拐枣七内生细菌溶磷相关基因的鉴定
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摘要
为了鉴定拐枣七内生细菌Pseudomonas sp.GZJR-8的pqqE和GDH基因在溶磷方面的功能,该研究通过同源重组技术分别获得pqqE和GDH基因的缺失突变体ΔpqqE和ΔGDH,采用电转法获得它们的互补菌株ΔpqqE(pqqE)和ΔGDH(GDH);难溶性无机磷培养基(PKO)定性检测结果显示,与野生型菌株(WT)相比,ΔpqqE和ΔGDH不能产生溶磷圈,ΔpqqE(pqqE)和ΔGDH(GDH)能够产生溶磷圈;抗坏血酸-钼蓝显色定磷法定量检测结果显示,WT、ΔpqqE、ΔpqqE(pqqE)、ΔGDH、ΔGDH(GDH)产生的有效磷总量分别为1 939.000 mg/L、1 279.000mg/L、1 999.000mg/L、439.000mg/L、2 314.000mg/L,与WT产生的有效磷总量相比,ΔpqqE降低1.52倍,ΔpqqE(pqqE)增加1.03倍,ΔGDH降低4.42倍,ΔGDH(GDH)增加1.19倍;培养液离心后上清的pH测定结果显示,WT、ΔpqqE、ΔpqqE(pqqE)、ΔGDH、ΔGDH(GDH)的pH分别为4.08、4.34、4.03、4.71、4.00,与WT的pH相比,ΔpqqE上升1.06倍,ΔpqqE(pqqE)降低0.27倍,ΔGDH上升1.15倍,ΔGDH(GDH)降低1.02倍。研究表明:pqqE和GDH基因具有溶磷能力,其中ΔpqqE和ΔGDH较WT的溶磷能力下降,但并未完全丧失溶磷能力,而ΔpqqE(pqqE)和ΔGDH(GDH)可以恢复到WT的溶磷能力,拐枣七内生细菌Pseudomonas sp.GZJR-8是通过产生酸性物质来溶磷,在农业生产方面具有潜在应用价值。
In order to identify the function of pqqE and GDH genes of Pseudomonas sp.GZJR-8 isolated from Hylomecon japonicain the case of dissolved phosphorus,we knocked out pqqE and GDH genes by the homologous recombination technology,marked asΔpqqE and ΔGDH, and we constructed the complementary strain by electro transformation,marked asΔpqqE(pqqE) and ΔGDH(GDH).The results of qualitative analysis with insoluble inorganic phosphorus medium(PKO)showed thatΔpqqE and ΔGDH could not produce dissolved phosphorus ring,ΔpqqE(pqqE) and ΔGDH(GDH)could produce dissolved phosphorus ring,compared with the wild type strain(WT).The total amount of available phosphorus produced by WT,ΔpqqE,ΔpqqE(pqqE),ΔGDH and ΔGDH(GDH)was 1 939.000 mg/L,1 279.000mg/L,1 999.000mg/L,439.000mg/L and 2 314.000mg/L,respectively.Compared with WT,ΔpqqE decreased by 1.52 times,ΔpqqE(pqqE)increased by 1.03 times,ΔGDH decreased by 4.42 times, and ΔGDH(GDH)increased by 1.19 times.The pH of the supernatant after centrifugation of the culture medium showed that the pH of WT,ΔpqqE,ΔpqqE(pqqE),ΔGDH and ΔGDH(GDH)was 4.08,4.34,4.03,4.71 and 4.00,respectively.Compared with WT,ΔpqqEincreased by 1.06 times,ΔpqqE(pqqE)decreased by 0.27 times,ΔGDHincreased by 1.15 times,ΔGDH(GDH)decreased by 1.02 times.The research showed that pqqE and GDH genes had the ability to dissolve phosphorus,in which ΔpqqE 和ΔGDH were lower than WT,but they did not completely lose it,while the complementary strains could restore the ability.The Pseudomonas sp.GZJR-8 isolated from Hylomecon japonica could dissolve phosphorus through the acid-producing mechanism.It had potential application value in agricultural production.
In order to identify the function of pqqE and GDH genes of Pseudomonas sp.GZJR-8 isolated from Hylomecon japonicain the case of dissolved phosphorus,we knocked out pqqE and GDH genes by the homologous recombination technology,marked asΔpqqE and ΔGDH, and we constructed the complementary strain by electro transformation,marked asΔpqqE(pqqE) and ΔGDH(GDH).The results of qualitative analysis with insoluble inorganic phosphorus medium(PKO)showed thatΔpqqE and ΔGDH could not produce dissolved phosphorus ring,ΔpqqE(pqqE) and ΔGDH(GDH)could produce dissolved phosphorus ring,compared with the wild type strain(WT).The total amount of available phosphorus produced by WT,ΔpqqE,ΔpqqE(pqqE),ΔGDH and ΔGDH(GDH)was 1 939.000 mg/L,1 279.000mg/L,1 999.000mg/L,439.000mg/L and 2 314.000mg/L,respectively.Compared with WT,ΔpqqE decreased by 1.52 times,ΔpqqE(pqqE)increased by 1.03 times,ΔGDH decreased by 4.42 times, and ΔGDH(GDH)increased by 1.19 times.The pH of the supernatant after centrifugation of the culture medium showed that the pH of WT,ΔpqqE,ΔpqqE(pqqE),ΔGDH and ΔGDH(GDH)was 4.08,4.34,4.03,4.71 and 4.00,respectively.Compared with WT,ΔpqqEincreased by 1.06 times,ΔpqqE(pqqE)decreased by 0.27 times,ΔGDHincreased by 1.15 times,ΔGDH(GDH)decreased by 1.02 times.The research showed that pqqE and GDH genes had the ability to dissolve phosphorus,in which ΔpqqE 和ΔGDH were lower than WT,but they did not completely lose it,while the complementary strains could restore the ability.The Pseudomonas sp.GZJR-8 isolated from Hylomecon japonica could dissolve phosphorus through the acid-producing mechanism.It had potential application value in agricultural production.
引文
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[13]杨璐,熊向华,王建华,等.吡咯喹啉醌研究进展[J].生物技术通讯,2009,20(6):874-879.YANG L,XIONG X H,WANG J Het al.Advances on the research of Pyrroloquinoline Quinone[J].Letters in Biotechnology,2009,20(6):874-879.
[14]ZHANG Y,ROSENBERG P A.The essential nutrient pyrroloquinoline quinone may act as a neuroprotectant by suppressing peroxynitrite formation[J].European Journal of Neuroscience,2002,16(6):1 015-1 024.
[15]MORITZ M,SANDRA P,STUART J.FISHER.Crystal structure of PqqB from Pseudomonas putida at 2.2resolution[J].Journal of Biophysical Chemistry,2012,2012:206-210.
[16]周怡雯,陈建华.新辅酶吡咯喹啉醌研究进展[J].中国生化药物杂志,2008,29(4):279-282.ZHOU Y W,CHEN J H.Progress in the research of Pyrroloquinoline Quinone[J].Chinese Journal of Biochemical and Pharmaceutics,2008,29(4):279-282.
[17]NAUTIYAL C S.An efficient microbiological growth medium for screening phosphate solubilizing microorganisms[J].FEMS Microbiology Letters,1999,170(1):265-270.
[18]HU Y,LU P,WANG Y,et al.OmpR positively regulates urease expression to enhance acid survival of Yersinia pseudotuberculosis[J].Microbiology,2009,155(8):2 522-2 531.
[19]ZHANG W,WANG Y,SONG Y,et al.A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis[J].Manual of Environmental Microbiology,2013,15(2):557-569.
[20]ZHNG J,LEUNG K Y.Dissection of a type VI secretion system in Edwardsiella tarda[J].Molecular Microbiology,2007,66(5):1 192-1 206.
[21]王春林,曾梅珍,宋荣钊.分光光度法测定有机磷的研究[J].广州化学,1993,(1):65-69.WANG C L,ZENG M Z,SONG R Z.Spectrophotometric determination of phousphorus in organic compound[J].Guangzhou Chemistry,1993,(1):65-69.
[22]KHAIRNAR NP,MISRA HS,APTE SK.Pyrroloquinolinequinone synthesized in Escherichia coli by pyrroloquinolinequinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization[J].Biochemical and Biophysical Research Communications,2003,312(2):303-308.
[23]CHOI O,KIM J,KIM J G,et al.Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16[J].Plant Physiology,2008,146(2):657-668.
[2]RODRIGUEZ H,FRAGA R.Phosphate solubilizing bacteria and theirrole in plant growth promotion[J].Biotechnology Advances,1999,17(4):319-339.
[3]STALSTROM V A.Beitrag zur Kennturs de rein-wisking steriler und in Garung bonfindlicher organischer stroffe auf dil Looslichkeit des phosphoresen der TCP[J].Zentralbl Bakteriol Mikrobiol Hyg B,1903,11:724-732.
[4]SACKETT W G,PATTEN A J,BROWN C W.The solvent action of soil bacteria upon the insoluble phosphates of raw bonemeal and natural raw rock phosphate[J].Centralbl Bakteriol,1908,202:688-703.
[5]GUINAZ U'L B,ANDRES J A,Del Papa M F,et al.Response of alfalfa(Medicago sativa L.)to single and mixed inoculation with phosphate-solubilizing bacteria and Sinorhizobium meliloti[J].Biology and Fertility of Soils,2010,46(2):185-190.
[6]毕江涛,孙权,李素剑,等.解磷微生物研究进展[J].农业科学研究,2009,(4):58-64.BI J T,SUN Q,LI S J,et al.Research advances in phosphorous solubilizing microorganisms[J].Journal of Agricultural Sciences,2009,(4):58-64.
[7]唐朝军,董发勤,代群威,等.嗜酸氧化硫硫杆菌对中低品位磷矿的溶磷效果研究[J].矿物学报,2010,S1:111-112.TANG C J,DONG F Q,DAI Q W,et al.A study on leaching effect of acidithilbacillus thiooxidans to phosphor from low-grade phosphate Ores[J].Acta Mineralogica Sinica,2010,S1:111-112.
[8]陆俊锟,陈俊,康丽华.四株红树林促生菌的遗传分析鉴定及其促生能力[J].微生物学报,2010,10:1 358-1 365.LU J K,CHEN J,KANG L H.Genotypic analysis and plant growth-promoting ability of four plant growth-promoting bacteria from mangrove[J].Acta Microbiologica Sinica,2010,10:1 358-1 365.
[9]NAIK P R,RAMAN G,NARAYANAN K B,et al.Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil[J].BMC Microbiology,2008,8(1):230.
[10]钟传青,黄为一.不同种类解磷微生物的溶磷效果及其磷酸酶活性的变化[J].土壤学报,2005,(2):286-294.ZHONG C Q,HUANG W Y.Phosphorus-solubilizing effect and phosphatase activity of different types of phosphorus-solubilizing microorganisms[J].Acta Pedologica Sinica,2005,(2):286-294.
[11]ILLMER P,SCHINNERr F.Solubilization of inorganic phosphates by microorganisms isolated from forest soils[J].Soil Biology and Biochemistry,1992,24(4):389-395.
[12]BASHAN Y,KAMNEV A A,DE-BASHAN L E.Tricalcium phosphate is inappropriate as a universal selection factor for isolating and testing phosphate-solubilizing bacteria that enhance plant growth:aproposal for an alternative procedure[J].Biology and Fertility of Soils,2013,49(4):465-479.
[13]杨璐,熊向华,王建华,等.吡咯喹啉醌研究进展[J].生物技术通讯,2009,20(6):874-879.YANG L,XIONG X H,WANG J Het al.Advances on the research of Pyrroloquinoline Quinone[J].Letters in Biotechnology,2009,20(6):874-879.
[14]ZHANG Y,ROSENBERG P A.The essential nutrient pyrroloquinoline quinone may act as a neuroprotectant by suppressing peroxynitrite formation[J].European Journal of Neuroscience,2002,16(6):1 015-1 024.
[15]MORITZ M,SANDRA P,STUART J.FISHER.Crystal structure of PqqB from Pseudomonas putida at 2.2resolution[J].Journal of Biophysical Chemistry,2012,2012:206-210.
[16]周怡雯,陈建华.新辅酶吡咯喹啉醌研究进展[J].中国生化药物杂志,2008,29(4):279-282.ZHOU Y W,CHEN J H.Progress in the research of Pyrroloquinoline Quinone[J].Chinese Journal of Biochemical and Pharmaceutics,2008,29(4):279-282.
[17]NAUTIYAL C S.An efficient microbiological growth medium for screening phosphate solubilizing microorganisms[J].FEMS Microbiology Letters,1999,170(1):265-270.
[18]HU Y,LU P,WANG Y,et al.OmpR positively regulates urease expression to enhance acid survival of Yersinia pseudotuberculosis[J].Microbiology,2009,155(8):2 522-2 531.
[19]ZHANG W,WANG Y,SONG Y,et al.A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis[J].Manual of Environmental Microbiology,2013,15(2):557-569.
[20]ZHNG J,LEUNG K Y.Dissection of a type VI secretion system in Edwardsiella tarda[J].Molecular Microbiology,2007,66(5):1 192-1 206.
[21]王春林,曾梅珍,宋荣钊.分光光度法测定有机磷的研究[J].广州化学,1993,(1):65-69.WANG C L,ZENG M Z,SONG R Z.Spectrophotometric determination of phousphorus in organic compound[J].Guangzhou Chemistry,1993,(1):65-69.
[22]KHAIRNAR NP,MISRA HS,APTE SK.Pyrroloquinolinequinone synthesized in Escherichia coli by pyrroloquinolinequinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization[J].Biochemical and Biophysical Research Communications,2003,312(2):303-308.
[23]CHOI O,KIM J,KIM J G,et al.Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16[J].Plant Physiology,2008,146(2):657-668.