广西红壤土壤微生物溶磷基因的克隆与表达
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摘要
本研究对广西红壤土壤微生物溶磷基因进行克隆,并在大肠杆菌中诱导表达。从广西红壤中提取宏基因组DNA,克隆了GabY基因,构建大肠杆菌原核表达载体p ETDuet-1,转化大肠杆菌DH5α得到重组工程菌。通过酶切验证重组体。重组体在诱导条件下24 h对难溶矿物质磷Ca_3(PO_4)_2的溶磷量为(40.73±1.32)μg/mL,而对照组仅为(5.11±0.08)μg/mL。重组体和对照随着诱导时间的增加,培养基pH值均有下降趋势,但重组体培养基pH值变化显著大于对照组,产酸能力显著优于对照组。证明成功克隆得到土壤微生物溶磷基因,并在大肠杆菌中得到表达。
The study cloned the phosphate-solubilizing gene of soil microorganisms from red soil in Guangxi and induced expression in E. coli. Metagenomic DNA was extracted from red soil in Guangxi, and GabY gene was cloned to construct the prokaryotic expression vector p ETDuet-1 of E. coli. E. coli DH5α was later transformed to obtain the recombinant engineering bacteria. The transformants were screened and identified by restriction endonuclease digestion analysis. Phosphate dissolving capacity of the transformants on insoluble mineral phosphorus Ca_3(PO_4)_2 under induction condition of 24 h was reached to(40.73±1.32) μg/mL, while that of the control group was only(5.11±0.08) μg/mL. Accompany with the increase of induction time, pH of transformants and control mediums both showed the trend of decrease, but the variation of p H in transformants medium was significantly greater than that in the control medium, and acid-producing ability was superior to the control. Those results indicated the successful cloning of phosphate-solubilizing gene of soil microorganisms and the expression in E. coli.
The study cloned the phosphate-solubilizing gene of soil microorganisms from red soil in Guangxi and induced expression in E. coli. Metagenomic DNA was extracted from red soil in Guangxi, and GabY gene was cloned to construct the prokaryotic expression vector p ETDuet-1 of E. coli. E. coli DH5α was later transformed to obtain the recombinant engineering bacteria. The transformants were screened and identified by restriction endonuclease digestion analysis. Phosphate dissolving capacity of the transformants on insoluble mineral phosphorus Ca_3(PO_4)_2 under induction condition of 24 h was reached to(40.73±1.32) μg/mL, while that of the control group was only(5.11±0.08) μg/mL. Accompany with the increase of induction time, pH of transformants and control mediums both showed the trend of decrease, but the variation of p H in transformants medium was significantly greater than that in the control medium, and acid-producing ability was superior to the control. Those results indicated the successful cloning of phosphate-solubilizing gene of soil microorganisms and the expression in E. coli.
引文
Ahuja A.,Ghosh S.B.,and D'Souza S.F.,2007,Isolation of a starch utilizing,phosphate solubilizing fungus on buffered medium and its characterization,Bioresource Technology,98(17):3408
Anzuay M.S.,Frola O.,Angelini J.G.,Ludue觡a L.M.,Iba觡ez F.,Fabra A.,and Taurian T.,2015,Effect of pesticides applica tion on peanut(Arachis hypogaea L.)associated phosphate solubilizing soil bacteria,Applied Soil Ecology,95:31-37
Babu-Khan S.,Yeo T.C.,Martin W.L.,Duron M.R.,Rogers R.D.,and Goldstein A.H.,1995,Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia,Appliedand Environmental Microbiology,61(3):972-978
Chen Z.,Wu M.N.,Qin H.L.,and Wei W.X.,2009,Advances in research on molecular mechanisms of phosphate-solubilizing microorganisms in soil,Turang Xuebao(Acta Pedologica Sinica),46(5):925-931(陈哲,吴敏娜,秦红灵,魏文学,2009,土壤微生物溶磷分子机理研究进展,土壤学报,46(5):925-931)
Glassop D.,Smith S.E.,and Smith F.W.,2005,Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots,Planta,222(4):688-698
Goosen N.,Horsman H.P.,Huinen R.G.,and Putte P.V.D.,1989,Acinetobacter calcoaceticus genes involved in biosynthesis of the coenzyme pyrrolo-quinoline-quinone:Nucleotide sequence and expression in Escherichia coli K-12,Journal of Bacteriology,171(1):447-455
Harrison M.J.,Dewbre G.R.,and Liu J.Y.,2002,A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by Arbuscular mycorrhizal fungi,Plant Cell,14(10):2413-2429
Illmer P.,and Schinner F.,1995,Solubilization of inorganic calcium phosphates-solubilization mechanisms,Soil Biology&Biochemistry,27(3):257-263
Kim K.Y.,Jordan D.,and Krishnan H.B.,1998,Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli,FEMS Microbiology Letters,159(1):121-127
Nautiyal C.S.,1999,An efficient microbiological growth medium for screening phosphate solubilizing microorganisms,FEMSMicrobiology Letters,170(1):265-270
Shahid M.,Hameed S.,Imran A.,Ali S.,and van Elsas J.D.,2012,Root colonization and growth promotion of sunflower(Helianthus annuus L.)by phosphate solubilizing Enterobac-%ter sp.Fs-11,World Journal of Microbiology and Biotechnology,28(8):2749-2758
Shao J.S.,Pu M.Y.,and Gu Z.J.,2013,Comparison of the three methods of extracting the genome DNA of microorganism of soil,Nanjing Xiaozhuang Xueyuan Xuebao(Journal of Nanjing Xiaozhuang University),29(3):64-67(邵劲松,浦牧野,顾祝军,2013,3种土壤微生物基因组DNA提取方法的比较,南京晓庄学院学报,29(3):64-67)
Tripura C.,Sudhakar R.P.,Reddy M.K.,Sashidhar B.,and Podile A.R.,2007,Glucose dehydrogenase of a rhizobacterial strain of Enterobacter asburiae involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae,Indian Journal of Microbiology,47(2):126-131
Wang X.,Wang Y.,Tian J.,Lim B.L.,Yan X.,and Liao H.,2009,Overexpressing At PAP15 enhances phosphorus efficiency in soybean,Plant Physiology,151(1):233-240
Wang Y.,He C.P.,Zheng X.L.,Yang Q.,and Zheng F.C.,2009,Research progress on phosphate-solubilizing microorganisms in soil,Anhui Nongxue Tongbao(Anhui Agricultural Science Bulletin),15(9):60-64(王义,贺春萍,郑肖兰,杨芩,郑服从,2009,土壤解磷微生物研究进展,安徽农学通报,15(9):60-64)
Xing F.F.,Gao M.F.,Zuo Y.Y.,Hu Z.P.,and Li X.Z.,2016,Screening and identification of phosphate solubilizing bacteria in Maize rhizosphere and their characteristics of phosphate solubilizing,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),32(9):119-124(邢芳芳,高明夫,禚优优,胡兆平,李新柱,2016,玉米根际高效溶磷菌的筛选、鉴定及溶磷特性研究,中国农学通报,32(9):119-124)
Xu G.H.,Chague V.,Melamed-bessudo C.,Kapulnik Y.,Jain A.,Raghothama K.G.,Levy A.A.,and Silber A.,2007,Functional characterization of LePT4:a phosphate transporter in tomato with mycorrhiza-enhanced expression,Journal of Experimental Botany,58(10):2491-2501
Yang F.,He Y.Q.,Li C.L.,Wang Y.L.,and Lin T.,2006,Effect of fertilization on phosphorus fixation in upland red soil and its affecting factors,Turang Xuebao(Acta Pedologica Sinica),43(2):267-272(杨芳,何园球,李成亮,王艳玲,林天,2006,不同施肥条件下旱地红壤磷素固定及影响因素的研究,土壤学报,43(2):267-272)
Zhang F.L.,2008,Regulation of mitochondrial ATPase activity by type 2C protein phosphatase in Saccharomyces cerevisiae,Thesis for M.S.,Tianjin University,Supervisor:Jiang L.H.,pp.1-66(张凤莲,2008,酿酒酵母PP2C类蛋白磷酸酯酶对线粒体ATP酶活性的调控,硕士学位论文,天津大学,导师:蒋伶活,pp.1-66)
Zhao K.,2007,Phosphate-solublizing characteristics of bacteria YM3-2S and cloning of phosphate-solublizing gene,Thesis for M.S.,Sichuan Agricultural University,Supervisor:Chen Q.,pp.1-57(赵珂,2007,溶磷菌YM3-2S溶磷特性及溶磷基因的克隆,硕士学位论文,四川农业大学,导师:陈强,pp.1-57)
Anzuay M.S.,Frola O.,Angelini J.G.,Ludue觡a L.M.,Iba觡ez F.,Fabra A.,and Taurian T.,2015,Effect of pesticides applica tion on peanut(Arachis hypogaea L.)associated phosphate solubilizing soil bacteria,Applied Soil Ecology,95:31-37
Babu-Khan S.,Yeo T.C.,Martin W.L.,Duron M.R.,Rogers R.D.,and Goldstein A.H.,1995,Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia,Appliedand Environmental Microbiology,61(3):972-978
Chen Z.,Wu M.N.,Qin H.L.,and Wei W.X.,2009,Advances in research on molecular mechanisms of phosphate-solubilizing microorganisms in soil,Turang Xuebao(Acta Pedologica Sinica),46(5):925-931(陈哲,吴敏娜,秦红灵,魏文学,2009,土壤微生物溶磷分子机理研究进展,土壤学报,46(5):925-931)
Glassop D.,Smith S.E.,and Smith F.W.,2005,Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots,Planta,222(4):688-698
Goosen N.,Horsman H.P.,Huinen R.G.,and Putte P.V.D.,1989,Acinetobacter calcoaceticus genes involved in biosynthesis of the coenzyme pyrrolo-quinoline-quinone:Nucleotide sequence and expression in Escherichia coli K-12,Journal of Bacteriology,171(1):447-455
Harrison M.J.,Dewbre G.R.,and Liu J.Y.,2002,A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by Arbuscular mycorrhizal fungi,Plant Cell,14(10):2413-2429
Illmer P.,and Schinner F.,1995,Solubilization of inorganic calcium phosphates-solubilization mechanisms,Soil Biology&Biochemistry,27(3):257-263
Kim K.Y.,Jordan D.,and Krishnan H.B.,1998,Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli,FEMS Microbiology Letters,159(1):121-127
Nautiyal C.S.,1999,An efficient microbiological growth medium for screening phosphate solubilizing microorganisms,FEMSMicrobiology Letters,170(1):265-270
Shahid M.,Hameed S.,Imran A.,Ali S.,and van Elsas J.D.,2012,Root colonization and growth promotion of sunflower(Helianthus annuus L.)by phosphate solubilizing Enterobac-%ter sp.Fs-11,World Journal of Microbiology and Biotechnology,28(8):2749-2758
Shao J.S.,Pu M.Y.,and Gu Z.J.,2013,Comparison of the three methods of extracting the genome DNA of microorganism of soil,Nanjing Xiaozhuang Xueyuan Xuebao(Journal of Nanjing Xiaozhuang University),29(3):64-67(邵劲松,浦牧野,顾祝军,2013,3种土壤微生物基因组DNA提取方法的比较,南京晓庄学院学报,29(3):64-67)
Tripura C.,Sudhakar R.P.,Reddy M.K.,Sashidhar B.,and Podile A.R.,2007,Glucose dehydrogenase of a rhizobacterial strain of Enterobacter asburiae involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae,Indian Journal of Microbiology,47(2):126-131
Wang X.,Wang Y.,Tian J.,Lim B.L.,Yan X.,and Liao H.,2009,Overexpressing At PAP15 enhances phosphorus efficiency in soybean,Plant Physiology,151(1):233-240
Wang Y.,He C.P.,Zheng X.L.,Yang Q.,and Zheng F.C.,2009,Research progress on phosphate-solubilizing microorganisms in soil,Anhui Nongxue Tongbao(Anhui Agricultural Science Bulletin),15(9):60-64(王义,贺春萍,郑肖兰,杨芩,郑服从,2009,土壤解磷微生物研究进展,安徽农学通报,15(9):60-64)
Xing F.F.,Gao M.F.,Zuo Y.Y.,Hu Z.P.,and Li X.Z.,2016,Screening and identification of phosphate solubilizing bacteria in Maize rhizosphere and their characteristics of phosphate solubilizing,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),32(9):119-124(邢芳芳,高明夫,禚优优,胡兆平,李新柱,2016,玉米根际高效溶磷菌的筛选、鉴定及溶磷特性研究,中国农学通报,32(9):119-124)
Xu G.H.,Chague V.,Melamed-bessudo C.,Kapulnik Y.,Jain A.,Raghothama K.G.,Levy A.A.,and Silber A.,2007,Functional characterization of LePT4:a phosphate transporter in tomato with mycorrhiza-enhanced expression,Journal of Experimental Botany,58(10):2491-2501
Yang F.,He Y.Q.,Li C.L.,Wang Y.L.,and Lin T.,2006,Effect of fertilization on phosphorus fixation in upland red soil and its affecting factors,Turang Xuebao(Acta Pedologica Sinica),43(2):267-272(杨芳,何园球,李成亮,王艳玲,林天,2006,不同施肥条件下旱地红壤磷素固定及影响因素的研究,土壤学报,43(2):267-272)
Zhang F.L.,2008,Regulation of mitochondrial ATPase activity by type 2C protein phosphatase in Saccharomyces cerevisiae,Thesis for M.S.,Tianjin University,Supervisor:Jiang L.H.,pp.1-66(张凤莲,2008,酿酒酵母PP2C类蛋白磷酸酯酶对线粒体ATP酶活性的调控,硕士学位论文,天津大学,导师:蒋伶活,pp.1-66)
Zhao K.,2007,Phosphate-solublizing characteristics of bacteria YM3-2S and cloning of phosphate-solublizing gene,Thesis for M.S.,Sichuan Agricultural University,Supervisor:Chen Q.,pp.1-57(赵珂,2007,溶磷菌YM3-2S溶磷特性及溶磷基因的克隆,硕士学位论文,四川农业大学,导师:陈强,pp.1-57)