大豆HMGR基因家族的鉴定与表达分析
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摘要
[目的]3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是大豆皂苷合成途径的关键酶之一。从全基因组水平鉴定大豆HMGR基因家族并进行生物信息学和表达模式分析,为研究和利用大豆HMGR基因家族生物学功能奠定基础。[方法]根据Pfam数据库中HMGR基因的保守结构域(编号PF00368),利用HMMER3.0、PFAM和SMART软件鉴定大豆基因组中的HMGR基因。采用ProtParam等软件对其基因和蛋白序列进行生物信息学分析。利用qRT-PCR方法分析GmHMGR基因在苗期根、茎、叶等不同组织中和籽粒不同发育时期的表达情况。[结果]大豆基因组中鉴定得到8个GmHMGR基因,分布于8条染色体上,编码80～608个氨基酸,相对分子质量介于8.23～64.96kD,理论等电点变化为4.89～8.24,二级结构中α螺旋和无规则卷曲所占比重较大,具有保守的基因结构和蛋白功能域。qRT-PCR分析表明,相比于其它GmHMGR基因,GmHMGR6在苗期根、茎、叶中高度表达,籽粒发育过程中GmHMGR3基因在开花后40d表达水平最高,开花后70d大豆的成熟籽粒中GmHMGR6的表达丰度最高。[结论]大豆基因组中含有8个HMGR基因,具有保守的功能结构域和基因结构。GmHMGR基因家族在苗期根、茎、叶及不同发育时期籽粒中具有多样化的组织表达模式和时空表达特征。
[Objective]3-hydroxy-3-methylglutaryl-coenzyme Areductase(HMGR)is one of the key enzymes involvedin the soybean saponin biosynthesis.Present study was aimed to identify and analyze HMGRfamily genes in soybean on the whole genome level which could support the in-depth study and utilization of HMGRfamily genes in the soybean.[Methods]HMMER3.0,PFAM and SMART software were applied to identify HMGR gene in the soybean genome based on the conserved domain of the HMGR genes of the Pfam database(No.PF00368).Bioinformatics analysis of HMGR gene and protein sequences were conducted with ProtParam and other software.Quantitative real time PCR was used to analyze the relative expression of HMGRgenes in roots,stems,leaves at seedling stage,and seeds at different developmental stages.[Results]A total of eight GmHMGR genes were identified which distributed in eight chromosomes.Proteins encodedby these genes contained 80～608 amino acids with the molecular weight ranged from 8.23～64.96 kD,and theoretical isoelectric point varied from 4.89～8.24.The main secondary structureof these proteins wereαhelix and random coils.Quantitative real time PCR analysisshowed that GmHMGR6 was highly expressed in various tissues at seedling stage.GmHMGR3 expressed at the highest level in 40 days after flowering,while GmHMGR6 produced the highest gene expression level among all HMGR genes,and it was highly expressed in 70 days after flowering when seeds was at matured stage.[Conclusion]Eight HMGRgenes existed in soybeans with conserved functional domains and gene structure.GmHMGR gene family owns diverse tissue and spatiotemporal expression characteristics.
[Objective]3-hydroxy-3-methylglutaryl-coenzyme Areductase(HMGR)is one of the key enzymes involvedin the soybean saponin biosynthesis.Present study was aimed to identify and analyze HMGRfamily genes in soybean on the whole genome level which could support the in-depth study and utilization of HMGRfamily genes in the soybean.[Methods]HMMER3.0,PFAM and SMART software were applied to identify HMGR gene in the soybean genome based on the conserved domain of the HMGR genes of the Pfam database(No.PF00368).Bioinformatics analysis of HMGR gene and protein sequences were conducted with ProtParam and other software.Quantitative real time PCR was used to analyze the relative expression of HMGRgenes in roots,stems,leaves at seedling stage,and seeds at different developmental stages.[Results]A total of eight GmHMGR genes were identified which distributed in eight chromosomes.Proteins encodedby these genes contained 80～608 amino acids with the molecular weight ranged from 8.23～64.96 kD,and theoretical isoelectric point varied from 4.89～8.24.The main secondary structureof these proteins wereαhelix and random coils.Quantitative real time PCR analysisshowed that GmHMGR6 was highly expressed in various tissues at seedling stage.GmHMGR3 expressed at the highest level in 40 days after flowering,while GmHMGR6 produced the highest gene expression level among all HMGR genes,and it was highly expressed in 70 days after flowering when seeds was at matured stage.[Conclusion]Eight HMGRgenes existed in soybeans with conserved functional domains and gene structure.GmHMGR gene family owns diverse tissue and spatiotemporal expression characteristics.
引文
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[3]Papadopoulou K,Melton R E,Leggett M,et al.Compromised disease resistance in saponin-deficient plants[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(22):12923-12928.
[4]Kuzina V,Ekstrom C T,Andersen S B,et al.Identification of defense compounds in barbarea vulgaris against the herbivore phyllotreta nemorum by an ecometabolomic approach[J].Plant Physiology,2009,151(4):1977-1990.
[5]Ellington A A,Berhow M,Singletary K W.Induction of macroautophagy in human colon cancer cells by soybean B-group triterpenoid saponins[J].Carcinogenesis,2005,26(1):159-167.
[6]Ellington A A,Berhow M A,Singletary K W.Inhibition of Akt signaling and enhanced ERK1/2activity are involved in induction of macroautophagy by triterpenoid B-group soyasaponins in colon cancer cells[J].Carcinogenesis,2005,27(2):298-306.
[7]张国红,肖纫秋.大豆皂甙对动脉粥样硬化的影响[J].吉林中医药,1993(3):43-44.
[8]Ishii Y,Tanizawa H.Effects of soyasaponins on lipid peroxidation through the secretion of thyroid hormones[J].Biological and Pharmaceutical Bulletin,2006,29(8):1759-1763.
[9]王章存.大豆皂甙研究进展[J].大豆科学,1996,15(1):74-79.
[10]Rouhi A M.Researchers unlocking potential of diverse,widely distributed saponins[J].Chemical and Engineering News,1995,11(37):28-43.
[11]Haralampidis K,Trojanowska M,Osbourn A E.Biosynthesis of triterpenoid saponins in plants[J].Advances in Biochemical Engineering Biotechnology,2002,75:31-49.
[12]Kim Y K,Kim Y B,Kim J K,et al.Molecular cloning and characterization of mevalonic acid(MVA)pathway genes and triterpene accumulation in Panax ginseng[J].Journal of the Korean Society for Applied Biological Chemistry,2014,57(3):289-295.
[13]Enjuto M,Lumbreras V,Marín C,et al.Expression of the Arabidopsis HMG2 gene,encoding 3-hydroxy-3-methylglutaryl coenzyme Areductase,is restricted to meristematic and floral tissues[J].The Plant Cell,1995,7(5):517-527.
[14]Ha S H,Lee S W,Kim Y M,et al.Molecular characterization of Hmg2 gene encoding a 3-hydroxy-methylglutaryl-CoA reductase in rice[J].Molecules and Cells,2001,11(3):295-302.
[15]Katoemori S,Higashi K,Hosoya K,et al.Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme Areductase in melon(Cucumis meloL.reticulatus)[J].Molecular Genetics and Genomics,2001,265(1):135-142.
[16]Wang Y,Guo B,Zhang F,et al.Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Hazel(Corylus avellana L.Gasaway)[J].Journal of Biochemistry and Molecular Biology,2007,40(6):861-869.
[17]Maldonado-Mendoza I E,Burnett R J,Nessler C L.Nucleotide sequence of a cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme Areductase from Catharanthus roseus[J].Plant Physiology,1992,100(3):1613-1614.
[18]Liao Z,Tan Q,Chai Y,et al.Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast[J].Functional Plant Biology,2004,31(1):73-81.
[19]Dai Z,Cui G,Zhou S F,et al.Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation[J].Journal of Plant Physiology,2011,168(2):148-157.
[20]Kim Y J,Lee O R,Oh J Y,et al.Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding genes in triterpene saponin-producing ginseng[J].Plant Physiology,2014,165(1):373-387.
[21]Ratna K,Lochana P,Ajit K S,et al.Molecular cloning,characterization and expression analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Centella asiatica L[J].Molecular Biology Reports,2015,42(9):1431-1439.
[22]Bhambhani S,Lakhwani D,Shukla T,et al.Genes encoding members of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)gene family from Azadirachta indica,and correlation with azadirachtin biosynthesis[J].Acta Physiologiae Plantarum,2017,39(2):65.
[23]Venkatachalam P,Priya P,Jayashree R,et al.Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme Areductase 1(hmgr1)gene from rubber tree(Hevea brasiliensisMuell.Arg.):A key gene involved in isoprenoid biosynthesis[J].Physiology and Molecular Biology of Plants,2009,15(2):133-143.
[24]李威.雷蒙德氏棉和亚洲棉萜类化合物合成关键基因家族的全基因组鉴定和系统发育研究[D].杭州:浙江大学,2014.
[25]Roberts S C.Production and engineering of terpenoids in plant cell culture[J].Nature Chemical Biology,2007,3(7):387-395.
[26]Darabi M,Masoudi-Nejad A,Nemat-Zadeh G.Bioinformatics study of the 3-hydroxy-3-methylglotaryl-coenzyme A reductase(HMGR)gene in Gramineae[J].Molecular Biology Reports,2012,39(9):8925-8935.
[27]Wei L,Wei L,Hengling W,et al.Species-specific expansion and molecular evolution of the 3-hydroxy-3-methylglutaryl coenzyme Areductase(HMGR)gene family in plants[J].Plos One,2014,9(4):e94172.
[28]罗红梅,宋经元,李雪莹,等.人参皂苷合成生物学关键元件HMGR基因克隆与表达分析[J].药学学报,2013,48(2):219-227.
[2]Osbourn A.Saponins and plant defence-a soap story[J].Trends in Plant Science,1996,1(1):4-9.
[3]Papadopoulou K,Melton R E,Leggett M,et al.Compromised disease resistance in saponin-deficient plants[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(22):12923-12928.
[4]Kuzina V,Ekstrom C T,Andersen S B,et al.Identification of defense compounds in barbarea vulgaris against the herbivore phyllotreta nemorum by an ecometabolomic approach[J].Plant Physiology,2009,151(4):1977-1990.
[5]Ellington A A,Berhow M,Singletary K W.Induction of macroautophagy in human colon cancer cells by soybean B-group triterpenoid saponins[J].Carcinogenesis,2005,26(1):159-167.
[6]Ellington A A,Berhow M A,Singletary K W.Inhibition of Akt signaling and enhanced ERK1/2activity are involved in induction of macroautophagy by triterpenoid B-group soyasaponins in colon cancer cells[J].Carcinogenesis,2005,27(2):298-306.
[7]张国红,肖纫秋.大豆皂甙对动脉粥样硬化的影响[J].吉林中医药,1993(3):43-44.
[8]Ishii Y,Tanizawa H.Effects of soyasaponins on lipid peroxidation through the secretion of thyroid hormones[J].Biological and Pharmaceutical Bulletin,2006,29(8):1759-1763.
[9]王章存.大豆皂甙研究进展[J].大豆科学,1996,15(1):74-79.
[10]Rouhi A M.Researchers unlocking potential of diverse,widely distributed saponins[J].Chemical and Engineering News,1995,11(37):28-43.
[11]Haralampidis K,Trojanowska M,Osbourn A E.Biosynthesis of triterpenoid saponins in plants[J].Advances in Biochemical Engineering Biotechnology,2002,75:31-49.
[12]Kim Y K,Kim Y B,Kim J K,et al.Molecular cloning and characterization of mevalonic acid(MVA)pathway genes and triterpene accumulation in Panax ginseng[J].Journal of the Korean Society for Applied Biological Chemistry,2014,57(3):289-295.
[13]Enjuto M,Lumbreras V,Marín C,et al.Expression of the Arabidopsis HMG2 gene,encoding 3-hydroxy-3-methylglutaryl coenzyme Areductase,is restricted to meristematic and floral tissues[J].The Plant Cell,1995,7(5):517-527.
[14]Ha S H,Lee S W,Kim Y M,et al.Molecular characterization of Hmg2 gene encoding a 3-hydroxy-methylglutaryl-CoA reductase in rice[J].Molecules and Cells,2001,11(3):295-302.
[15]Katoemori S,Higashi K,Hosoya K,et al.Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme Areductase in melon(Cucumis meloL.reticulatus)[J].Molecular Genetics and Genomics,2001,265(1):135-142.
[16]Wang Y,Guo B,Zhang F,et al.Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Hazel(Corylus avellana L.Gasaway)[J].Journal of Biochemistry and Molecular Biology,2007,40(6):861-869.
[17]Maldonado-Mendoza I E,Burnett R J,Nessler C L.Nucleotide sequence of a cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme Areductase from Catharanthus roseus[J].Plant Physiology,1992,100(3):1613-1614.
[18]Liao Z,Tan Q,Chai Y,et al.Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast[J].Functional Plant Biology,2004,31(1):73-81.
[19]Dai Z,Cui G,Zhou S F,et al.Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation[J].Journal of Plant Physiology,2011,168(2):148-157.
[20]Kim Y J,Lee O R,Oh J Y,et al.Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding genes in triterpene saponin-producing ginseng[J].Plant Physiology,2014,165(1):373-387.
[21]Ratna K,Lochana P,Ajit K S,et al.Molecular cloning,characterization and expression analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Centella asiatica L[J].Molecular Biology Reports,2015,42(9):1431-1439.
[22]Bhambhani S,Lakhwani D,Shukla T,et al.Genes encoding members of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)gene family from Azadirachta indica,and correlation with azadirachtin biosynthesis[J].Acta Physiologiae Plantarum,2017,39(2):65.
[23]Venkatachalam P,Priya P,Jayashree R,et al.Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme Areductase 1(hmgr1)gene from rubber tree(Hevea brasiliensisMuell.Arg.):A key gene involved in isoprenoid biosynthesis[J].Physiology and Molecular Biology of Plants,2009,15(2):133-143.
[24]李威.雷蒙德氏棉和亚洲棉萜类化合物合成关键基因家族的全基因组鉴定和系统发育研究[D].杭州:浙江大学,2014.
[25]Roberts S C.Production and engineering of terpenoids in plant cell culture[J].Nature Chemical Biology,2007,3(7):387-395.
[26]Darabi M,Masoudi-Nejad A,Nemat-Zadeh G.Bioinformatics study of the 3-hydroxy-3-methylglotaryl-coenzyme A reductase(HMGR)gene in Gramineae[J].Molecular Biology Reports,2012,39(9):8925-8935.
[27]Wei L,Wei L,Hengling W,et al.Species-specific expansion and molecular evolution of the 3-hydroxy-3-methylglutaryl coenzyme Areductase(HMGR)gene family in plants[J].Plos One,2014,9(4):e94172.
[28]罗红梅,宋经元,李雪莹,等.人参皂苷合成生物学关键元件HMGR基因克隆与表达分析[J].药学学报,2013,48(2):219-227.