the function of psy gene and its molecular mechanism in petal coloring. A psy gene was cloned from the Rhododendron molle cDNA by using RT-PCR and RACE methods. The full length of the psy gene was 1638 bp, named RmPSY (GenBank accession No. KX230461), which contained a 1296 bp open reading frame encoding 432 amino acid residues. RmPSY, a hydrophilic unstable protein with no signal peptide, was a non-secreted protein. The putative protein molecular weight was 48.91 kD and its theoretical isoelectric point was 8.78. Homologous alignment showed that it shared 81% amino acid identities with PSY from other plants in GenBank. The phylogenetic tree constructed on the basis of amino acid sequences of PSY suggested that the relationship between R. molle and Vitis vinifera was the most intimate. The results showed that the RmPSY gene was expressed in the petal during the flower development. The RmPSY transcripts were the most abundant in the initial opening period of flower, low in the bud stage, and decreased during the blooming period of flower. This study may provide references for further research on the molecular mechanism of RmPSY involved in the formation of R. molle flower color.

CLC Number:

S685.21

References

[1]顾地周,陆爽,巴春影,等. 羊踯躅嫩叶离体培养和植株高效再生技术研究[J]. 西北农林科技大学学报(自然科学版),2012,40(6): 189-194.
[2]王玉萍,刘庆昌,翟红. 植物类胡萝卜素生物合成相关基因的表达调控及其在植物基因工程中的应用[J]. 分子植物育种,2006, 4(1): 103-110.
[3]Miller R, Owens S J, Rrslett B. Plants and colour: Flowers and pollination[J]. Optics Laser Technology, 2011, 43(2): 282-294.
[4]Fraser P D, Bramley P M. The biosynthesis and nutritional uses of carotenoids[J]. Progress in Lipid Research, 2004, 43(3): 228-265.
[5]Burkhardt P K, Beyer P, Wunn J, et al. Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis[J]. The Plant journal, 1997, 11(5): 1071-1078.
[6]Gayen D, Ghosh S, Paul S, et al. Metabolic Regulation of Carotenoid-Enriched Golden Rice Line[J]. Frontiers in Plant Science, 2016, 7: 12.
[7]Kaur N, Pandey A, Shivani, et al. Regulation of Banana Phytoene Synthase (MaPSY) Expression, Characterization and Their Modulation under Various Abiotic Stress Conditions[J]. Frontiers in Plant Science, 2017, 8:462.
[8]卢萍,王柬人,李锡香,等. 西双版纳黄瓜Cs-Psy1基因的序列特征与表达分析[J]. 植物遗传资源学报,2016, 17(6): 1050-1057.
[9]黄建峰,高爱平,赵志常,等. 杧果类胡萝卜素合成酶基因PSY的表达特性分析[J]. 中国南方果树,2016, 45(5): 18-22.
[10]Vargas V H, Schulthess A, Royo C, et al. Transcripts levels of Phytoene synthase 1 (Psy-1) are associated to semolina yellowness variation in durum wheat (Triticum turgidum L. ssp durum)[J]. Journal of Cereal Science, 2016, 68: 155-163.
[11]Lopez-Emparan A, Quezada-Martinez D, Zuniga-Bustos M, et al. Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family[J]. Plos One, 2014, 9(12): 19.
[12]朱海生,陈敏氡,温庆放. 草莓psy、pds和zds基因融合植物表达载体的构建[J]. 福建农林大学学报(自然科学版),2013, 42(4): 391-397.
[13]Salvini M, Bernini A, Fambrini M, et al. cDNA cloning and expression of the phytoene synthase gene in sunflower[J]. Journal of Plant physiology, 2005, 162(4): 479-484.
[14]邹清成,庄晓英,卢钢,等. 反义PSY基因植物表达载体的构建及其对中国水仙的转化[J]. 浙江林业科技, 2006, 26(3): 25-30.
[15]高蓝,朱碧云. 栀子GjPSY基因的克隆及原核表达[J]. 生物技术,2013(3): 12-17.
[16] Walker J M. The Proteomics Protocols Handbook[M]. Humana Press, 2005: 571-607
[17]Bendtsen J D, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides: SignalP 3.0[J]. Journal of molecular biology, 2004, 340(4): 783-795.
[18]Guermeur Y, Pollastri G, Elisseeff A, et al. Combining protein secondary structure prediction models with ensemble methods of optimal complexity[J]. Neuro computing, 2004, 56: 305-327.
[19]Tamura K, Dudley J, Nei M. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular biology and evolution, 2007, 24(8): 1596-1599.
[20]Xiao Z, Sun X, Liu X, et al. Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don. Frontiers in Plant Science, 2016, 7(e0141853): 1547.
[21]吴彦庆,赵大球,王静,等. 芍药查尔酮异构酶基因 (CHI) 克隆、密码子偏好性分析以及蛋白结构功能预测[J]. 华北农学报,2016, 31(2): 71-80.
[22]Marchler-Bauer A, Anderson J B, Chitsaz F, et al. CDD: specific functional annotation with the Conserved Domain Database[J]. Nucleic Acids Res, 2009, 37(Suppl 1): D205-D210.
[23]Duc T, James H, Qiu W, et al. An update on carotenoid biosynthesis in algae: phylogenetic evidence for the existence of two classes of phytoene synthase[J]. Planta, 2009, 229(3): 723-729.
[24]Fraser P D, Romer S, Shipton C A, et al. Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner[J]. PNAS, 2002, 99(2): 1092-1097.
[25]Busch M, Seuter A, Hain R. Functional analysis of the early steps of carotenoid biosynthesis in tobacco[J]. Plant physiology, 2002, 128(2): 439-453.
[26]Just B J, Santos C A F, Fonseca M E N, et al. Carotenoid biosynthesis structural genes in carrot (Daucus carota): isolation, sequence-characterization, single nucleotide polymorphism (SNP) markers and genome mapping[J]. Theoretical and Applied Genetics, 2007, 114(4): 693-704.
[27]Welsch R, Wust F, Bar C, et al. A third phytoene synthase is devoted to abiotic stress-induced abscisic acid formation in rice and defines functional diversification of phytoene synthase genes[J]. Plant physiology, 2008, 147(1): 367-380.
[28]Li F, Vallabhaneni R, Wurtzel E T. PSY3, a New Member of the Phytoene Synthase Gene Family Conserved in the Poaceae and Regulator of Abiotic Stress-Induced Root Carotenogenesis[J]. Plant physiology, 2008, 146(3): 1333-1345.
[29]Jacobo A, Florian W, Peter B, et al. Characterization of phytoene synthases from cassava and their involvement in abiotic stress-mediated responses[J]. Planta, 2010, 232(5): 1251-1262.
[30]Bramley P, Teulieres C, Blain I, et al. Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5[J]. The Plant journal, 1992, 2(3): 343-349.
[31]Fraser P D, Truesdale M R, Bird C R, et al. Carotenoid biosynthesis during tomato fruit development[J]. Plant physiology, 1994, 105(1): 405-413.
[32]Fray R G, Wallace A, Fraser P D, et al. Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway[J]. The Plant journal, 1995, 8(5): 693-701.
[33]马乐园, 于喜艳. 甜瓜果实八氢番茄红素合成酶基因的克隆及正义表达载体的构建[J]. 山东农业科学, 2011(8): 4-7.
[34]Zhu C, Yamamura S, Koiwa H, et al. cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea[J]. Plant Molecular Biology, 2002, 48(3): 277-285.
[35]Ye X, Al-Babili S. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm[J]. Science, 2000, 287(5451): 303-305.
[36]Paine JA, Shipton CA, Chaggar S, et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content[J]. Nature biotechnology, 2005, 23(4): 482-487.