木薯MeHXK1酵母功能互补鉴定
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摘要
已糖激酶是植物催化已糖磷酸化生成磷酸已糖的一类重要的酶,参与植物生长发育及信号转导的过程。在前期研究中已从木薯基因组克隆获得7个已糖激酶基因,其中木薯已糖激酶基因MeHXK1主要在叶片、块根韧皮部、雄花和雌花中表达。本研究利用已糖激酶酵母突变菌株YSH7.4-3C鉴定酵母功能,研究已糖激酶是否具有催化已糖磷酸化的作用。结果发现:转pDR195-MeHXK1载体的YSH7.4-3C酵母在以葡萄糖或果糖为唯一碳源的培养基中能生长,说明木薯已糖激酶MeHXK1可通过催化己糖磷酸化为酵母的生长提供碳水化合物和能量。这些结果为进一步研究MeHXK1蛋白的酶学特性及生理功能提供参考。
Hexokinase is an important class of enzymes in plants that catalyze the phosphorylation of hexose to phosphoselated sugars, which is involved in the process of plant growth and signal transduction. In the previous study, 7 hexokinase genes have been cloned from cassava genome, of which MeHXK1 is mainly expressed in leaves, root phloem, male flowers and female flowers. This study mainly used yeast mutant strain YSH7.4-3 C to test the function of hexokinase catalyzed glucose phosphorylation by yeast functional complementation experiments. The results showed that YSH7.4-3 C yeast transformed with pDR195-MeHXK1 vector could grow in the medium with glucose or fructose as sole carbon source, indicating that MeHXK1 protein can provide carbohydrate and energy for the growth of yeast by catalyzing hexose phosphorylation. These results provide reference for the further study of the enzymatic and physiological functions of MeHXK1 protein.
Hexokinase is an important class of enzymes in plants that catalyze the phosphorylation of hexose to phosphoselated sugars, which is involved in the process of plant growth and signal transduction. In the previous study, 7 hexokinase genes have been cloned from cassava genome, of which MeHXK1 is mainly expressed in leaves, root phloem, male flowers and female flowers. This study mainly used yeast mutant strain YSH7.4-3 C to test the function of hexokinase catalyzed glucose phosphorylation by yeast functional complementation experiments. The results showed that YSH7.4-3 C yeast transformed with pDR195-MeHXK1 vector could grow in the medium with glucose or fructose as sole carbon source, indicating that MeHXK1 protein can provide carbohydrate and energy for the growth of yeast by catalyzing hexose phosphorylation. These results provide reference for the further study of the enzymatic and physiological functions of MeHXK1 protein.
引文
Ceballos H.,Kawuki R.S.,Gracen V.E.,Yencho G.C.,and Hershey C.H.,2015,Conventional breeding,marker-assisted selection,genomic selection and inbreeding in clonally propagated crops:a case study for cassava,Theor.Appl.Genet.,128(9):1647-1667
Cheng Y.E.,Xu Y.,Pan X.Q.,Feng Y.,and Li Y.Z.,2017,Activiities of related enzymes associated with high starch accumulation in cassava tuberous roots,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(1):376-381(程艳娥,徐勇,潘晓芹,冯燕,李有志,2017,木薯块根淀粉高累积的相关酶的活性特征,基因组学与应用生物学,36(1):376-381)
Dai N.,Schaffer A.,Petreikov M.,Shahak Y.,Giller Y.,Ratner K.,Levine A.,and Granot D.,1999,Overexpression of arabidopsis hexokinase in tomato plants inhibits growth,reduces photosynthesis,a nd induces rapid senescence,Plant Cell.11(7):1253-1266
Geng M.T.,Yao Y.,Wang Y.L.,Wu X.H.,Wu X.H.,Sun C.,Li R.M.,Fu S.P.,Duan R.J.,Liu J.,Hu X.W.,and Guo J.C.,2017,Structure,expression,and functional analysis of the hexokinase gene family in cassava,Int.J.Mol.Sci.,18(5):1041
Granot D.,David-Schwartz R.,and Kelly G.,2013,Hexose kinases and their role in sugar-sen sing and plant development,Front.Plant Sci.,4(1):44
Granot D.,Kelly G.,Stein O.,and David-Schwartz R.,2014,Substantial roles of hexokinase and fructokinase in the effects of sugars on plant physiology and develo pment,J.Exp.Bot.,65(3):809-819
Ihemere U.,Arias-Garzon D.,Lawrence S.,and Sayre R.,2006,Genetic modification of cassava for enhanced starch production,Plant Biotechnol.J.,4(4):453-465
Jang J.C.,León P.,Zhou L.,and Sheen J.,1997,Hexokinase as a sugar sensor in higher plants,Plant Cell,9(1):5-19
Jaquinod M.,Villiers F.,Kieffer-Jaquinod S.,Hugouvieux V.,Bruley C.,Garin J.,and Bourguignon J.,2007,A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture,Mol.Cell.Proteomics,6(3):394-412
Kandel-Kfir M.,Damari-Weissler H.,German M.A.,Gidoni D.,Mett A.,Belausov E.,Petreikov M.,Adir N.,and Granot D.,2006,Two newly identified membrane-associated and plastidic tomato HXKs:characteristics,predicted structure and intracellular localization,Planta,224(6):1341-1352
Kelly G.,Sade N.,Attia Z.,Secchi F.,Zwieniecki M.,Holbrook N.M.,Levi A.,Alchanatis V.,Moshelion M.,and Granot D.,2014,Relationship between hexokinase and the aquaporin PIP1 in the regulation of photosynthesis and plant growth.,PLoS One,9(2):e87888
Kim M.,Lim J.H.,Ahn C.S.,Park K.,Kim G.T.,Kim W.T.,and Pai H.S.,2006,Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana,Plant Cell,18(9):2341-2355
Li J.,Baroja-Fernández E.,Bahaji A.,Munoz F.J.,Ovecka M.,Montero M.,Sesma M.T.,Alonso-Casajns N.,Almagro G.,Sánchez-López A.M.,Hidalgo M.,Zamarbide M.,and Pozueta-Romero J.,2013,Enhancing sucrose synthase activity results in increased levels of starch and ADP-glucose in maize(Zea mays L.)seed endosperms,Plant Cell Physiol.,54(2):282-294
Minet M.,Dufour M.E.,and Lacroute F.,1992,Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs,Plant J.,2(3):417-422
Moore B.,Zhou L.,Rolland F.,Hall Q.,Cheng W.H.,Liu Y.X.,Hwang I.,Jones T.,and Sheen J.,2003,Role of the Arabidopsis glucose sensor HXK1 in nutrient,light,and hormonal signaling,Science,300(5617):332-336
Nilsson A.,Olsson T.,Ulfstedt M.,Thelander M.,and Ronne H.,2011,Two novel types of hexokinases in the moss Physcomitrella patens,BMC Plant Biol.,11(1):32
Olsson T.,Thelander M.,and Ronne H.,2003,A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens,J.Biol.Chem.,278(45):44439-44447
Wu X.H.,2014,Cloning,structure evolution and expressing analysis of the hexokinase family genes in cassava(Manihot esculenta Crantz),Thesis for M.S.,College of Agriculture,Hainan University,Supervisor:Hu X.W.,and Guo J.C.,pp.57-69(吴晓慧,2014,木薯己糖激酶基因家族克隆、结构进化及表达分析,硕士学位论文,海南大学热带农林学院,导师:胡新文,郭建春,pp.57-69)
Xu F.Q.,Li X.R.,and Ruan Y.L.,2008,RNAi-mediated suppression of hexokinase gene OsHXK10 in rice leads to non-dehiscent anther and reduction of pollen germination,Plant Sci.,175(5):674-684
Yao Y.,Sun C.,Lu X.H.,Wang Y.L.,Geng M.T.,Wu X.H.,Shang L.,Li Z.,Fu S.P.,Li R.M.,Duan R.J.,Liu J.,Hu X.W.,and Guo J.C.,2017,Catalytic functional identification of cassava hexokinase MeHXK5,Guangxi Zhiwu(Guihaia),37(12):1545-1550(姚远,孙冲,陆小花,王运林,耿梦婷,吴晓慧,尚璐,李崭,符少萍,李瑞梅,段瑞军,刘姣,胡新文,郭建春,2017,木薯已糖激酶MeHXK5的催化功能鉴定,广西植物,37(12):1545-1550)
Yu S.M.,Lee Y.C.,Fang S.C.,Chan M.T.,Hwa S.F.,and Liu L.F.,1996,Sugars act as signal molecules and osmotica to regulate the expression of alpha-amylase genes and metabolic activities in germinating cereal grains,Plant Mol.Biol.,30(6):1277-1289
Zhang Z.,Zhang J.,Chen Y.,Li R.,Wang H.,Ding L.,and Wei J.,2014,Isolation,structural analysis,and expression characteristics of the maize(Zea mays L.)hexokinase gene family,Mol.Biol.Rep.,41(9):6157-6166
Zhou Y.,2011,Fuctional characterization of wheat Na+/H+antiporter TaNHX2 and study of TaNHX2 C-terminus domain,Thesis for M.S.,Henan Agricultural University,Supervisor:Cui D.Q.,and Xu H.X.,pp.27-28(周扬,2011,小麦Na+/H+逆转运蛋白TaNHX2的功能验证及功能域分析,硕士学位论文,河南农业大学,导师:崔党群,许海霞,pp.27-28)
Cheng Y.E.,Xu Y.,Pan X.Q.,Feng Y.,and Li Y.Z.,2017,Activiities of related enzymes associated with high starch accumulation in cassava tuberous roots,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(1):376-381(程艳娥,徐勇,潘晓芹,冯燕,李有志,2017,木薯块根淀粉高累积的相关酶的活性特征,基因组学与应用生物学,36(1):376-381)
Dai N.,Schaffer A.,Petreikov M.,Shahak Y.,Giller Y.,Ratner K.,Levine A.,and Granot D.,1999,Overexpression of arabidopsis hexokinase in tomato plants inhibits growth,reduces photosynthesis,a nd induces rapid senescence,Plant Cell.11(7):1253-1266
Geng M.T.,Yao Y.,Wang Y.L.,Wu X.H.,Wu X.H.,Sun C.,Li R.M.,Fu S.P.,Duan R.J.,Liu J.,Hu X.W.,and Guo J.C.,2017,Structure,expression,and functional analysis of the hexokinase gene family in cassava,Int.J.Mol.Sci.,18(5):1041
Granot D.,David-Schwartz R.,and Kelly G.,2013,Hexose kinases and their role in sugar-sen sing and plant development,Front.Plant Sci.,4(1):44
Granot D.,Kelly G.,Stein O.,and David-Schwartz R.,2014,Substantial roles of hexokinase and fructokinase in the effects of sugars on plant physiology and develo pment,J.Exp.Bot.,65(3):809-819
Ihemere U.,Arias-Garzon D.,Lawrence S.,and Sayre R.,2006,Genetic modification of cassava for enhanced starch production,Plant Biotechnol.J.,4(4):453-465
Jang J.C.,León P.,Zhou L.,and Sheen J.,1997,Hexokinase as a sugar sensor in higher plants,Plant Cell,9(1):5-19
Jaquinod M.,Villiers F.,Kieffer-Jaquinod S.,Hugouvieux V.,Bruley C.,Garin J.,and Bourguignon J.,2007,A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture,Mol.Cell.Proteomics,6(3):394-412
Kandel-Kfir M.,Damari-Weissler H.,German M.A.,Gidoni D.,Mett A.,Belausov E.,Petreikov M.,Adir N.,and Granot D.,2006,Two newly identified membrane-associated and plastidic tomato HXKs:characteristics,predicted structure and intracellular localization,Planta,224(6):1341-1352
Kelly G.,Sade N.,Attia Z.,Secchi F.,Zwieniecki M.,Holbrook N.M.,Levi A.,Alchanatis V.,Moshelion M.,and Granot D.,2014,Relationship between hexokinase and the aquaporin PIP1 in the regulation of photosynthesis and plant growth.,PLoS One,9(2):e87888
Kim M.,Lim J.H.,Ahn C.S.,Park K.,Kim G.T.,Kim W.T.,and Pai H.S.,2006,Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana,Plant Cell,18(9):2341-2355
Li J.,Baroja-Fernández E.,Bahaji A.,Munoz F.J.,Ovecka M.,Montero M.,Sesma M.T.,Alonso-Casajns N.,Almagro G.,Sánchez-López A.M.,Hidalgo M.,Zamarbide M.,and Pozueta-Romero J.,2013,Enhancing sucrose synthase activity results in increased levels of starch and ADP-glucose in maize(Zea mays L.)seed endosperms,Plant Cell Physiol.,54(2):282-294
Minet M.,Dufour M.E.,and Lacroute F.,1992,Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs,Plant J.,2(3):417-422
Moore B.,Zhou L.,Rolland F.,Hall Q.,Cheng W.H.,Liu Y.X.,Hwang I.,Jones T.,and Sheen J.,2003,Role of the Arabidopsis glucose sensor HXK1 in nutrient,light,and hormonal signaling,Science,300(5617):332-336
Nilsson A.,Olsson T.,Ulfstedt M.,Thelander M.,and Ronne H.,2011,Two novel types of hexokinases in the moss Physcomitrella patens,BMC Plant Biol.,11(1):32
Olsson T.,Thelander M.,and Ronne H.,2003,A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens,J.Biol.Chem.,278(45):44439-44447
Wu X.H.,2014,Cloning,structure evolution and expressing analysis of the hexokinase family genes in cassava(Manihot esculenta Crantz),Thesis for M.S.,College of Agriculture,Hainan University,Supervisor:Hu X.W.,and Guo J.C.,pp.57-69(吴晓慧,2014,木薯己糖激酶基因家族克隆、结构进化及表达分析,硕士学位论文,海南大学热带农林学院,导师:胡新文,郭建春,pp.57-69)
Xu F.Q.,Li X.R.,and Ruan Y.L.,2008,RNAi-mediated suppression of hexokinase gene OsHXK10 in rice leads to non-dehiscent anther and reduction of pollen germination,Plant Sci.,175(5):674-684
Yao Y.,Sun C.,Lu X.H.,Wang Y.L.,Geng M.T.,Wu X.H.,Shang L.,Li Z.,Fu S.P.,Li R.M.,Duan R.J.,Liu J.,Hu X.W.,and Guo J.C.,2017,Catalytic functional identification of cassava hexokinase MeHXK5,Guangxi Zhiwu(Guihaia),37(12):1545-1550(姚远,孙冲,陆小花,王运林,耿梦婷,吴晓慧,尚璐,李崭,符少萍,李瑞梅,段瑞军,刘姣,胡新文,郭建春,2017,木薯已糖激酶MeHXK5的催化功能鉴定,广西植物,37(12):1545-1550)
Yu S.M.,Lee Y.C.,Fang S.C.,Chan M.T.,Hwa S.F.,and Liu L.F.,1996,Sugars act as signal molecules and osmotica to regulate the expression of alpha-amylase genes and metabolic activities in germinating cereal grains,Plant Mol.Biol.,30(6):1277-1289
Zhang Z.,Zhang J.,Chen Y.,Li R.,Wang H.,Ding L.,and Wei J.,2014,Isolation,structural analysis,and expression characteristics of the maize(Zea mays L.)hexokinase gene family,Mol.Biol.Rep.,41(9):6157-6166
Zhou Y.,2011,Fuctional characterization of wheat Na+/H+antiporter TaNHX2 and study of TaNHX2 C-terminus domain,Thesis for M.S.,Henan Agricultural University,Supervisor:Cui D.Q.,and Xu H.X.,pp.27-28(周扬,2011,小麦Na+/H+逆转运蛋白TaNHX2的功能验证及功能域分析,硕士学位论文,河南农业大学,导师:崔党群,许海霞,pp.27-28)