异源表达芸薹生链格孢谷氨酸脱氢酶基因AbGDH提高水稻氮素利用率的研究（英文）
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摘要
氮元素是植物生长发育所必需的重要元素之一。高等植物中的NADP(H)型谷氨酸脱氢酶(glutamate dehydrogenase, GDH)对NH4~+亲和力较低,因此植物主要通过谷氨酰胺合成酶(GS)/谷氨酸合酶(GOGAT)途径吸收NH4~+。真菌等低等生物中的GDH对NH4~+亲和力较高,所以它们在对NH4~+的利用途径中起着重要作用。本研究克隆了来自芸薹生链格孢(Alternaria brassicicola)的谷氨酸脱氢酶基因(AbGDH),并在水稻(Oryza sativa L. cv. Kitaake)中成功表达。体外酶活性分析表明, AbGDH对NH4~+、α-酮戊二酸和谷氨酸的K_m值分别为2.144±0.141 mmol/L、2.690±0.233 mmol/L和96.772±0.542 mmol/L。体内酶活性测定显示,与野生型相比,过表达AbGDH的水稻有更高的NH4~+亲和力和氨同化能力。此外,水培实验表明,与对照植物相比,转基因幼苗在低氮条件下植物高度和干重显著增加。这些结果说明,在水稻中异源表达AbGDH能促进α-酮戊二酸转化为谷氨酸,并在低氮条件下促进水稻的氨同化,从而提高氮素利用效率。
Nitrogen is one of the most important elements in agriculture industry. NADP(H)-dependent glutamate dehydrogenase(GDH) has a lower affinity for ammonium in higher plants, whose nitrogen is thus main-ly assimilated by the glutamine synthetase(GS)/glutamate synthase(GOGAT) pathway. But, in lower organ isms such as fungi, GDHs play an important role in nitrogen assimilation due to their higher affinity for ammonium. In this study, a glutamate dehydrogenase gene AbGDH from Alternaria brassicicola was cloned and successfully over-expressed in rice(Oryza sativa L. cv. Kitaake). Enzyme activity assay in vitro demonstrated that the K_m values of Ab GDH for NH4+, 2-oxoglutarate and glutamate were 2.144 ±0.141 mmol/L, 2.690 ±0.233 mmol/L and 96.772 ±0.542 mmol/L, respectively. Meanwhile, the enzyme activity assay in vivo revealed that, compared with the nontransgenic plants, the AbGDH over-expressing plants showed a higher aminating activity and a lower Kmvalue for NH4+. Additionally, the hydroponic experiments revealed that,compared with control plants, the transgenic seedlings exhibited a significant increase in plant height and dry weight at lower nitrogen conditions. These results suggested that ectopic expression of Ab GDH was inclined to convert 2-oxoglutarate to glutamate, which subsequently improved the ammonium assimilation and enhanced the nitrogen use efficiency particularly under low nitrogen fertility.
Nitrogen is one of the most important elements in agriculture industry. NADP(H)-dependent glutamate dehydrogenase(GDH) has a lower affinity for ammonium in higher plants, whose nitrogen is thus main-ly assimilated by the glutamine synthetase(GS)/glutamate synthase(GOGAT) pathway. But, in lower organ isms such as fungi, GDHs play an important role in nitrogen assimilation due to their higher affinity for ammonium. In this study, a glutamate dehydrogenase gene AbGDH from Alternaria brassicicola was cloned and successfully over-expressed in rice(Oryza sativa L. cv. Kitaake). Enzyme activity assay in vitro demonstrated that the K_m values of Ab GDH for NH4+, 2-oxoglutarate and glutamate were 2.144 ±0.141 mmol/L, 2.690 ±0.233 mmol/L and 96.772 ±0.542 mmol/L, respectively. Meanwhile, the enzyme activity assay in vivo revealed that, compared with the nontransgenic plants, the AbGDH over-expressing plants showed a higher aminating activity and a lower Kmvalue for NH4+. Additionally, the hydroponic experiments revealed that,compared with control plants, the transgenic seedlings exhibited a significant increase in plant height and dry weight at lower nitrogen conditions. These results suggested that ectopic expression of Ab GDH was inclined to convert 2-oxoglutarate to glutamate, which subsequently improved the ammonium assimilation and enhanced the nitrogen use efficiency particularly under low nitrogen fertility.
引文
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[16]WANG F,TIAN B.Neurospora NADP glutamate dehydrogenases and its expression in E.coli and transgenic plant[J].Chinese Science Bulletin,2001,46(12):1029-1032.
[17]NOOR S,PUNEKAR N S.Allosteric NADP-glutamate dehydrogenase from Aspergilli:purification,characterization and implications for metabolic regulation at the carbon nitrogen interface[J].Microbiology,2005,151(Pt 5):1409-1419.
[18]DU C,LIN J,YANG Y,et al.Molecular cloning,characterization and function analysis of a GDH gene from Sclerotinia sclerotiorum in rice[J].Molecular Biology Reports,2014,41(6):3683-3693.
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[23]AMEZIANE R,BERNHARD K,LIGHTFOOT D A.Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development[J].Plant and Soil,2000,221(1):47-57.
[24]ABIKO T,WAKAYAMA M,KAWAKAMI A,et al.Changes in nitrogen assimilation,metabolism,and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase(gdhA)[J].Planta,2010,232(2):299-311.
[25]ZHOU X,LIN J,ZHOU Y,et al.Overexpressing a fungal gene improves nitrogen utilization and growth in rice[J].Crop Science,2015,55(2):811-820.
[26]KOBAYASHI T,ISHIGURO K,NAKAJIMA T,et al.Effects of elevated atmospheric CO2concentration on the infection of rice blast and sheath blight[J].Phytopathology,2006,96(4):425-431.
[27]LIN J Z,YANG Y Z,ZHOU B,et al.A new and effective somatic mutagenesis rice breeding method[J].Journal of Hunan University(Natural Sciences),2013,40(9):79-85.
[28]ZHOU B,LIN J Z,PENG D,et al.Plant architecture and grain yield are regulated by the novel DHHC-type zinc finger protein genes in rice(Oryza sativa L.)[J].Plant Science,2016,254:12-21.
[29]CHEN Q J,ZHOU H M,CHEN J.Using a modified TA cloning method to create entry clones[J].Analytical Biochemistry,2006,358:120-125.
[30]MURRAY M G,THOMPSON W F.Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4321-4325.
[31]ZHOU Y B,LIU C,TANG D Y,et al.The receptor-like cytoplasmic kinase STRK1 phosphorylates and activates CatC,thereby regulating H2O2homeostasis and improving salt tolerance in rice[J].The Plant Cell,2018,30(5):1100-1118.
[32]TANG D,PENG Y,LIN J,et al.Ectopic expression of fungal EcGDH improves nitrogen assimilation and grain yield in rice[J].Journal of Integrative Plant Biology,2018,60(2):85-88.
[33]FERRARO G,BORTOLOTTI S,MORTERA P,et al.Novel glutamate dehydrogenase genes show increased transcript and protein abundances in mature tomato fruits[J].Journal of Plant Physiology,2012,169(9):899-907.
[34]ZHOU Y,ZHANG C,LIN J,et al.Over-expression of a glutamate dehydrogenase gene,MgGDH,from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice[J].Planta,2015,241(3):727-740.
[35]SRIVASTAVA H S,SINGH R P.Role and regulation of L-glutamate dehydrogenase activity in higher plants[J].Phytochemistry,1987,26(3):597-610.
[36]LIGHTFOOT D A,MUNGUR R,AMEZIANE R,et al.Improved drought tolerance of transgenic Zea mays plants that express the glutamate dehydrogenase gene(gdhA)of E.coli[J].Euphytica,2007,156(1-2):103-116.
[37]OBARA M,SATO T,YAMAYA T.High contents of glutamine synthetase does not accompany a high activity of the enzyme in rice(Oryza sativa)leaves of indica cultivars[J].Physiologia Plantarum,2000,108(1):11-18.
[38]ZHANG H,LIANG C,AOKI N,et al.Introduction of a fungal NADP(H)-dependent glutamate dehydrogenase(gdhA)improves growth,grain weight and salt resistance by enhancing the nitrogen uptake efficiency in forage rice[J].Plant Production Science,2016,19(2):267-278.
[2]MICHAEL BEMAN J,ARRIGO K R,MATSON P A.Agricultural runoff fuels large phytoplankton blooms in vulnerable areas of the ocean[J].Nature,2005,434(7030):211-214.
[3]FAN X,FENG H,TAN Y,et al.A putative 6 transmembrane nitrate transporter OsNRT1.1b plays a key role in rice under low nitrogen[J].Journal of Integrative Plant Biology,2016,58(6):590-599.
[4]LIU Q,CHEN X,WU K,et al.Nitrogen signaling and use efficiency in plants:what’s new?[J]Current Opinion in Plant Biology,2015,27:192-198.
[5]SKOPELITIS D S,PARANYCHIANAKIS N V,KOUVARAKISA,et al.The isoenzyme 7 of tobacco NAD(H)-dependent glutamate dehydrogenase exhibits high deaminating and low aminating activities in vivo[J].Plant Physiology,2007,145(4):1726-1734.
[6]LEA P J,MIFLIN B J.Alternative route for nitrogen assimilation in higher plants[J].Nature,1974,251:614-616.
[7]IRELAND R J,LEA P J.The enzymes of glutamine,glutamate,asparagine,and aspartate metabolism[M]//SINGH B K.Plant Amino Acids:Biochemistry&Biotechnology.New York:Marcel Dekker,1999:49-109.
[8]MIFLIN B J,LEA P J.Ammonia assimilation[M]//MIFLIN B J.The Biochemistry of Plants.New York:Academic Press,1980:169-202.
[9]TOBIN A K,YAMAYA T.Cellular compartmentation of ammonia assimilation in rice and barley[J].Journal of Experimental Botany,2001,52(356):591-604.
[10]MASCLAUX-DAUBRESSE C,REISDORF-CREN M,PAGEAUK,et al.Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sinksource nitrogen cycle in tobacco[J].Plant Physiology,2006,140(2):444-456.
[11]WOOTTON J C.Re-assessment of ammonium-ion affinities of NADP-specific glutamate dehydrogenases.Activation of the Neurospora crassa enzyme by ammonium and rubidium ions[J].Biochemical Journal,1983,209(2):527-531.
[12]OAKS A.Primary nitrogen assimilation in higher plants and its regulation[J].Canadian Journal of Botany-Revue Canadienne De Botanique,1994,72:739-750.
[13]BUCHANAN B B,GRUISSEM W,JONES R L.Biochemistry and Molecular Biology of Plants.2nd Edition[M].Hoboken:John Wiley&Sons,Inc,2015:16.1-16.2.
[14]ZHOU Y,LIU H,ZHOU X,et al.Over-expression of a fungal NADP(H)-dependent glutamate dehydrogenase Pc GDH improves nitrogen assimilation and growth quality in rice[J].Molecular Breeding,2014,34(2):335-349.
[15]HARRISON J,BRUGIRE N,PHILLIPSON B,et al.Manipulating the pathway of ammonia assimilation through genetic engineering and breeding:consequences to plant physiology and plant development[J].Plant and Soil,2000,221(1):81-93.
[16]WANG F,TIAN B.Neurospora NADP glutamate dehydrogenases and its expression in E.coli and transgenic plant[J].Chinese Science Bulletin,2001,46(12):1029-1032.
[17]NOOR S,PUNEKAR N S.Allosteric NADP-glutamate dehydrogenase from Aspergilli:purification,characterization and implications for metabolic regulation at the carbon nitrogen interface[J].Microbiology,2005,151(Pt 5):1409-1419.
[18]DU C,LIN J,YANG Y,et al.Molecular cloning,characterization and function analysis of a GDH gene from Sclerotinia sclerotiorum in rice[J].Molecular Biology Reports,2014,41(6):3683-3693.
[19]KINGHORN J R,PATEMAN J A.NAD and NADP L-glutamate dehydrogenase activity and ammonium regulation in Aspergillus nidulans[J].Journal of General and Applied Microbiology,1973,78(1):39-46.
[20]MACHEDA M L,HYNES M J,DAVIS M A.The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase[J].Current Genetics,1999,34(6):467-471.
[21]WINDASS J D,WORSEY M J,PIOLI E M,et al.Improved conversion of methanol to single-cell protein by Methylophilus methylotrophus[J].Nature,1980,287(5781):396-401.
[22]HELLING R B.Pathway choice in glutamate synthesis in Escherichia coli[J].Journal of Bacteriology,1998,180(17):4571-4575.
[23]AMEZIANE R,BERNHARD K,LIGHTFOOT D A.Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development[J].Plant and Soil,2000,221(1):47-57.
[24]ABIKO T,WAKAYAMA M,KAWAKAMI A,et al.Changes in nitrogen assimilation,metabolism,and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase(gdhA)[J].Planta,2010,232(2):299-311.
[25]ZHOU X,LIN J,ZHOU Y,et al.Overexpressing a fungal gene improves nitrogen utilization and growth in rice[J].Crop Science,2015,55(2):811-820.
[26]KOBAYASHI T,ISHIGURO K,NAKAJIMA T,et al.Effects of elevated atmospheric CO2concentration on the infection of rice blast and sheath blight[J].Phytopathology,2006,96(4):425-431.
[27]LIN J Z,YANG Y Z,ZHOU B,et al.A new and effective somatic mutagenesis rice breeding method[J].Journal of Hunan University(Natural Sciences),2013,40(9):79-85.
[28]ZHOU B,LIN J Z,PENG D,et al.Plant architecture and grain yield are regulated by the novel DHHC-type zinc finger protein genes in rice(Oryza sativa L.)[J].Plant Science,2016,254:12-21.
[29]CHEN Q J,ZHOU H M,CHEN J.Using a modified TA cloning method to create entry clones[J].Analytical Biochemistry,2006,358:120-125.
[30]MURRAY M G,THOMPSON W F.Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4321-4325.
[31]ZHOU Y B,LIU C,TANG D Y,et al.The receptor-like cytoplasmic kinase STRK1 phosphorylates and activates CatC,thereby regulating H2O2homeostasis and improving salt tolerance in rice[J].The Plant Cell,2018,30(5):1100-1118.
[32]TANG D,PENG Y,LIN J,et al.Ectopic expression of fungal EcGDH improves nitrogen assimilation and grain yield in rice[J].Journal of Integrative Plant Biology,2018,60(2):85-88.
[33]FERRARO G,BORTOLOTTI S,MORTERA P,et al.Novel glutamate dehydrogenase genes show increased transcript and protein abundances in mature tomato fruits[J].Journal of Plant Physiology,2012,169(9):899-907.
[34]ZHOU Y,ZHANG C,LIN J,et al.Over-expression of a glutamate dehydrogenase gene,MgGDH,from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice[J].Planta,2015,241(3):727-740.
[35]SRIVASTAVA H S,SINGH R P.Role and regulation of L-glutamate dehydrogenase activity in higher plants[J].Phytochemistry,1987,26(3):597-610.
[36]LIGHTFOOT D A,MUNGUR R,AMEZIANE R,et al.Improved drought tolerance of transgenic Zea mays plants that express the glutamate dehydrogenase gene(gdhA)of E.coli[J].Euphytica,2007,156(1-2):103-116.
[37]OBARA M,SATO T,YAMAYA T.High contents of glutamine synthetase does not accompany a high activity of the enzyme in rice(Oryza sativa)leaves of indica cultivars[J].Physiologia Plantarum,2000,108(1):11-18.
[38]ZHANG H,LIANG C,AOKI N,et al.Introduction of a fungal NADP(H)-dependent glutamate dehydrogenase(gdhA)improves growth,grain weight and salt resistance by enhancing the nitrogen uptake efficiency in forage rice[J].Plant Production Science,2016,19(2):267-278.