橡胶树中碱性转化酶基因HbNIN7的克隆与表达分析
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摘要
蔗糖主要由植物光合作用产生,其水解依赖于转化酶和蔗糖合成酶。其中,转化酶催化蔗糖不可逆分解为葡萄糖与果糖。本研究基于橡胶树的基因组和转录组数据库,采用RT-PCR技术克隆得到一个中碱性转化酶基因HbNIN7基因,预测编码683个氨基酸。同源与亚细胞定位分析表明HbNIN7属于α类中碱性转化酶,定位于线粒体。通过原核表达获得其重组蛋白,酶活性分析表明,HbNIN7重组蛋白最适酶活pH值7.2;最适酶活温度45℃;最大反应速率32.69 mmol hexose·mg-1protein·h-1;Km值25.04 mmol/L。利用荧光定量方法分析基因的表达特征,结果显示:HbNIN7基因在雄花中表达水平最高,且在雄花发育的不同阶段差异显著,说明Hb NIN7很可能参与橡胶树花组织中糖利用及糖信号调控。
Sucrose is an important storage form of photosynthetic products in higher plants, where invertases and sucrose synthases are the main enzymes to sucrose hydrolysis. Invertases catalyze the decomposition of sucrose into glucose and fructose irreversibly. Based on available genome and transcriptome data, the cDNA of HbNIN7, a neutral/alkaline invertase gene was cloned from Hevea brasiliensis, which was predicted to encode a protein of 683 amino acids. HbNIN7 was predicted to locate in mitochondria, belonging to the α group of neutral/alkaline type of invertases. The enzymatic properties of recombinant HbNIN7 protein were analyzed, showing an optimum pH at7.2, an optimum temperature at 45℃, maximum reaction rate of 32.69 mmol hexose·mg-1 protein·h-1, and a Km value of 25.04 mmol/L. The expression pattern was analyzed by qRT-PCR. The results showed that HbNIN7 was mainly expressed in male flowers and changed significantly at different stages of male flower development. Therefore, HbNIN7 might play a role in regulating the development of flower tissues in Hevea brasiliensis.
Sucrose is an important storage form of photosynthetic products in higher plants, where invertases and sucrose synthases are the main enzymes to sucrose hydrolysis. Invertases catalyze the decomposition of sucrose into glucose and fructose irreversibly. Based on available genome and transcriptome data, the cDNA of HbNIN7, a neutral/alkaline invertase gene was cloned from Hevea brasiliensis, which was predicted to encode a protein of 683 amino acids. HbNIN7 was predicted to locate in mitochondria, belonging to the α group of neutral/alkaline type of invertases. The enzymatic properties of recombinant HbNIN7 protein were analyzed, showing an optimum pH at7.2, an optimum temperature at 45℃, maximum reaction rate of 32.69 mmol hexose·mg-1 protein·h-1, and a Km value of 25.04 mmol/L. The expression pattern was analyzed by qRT-PCR. The results showed that HbNIN7 was mainly expressed in male flowers and changed significantly at different stages of male flower development. Therefore, HbNIN7 might play a role in regulating the development of flower tissues in Hevea brasiliensis.
引文
Borisjuk L.,Rolletschek H.,Wobus U.,and Weber H.,2003,Differentiation of legume cotyledons as related tometabolic gradients and assimilate transport into seeds,J.Exp.Bot.,54(382):503-506
Chen J.Q.,and Black C.C.,1992,Biochemical and immunological properties of alkaline invertases isolated from sprouting soybean hypocotyls,Archives of Biochemistry and Biophysics,295(1):61-69
Dong J.,Zhou J.,Xin P.Y.,Xu Y.L.,Liu Y.,Wei Y.J.,and Fu H.H.,2010,Bioinformatics analysis of LDOX/ANS gene in different plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(5):815-822(董娇,周军,辛培尧,许玉兰,刘岩,韦援教,付海辉,2010,不同植物LDOX/ANS基因的生物信息学分析,基因组学与应用生物学,29(5):815-822)
Emanuelsson O.,Brunak S.,Von Heijne G.,and Nielsen H.,2007,Locating proteins in the cell using targetp,signalp and related tools,Nat.Protoc.,2(4):953-971
Gao Y.,Xi M.L.,Wang G.F.,Yang L.W.,and Shi J.S.,2010,Molecular characterization and expression analysis of PmSERK1 during somatic embryogenesis in masson pine,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(1):53-58(高燕,席梦利,王桂凤,杨立伟,施季森,2010,马尾松体细胞胚胎发生相关基因PmSERK1的克隆与表达分析,分子植物育种,8(1):53-58)
Ji X.,Ende W.V.,Laere A.V.,Cheng S.H.,and Bennett J.,2005,Structure,evolution,and expression of the two invertase gene families of rice,J.Mol.Evol.,60(5):615-634
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets,Mol.Biol.Evol.,33(7):1870
Li H.,Qin Y.X.,Xiao X.H.,and Tang C.R.,2011,Screening of valid reference genes for real-time RT-PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3,Plant Sci.,181(2):132-139
Liu S.J.,Lan J.X.,Zhou B.H.,Qin Y.X.,Xiao X.H.,Yang J.H.,Guo J.P.,Qi J.Y.,Huang Y.C.,and Tang C.R.,2015,HbNIN2,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
Long X.Y.,He B.,Gao X.S.,Qin Y.X.,Yang J.H.,Fang Y.J.,Qi J.Y.,and Tang C.R.,2015,Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree,Gene,563(2):190-195
Lou Y.,Gou J.Y.,and Xue H.W.,2007,PIP5K9,an Arabidopsis phosphatidylinositol monophosphate kinase,interacts with a cytosolic invertase to negatively regulate sugar-mediated root growth,Plant Cell,19(1):163-181
Roitsch T.,and Gonzalez M.,2004,Function and regulation of plant invertases:sweet sensations,Trends Plant Sci.,9(12):606-613
Smeekens S.,Hanson J.,and Rolland F.,2010,Sugar signals and molecular networks controlling plant growth,Curr.Opin.Plant Biol.,13(3):274-279
Tymowska-Lalanne Z.,and Kreis M.,1998,The plant invertases physiology,biochemistry and molecular biology,Advance in Botany Research,(28):71-117
Vargas W.A.,and Salerno G.L.,2010,The Cinderella story of sucrose hydrolysis:alkaline/neutral invertases,from cyanobacteria to unforeseen roles in plant cytosol and organelles,Plant Sci.,178(1):1-8
Vargas W.A.,Pontis H.G.,and Salerno G.L.,2008,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
Weschke W.,Panitz R.,Gubatz S.,Wang Q.,Radchuk R.,and Weber H.,2003,The role of invertases and hexose transporters in controlling sugar rations in maternal and filiar tissues of barley caryopses during early development,Plant J.,33(2):395-411
Xiang L.,Le Roy K.,Bolouri-Moghaddam M.R.,Vanhaecke M.,Lammens W.,and Rolland F.,2011,Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana,J.Exp.Bot.,62(11):3849-3862
Xiao X.H.,2013,Cloning,structural evolution and expression analysis of a gene family related to sucrose metabolism in Hevea brasiliensis,Dissertation for Ph.D.,Hainan University,Supervisor:Tang C.R.,pp.1-12(肖小虎,2013,巴西橡胶树蔗糖代谢相关基因家族的克隆、结构进化和表达分析,博士学位论文,海南大学,导师:唐朝荣,pp.1-12)
Zhang W.L.,Cai J.,Niu J.,Pu J.Y.,Zhu M.Y.,Hu H.W.,Zhang Z.X.,and Lin S.Z.,2013,Cloning of malic enzyme gene from Prunus armeniaca and its bioinformatics analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(3):332-338(章文乐,蔡兼,钮俊,蒲婧懿,朱梦媛,胡惠雯,张志翔,林善枝,2013,山杏苹果酸酶基因的克隆与生物信息学分析,基因组学与应用生物学,32(3):332-338)
Chen J.Q.,and Black C.C.,1992,Biochemical and immunological properties of alkaline invertases isolated from sprouting soybean hypocotyls,Archives of Biochemistry and Biophysics,295(1):61-69
Dong J.,Zhou J.,Xin P.Y.,Xu Y.L.,Liu Y.,Wei Y.J.,and Fu H.H.,2010,Bioinformatics analysis of LDOX/ANS gene in different plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(5):815-822(董娇,周军,辛培尧,许玉兰,刘岩,韦援教,付海辉,2010,不同植物LDOX/ANS基因的生物信息学分析,基因组学与应用生物学,29(5):815-822)
Emanuelsson O.,Brunak S.,Von Heijne G.,and Nielsen H.,2007,Locating proteins in the cell using targetp,signalp and related tools,Nat.Protoc.,2(4):953-971
Gao Y.,Xi M.L.,Wang G.F.,Yang L.W.,and Shi J.S.,2010,Molecular characterization and expression analysis of PmSERK1 during somatic embryogenesis in masson pine,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(1):53-58(高燕,席梦利,王桂凤,杨立伟,施季森,2010,马尾松体细胞胚胎发生相关基因PmSERK1的克隆与表达分析,分子植物育种,8(1):53-58)
Ji X.,Ende W.V.,Laere A.V.,Cheng S.H.,and Bennett J.,2005,Structure,evolution,and expression of the two invertase gene families of rice,J.Mol.Evol.,60(5):615-634
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets,Mol.Biol.Evol.,33(7):1870
Li H.,Qin Y.X.,Xiao X.H.,and Tang C.R.,2011,Screening of valid reference genes for real-time RT-PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3,Plant Sci.,181(2):132-139
Liu S.J.,Lan J.X.,Zhou B.H.,Qin Y.X.,Xiao X.H.,Yang J.H.,Guo J.P.,Qi J.Y.,Huang Y.C.,and Tang C.R.,2015,HbNIN2,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
Long X.Y.,He B.,Gao X.S.,Qin Y.X.,Yang J.H.,Fang Y.J.,Qi J.Y.,and Tang C.R.,2015,Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree,Gene,563(2):190-195
Lou Y.,Gou J.Y.,and Xue H.W.,2007,PIP5K9,an Arabidopsis phosphatidylinositol monophosphate kinase,interacts with a cytosolic invertase to negatively regulate sugar-mediated root growth,Plant Cell,19(1):163-181
Roitsch T.,and Gonzalez M.,2004,Function and regulation of plant invertases:sweet sensations,Trends Plant Sci.,9(12):606-613
Smeekens S.,Hanson J.,and Rolland F.,2010,Sugar signals and molecular networks controlling plant growth,Curr.Opin.Plant Biol.,13(3):274-279
Tymowska-Lalanne Z.,and Kreis M.,1998,The plant invertases physiology,biochemistry and molecular biology,Advance in Botany Research,(28):71-117
Vargas W.A.,and Salerno G.L.,2010,The Cinderella story of sucrose hydrolysis:alkaline/neutral invertases,from cyanobacteria to unforeseen roles in plant cytosol and organelles,Plant Sci.,178(1):1-8
Vargas W.A.,Pontis H.G.,and Salerno G.L.,2008,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
Weschke W.,Panitz R.,Gubatz S.,Wang Q.,Radchuk R.,and Weber H.,2003,The role of invertases and hexose transporters in controlling sugar rations in maternal and filiar tissues of barley caryopses during early development,Plant J.,33(2):395-411
Xiang L.,Le Roy K.,Bolouri-Moghaddam M.R.,Vanhaecke M.,Lammens W.,and Rolland F.,2011,Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana,J.Exp.Bot.,62(11):3849-3862
Xiao X.H.,2013,Cloning,structural evolution and expression analysis of a gene family related to sucrose metabolism in Hevea brasiliensis,Dissertation for Ph.D.,Hainan University,Supervisor:Tang C.R.,pp.1-12(肖小虎,2013,巴西橡胶树蔗糖代谢相关基因家族的克隆、结构进化和表达分析,博士学位论文,海南大学,导师:唐朝荣,pp.1-12)
Zhang W.L.,Cai J.,Niu J.,Pu J.Y.,Zhu M.Y.,Hu H.W.,Zhang Z.X.,and Lin S.Z.,2013,Cloning of malic enzyme gene from Prunus armeniaca and its bioinformatics analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(3):332-338(章文乐,蔡兼,钮俊,蒲婧懿,朱梦媛,胡惠雯,张志翔,林善枝,2013,山杏苹果酸酶基因的克隆与生物信息学分析,基因组学与应用生物学,32(3):332-338)