白木香倍半萜合酶基因AsVS的克隆与表达分析
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摘要
法尼基焦磷酸(farnesyl diphosphate, FPP)是植物萜类化合物合成途径的重要前体之一,经不同的酶催化可形成各类萜类化合物,Vetispiradiene合酶可催化FPP形成Solavetivone和Lubimin的前体Vetispiradiene。本研究利用白木香转录组数据,首次克隆了编码白木香Vetispiradiene合酶的基因(命名为As VS, Genbank登录号为MH378283),AsVS基因序列全长为1 632 bp,编码543个氨基酸,该氨基酸序列与马来沉香倍半萜合酶聚类于同一分支,具有较近的亲缘关系,该蛋白植物倍半萜的保守性结构域DDXXD,NST/DTE和R(R)X8W,相对分子量为62.73 kD,理论等电点为5.51,包含40个磷酸化位点;二级结构以α螺旋和无规则卷曲为主;不含跨膜结构域。RT-qPCR分析结果表明AsVS基因在结香剂处理过程中于白木香茎干样品中持续上调表达,其第6天及第9天的表达量分别为对照处理的2.36和5.02倍。此结果为进一步研究As VS基因在白木香萜类化合物的合成及沉香致香成分富集中的功能提供了理论基础。
FPP(Farnesyl diphosphate), one of most pivotal precursor in plant terpene synthase pathway, can be converted to various terpenes via plant enzyme systems. EAS and its isozyme, VS, can catalyze FFP to 5-epiaristolochene and vetispiradiene. In this study, we cloned a full length of cDNA coding vetispiradiene synthase in Aquilaria sinensis based on the annotation of its transcriptome database. This gene was named it for AsVS and its accession number in GenBank is MH378283. AsVS gene inculding 1 632 base pairs which encoded 543 amino acid residues. There was more homology of protein between AsVS and sesquiterpene synthase from Aquilaria malaccensis and this result was then identified by evolution analysis with MEGA X. In addition, As VS protein contained three conservative structures which were DDXXD,NST/DTE and R(R)X8 W. Bioinformatic analysis reveals that MW and pI of AsVS were 62.73 kD and 5.51, and it is a non-secretory protein and contained 40 phosphorylation sites. The secondary structure of AsVS were mainly mixed α-helix and random coil. The transcript profiles of demonstrated that the relative expression of AsVS was continuous increased in stem of A. sinensis during artificial induce agarwood formation, its relative expression in stem of A. sinensis after treatment with 6 day and 9 day were increased 2.36 and 5.02 than the control treatment. This study offered the functions for indicating AsVS in multi-types terpenes synthesis and regulatory mechanism of aroma components of eaglewood formation in Aquilaria spp.
FPP(Farnesyl diphosphate), one of most pivotal precursor in plant terpene synthase pathway, can be converted to various terpenes via plant enzyme systems. EAS and its isozyme, VS, can catalyze FFP to 5-epiaristolochene and vetispiradiene. In this study, we cloned a full length of cDNA coding vetispiradiene synthase in Aquilaria sinensis based on the annotation of its transcriptome database. This gene was named it for AsVS and its accession number in GenBank is MH378283. AsVS gene inculding 1 632 base pairs which encoded 543 amino acid residues. There was more homology of protein between AsVS and sesquiterpene synthase from Aquilaria malaccensis and this result was then identified by evolution analysis with MEGA X. In addition, As VS protein contained three conservative structures which were DDXXD,NST/DTE and R(R)X8 W. Bioinformatic analysis reveals that MW and pI of AsVS were 62.73 kD and 5.51, and it is a non-secretory protein and contained 40 phosphorylation sites. The secondary structure of AsVS were mainly mixed α-helix and random coil. The transcript profiles of demonstrated that the relative expression of AsVS was continuous increased in stem of A. sinensis during artificial induce agarwood formation, its relative expression in stem of A. sinensis after treatment with 6 day and 9 day were increased 2.36 and 5.02 than the control treatment. This study offered the functions for indicating AsVS in multi-types terpenes synthesis and regulatory mechanism of aroma components of eaglewood formation in Aquilaria spp.
引文
Back K.,and Chappell J.,1995,Cloning and bacterial expression of a sesquiterpene cyclase from Hyoscyamus muticus and its molecular comparison to related terpene cyclases.,J.Biol.Chem.,270(13):7375-7381
Back K.,and Chappell J.,1996,Identifying functional domains within terpene cyclases using a domain-swapping strategy,Proc.Natl.Acad.Sci.USA,93(13):6841-6845
Back K.,Yin S.,and Chappell J.,1994,Expression of a plant sesquiterpene cyclase gene in Escherichia coli,Arch.Biochem.Biophys.,315(2):527-532
Bleeker P.M.,Spyropoulou E.A.,Diergaarde P.J.,Volpin H.,De Both M.T.,Zerbe P.,Bohlmann J.,and Schuurink R.C.,2011,RNA-seq d iscovery,functional characterization,and comparison of sesquiterpene synthases from Solanum lycopersicum and Solanum habrochaites trichomes,Plant Mol.Biol.,77(4-5):323-336
Bohlmann J.,Meyer-Gauen G.,and Croteau R.,1998,Plant terpenoid synthases:molecular biology and phylogenetic analysis,Proc.Natl.Acad.Sci.USA,95(8):4126-4133
Chase M.W.,and Reveal J.L.,2009,A phylogenetic classification of the land plants to accompany APGⅢ,Bot.J.the Linn.Soc.,161(2):122-127
Cheng A.X.,Lou Y.G.,Mao Y.B.,Lu S.,Wang L.J.,and Chen X.Y.,2007,Plant terpenoids:biosynthesis and ecological functions,J.Integr.Plant Biol.,49(2):179-186
Dai H.F.,Liu J.,Han Z.,Zeng Y.B.,Wang H.,and Mei W.L.,2010,Two new 2-(2-phenylethyl)chromones from Chinese eaglewood,J.Asian Nat.Prod.Res.,12(2):134-137
Ding X.P.,Wu C.L.,Zhu J.H.,Peng S.Q.,Mei W.L.,and Dai H.F.,2018,Identification and expression analysis of jasmonic acid receptor gene DcCOI1 in Dracaena cambodiana,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(10):4423-4430(丁旭坡,吴春来,朱家红,彭世清,梅文莉,戴好富,2018,海南龙血树茉莉酸受体基因DcCOI1的鉴定和表达分析,基因组学与应用生物学,37(10):4423-4430)
Facchini P.J.,and Chappell J.,1992,Gene family for an elicitor-induced sesquiterpene cyclase in tobacco,Proc.Natl.A-cad.Sci.USA,89(22):11088-11092
Hashim Y.Z.,Kerr P.G.,Abbas P.,and Mohd S.H.,2016,Aquilaria spp.(agarwood)as source of health beneficial compounds:a review of traditional use,phytochemistry and pharmacology,J.Ethnopharmacol.,189:331-360
Jin J.,Kim M.J.,Dhandapani S.,Tjhang J.G.,Yin J.L.,Wong L.,Sarojam R.,Chua N.H.,and Jang I.C.,2015,The floral transcriptome of ylang ylang(Cananga odorata var.fruticosa)uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase,J.Exp.Bot.,66(13):3959-3975
Kumar S.,Stecher G.,Li M.,Knyaz C.,and Tamura K.,2018,Mega X:molecular evolutionary genetics analysis across computing platforms,Mol.Biol.Evol.,35(6):1547-1549
Liao G.,Dong W.H.,Yang J.L.,Li W.,Wang J.,Mei W.L.,and Dai H.F.,2018,Monitoring the chemical profile in agarwood formation within one year and speculating on the biosynthesis of 2-(2-phenylethyl)chromones,Molecules,23(6):1261
Martin D.M.,F覿ldt J.,and Bohlmann J.,2004,Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily,Plant Physiol.,135(4):1908-1927
Paoli G.D.,Peart D.R.,Leighton M.,and Samsoedin I.,2001,An ecological and economic assessment of the nontimber forest product gaharu wood in Gunung Palung National Park,West Kalimantan,Indonesia.,Cons.Biol.,15(6):1721-1732
Srivastava P.L.,Daramwar P.P.,Krithika R.,Pandreka A.,Shankar S.S.,and Thulasiram H.V.,2015,Functional characterization of novel sesquiterpene synthases from Indian sandalwood,Santalum album.,Sci.Rep.,5:10095
Starks C.M.,Back K.,Chappell J.,and Noel J.P.,1997,Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase,Science,277(5333):1815-1820
Yu F.,and Utsumi R.,2009,Diversity,regulation,and genetic manipulation of plant mono-and sesquiterpenoid biosynthesis,Cell Mol.Life Sci.,66(18):3043-3052
Zhao W.,Zhu J.H.,Dai H.F.,Wang H.,Mei W.L.,Peng S.Q.,and Zhao R.,2016,Cloning and expression analysis of Leucoanthocyanidin reductase gene DcLAR1 in Dracaena cambod iana,Jiyinzuxue Yu Yingyong Sheng wuxue(Genomics and Applied Biology),35(9):2475-2479(赵婉,朱家红,戴好富,王辉,梅文莉,彭世清,赵蕊,2016,海南龙血树无色花色素还原酶基因DcLAR1的克隆和表达分析,基因组学与应用生物学,35(9):2475-2479)
Zhu J.H.,Cao T.J.,Dai H.F.,Li H.L.,Guo D.,Mei W.L.,and Peng S.Q.,2016,De Novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon's blood,Sci.Rep.,6:38315
Back K.,and Chappell J.,1996,Identifying functional domains within terpene cyclases using a domain-swapping strategy,Proc.Natl.Acad.Sci.USA,93(13):6841-6845
Back K.,Yin S.,and Chappell J.,1994,Expression of a plant sesquiterpene cyclase gene in Escherichia coli,Arch.Biochem.Biophys.,315(2):527-532
Bleeker P.M.,Spyropoulou E.A.,Diergaarde P.J.,Volpin H.,De Both M.T.,Zerbe P.,Bohlmann J.,and Schuurink R.C.,2011,RNA-seq d iscovery,functional characterization,and comparison of sesquiterpene synthases from Solanum lycopersicum and Solanum habrochaites trichomes,Plant Mol.Biol.,77(4-5):323-336
Bohlmann J.,Meyer-Gauen G.,and Croteau R.,1998,Plant terpenoid synthases:molecular biology and phylogenetic analysis,Proc.Natl.Acad.Sci.USA,95(8):4126-4133
Chase M.W.,and Reveal J.L.,2009,A phylogenetic classification of the land plants to accompany APGⅢ,Bot.J.the Linn.Soc.,161(2):122-127
Cheng A.X.,Lou Y.G.,Mao Y.B.,Lu S.,Wang L.J.,and Chen X.Y.,2007,Plant terpenoids:biosynthesis and ecological functions,J.Integr.Plant Biol.,49(2):179-186
Dai H.F.,Liu J.,Han Z.,Zeng Y.B.,Wang H.,and Mei W.L.,2010,Two new 2-(2-phenylethyl)chromones from Chinese eaglewood,J.Asian Nat.Prod.Res.,12(2):134-137
Ding X.P.,Wu C.L.,Zhu J.H.,Peng S.Q.,Mei W.L.,and Dai H.F.,2018,Identification and expression analysis of jasmonic acid receptor gene DcCOI1 in Dracaena cambodiana,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(10):4423-4430(丁旭坡,吴春来,朱家红,彭世清,梅文莉,戴好富,2018,海南龙血树茉莉酸受体基因DcCOI1的鉴定和表达分析,基因组学与应用生物学,37(10):4423-4430)
Facchini P.J.,and Chappell J.,1992,Gene family for an elicitor-induced sesquiterpene cyclase in tobacco,Proc.Natl.A-cad.Sci.USA,89(22):11088-11092
Hashim Y.Z.,Kerr P.G.,Abbas P.,and Mohd S.H.,2016,Aquilaria spp.(agarwood)as source of health beneficial compounds:a review of traditional use,phytochemistry and pharmacology,J.Ethnopharmacol.,189:331-360
Jin J.,Kim M.J.,Dhandapani S.,Tjhang J.G.,Yin J.L.,Wong L.,Sarojam R.,Chua N.H.,and Jang I.C.,2015,The floral transcriptome of ylang ylang(Cananga odorata var.fruticosa)uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase,J.Exp.Bot.,66(13):3959-3975
Kumar S.,Stecher G.,Li M.,Knyaz C.,and Tamura K.,2018,Mega X:molecular evolutionary genetics analysis across computing platforms,Mol.Biol.Evol.,35(6):1547-1549
Liao G.,Dong W.H.,Yang J.L.,Li W.,Wang J.,Mei W.L.,and Dai H.F.,2018,Monitoring the chemical profile in agarwood formation within one year and speculating on the biosynthesis of 2-(2-phenylethyl)chromones,Molecules,23(6):1261
Martin D.M.,F覿ldt J.,and Bohlmann J.,2004,Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily,Plant Physiol.,135(4):1908-1927
Paoli G.D.,Peart D.R.,Leighton M.,and Samsoedin I.,2001,An ecological and economic assessment of the nontimber forest product gaharu wood in Gunung Palung National Park,West Kalimantan,Indonesia.,Cons.Biol.,15(6):1721-1732
Srivastava P.L.,Daramwar P.P.,Krithika R.,Pandreka A.,Shankar S.S.,and Thulasiram H.V.,2015,Functional characterization of novel sesquiterpene synthases from Indian sandalwood,Santalum album.,Sci.Rep.,5:10095
Starks C.M.,Back K.,Chappell J.,and Noel J.P.,1997,Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase,Science,277(5333):1815-1820
Yu F.,and Utsumi R.,2009,Diversity,regulation,and genetic manipulation of plant mono-and sesquiterpenoid biosynthesis,Cell Mol.Life Sci.,66(18):3043-3052
Zhao W.,Zhu J.H.,Dai H.F.,Wang H.,Mei W.L.,Peng S.Q.,and Zhao R.,2016,Cloning and expression analysis of Leucoanthocyanidin reductase gene DcLAR1 in Dracaena cambod iana,Jiyinzuxue Yu Yingyong Sheng wuxue(Genomics and Applied Biology),35(9):2475-2479(赵婉,朱家红,戴好富,王辉,梅文莉,彭世清,赵蕊,2016,海南龙血树无色花色素还原酶基因DcLAR1的克隆和表达分析,基因组学与应用生物学,35(9):2475-2479)
Zhu J.H.,Cao T.J.,Dai H.F.,Li H.L.,Guo D.,Mei W.L.,and Peng S.Q.,2016,De Novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon's blood,Sci.Rep.,6:38315