大豆快生根瘤菌SfHH103＿03182基因突变体的构建和共生固氮表型分析
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摘要
以大豆快生根瘤菌HH103中glnK同源基因SfHH103_03182为研究对象,采用pK19mob单插入失活技术构建该基因的插入失活突变体,菌落PCR和测序分析表明突变体构建正确。植物盆栽试验结果显示,与接种野生型菌株HH103相比,突变体接种植株的根瘤数量增多,植株鲜质量和根瘤鲜质量增加,根瘤固氮酶活无显著差异。在不同浓度氮素处理和盆栽条件下,对共生表型的检测表明突变体接种植物的根瘤数量没有显著变化。
The GlnK protein is a PⅡfamily protein existing widely in bacteria,archaea and plants and involved in regulating nitrogen metabolism.The insertion mutant of glnK homologue gene SfHH103_03182 in Sinorhizobium fredii HH103 was constructed.The results of PCR and sequence analyses confirmed that the mutant was correctly constructed.The results of pot plant experiments showed that the nodule number of the plant inoculated with the mutant,the fresh weight of plants and the fresh weight of root nodules increased compared with inoculated with wild-type strain HH103.There was no significant difference in root nodule nitrogenase activity.The results of symbiotic phenotype tested on host plant soybean at different concentrations of nitrogen levels showed that there was no significant changes in the number of root nodules in the mutant inoculated plants.
The GlnK protein is a PⅡfamily protein existing widely in bacteria,archaea and plants and involved in regulating nitrogen metabolism.The insertion mutant of glnK homologue gene SfHH103_03182 in Sinorhizobium fredii HH103 was constructed.The results of PCR and sequence analyses confirmed that the mutant was correctly constructed.The results of pot plant experiments showed that the nodule number of the plant inoculated with the mutant,the fresh weight of plants and the fresh weight of root nodules increased compared with inoculated with wild-type strain HH103.There was no significant difference in root nodule nitrogenase activity.The results of symbiotic phenotype tested on host plant soybean at different concentrations of nitrogen levels showed that there was no significant changes in the number of root nodules in the mutant inoculated plants.
引文
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[11]谭茜,王龙祥,张先鹏,等.CRISPR/Cas9在豆科植物毛根转化中的应用及共生基因的编辑[J].华中农业大学学报,2017,36(5):45-51.
[12]周向珍,朱辉.百脉根根瘤菌MAFF303099Tn5转座子插入突变体库的构建及筛选[J].华中农业大学学报,2018,37(2):16-22.
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[14]杨倩倩,刘元,陈大松,等.蒺藜苜蓿中1个聚半乳糖醛酸酶基因突变体的筛选及共生固氮表型的初步鉴定[J].华中农业大学学报,2017,36(4):62-70.
[15]SANT’ANNA F H,TRENTINI D B B,SHANA-DE-SOUTOW,et al.The PII superfamily revised:a novel group and evolutionary insights[J].Journal of molecular evolution,2009,68(4):322-336.
[16]D’APUZZO E,VALKOV V T,PARLATI A,et al.PII overexpression in Lotus japonicus affects nodule activity in permissive low-nitrogen conditions and increases nodule numbers in high nitrogen treated plants[J].Mol Plant Microbe Interact,2015,28(4):432-442.
[17]DIXON R,KAHN D.Genetic regulation of biological nitrogen fixation[J].Nat Rev Microbiol,2004,2(8):621-631.
[18]BLAUWKAMP T A,NINFA A J.Physiological role of the GlnK signal transduction protein of Escherichia coli:survival of nitrogen starvation[J].Mol Microbiol,2002,46(1):203-214.
[19]GOSZTOLAI A,SCHUMACHER J,BEHRENDS V,et al.GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation[J].Biophys J,2017,112(10):2219-2230.
[20]PEDRO-ROIG L,CAMACHO M,BONETE M J.Regulation of ammonium assimilation in Haloferax mediterranei:interaction between glutamine synthetase and two GlnK proteins[J].Biochim Biophys Acta,2013,1834:16-23.
[21]HE S,CHEN M,XIE Z,et al.Involvement of GlnK,a PⅡprotein,in control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1501[J].Arch Microbiol,2008,190(1):1-10.
[22]YURGEL S N,RICE J,MULDER M,et al.GlnB/GlnK PⅡproteins and regulation of the Sinorhizobium meliloti Rm1021nitrogen stress response and symbiotic function[J].J Bacteriol,2010,192(10):2473-2481.
[23]PENG J,HAO B,LIU L,et al.RNA-Seq and microarrays analyses reveal global differential transcriptomes of Mesorhizobium huakuii 7653Rbetween bacteroids and free-living cells[J].PLoS One,2014,9(4):e93626.
[24]AMPE F,KISS E,SABOURDY F,et al.Transcriptome analysis of Sinorhizobium meliloti during symbiosis[J].Genome biology,2003,4(2):R15.
[25]ERCOLANO E,MIRABELLA R,MERRICK M,et al.The Rhizobium leguminosarumglnB gene is down-regulated during symbiosis[J].Mol Gen Genet,2001,264(5):555-564.
[2]WEIDNERA S,BECKER A,BONILLA I,et al.Genome sequence of the soybean symbiont Sinorhizobium fredii HH103[J].J Bacteriol,2012,194(6):1617-1618.
[3]SHAPIRO B M.The glutamine synthetase deadenylylating enzyme system fromEscherichia coli resolution into two components,specific nucleotide stimulation,and cofactor requirements[J].Biochemistry,1969,8(2):659-670.
[4]TRUAN D,BJELIC S,LI X D,et al.Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PⅡprotein GlnZ from Azospirillum brasilense[J].J Mol Biol,2014,426(15):2783-2799.
[5]HUERGO L F,CHANDRA G,MERRICK M.P(Ⅱ)signal transduction proteins:nitrogen regulation and beyond[J].FEMS Microbiol Rev,2013,37(2):251-283.
[6]JIANG P,MAYO A E,NINFA A J.Escherichia coli glutamine synthetase adenylyltransferase(ATase,EC 2.7.7.49):kinetic characterization of regulation by PⅡ,PⅡ-UMP,glutamine,and alpha-ketoglutarate[J].Biochemistry,2007,46(13):4133-4146.
[7]JAVELLE A,SEVERI E,THORNTON J,et al.Ammonium sensing in Escherichia coli role of the ammonium transporter AmtB and AmtB-GlnK complex formation[J].J Biol Chem,2004,279(10):8530-8538.
[8]刘莉,周俊初,陈华癸.不同化合态氮浓度对大豆根瘤菌结瘤和固氮作用的影响[J].中国农业科学,1998,31(1):87-89.
[9]LIM C W,LEE Y W,LEE S C,et al.Nitrate inhibits soybean nodulation by regulating expression of CLE genes[J].Plant Sci,2014,229:1-9.
[10]萨姆布鲁克J,塞尔D W.分子克隆实验指南版[M].3版.北京:科学出版社,2002.
[11]谭茜,王龙祥,张先鹏,等.CRISPR/Cas9在豆科植物毛根转化中的应用及共生基因的编辑[J].华中农业大学学报,2017,36(5):45-51.
[12]周向珍,朱辉.百脉根根瘤菌MAFF303099Tn5转座子插入突变体库的构建及筛选[J].华中农业大学学报,2018,37(2):16-22.
[13]SCHFER A,TAUCH A,GER W J,et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum[J].Gene,1994,145(1):69-73.
[14]杨倩倩,刘元,陈大松,等.蒺藜苜蓿中1个聚半乳糖醛酸酶基因突变体的筛选及共生固氮表型的初步鉴定[J].华中农业大学学报,2017,36(4):62-70.
[15]SANT’ANNA F H,TRENTINI D B B,SHANA-DE-SOUTOW,et al.The PII superfamily revised:a novel group and evolutionary insights[J].Journal of molecular evolution,2009,68(4):322-336.
[16]D’APUZZO E,VALKOV V T,PARLATI A,et al.PII overexpression in Lotus japonicus affects nodule activity in permissive low-nitrogen conditions and increases nodule numbers in high nitrogen treated plants[J].Mol Plant Microbe Interact,2015,28(4):432-442.
[17]DIXON R,KAHN D.Genetic regulation of biological nitrogen fixation[J].Nat Rev Microbiol,2004,2(8):621-631.
[18]BLAUWKAMP T A,NINFA A J.Physiological role of the GlnK signal transduction protein of Escherichia coli:survival of nitrogen starvation[J].Mol Microbiol,2002,46(1):203-214.
[19]GOSZTOLAI A,SCHUMACHER J,BEHRENDS V,et al.GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation[J].Biophys J,2017,112(10):2219-2230.
[20]PEDRO-ROIG L,CAMACHO M,BONETE M J.Regulation of ammonium assimilation in Haloferax mediterranei:interaction between glutamine synthetase and two GlnK proteins[J].Biochim Biophys Acta,2013,1834:16-23.
[21]HE S,CHEN M,XIE Z,et al.Involvement of GlnK,a PⅡprotein,in control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1501[J].Arch Microbiol,2008,190(1):1-10.
[22]YURGEL S N,RICE J,MULDER M,et al.GlnB/GlnK PⅡproteins and regulation of the Sinorhizobium meliloti Rm1021nitrogen stress response and symbiotic function[J].J Bacteriol,2010,192(10):2473-2481.
[23]PENG J,HAO B,LIU L,et al.RNA-Seq and microarrays analyses reveal global differential transcriptomes of Mesorhizobium huakuii 7653Rbetween bacteroids and free-living cells[J].PLoS One,2014,9(4):e93626.
[24]AMPE F,KISS E,SABOURDY F,et al.Transcriptome analysis of Sinorhizobium meliloti during symbiosis[J].Genome biology,2003,4(2):R15.
[25]ERCOLANO E,MIRABELLA R,MERRICK M,et al.The Rhizobium leguminosarumglnB gene is down-regulated during symbiosis[J].Mol Gen Genet,2001,264(5):555-564.