拐枣七内生细菌溶磷相关基因的鉴定

Identification of Phosphate Solubilizing-related Genes in Endophytic Strains Isolated from Hylomecon japonica

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.