橡胶树中碱性转化酶HbNIN9表达及酶学特性分析
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摘要
转化酶作为蔗糖分解代谢的关键酶,在植物生长发育、生物与非生物胁迫响应等生理过程中发挥重要作用。基于对橡胶树不同组织转录组数据库的分析,本研究克隆了一个在橡胶树种子中有较高丰度表达的中碱性转化酶基因HbNIN9;系统进化分析和在线软件预测均显示其亚细胞定位为线粒体,属于中碱性转化酶α亚族成员;利用原核表达体系成功获得Hb NIN9优化表达的重组蛋白,优化表达的诱导条件为0.5 mmol/L IPTG,25℃培养6 h;酶学特性分析表明,HbNIN9蛋白酶活反应的最适温度为45℃,最适pH值为7.5,米氏常数Km值为18.09 mmol/L。本研究从蛋白水平证实了Hb NIN9为典型的中碱性转化酶,有助于深入揭示线粒体型转化酶在橡胶树种子糖代谢中生理功能,丰富橡胶树中碱性转化酶家族成员的生理生化研究。
Invertases, as a kind of key enzymes of sucrose metabolism in higher plants, play an important role in plants growth, development and stress response. Basing on transcriptome data of tissues from Hevea brasiliensis,HbNIN9, a new neutral/alkaline invertase gene mainly expressed in seeds, was cloned. Phylogenetic analysis and subcellular location prediction showed that Hb NIN9 was located in mitochondria and belonged to a member of αgroup of neutral/alkaline invertases. HbNIN9 was successfully expressed in E. coli, and the expression conditions were optimized as 6 h induction with 0.5 mmol/L IPTG at 25℃. The biochemical properties of HbNIN9 enzymatic activity were investigated. HbNIN9 showed the highest enzymatic activity at pH 7.5 and 45℃, and had a Km of18.09 mmol/L for its substrate of sucrose. The study shows that Hb NIN9 is a typical neural/alkaline invertase in the protein level, which can help to reveal the physiological function of the mitochondrial-type invertase in the seed sugar metabolism of the rubber tree, and enriched the physiological and biochemical study of alkaline transforming enzyme family members in Hevea brasiliensis.
Invertases, as a kind of key enzymes of sucrose metabolism in higher plants, play an important role in plants growth, development and stress response. Basing on transcriptome data of tissues from Hevea brasiliensis,HbNIN9, a new neutral/alkaline invertase gene mainly expressed in seeds, was cloned. Phylogenetic analysis and subcellular location prediction showed that Hb NIN9 was located in mitochondria and belonged to a member of αgroup of neutral/alkaline invertases. HbNIN9 was successfully expressed in E. coli, and the expression conditions were optimized as 6 h induction with 0.5 mmol/L IPTG at 25℃. The biochemical properties of HbNIN9 enzymatic activity were investigated. HbNIN9 showed the highest enzymatic activity at pH 7.5 and 45℃, and had a Km of18.09 mmol/L for its substrate of sucrose. The study shows that Hb NIN9 is a typical neural/alkaline invertase in the protein level, which can help to reveal the physiological function of the mitochondrial-type invertase in the seed sugar metabolism of the rubber tree, and enriched the physiological and biochemical study of alkaline transforming enzyme family members in Hevea brasiliensis.
引文
Barratt D.H.P.,Derbyshire P.,Findlay K.,Pike M.,Wellner N.,Lunn J.,Feil R.,Simpson C.,Maule A.J.,and Smith A.M.,2009,Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase,Proc.Natl.Acad.Sci.USA,106(31):13124-13129
Huang Y.,Niu J.Z.,Tang J.F.,Lu Y.S.,Wang Z.W.,Zheng Q.,Cai J.,and Jian J.C.,2018,Prokaryotic expression and condition optimization of NCCRP-1 gene in nile tilapia,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(9):3753-3758(黄瑜,牛金中,汤菊芬,鲁义善,汪志文,郑琦,蔡佳,简纪常,2018,尼罗罗非鱼NCC-RP-1基因的原核表达及条件优化,基因组学与应用生物学,37(9):3753-3758)
Ji X.M.,Van den Ende W.,Van Laere A.,Cheng S.H.,and Bennett J.,2005,Structure,evolution and expression of the two invertase gene families of rice,Journal of Molecular Evolution,60(5):615-634
Liu S.J.,Lan J.X.,Zhou B.H.,Qin Y.X.,Zhou Y.H.,Xiao X.H.,Yang J.H.,Gou J.Q.,Qi J.Y.,Huang Y.C.,and Tang C.R.,2015,Hb NIN2,a cytosolic alkaline/neutral-invertase,is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis(para rubber tree),New Phytologist,206(2):709-725
Mariana L.M.,Leandra L.,Eduardo J.Z.,and Graciela L.S.,2013,A mitochondrial alkaline/neutral invertase isoform(A/N-Inv C)functions in developmental energy-demanding processes in Arabidopsis,Planta,237(3):813-822
Murayama S.,and Handa H.,2006,Genes for alkaline/neutral invertase in rice:alkaline/neutral invertases are located in plant mitochondria and also in plastids,Planta,225(5):1193-1203
Nonis A.,Ruperti B.,Pierasco A.,Canaguier A.,Adam-Blondon A.F.,Gaspero G.D.,and Vizzotto G.,2008,Neutral invertases in grapevine and comparative analysis with Arabidopsis,poplar and rice,Planta,229(1):129-142
Qian W.J.,Yue C.,Cao H.L.,Hao X.Y,Wang L.,Wang Y.C.,Huang Y.T.,Wang B.,Wang X.C.,Xiao B.,and Yang Y.J.,2016,Cloning and expression analysis of a neutral/alkaline invertase gene(CsINV10)in tea plant(Camellia sinensis L.O.Kuntze),Zuowu Xuebao(Acta Agronomica Sinica),42(3):376-388(钱文俊,岳川,曹红利,郝心愿,王璐,王玉春,黄玉婷,王博,王新超,肖斌,杨亚军,2016,茶树中性/碱性转化酶基因CsINV10的克隆与表达分析,作物学报,42(3):376-388)
Ruan Y.L.,Jin Y.,and Huang J.,2009,Capping invertase activity by its inhibitor:roles and implications in sugar signaling,carbon allocation,senescence and evolution,Plant Signaling and Behavior,4(10):983-985
Sturm A.,1999,Invertases,primary structures,functions and roles in plant development and sucrose partitioning,Plant Physiol.,121(1):1-8
Szarka A.,Horemans N.,Passarella S.,Tarcsay魣.,魻rsi F.,SalgóA.,and Bánhegyi G.,2008,Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke(Helianthus tuberosus L.)tubers,Planta,228(5):765-775
Tup伥J.,d'Auzac J.,and Chrestin H.,eds.,1988,Sucrose supply and utilization for latex production,In:Physiology of Rubber Tree Latex,CRC Press,Boca Raton,pp.275-332
Tup伥J.,and Primot L.,1976,Control of carbohydrate metabolism by ethylene in latex vessels of Hevea brasiliensis Muel.ARCin relation to rubber production,Biologia Plantarum,18(5):373-384
Tup伥J.,and Primot L.,1982,Sucrose synthetase in the latex of Hevea brasiliensis Muell,J.Exp.Bot.,33(136):988-995
Tup伥J.,1973,The regulation of invertase activity in the latex of Hevea brasiliensis Muell,J.Exp.Bot.,24(3):516-524
Tymowska-Lalanne Z.,and Kreis M.,1998,The plant invertases:physiology,biochemistry and molecular biology,Advances in Botanical Research,28:71-117
Vargas W.A.,Cumino A.,and Salerno G.L.,2003,Cyanobacterial alkaline/neutral invertases,Origin of sucrose hydrolysis in the plant cytosol?Planta,216(6):951-960
Vargas W.A.,Pontis H.G.,and Salerno G.L.,2007,New insights on sucrose metabolism:evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking,Planta,227(4):795-807
Huang Y.,Niu J.Z.,Tang J.F.,Lu Y.S.,Wang Z.W.,Zheng Q.,Cai J.,and Jian J.C.,2018,Prokaryotic expression and condition optimization of NCCRP-1 gene in nile tilapia,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(9):3753-3758(黄瑜,牛金中,汤菊芬,鲁义善,汪志文,郑琦,蔡佳,简纪常,2018,尼罗罗非鱼NCC-RP-1基因的原核表达及条件优化,基因组学与应用生物学,37(9):3753-3758)
Ji X.M.,Van den Ende W.,Van Laere A.,Cheng S.H.,and Bennett J.,2005,Structure,evolution and expression of the two invertase gene families of rice,Journal of Molecular Evolution,60(5):615-634
Liu S.J.,Lan J.X.,Zhou B.H.,Qin Y.X.,Zhou Y.H.,Xiao X.H.,Yang J.H.,Gou J.Q.,Qi J.Y.,Huang Y.C.,and Tang C.R.,2015,Hb NIN2,a cytosolic alkaline/neutral-invertase,is responsible for sucrose catabolism in rubber-producing laticifers of Hevea brasiliensis(para rubber tree),New Phytologist,206(2):709-725
Mariana L.M.,Leandra L.,Eduardo J.Z.,and Graciela L.S.,2013,A mitochondrial alkaline/neutral invertase isoform(A/N-Inv C)functions in developmental energy-demanding processes in Arabidopsis,Planta,237(3):813-822
Murayama S.,and Handa H.,2006,Genes for alkaline/neutral invertase in rice:alkaline/neutral invertases are located in plant mitochondria and also in plastids,Planta,225(5):1193-1203
Nonis A.,Ruperti B.,Pierasco A.,Canaguier A.,Adam-Blondon A.F.,Gaspero G.D.,and Vizzotto G.,2008,Neutral invertases in grapevine and comparative analysis with Arabidopsis,poplar and rice,Planta,229(1):129-142
Qian W.J.,Yue C.,Cao H.L.,Hao X.Y,Wang L.,Wang Y.C.,Huang Y.T.,Wang B.,Wang X.C.,Xiao B.,and Yang Y.J.,2016,Cloning and expression analysis of a neutral/alkaline invertase gene(CsINV10)in tea plant(Camellia sinensis L.O.Kuntze),Zuowu Xuebao(Acta Agronomica Sinica),42(3):376-388(钱文俊,岳川,曹红利,郝心愿,王璐,王玉春,黄玉婷,王博,王新超,肖斌,杨亚军,2016,茶树中性/碱性转化酶基因CsINV10的克隆与表达分析,作物学报,42(3):376-388)
Ruan Y.L.,Jin Y.,and Huang J.,2009,Capping invertase activity by its inhibitor:roles and implications in sugar signaling,carbon allocation,senescence and evolution,Plant Signaling and Behavior,4(10):983-985
Sturm A.,1999,Invertases,primary structures,functions and roles in plant development and sucrose partitioning,Plant Physiol.,121(1):1-8
Szarka A.,Horemans N.,Passarella S.,Tarcsay魣.,魻rsi F.,SalgóA.,and Bánhegyi G.,2008,Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke(Helianthus tuberosus L.)tubers,Planta,228(5):765-775
Tup伥J.,d'Auzac J.,and Chrestin H.,eds.,1988,Sucrose supply and utilization for latex production,In:Physiology of Rubber Tree Latex,CRC Press,Boca Raton,pp.275-332
Tup伥J.,and Primot L.,1976,Control of carbohydrate metabolism by ethylene in latex vessels of Hevea brasiliensis Muel.ARCin relation to rubber production,Biologia Plantarum,18(5):373-384
Tup伥J.,and Primot L.,1982,Sucrose synthetase in the latex of Hevea brasiliensis Muell,J.Exp.Bot.,33(136):988-995
Tup伥J.,1973,The regulation of invertase activity in the latex of Hevea brasiliensis Muell,J.Exp.Bot.,24(3):516-524
Tymowska-Lalanne Z.,and Kreis M.,1998,The plant invertases:physiology,biochemistry and molecular biology,Advances in Botanical Research,28:71-117
Vargas W.A.,Cumino A.,and Salerno G.L.,2003,Cyanobacterial alkaline/neutral invertases,Origin of sucrose hydrolysis in the plant cytosol?Planta,216(6):951-960
Vargas W.A.,Pontis H.G.,and Salerno G.L.,2007,New insights on sucrose metabolism:evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking,Planta,227(4):795-807