桂花萜烯合酶(TPS)的生物信息学与原核表达分析
|
摘要
挥发性萜烯类化合物是植物花和果实中重要的香气活性物质,萜烯合酶(TPS)的种类和功能决定物种中萜烯类物质的多样性。桂花作为重要的香花植物,萜烯类物质是其花香的重要成分,但有关桂花萜烯合酶的研究却并不多。为揭示桂花萜烯类物质的生物合成机理,该研究利用生物信息学分析了4个TPS蛋白的理化性质、亚细胞定位及其结构,在原核表达系统中进行4个TPS蛋白的体外表达,并对可溶性的TPS4重组蛋白进行了体外酶反应功能分析。结果表明:(1) 4个TPS蛋白的理化性质差异较小,但仅有TPS4蛋白定位于其他靶向,不含信号肽,延伸链的比例较低,在靠近氨基端的区域内不含延伸链。(2) 4个TPS蛋白在原核系统中均可成功表达,但仅有TPS4获得了可溶性重组蛋白。(3)将纯化的TPS4重组蛋白分别与GPP、NDP和FPP进行反应,均只检测到一种产物,分别为反式-β-罗勒烯、β-水芹烯和α-法呢烯。这为揭示植物萜烯类物质生物合成的分子机理提供了参考,对从蛋白水平研究桂花花香基因功能奠定了基础。
Volatile terpenes are important aroma active compounds in flowers and fruits of plant. Diversities of terpenes are usually determined by type and function of terpene synthase in different species. Osmanthus fragrans is an important fragrant plant,in which terpenes are the important components of floral scent. But there are few studies on terpene synthase in O. fragrans. To reveal the biosynthesis mechanism of terpenes in O. fragrans,we predicted the physicochemical properties,subcellular localization and structure of four TPS proteins by bioinformatics,and expressed them in prokaryotic expression system. Finally,the function of soluble TPS4 recombinant protein was analyzed by enzyme reaction in vitro. The results were as follows:( 1) The physicochemical properties of the four TPS proteins had relatively little difference. Only TPS4 protein locating in other targets without signal peptide had low proportional extended strand and no extended strand near amidogen terminal.( 2) All of the four TPS proteins were successfully expressed in prokaryotic system,but only TPS4 obtained soluble recombinant protein.( 3) The purified TPS4 recombinant protein was reacted with GPP,NDP and FPP respectively,and only one product( trans-β-ocimene,β-phellandrene and α-farnesene) was detected. The results provide reference for functional analysis of floral scent related gene at protein level in O. fragrans and for revealing the molecular mechanism of terpenes biosynthesis in plant.
Volatile terpenes are important aroma active compounds in flowers and fruits of plant. Diversities of terpenes are usually determined by type and function of terpene synthase in different species. Osmanthus fragrans is an important fragrant plant,in which terpenes are the important components of floral scent. But there are few studies on terpene synthase in O. fragrans. To reveal the biosynthesis mechanism of terpenes in O. fragrans,we predicted the physicochemical properties,subcellular localization and structure of four TPS proteins by bioinformatics,and expressed them in prokaryotic expression system. Finally,the function of soluble TPS4 recombinant protein was analyzed by enzyme reaction in vitro. The results were as follows:( 1) The physicochemical properties of the four TPS proteins had relatively little difference. Only TPS4 protein locating in other targets without signal peptide had low proportional extended strand and no extended strand near amidogen terminal.( 2) All of the four TPS proteins were successfully expressed in prokaryotic system,but only TPS4 obtained soluble recombinant protein.( 3) The purified TPS4 recombinant protein was reacted with GPP,NDP and FPP respectively,and only one product( trans-β-ocimene,β-phellandrene and α-farnesene) was detected. The results provide reference for functional analysis of floral scent related gene at protein level in O. fragrans and for revealing the molecular mechanism of terpenes biosynthesis in plant.
引文
BLEEKER PM,SPYROPOULOU EA,DIERGAARDE PJ,et al.,2011.RNA-seq discovery,functional characterization,and comparison of sesquiterpene synthases from Solanum lycopersicum and Solanum habrochaites trichomes[J].Plant Mol Biol,77:323-336.
BOHLMANN J,MEYERGAUEN G,CROTEAU R,1998.INAU-GURAL ARTICLE by a recently elected academy member:Plant terpenoid synthases:Molecular biology and phylogenetic analysis[J].Proc Natl Acad Sci USA,95:4126-4133.
CABRITA LD,BOTTOMLEY SP,2004.Protein expression and refolding-A practical guide to getting the most out of inclusion bodiprotein expression and refolding-A practical guide to getting the most out of inclusion bodieses[J].Biotechnol Ann Rev,10(4):31-50.
CAI X,MAI RZ,ZOU JJ,et al.,2014.Analysis of aroma-active compounds in three sweet osmanthus(Osmanthus fragrans)cultivars by gas-chromatography-olfactometry and GC-mass spectrometry[J].J Zhejiang Univ Sci B,15(7):638-648.
CAO H,LI ZG,SHEN DL,2009.GC/MS fingerprint analysis of Osmanthus fragrans Lour.in different varieties[J].Acta Hortic Sin,36(3):391-398.[曹慧,李祖光,沈德隆,2009.桂花品种香气成分的GC/MS指纹图谱研究[J].园艺学报,36(3):391-398.]
CHEN F,THOLL D,BOHLMANN J,et al.,2011.The family of terpene synthases in plants:A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[J].Plant J,66:212-229.
CROWELL AL,WILLIAMS DC,DAVIS EM,et al.,2002.Molecular cloning and characterization of a new linalool synthase[J].Arch Biochem Biophys,405(1):112-121.
DEGENHARDT J,K?LLNER TG,GERSHENZON J,2009.Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants[J].Phytochemistry,70:1621-1637.
DU YQ,TIAN XL,GAN JZ,et al.,2013.Essential oils extracted from different varieties of wintersweet(Chimonanthus praecox)[J].J Beijing For Univ,35(Suppl.1):81-85.[杜永芹,田晓龙,甘建忠,等,2013.不同品种蜡梅花精油成分的研究[J].北京林业大学学报,35(增刊1):81-85.]
DUDAREVA N,KLEMPIEN A,MUHLEMANN JK,et al.,2013.Biosynthesis,function and metabolic engineering of plant volatile organic compounds[J].New Phytol,198(1):16-32.
GARMS S,KOLLNER TG,BOLAND W,2010.A multiproduct terpenesynthase from Medicago truncatula generates cadalane sesquiterpenes via two different mechanisms[J].J Organ Chem,75:5590-5600.
MARTIN DM,AUBOURG S,SCHOUWEY M,et al.,2010.Functional annotation,genome organization and phylogeny of the grapevine(Vitis vinifera)terpene synthase gene family based on genome assembly,FLc DNA cloning,and enzyme assays[J].BMC Plant Biol,10:226.
MOSES T,POLLIER J,THEVELEIN JM,et al.,2013.Bioengineering of plant(tri)terpenoids:From metabolic engineering of plants to synthetic biology in vivo and in vitro[J].New Phytol,200(1):27-43.
NAGEGOWDA DA,2010.Plant volatile terpenoid metabolism:Biosynthetic genes,transcriptional regulation and subcellular compartmentation[J].FEBS Lett,584(14):2965-2973.
SHIMADA K,NAGANO M,KAWAI M,et al.,2005.Influences of amino acid features of glutathione S-transferase fusion proteins on their solubility[J].Proteomics,5(15):3859-3863.
TERPE K,2006.Overview of bacterial expression systems for heterologous protein production:From molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biotechnol,72(2):211-222.
THOLL D,CHEN F,PETRI J,et al.,2005.Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers[J].Plant J,42(5):757-771.
THOLL D,2006.Terpene synthases and the regulation,diversity and biological roles of terpene metabolism[J].Curr Opin Plant Biol,9:297-304.
WILKINSON DL,HARRISON RG,1991.Predicting the solubility of recombinant proteins in Escherichia coli[J].Nat Biotechnol,9:443-448.
XIN HP,WU BH,ZHANG HH,et al.,2013.Characterization of volatile compounds in flowers from four groups of sweet osmanthus(Osmanthus fragrans)cultivars[J].Can J Plant Sci,93:923-931.
XIONG M,ZHOU MQ,XIANG L,et al.,2012.Analysis of essential oil in Chimonanthus praecox by GC-MS[J].J Huazhong Agric Univ,31(2):182-186.[熊敏,周明芹,向林,等,2012.蜡梅花挥发油成分的GC-MS分析[J].华中农业大学学报,31(2):182-186.]
YANG XL,SHI TT,WEN AL,et al.,2015.Variance analysis of aromatic components from different varieties of Osmanthus fragrans[J].J NE For Univ,43(1):83-87.[杨秀莲,施婷婷,文爱林,等,2015.不同桂花品种香气成分的差异分析[J].东北林业大学学报,43(1):83-87.]
ZENG XL,LIU C,ZHENG RR,et al.,2015.Emission and accumμlation of monoterpene and the key terpene synthase(TPS)associated with monoterpene biosynthesis in Osmanthus fragrans Lour[J].Front Plant Sci,6:1232.
ZHANG Y,LI XL,WANG Y,et al.,2011.Changes of aroma components in Oncidium Sharry Baby in different florescence and flower parts[J].Sci Agric Sin,44(1):110-117.[张莹,李辛雷,王雁,等,2011.文心兰不同花期及花朵不同部位香气成分的变化[J].中国农业科学,44(1):110-117.]
ZHAO YQ,ZHOU SJ,PENG PH,et al.,2011.Analysis of volatile compounds from Prunus mume flowers[J].Guihaia,31(4):554-558.[赵印泉,周斯建,彭培好,等,2011.三轮玉蝶梅花朵挥发性成分的分析[J].广西植物,31(4):554-558.]
BOHLMANN J,MEYERGAUEN G,CROTEAU R,1998.INAU-GURAL ARTICLE by a recently elected academy member:Plant terpenoid synthases:Molecular biology and phylogenetic analysis[J].Proc Natl Acad Sci USA,95:4126-4133.
CABRITA LD,BOTTOMLEY SP,2004.Protein expression and refolding-A practical guide to getting the most out of inclusion bodiprotein expression and refolding-A practical guide to getting the most out of inclusion bodieses[J].Biotechnol Ann Rev,10(4):31-50.
CAI X,MAI RZ,ZOU JJ,et al.,2014.Analysis of aroma-active compounds in three sweet osmanthus(Osmanthus fragrans)cultivars by gas-chromatography-olfactometry and GC-mass spectrometry[J].J Zhejiang Univ Sci B,15(7):638-648.
CAO H,LI ZG,SHEN DL,2009.GC/MS fingerprint analysis of Osmanthus fragrans Lour.in different varieties[J].Acta Hortic Sin,36(3):391-398.[曹慧,李祖光,沈德隆,2009.桂花品种香气成分的GC/MS指纹图谱研究[J].园艺学报,36(3):391-398.]
CHEN F,THOLL D,BOHLMANN J,et al.,2011.The family of terpene synthases in plants:A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom[J].Plant J,66:212-229.
CROWELL AL,WILLIAMS DC,DAVIS EM,et al.,2002.Molecular cloning and characterization of a new linalool synthase[J].Arch Biochem Biophys,405(1):112-121.
DEGENHARDT J,K?LLNER TG,GERSHENZON J,2009.Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants[J].Phytochemistry,70:1621-1637.
DU YQ,TIAN XL,GAN JZ,et al.,2013.Essential oils extracted from different varieties of wintersweet(Chimonanthus praecox)[J].J Beijing For Univ,35(Suppl.1):81-85.[杜永芹,田晓龙,甘建忠,等,2013.不同品种蜡梅花精油成分的研究[J].北京林业大学学报,35(增刊1):81-85.]
DUDAREVA N,KLEMPIEN A,MUHLEMANN JK,et al.,2013.Biosynthesis,function and metabolic engineering of plant volatile organic compounds[J].New Phytol,198(1):16-32.
GARMS S,KOLLNER TG,BOLAND W,2010.A multiproduct terpenesynthase from Medicago truncatula generates cadalane sesquiterpenes via two different mechanisms[J].J Organ Chem,75:5590-5600.
MARTIN DM,AUBOURG S,SCHOUWEY M,et al.,2010.Functional annotation,genome organization and phylogeny of the grapevine(Vitis vinifera)terpene synthase gene family based on genome assembly,FLc DNA cloning,and enzyme assays[J].BMC Plant Biol,10:226.
MOSES T,POLLIER J,THEVELEIN JM,et al.,2013.Bioengineering of plant(tri)terpenoids:From metabolic engineering of plants to synthetic biology in vivo and in vitro[J].New Phytol,200(1):27-43.
NAGEGOWDA DA,2010.Plant volatile terpenoid metabolism:Biosynthetic genes,transcriptional regulation and subcellular compartmentation[J].FEBS Lett,584(14):2965-2973.
SHIMADA K,NAGANO M,KAWAI M,et al.,2005.Influences of amino acid features of glutathione S-transferase fusion proteins on their solubility[J].Proteomics,5(15):3859-3863.
TERPE K,2006.Overview of bacterial expression systems for heterologous protein production:From molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biotechnol,72(2):211-222.
THOLL D,CHEN F,PETRI J,et al.,2005.Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers[J].Plant J,42(5):757-771.
THOLL D,2006.Terpene synthases and the regulation,diversity and biological roles of terpene metabolism[J].Curr Opin Plant Biol,9:297-304.
WILKINSON DL,HARRISON RG,1991.Predicting the solubility of recombinant proteins in Escherichia coli[J].Nat Biotechnol,9:443-448.
XIN HP,WU BH,ZHANG HH,et al.,2013.Characterization of volatile compounds in flowers from four groups of sweet osmanthus(Osmanthus fragrans)cultivars[J].Can J Plant Sci,93:923-931.
XIONG M,ZHOU MQ,XIANG L,et al.,2012.Analysis of essential oil in Chimonanthus praecox by GC-MS[J].J Huazhong Agric Univ,31(2):182-186.[熊敏,周明芹,向林,等,2012.蜡梅花挥发油成分的GC-MS分析[J].华中农业大学学报,31(2):182-186.]
YANG XL,SHI TT,WEN AL,et al.,2015.Variance analysis of aromatic components from different varieties of Osmanthus fragrans[J].J NE For Univ,43(1):83-87.[杨秀莲,施婷婷,文爱林,等,2015.不同桂花品种香气成分的差异分析[J].东北林业大学学报,43(1):83-87.]
ZENG XL,LIU C,ZHENG RR,et al.,2015.Emission and accumμlation of monoterpene and the key terpene synthase(TPS)associated with monoterpene biosynthesis in Osmanthus fragrans Lour[J].Front Plant Sci,6:1232.
ZHANG Y,LI XL,WANG Y,et al.,2011.Changes of aroma components in Oncidium Sharry Baby in different florescence and flower parts[J].Sci Agric Sin,44(1):110-117.[张莹,李辛雷,王雁,等,2011.文心兰不同花期及花朵不同部位香气成分的变化[J].中国农业科学,44(1):110-117.]
ZHAO YQ,ZHOU SJ,PENG PH,et al.,2011.Analysis of volatile compounds from Prunus mume flowers[J].Guihaia,31(4):554-558.[赵印泉,周斯建,彭培好,等,2011.三轮玉蝶梅花朵挥发性成分的分析[J].广西植物,31(4):554-558.]