基因组步移技术克隆思茅松α-蒎烯合成酶基因及表达分析
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摘要
蒎烯合成酶(α-pinene synthase, APS)是α-蒎烯生物合成的关键酶。本研究利用同源克隆及基因组步移法从思茅松中克隆得到一个α-蒎烯合成酶基因,命名为PkAPS (GenBank登录号为KX394684),基因全长3 523 bp,含有10个内含子,其c DNA全长1 956 bp,编码651个氨基酸残基。生物信息学分析显示PkAPS属于萜烯合成酶家族蛋白;系统进化树分析显示PkAPS与马尾松α-蒎烯合成酶亲缘关系最近。基因表达分析显示:PkAPS在高产脂思茅松个体各组织的表达量均高于低产产脂思茅松个体;而在高产脂思茅松不同组织中的表达比较中,PkAPS在小枝中的表达量最高。本研究将有利于进一步研究思茅松α-蒎烯合成酶调控机理及培育高产α-蒎烯思茅松新品种的研究。
pinene synthase(APS) is a key enzyme during the process of α-pinene biosynthesis. The present study cloned an α-pinene synthase gene from Pinus kesiya var langbianensis with the methods of homologous gene cloning and genomic walking cloning, and named it Pk APS(GenBank ID: KX394684). There is 3 523 bp including 10 introns of Pk APS, its full length of c DNA is 1 956 bp and encodes 651 amino acids residue. The bioinformatics showed that Pk APS is assigned to terpene biosynthetic enzyme families, and the dendrogram revealed that Pk APS should be the closest genetic relationships with the α-pinene synthase gene from P. massoniana. The gene expression clarified the expression levels of gene Pk APS originated from all the individuals in the high-yield oleoresin P. kesiya are higher than that of in the low-yield oleoresin P. kesiya; and the Pk APS gene expression compared with the different tissues of high-yield oleoresin P. kesiya revealed the highest expression levels was in the branchlet. The results of this research has applied some theoretical basis for exploring the mechanism of α-pinene biosynthetic regulation and breeding a new variety of P. kesiya that could produce higher yield α-pinene annually.
pinene synthase(APS) is a key enzyme during the process of α-pinene biosynthesis. The present study cloned an α-pinene synthase gene from Pinus kesiya var langbianensis with the methods of homologous gene cloning and genomic walking cloning, and named it Pk APS(GenBank ID: KX394684). There is 3 523 bp including 10 introns of Pk APS, its full length of c DNA is 1 956 bp and encodes 651 amino acids residue. The bioinformatics showed that Pk APS is assigned to terpene biosynthetic enzyme families, and the dendrogram revealed that Pk APS should be the closest genetic relationships with the α-pinene synthase gene from P. massoniana. The gene expression clarified the expression levels of gene Pk APS originated from all the individuals in the high-yield oleoresin P. kesiya are higher than that of in the low-yield oleoresin P. kesiya; and the Pk APS gene expression compared with the different tissues of high-yield oleoresin P. kesiya revealed the highest expression levels was in the branchlet. The results of this research has applied some theoretical basis for exploring the mechanism of α-pinene biosynthetic regulation and breeding a new variety of P. kesiya that could produce higher yield α-pinene annually.
引文
Byun-Mckay A.,Godard K.A.,Toudefallah M.,Martin D.M.,Alfaro R.,King J.,Bohlmann J.,and Plant A.L.,2006,Woundinduced terpene synthase gene expression in Sitka spruce that exhibit resistance or susceptibility to attack by the white Pine weevil,Plant Physiol.,140(3):1009-1021
Cai N.H.,Xu Y.L.,Wang D.W.,Chen S.,and Li G.Q.,2017,Identification and characterization of microsatellite markers in Pinus kesiya var.langbianensis(Pinaceae),Appl.Plant Sci.,5(2):1600126
Jiang Y.D.,Li S.G.,and Liu Y.C.,2017,Review on the research progress of the oriented cultivation techniques for the high-resin-yield Pinus kesiya var.langbianensis,Xibu Linye Kexue(Journal of West China Forestry Science),46(2):32-37(蒋云东,李思广,刘云彩,2017,思茅松高产脂定向培育技术的研究进展,西部林业科学,46(2):32-37)
Kim Y.B.,Kim S.M.,Kang M.K.,Kuzuyama T.,Lee J.K.,Park S.C.,Shin S.C.,and Kim S.U.,2009,Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes,Tree Physiol.,29(5):737-749
Lange B.M.,and Rios-Estepa R.,2014,Kinetic modeling of plant metabolism and its predictive power:peppermint essential oil biosynthesis as an example,Methods Mol.Biol.,1083(1):287-311
Lei L.,Pan X.Q.,Zhang L.,Huang S.W.,Zhao H.,Yi M.,and Lai M.,2015,An analysi of cloning and bioinformatics of sinistral beta pinene synthetase gene PeTPS-(-)BPin in Pinus elliottii,Jiangxi Nongye Daxue Xuebao(Acta Agriculture Universitatis Jiangxiensis),37(2):205-211(雷蕾,潘显强,张露,黄少伟,赵衡,易敏,赖猛,2015,湿地松β-蒎烯合成酶基因PeTPS-(-)BPin的同源克隆及生物信息学分析,江西农业大学学报,37(2):205-211)
Li S.F.,Su J.R.,Liu W.D.,Lang X.D.,Zhang Z.J.,Su L.,Jia C.X.Z.,and Yang H.J.,2013,Phenotypic variations in cones and seeds of natural Pinus kesiya var.langbianensis populations in Yunnan Province,China,Zhiwu Shengtai Xuebao(Chinese Journal of Plant Ecology),37(11):998-1009(李帅锋,苏建荣,刘万德,郎学东,张志钧,苏磊,贾呈鑫卓,杨华景,2013,思茅松天然群体种实表型变异,植物生态学报,37(11):998-1009)
Li S.G.,Jiang Y.D.,He J.,and Li M.,2007,Cluster analysis of high resin yield grafting clones of Pinus kesiya var.langbianensis,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry and Technology),27(3):45-48(李思广,蒋云东,何俊,李明,2007,高产脂思茅松无性系的选育,中南林业科技大学学报,27(3):45-48)
Li Y.L.,Zhao F.C.,Zhang Y.Z.,and Wu H.S.,2009,Applications of molecular marker on the research of pine genetics and evolution,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(5):1004-1009(李义良,赵奋成,张应中,吴慧珊,2009,分子标记在松树遗传与进化研究中的应用,分子植物育种,7(5):1004-1009)
Liu G.B.,and Ji K.S.,2009,Designing Pinus massoniana SSRprimers from Pinus EST sequences,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(4):833-838(刘公秉,季孔庶,2009,基于松树EST序列的马尾松SSR引物开发,分子植物育种,7(4):833-838)
Liu Q.H.,Zhou Z.C.,Wei Y.C.,Shen D.Y.,Feng Z.P.,and Hong S.P.,2015,Genome-wide identification of differentially expressed genes associated with the high yielding of oleoresin in secondary xylem of Masson Pine(Pinus massoniana Lamb)by transcriptomic analysis,PLoS One,10(7):e0132624
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
McKay S.A.,Hunter W.L.,Godard K.A.,Wang S.X.,Martin D.M.,Bohlmann J.,and Plant A.L.,2003,Insect attack and wounding induce traumatic resin duct development and gene expression of(-)-pinene synthase in Sitka spruce,Plant physiol.,133(1):368-378
Miao F.J.,Li J.,Xiong Z.,Yuan X.L.,Yang Y.M.,and Wang J.,2016,Analysis on resin-producing capacity of Pinus kesiya,Fujian Nonglin Daxue Xuebao(Ziranban)(Journal of Fujian Agriculture and Forestry University(Natural Science Edition)),45(4):405-408(缪福俊,李江,熊智,原晓龙,杨宇明,王娟,2016,思茅松产脂力分析,福建农林大学学报(自然版),45(4):405-408)
Phillips M.A.,Wildung M.R.,Williams D.C.,Hyatt D.C.,and Croteau R.,2003,cDNA isolation,functional expression,and characterization of(+)-α-pinene synthase and(-)-α-pinene synthase from loblolly pine(Pinus taeda):stereocontrol in pinene biosynthesis,Arch.Biochem.Biophys.,411(2):267-276
Wang Y.,Liu Z.Y.,Lv Q.,and Zhang X.Y.,2014,Cloning and sequence analysis ofα-pinene synthase gene from Pinus massoniana,Anhui Nongye Kexue(Journal of Anhui Agriculture Science),42(13):3808-3811(王颖,刘振宇,吕全,张星耀,2014,马尾松α-蒎烯合成酶基因cDNA全长克隆及序列分析,安徽农业科学,42(13):3808-3811)
Wang Y.,Zhou X.,Bi W.,Yang Y.M.,Li J.,and Wang J.,2015a,Identification and characterization of a 1-deoxy-D-xylulose5-phosphate synthase gene from Pinus kesiya var.langbianensis,Linye Kexue Yanjiu(Forest Research),28(6):833-838(王毅,周旭,毕玮,杨宇明,李江,王娟,2015a,思茅松1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析,林业科学研究,28(6):833-838)
Wang Y.,Zhou X.,Bi W.,Yang Y.M.,Li J.,and Wang J.,2015b,Cloning and sequence analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene cDNA from Pi nus kesiya var.langbianensis,Guangxi Zhiwu(Guihaia),35(5):721-727(王毅,周旭,毕玮,杨宇明,李江,王娟,2015b,思茅松HDR基因全长cDNA克隆与序列分析,广西植物,35(5):721-727)
Weng H.L.,Chen H.W.,and Duan A.A.,2010,Selection of plus-tree with high-rosin production for Pinus kesiya var.langbianensis,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),25(3):71-74(翁海龙,陈宏伟,段安安,2010,思茅松高产脂优树选择,西北林学院学报,25(3):71-74)
Westbrook J.W.,Resende M.F.R.,Munoz P.,Walker A.R.,Wegrzyn J.L.,Nelson C.D.,Neale D.B.,Kirst M.,Huber D.A.,Gezan S.A.,Peter G.F.,and Davis J.M.,2013,Association genetics of oleoresin flow in loblolly pine:discovering genes and predicting phenotype for improved resistance to bark beetles and bioenergy potential,New Phytol.,199(1):89-100
Wu S.R.,Ma Y.F.,Li Z.Y.,Zhou P.Y.,Yuan S.Y.,and Xiao C.,2009,Analysis of chemical composition of turpentine in Pinus kesiya var.langbianensis,Xibu Linye Kexue(Journal of West China Forestry Science),38(3):90-92(伍苏然,马艳粉,李正跃,周平阳,袁盛勇,肖春,2009,思茅松松节油化学成分分析,西部林业科学,38(3):90-92)
Xu L.H.,Jiang Y.D.,Fu Y.P.,Zhang K.F.,Zhao Y.H.,and Li S.G.,2014,Selection of half-sib Pinus kesiya var.langbianensis with high-resin-yield,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),29(3):109-112(许林红,蒋云东,付玉嫔,张快富,赵永红,李思广,2014,思茅松高产脂半同胞家系选育,西北林学院学报,29(3):109-112)
Yan M.M.,Dai X.G.,Li S.X.,and Yin T.M.,2011,Sequence analysis and comparison of EST-SSRs in Pine,Polar and Eucalyptus,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(1):103-109(阎毛毛,戴晓港,李淑娴,尹佟明,2011,松树、杨树及桉树表达基因序列微卫星比对分析,基因组学与应用生物学,30(1):103-109)
Yang T.,and Zeng Y.,2005,Plant terpenoid synthases:molecular biology and genetic engineering,Yunnan Zhiwu Yanjiu(Acta Botanica Yunnanica),27(1):1-10(杨涛,曾英,2005,植物萜类合酶研究进展,云南植物研究,27(1):1-10)
Zhao N.,Yuan X.L.,Miao F.J.,Wang Y.,Chen W.,Li J.,Wu T.,and Wang J.,2017,Development of SSR molecular markers based on transcriptome data of Pinus kesiya var.langbianensis,Shengwu Jishu Tongbao(Biotechnology Bulletin),33(5):71-77(赵能,原晓龙,缪福俊,王毅,陈伟,李江,吴涛,王娟,2017,思茅松转录组SSR分析及标记开发,生物技术通报,33(5):71-77)
Zhu Y.F.,Chen S.Y.,Hao J.B.,and Wu T.,2015,RAPD analysis of genetic diversity of Pinus kesiya var.langbianensis from seed orchard,Xibu Linye Kexue(Journal of West China Forestry Science),44(2):141-146(朱云凤,陈少瑜,郝佳波,吴涛,2015,思茅松种子园遗传多样性的RAPD分析,西部林业科学,44(2):141-146)
Cai N.H.,Xu Y.L.,Wang D.W.,Chen S.,and Li G.Q.,2017,Identification and characterization of microsatellite markers in Pinus kesiya var.langbianensis(Pinaceae),Appl.Plant Sci.,5(2):1600126
Jiang Y.D.,Li S.G.,and Liu Y.C.,2017,Review on the research progress of the oriented cultivation techniques for the high-resin-yield Pinus kesiya var.langbianensis,Xibu Linye Kexue(Journal of West China Forestry Science),46(2):32-37(蒋云东,李思广,刘云彩,2017,思茅松高产脂定向培育技术的研究进展,西部林业科学,46(2):32-37)
Kim Y.B.,Kim S.M.,Kang M.K.,Kuzuyama T.,Lee J.K.,Park S.C.,Shin S.C.,and Kim S.U.,2009,Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes,Tree Physiol.,29(5):737-749
Lange B.M.,and Rios-Estepa R.,2014,Kinetic modeling of plant metabolism and its predictive power:peppermint essential oil biosynthesis as an example,Methods Mol.Biol.,1083(1):287-311
Lei L.,Pan X.Q.,Zhang L.,Huang S.W.,Zhao H.,Yi M.,and Lai M.,2015,An analysi of cloning and bioinformatics of sinistral beta pinene synthetase gene PeTPS-(-)BPin in Pinus elliottii,Jiangxi Nongye Daxue Xuebao(Acta Agriculture Universitatis Jiangxiensis),37(2):205-211(雷蕾,潘显强,张露,黄少伟,赵衡,易敏,赖猛,2015,湿地松β-蒎烯合成酶基因PeTPS-(-)BPin的同源克隆及生物信息学分析,江西农业大学学报,37(2):205-211)
Li S.F.,Su J.R.,Liu W.D.,Lang X.D.,Zhang Z.J.,Su L.,Jia C.X.Z.,and Yang H.J.,2013,Phenotypic variations in cones and seeds of natural Pinus kesiya var.langbianensis populations in Yunnan Province,China,Zhiwu Shengtai Xuebao(Chinese Journal of Plant Ecology),37(11):998-1009(李帅锋,苏建荣,刘万德,郎学东,张志钧,苏磊,贾呈鑫卓,杨华景,2013,思茅松天然群体种实表型变异,植物生态学报,37(11):998-1009)
Li S.G.,Jiang Y.D.,He J.,and Li M.,2007,Cluster analysis of high resin yield grafting clones of Pinus kesiya var.langbianensis,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry and Technology),27(3):45-48(李思广,蒋云东,何俊,李明,2007,高产脂思茅松无性系的选育,中南林业科技大学学报,27(3):45-48)
Li Y.L.,Zhao F.C.,Zhang Y.Z.,and Wu H.S.,2009,Applications of molecular marker on the research of pine genetics and evolution,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(5):1004-1009(李义良,赵奋成,张应中,吴慧珊,2009,分子标记在松树遗传与进化研究中的应用,分子植物育种,7(5):1004-1009)
Liu G.B.,and Ji K.S.,2009,Designing Pinus massoniana SSRprimers from Pinus EST sequences,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(4):833-838(刘公秉,季孔庶,2009,基于松树EST序列的马尾松SSR引物开发,分子植物育种,7(4):833-838)
Liu Q.H.,Zhou Z.C.,Wei Y.C.,Shen D.Y.,Feng Z.P.,and Hong S.P.,2015,Genome-wide identification of differentially expressed genes associated with the high yielding of oleoresin in secondary xylem of Masson Pine(Pinus massoniana Lamb)by transcriptomic analysis,PLoS One,10(7):e0132624
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
McKay S.A.,Hunter W.L.,Godard K.A.,Wang S.X.,Martin D.M.,Bohlmann J.,and Plant A.L.,2003,Insect attack and wounding induce traumatic resin duct development and gene expression of(-)-pinene synthase in Sitka spruce,Plant physiol.,133(1):368-378
Miao F.J.,Li J.,Xiong Z.,Yuan X.L.,Yang Y.M.,and Wang J.,2016,Analysis on resin-producing capacity of Pinus kesiya,Fujian Nonglin Daxue Xuebao(Ziranban)(Journal of Fujian Agriculture and Forestry University(Natural Science Edition)),45(4):405-408(缪福俊,李江,熊智,原晓龙,杨宇明,王娟,2016,思茅松产脂力分析,福建农林大学学报(自然版),45(4):405-408)
Phillips M.A.,Wildung M.R.,Williams D.C.,Hyatt D.C.,and Croteau R.,2003,cDNA isolation,functional expression,and characterization of(+)-α-pinene synthase and(-)-α-pinene synthase from loblolly pine(Pinus taeda):stereocontrol in pinene biosynthesis,Arch.Biochem.Biophys.,411(2):267-276
Wang Y.,Liu Z.Y.,Lv Q.,and Zhang X.Y.,2014,Cloning and sequence analysis ofα-pinene synthase gene from Pinus massoniana,Anhui Nongye Kexue(Journal of Anhui Agriculture Science),42(13):3808-3811(王颖,刘振宇,吕全,张星耀,2014,马尾松α-蒎烯合成酶基因cDNA全长克隆及序列分析,安徽农业科学,42(13):3808-3811)
Wang Y.,Zhou X.,Bi W.,Yang Y.M.,Li J.,and Wang J.,2015a,Identification and characterization of a 1-deoxy-D-xylulose5-phosphate synthase gene from Pinus kesiya var.langbianensis,Linye Kexue Yanjiu(Forest Research),28(6):833-838(王毅,周旭,毕玮,杨宇明,李江,王娟,2015a,思茅松1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析,林业科学研究,28(6):833-838)
Wang Y.,Zhou X.,Bi W.,Yang Y.M.,Li J.,and Wang J.,2015b,Cloning and sequence analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene cDNA from Pi nus kesiya var.langbianensis,Guangxi Zhiwu(Guihaia),35(5):721-727(王毅,周旭,毕玮,杨宇明,李江,王娟,2015b,思茅松HDR基因全长cDNA克隆与序列分析,广西植物,35(5):721-727)
Weng H.L.,Chen H.W.,and Duan A.A.,2010,Selection of plus-tree with high-rosin production for Pinus kesiya var.langbianensis,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),25(3):71-74(翁海龙,陈宏伟,段安安,2010,思茅松高产脂优树选择,西北林学院学报,25(3):71-74)
Westbrook J.W.,Resende M.F.R.,Munoz P.,Walker A.R.,Wegrzyn J.L.,Nelson C.D.,Neale D.B.,Kirst M.,Huber D.A.,Gezan S.A.,Peter G.F.,and Davis J.M.,2013,Association genetics of oleoresin flow in loblolly pine:discovering genes and predicting phenotype for improved resistance to bark beetles and bioenergy potential,New Phytol.,199(1):89-100
Wu S.R.,Ma Y.F.,Li Z.Y.,Zhou P.Y.,Yuan S.Y.,and Xiao C.,2009,Analysis of chemical composition of turpentine in Pinus kesiya var.langbianensis,Xibu Linye Kexue(Journal of West China Forestry Science),38(3):90-92(伍苏然,马艳粉,李正跃,周平阳,袁盛勇,肖春,2009,思茅松松节油化学成分分析,西部林业科学,38(3):90-92)
Xu L.H.,Jiang Y.D.,Fu Y.P.,Zhang K.F.,Zhao Y.H.,and Li S.G.,2014,Selection of half-sib Pinus kesiya var.langbianensis with high-resin-yield,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),29(3):109-112(许林红,蒋云东,付玉嫔,张快富,赵永红,李思广,2014,思茅松高产脂半同胞家系选育,西北林学院学报,29(3):109-112)
Yan M.M.,Dai X.G.,Li S.X.,and Yin T.M.,2011,Sequence analysis and comparison of EST-SSRs in Pine,Polar and Eucalyptus,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(1):103-109(阎毛毛,戴晓港,李淑娴,尹佟明,2011,松树、杨树及桉树表达基因序列微卫星比对分析,基因组学与应用生物学,30(1):103-109)
Yang T.,and Zeng Y.,2005,Plant terpenoid synthases:molecular biology and genetic engineering,Yunnan Zhiwu Yanjiu(Acta Botanica Yunnanica),27(1):1-10(杨涛,曾英,2005,植物萜类合酶研究进展,云南植物研究,27(1):1-10)
Zhao N.,Yuan X.L.,Miao F.J.,Wang Y.,Chen W.,Li J.,Wu T.,and Wang J.,2017,Development of SSR molecular markers based on transcriptome data of Pinus kesiya var.langbianensis,Shengwu Jishu Tongbao(Biotechnology Bulletin),33(5):71-77(赵能,原晓龙,缪福俊,王毅,陈伟,李江,吴涛,王娟,2017,思茅松转录组SSR分析及标记开发,生物技术通报,33(5):71-77)
Zhu Y.F.,Chen S.Y.,Hao J.B.,and Wu T.,2015,RAPD analysis of genetic diversity of Pinus kesiya var.langbianensis from seed orchard,Xibu Linye Kexue(Journal of West China Forestry Science),44(2):141-146(朱云凤,陈少瑜,郝佳波,吴涛,2015,思茅松种子园遗传多样性的RAPD分析,西部林业科学,44(2):141-146)
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