雷公藤单萜合酶基因TwMS的克隆及蛋白表达分析
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摘要
该研究克隆得到1条雷公藤单萜合酶基因TwMS。其完整开放阅读框为1797bp,编码579个氨基酸,相对分子质量为69.75kDa、理论等电点为5.37。生物信息学分析表明,TwMS具有单萜合酶的特征结构域,属于萜类合酶TPSb亚家族。实时荧光定量PCR检测显示,经茉莉酸甲酯(MeJA)诱导后,TwMS的相对表达量显著上调,并在24 h达到最高。此外,该研究在大肠杆菌BL21(DE3)中成功表达TwMS蛋白,为进一步研究该基因的功能奠定了基础。
In this study,we cloned a monoterpene synthases,TwMS from Tripterygium wilfordii suspension cells.TwMS gene contained a 1797 bp open reading frame(ORF),encoding a polypeptide of 579 amino acids,which deduced isoelectric point(pI) was6.10 and the calculated molecular weight was 69.75 kDa.Bioinformation analysis showed that the sequence of TwMS was consistent with the feature of monoterpene synthases.Differential expression analysis revealed that the relative expression level of TwMS increased significantly after being induced by methyl jasmonate(MeJA).The highest expression level occurred at 24 h.TwMS protein was successfully expressed in Escherichia coli BL21(DE3),which laid the foundation for identifying the function of T.wilfordii monoterpene synthases.
In this study,we cloned a monoterpene synthases,TwMS from Tripterygium wilfordii suspension cells.TwMS gene contained a 1797 bp open reading frame(ORF),encoding a polypeptide of 579 amino acids,which deduced isoelectric point(pI) was6.10 and the calculated molecular weight was 69.75 kDa.Bioinformation analysis showed that the sequence of TwMS was consistent with the feature of monoterpene synthases.Differential expression analysis revealed that the relative expression level of TwMS increased significantly after being induced by methyl jasmonate(MeJA).The highest expression level occurred at 24 h.TwMS protein was successfully expressed in Escherichia coli BL21(DE3),which laid the foundation for identifying the function of T.wilfordii monoterpene synthases.
引文
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[3]Chugh R,Sangwan V,Patil S P,et al.A preclinical evaluation of minnelide as a therapeutic agent against pancreatic cancer[J].Sci Transl Med,2012,4(156):4085.
[4]Lu L,Li F,Wang X.Novel anti-inflammatory and neuroprotective agents for Parkinson's disease[J].Cns Neurol Disord-Dr,2010,9(9):232.
[5]Wang X,Liang X B,Li F Q,et al.Therapeutic strategies for Parkinson's disease:the ancient meets the future-traditional Chinese herbal medicine,electroacupuncture,gene therapy and stem cells[J].Neurochem Res,2008,33(10):1956.
[6]Junli L,Jaemin L,Mario Andres S H,et al.Treatment of obesity with celastrol[J].Cell,2015,161(5):999.
[7]Bohlmann J,Meyer-Gauen G,Croteau R.Plant terpenoid synthases:molecular biology and phylogenetic analysis[J].Proc Natl Acad Sci USA,1998,95(8):4126.
[8]Lippert D,Chowrira S,Ralph S G,et al.Conifer defense against insects:proteome analysis of Sitka spruce(Picea sitchensis)bark induced by mechanical wounding or feeding by white pine weevils(Pissodes strobi)[J].Proteomics,2007,7(2):248.
[9]Holopainen J K.Multiple functions of inducible plant volatiles[J].Trends Plant Sci,2004,9(11):529
[10]Pichersky E,Gershenzon J.The formation and function of plant volatiles:perfumes for pollinator attraction and defense[J].Curr Opin Plant Biol,2002,5(3):237.
[11]Chen F,Tholl D,D'Auria J C,et al.Biosynthesis and emission of terpenoid volatiles from arabidopsis flowers[J].Plant Cell,2003,15(2):481.
[12]Chen F,Ro D K,Petri J,et al.Characterization of a root-specific Arabidopsis terpene synthase responsible for the formation of the volatile monoterpene 1,8-cineole[J].Plant Physiol,2004,135(4):1956.
[13]Steele C L,Katoh S,Bohlmann J,et al.Regulation of oleoresinosis in grand fir(Abies grandis).Differential transcriptional control of monoterpene,sesquiterpene,and diterpene synthase genes in response to wounding[J].Plant Physiol,1998,116(4):1497.
[14]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT,method[J].Methods,2001,25(4):402.
[15]Bohlmann J,Meyer-Gauen G,Croteau R.Plant terpenoid synthases:molecular biology and phylogenetic analysis[J].Proc Natl Acad Sci USA,1998,95(8):4126.
[16]Natalia Dudareva,Diane Martin,Christine M Kish,et al.(E)-β-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon:function and expression of three terpene synthase genes of a new terpene synthase subfamily[J].Plant Cell,2003,15(5):1227.
[17]Aharoni A,Giri A P,Verstappen F W,et al.Gain and loss of fruit flavour compounds produced by wild and cultivated strawberry species[J].Plant Cell,2004,16(11):3110.
[18]Hyatt D C,Youn B,Zhao Y,et al.Structure of limonene synthase,a simple model for terpenoid cyclase catalysis[J].Proc Natl Acad Sci USA,2007,104(13):5360.
[19]张萌,苏平,刘雨佳,等.雷公藤牻牛儿基牻牛儿基焦磷酸合酶基因全长c DNA的获得及生物信息学分析[J].中国中药杂志,2015,40(6):1066.