紫花苜蓿MsUGT73B2基因的克隆及表达
|
摘要
糖基化是植物体蛋白、激素等物质的常见修饰方式,对维持植物正常生长发育具有重要作用,在植物中糖基化反应由糖基转移酶催化。利用蒺藜苜蓿参考序列通过RACE法克隆得到紫花苜蓿基因MsUGT73B2的全长序列,利用生物信息学方法分析其氨基酸序列、二级结构、理化性质等,RT-qPCR分析基因表达情况,构建基因表达载体,研究亚细胞定位。结果表明,MsUGT73B2基因全长1 512bp,编码503个氨基酸,属于稳定的亲水性蛋白,序列比对结果显示其属于UGT家族基因,亚细胞定位于细胞质中。MSOGT73B2主要在紫花苜蓿叶片中表达,且干旱、10μmol/L ABA处理下在2h之前升高,之后下降;在150mmol/L NaCl胁迫处理下表现为4h之前表达水平升高,之后下降的趋势。
Glycosylation is a common modification of plant proteins,hormones and other substances,and plays an important role in maintaining the normal growth and development of plants.Glycosylation in plants is catalyzed by glycosyltransferases.The full-length sequence of MsUGT73 B2 gene was cloned by RACE-PCR using reference sequence of Medicago truncatula,and its amino acid sequence,secondary structure,and physicochemical properties were analyzed by bioinformatics.The gene expression was analyzed by qRT-PCR.By constructing gene expression vector,we studied gene's subcellular localization.The result showed that the full-length MsUGT73 B2 gene was 1 512 bp in length and encoded 503 amino acids.It was a stable hydrophilic protein.The sequence alignment showed that it belonged to the UGT family.The subcellular localization result showed that the MsUGT73 B2 gene played a role in the cytoplasm.The gene was mainly expressed in leaves.The expression of the gene increased in 2 hunder drought and 10μmol/L ABA treatments,and then decreased;the expression level of the gene increased in 4 hunder 150 mmol/L NaCl treatment,and then decreased.
Glycosylation is a common modification of plant proteins,hormones and other substances,and plays an important role in maintaining the normal growth and development of plants.Glycosylation in plants is catalyzed by glycosyltransferases.The full-length sequence of MsUGT73 B2 gene was cloned by RACE-PCR using reference sequence of Medicago truncatula,and its amino acid sequence,secondary structure,and physicochemical properties were analyzed by bioinformatics.The gene expression was analyzed by qRT-PCR.By constructing gene expression vector,we studied gene's subcellular localization.The result showed that the full-length MsUGT73 B2 gene was 1 512 bp in length and encoded 503 amino acids.It was a stable hydrophilic protein.The sequence alignment showed that it belonged to the UGT family.The subcellular localization result showed that the MsUGT73 B2 gene played a role in the cytoplasm.The gene was mainly expressed in leaves.The expression of the gene increased in 2 hunder drought and 10μmol/L ABA treatments,and then decreased;the expression level of the gene increased in 4 hunder 150 mmol/L NaCl treatment,and then decreased.
引文
[1]XUE F,GUO H,HU Y,et al.Expression of codon-optimized plant glycosyltransferase UGT72B14 in Escherichia coli enhances salidroside production[J].Biomed Research International,2016,2016:1-8.
[2]VAISTIJ F E,LIM E K,EDWARDS R,et al.Glycosylation of Secondary Metabolites and Xenobiotics[M]//OS-BOURN A,LANZOTTI V.Plant-derived Natural Products.New York:Springer,2009:209-228.
[3]JONES P,VOGT T.Glycosyltransferases in secondary plant metabolism:tranquilizers and stimulant controllers[J].Planta,2001,213(2):164-174.
[4]BOWLES D,ISAYENKOVA J,LIM E K,et al.Glycosyltransferases:managers of small molecules[J].Current Opinion in Plant Biology,2005,8(3):254-263.
[5]WANG J,HOU B.Glycosyltransferases:key players involved in the modification of plant secondary metabolites[J].Frontiers of Biology in China,2009,4(1):39-46.
[6]JONES P,MESSNER B,NAKAJIMA J,et al.UGT73C6and UGT78D1,glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana[J].Journal of Biological Chemistry,2003,278(45):43910-49918.
[7]WANG Y,JIN S,WANG B,et al.Over-expression of a putative poplar glycosyltransferase gene PtGTin tobacco increases lignin content and causes early flowering[J].Journal of Experimental Botany,2012,63(7):2799-2808.
[8]FUJIOKA S,YOKOTA T.Biosynthesis and metabolism of brassinosteroids[J].Physiologia Plantarum,1997,100(3):137-164.
[9]VON S P V,ZHANG W,KANAWATI B,et al.The Arabidopsis glucosyltransferase UGT76B1 conjugates isoleucic acid and modulates plant defense and senescence[J].Plant Cell,2011,23(11):4124-4145.
[10]TOGNETTI V B,BREUSEGEM F V.Perturbation of indole-3-butyric acid homeostasis by the UDP-Glucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance[J].Plant Cell,2010,22(8):2660-2679.
[11]SUN Y G,WANG B,JIN S H,et al.Ectopic expression of Arabidopsis glycosyltransferase UGT85A5enhances salt stress tolerance in tobacco[J].PLoS One,2013,8(3):e59924.
[12]ACHNINE L,HUHMAN D V,FARAG M A,et al.Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula[J].Plant Journal,2005,41(6):875-887.
[13]STAFFORD H A.Roles of flavonoids in symbiotic and defense functions in legume roots[J].Botanical Review,1997,63(1):27-39.
[14]SHIRLEY B W.Flavonoids in seeds and grains:physiological function,agronomic importance and the genetics of biosynthesis[J].Seed Science Research,1998,8(4):415-422.
[15]AOKI T,AKASHI T,AYABE S.Flavonoids of leguminous plants:structure,biological activity,and biosynthesis[J].Journal of Plant Research,2000,113(4):475-488.
[16]FORKMANN G,MARTENS S.Metabolic engineering and applications of flavonoids[J].Current Opinion in Biotechnology,2001,12(2):155-160.
[17]KANADASWAMI C,LEE L,LEE P H,et al.The antitumor activities of flavonoids[J].In Vivo,2005,19(5):895-909.
[18]卜耀军,徐伟洲,李强,等.14个紫花苜蓿品种在农牧交错区的生长特征及品质[J].西北农业学报,2017,26(10):1438-1445.BU Y J,XU W ZH,LI Q,et al.Growth characteristics and quality of 14alfalfa varieties in agro-pastoral ecotone[J].Acta Agriculture Boreali-occidentalis Sinica,2017,26(10):1438-1445.
[19]LONG R,WWANG H,SHEN Y,et al.Molecular cloning and functional analysis of a salt-induced gene encoding an RNA-binding protein in alfalfa[J].Molecular Breeding,2014,34(3):1465-1473.
[20]VELJANOVSKI V,CONSTABEL C P.Molecular cloning and biochemical characterization of two UDP-glycosyltransferases from poplar[J].Phytochemistry,2013,91:148-157.
[21]LAIRSON L L,HENRISSAT B,DAVIES G J,et al.Glycosyltransferases:structures,functions,and mechanisms[J].Annual Review of Biochemisty,2008,77:521-555.
[22]LI L,MODOLO L V,ESCAMILLATREVINO L L,et al.Crystal structure of Medicago truncatula UGT85H2-insights into the structural basis of a multifunctional(iso)flavonoid glycosyltransferase[J].Journal of Molecular Biology,2007,370(5):951-963.
[23]KUBO A,ARAI Y,NAGASHIMA S,et al.Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation[J].Archives of Biochemistry and Biophysics,2004,429(2):198-203.
[24]KIM J H,KIM B G,KO J H,et al.Molecular cloning,expression,and characterization of a flavonoid glycosyltransferase from Arabidopsis thaliana[J].Plant Science,2006,170(4):897-903.
[25]KIGASAWA K,SHIMIZU H,SAITO M,et al.Biopharmaceutical studies of drugs.II.The competitive inhibition of pentazocine glucuronidation by salicylamide in vitro(author’s transl)[J].Yakugaku Zasshi-journal of the Pharmaceutical Society of Japan,1980,100(3):249-255.
[26]ROSS J,LI Y,LIM E,et al.Higher plant glycosyltransferases[J].Genome Biology,2001,2(2):1-6.
[27]甘晓燕,吴明朝,巩檑,等.梭梭糖基转移酶基因克隆及序列分析[J].西北农业学报,2014,23(9):166-171.GAN X Y,WU M ZH,GONG L,et al.Clonging and sequence analysis of glycosyltransferase from Haloxylon ammodendron[J].Acta Agriculture Boreali-occidentalis Sinica,2014,23(9):166-171.
[28]邢朝斌,修乐山,吴鹏,等.刺五加葡萄糖基转移酶基因的克隆与表达分析[J].西北农业学报,2014,23(2):126-130.XING ZH B,XIU L SH,WU P,et al.Cloning and expression analysis of glucosyltransferase gene in Eleutherococcus senticosus[J].Acta Agriculture Boreali-occidentalis Sinica,2014,23(2):126-130.
[29]BRETON C,SNAJDROVA L,JEANNEAU C,et al.Structures and mechanisms of glycosyltransferases[J].Glycobiology,2006,16(2):29R-37R.
[30]MODOLO L V,BLOUNT J W,ACHNINE L,et al.A functional genomics approach to(iso)flavonoid glycosylation in the model legume Medicago truncatula[J].Plant Molecular Biology,2007,64(5):499-518.
[31]KIM I A,HEO J O,CHANG K S,et al.Overexpression and inactivation of UGT73B2 modulate tolerance to oxidative stress in Arabidopsis[J].Journal of Plant Biology,2010,53(3):233-239.
[2]VAISTIJ F E,LIM E K,EDWARDS R,et al.Glycosylation of Secondary Metabolites and Xenobiotics[M]//OS-BOURN A,LANZOTTI V.Plant-derived Natural Products.New York:Springer,2009:209-228.
[3]JONES P,VOGT T.Glycosyltransferases in secondary plant metabolism:tranquilizers and stimulant controllers[J].Planta,2001,213(2):164-174.
[4]BOWLES D,ISAYENKOVA J,LIM E K,et al.Glycosyltransferases:managers of small molecules[J].Current Opinion in Plant Biology,2005,8(3):254-263.
[5]WANG J,HOU B.Glycosyltransferases:key players involved in the modification of plant secondary metabolites[J].Frontiers of Biology in China,2009,4(1):39-46.
[6]JONES P,MESSNER B,NAKAJIMA J,et al.UGT73C6and UGT78D1,glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana[J].Journal of Biological Chemistry,2003,278(45):43910-49918.
[7]WANG Y,JIN S,WANG B,et al.Over-expression of a putative poplar glycosyltransferase gene PtGTin tobacco increases lignin content and causes early flowering[J].Journal of Experimental Botany,2012,63(7):2799-2808.
[8]FUJIOKA S,YOKOTA T.Biosynthesis and metabolism of brassinosteroids[J].Physiologia Plantarum,1997,100(3):137-164.
[9]VON S P V,ZHANG W,KANAWATI B,et al.The Arabidopsis glucosyltransferase UGT76B1 conjugates isoleucic acid and modulates plant defense and senescence[J].Plant Cell,2011,23(11):4124-4145.
[10]TOGNETTI V B,BREUSEGEM F V.Perturbation of indole-3-butyric acid homeostasis by the UDP-Glucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance[J].Plant Cell,2010,22(8):2660-2679.
[11]SUN Y G,WANG B,JIN S H,et al.Ectopic expression of Arabidopsis glycosyltransferase UGT85A5enhances salt stress tolerance in tobacco[J].PLoS One,2013,8(3):e59924.
[12]ACHNINE L,HUHMAN D V,FARAG M A,et al.Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula[J].Plant Journal,2005,41(6):875-887.
[13]STAFFORD H A.Roles of flavonoids in symbiotic and defense functions in legume roots[J].Botanical Review,1997,63(1):27-39.
[14]SHIRLEY B W.Flavonoids in seeds and grains:physiological function,agronomic importance and the genetics of biosynthesis[J].Seed Science Research,1998,8(4):415-422.
[15]AOKI T,AKASHI T,AYABE S.Flavonoids of leguminous plants:structure,biological activity,and biosynthesis[J].Journal of Plant Research,2000,113(4):475-488.
[16]FORKMANN G,MARTENS S.Metabolic engineering and applications of flavonoids[J].Current Opinion in Biotechnology,2001,12(2):155-160.
[17]KANADASWAMI C,LEE L,LEE P H,et al.The antitumor activities of flavonoids[J].In Vivo,2005,19(5):895-909.
[18]卜耀军,徐伟洲,李强,等.14个紫花苜蓿品种在农牧交错区的生长特征及品质[J].西北农业学报,2017,26(10):1438-1445.BU Y J,XU W ZH,LI Q,et al.Growth characteristics and quality of 14alfalfa varieties in agro-pastoral ecotone[J].Acta Agriculture Boreali-occidentalis Sinica,2017,26(10):1438-1445.
[19]LONG R,WWANG H,SHEN Y,et al.Molecular cloning and functional analysis of a salt-induced gene encoding an RNA-binding protein in alfalfa[J].Molecular Breeding,2014,34(3):1465-1473.
[20]VELJANOVSKI V,CONSTABEL C P.Molecular cloning and biochemical characterization of two UDP-glycosyltransferases from poplar[J].Phytochemistry,2013,91:148-157.
[21]LAIRSON L L,HENRISSAT B,DAVIES G J,et al.Glycosyltransferases:structures,functions,and mechanisms[J].Annual Review of Biochemisty,2008,77:521-555.
[22]LI L,MODOLO L V,ESCAMILLATREVINO L L,et al.Crystal structure of Medicago truncatula UGT85H2-insights into the structural basis of a multifunctional(iso)flavonoid glycosyltransferase[J].Journal of Molecular Biology,2007,370(5):951-963.
[23]KUBO A,ARAI Y,NAGASHIMA S,et al.Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation[J].Archives of Biochemistry and Biophysics,2004,429(2):198-203.
[24]KIM J H,KIM B G,KO J H,et al.Molecular cloning,expression,and characterization of a flavonoid glycosyltransferase from Arabidopsis thaliana[J].Plant Science,2006,170(4):897-903.
[25]KIGASAWA K,SHIMIZU H,SAITO M,et al.Biopharmaceutical studies of drugs.II.The competitive inhibition of pentazocine glucuronidation by salicylamide in vitro(author’s transl)[J].Yakugaku Zasshi-journal of the Pharmaceutical Society of Japan,1980,100(3):249-255.
[26]ROSS J,LI Y,LIM E,et al.Higher plant glycosyltransferases[J].Genome Biology,2001,2(2):1-6.
[27]甘晓燕,吴明朝,巩檑,等.梭梭糖基转移酶基因克隆及序列分析[J].西北农业学报,2014,23(9):166-171.GAN X Y,WU M ZH,GONG L,et al.Clonging and sequence analysis of glycosyltransferase from Haloxylon ammodendron[J].Acta Agriculture Boreali-occidentalis Sinica,2014,23(9):166-171.
[28]邢朝斌,修乐山,吴鹏,等.刺五加葡萄糖基转移酶基因的克隆与表达分析[J].西北农业学报,2014,23(2):126-130.XING ZH B,XIU L SH,WU P,et al.Cloning and expression analysis of glucosyltransferase gene in Eleutherococcus senticosus[J].Acta Agriculture Boreali-occidentalis Sinica,2014,23(2):126-130.
[29]BRETON C,SNAJDROVA L,JEANNEAU C,et al.Structures and mechanisms of glycosyltransferases[J].Glycobiology,2006,16(2):29R-37R.
[30]MODOLO L V,BLOUNT J W,ACHNINE L,et al.A functional genomics approach to(iso)flavonoid glycosylation in the model legume Medicago truncatula[J].Plant Molecular Biology,2007,64(5):499-518.
[31]KIM I A,HEO J O,CHANG K S,et al.Overexpression and inactivation of UGT73B2 modulate tolerance to oxidative stress in Arabidopsis[J].Journal of Plant Biology,2010,53(3):233-239.