荧光假单胞菌CLW17菌株pqqE和GDH基因的功能

Functional analysis of pqqE and GDH genes in Pseudomonas fluorescens CLW17 strain

摘要:

【背景】磷是植物生长所必需的大量元素，但绝大多数不能被植物吸收利用。然而溶磷微生物能够分泌有机酸来溶解土壤中难溶性磷，提高土壤中磷的利用率，促进植物生长，提高作物的产量和品质。【目的】探究高效解磷荧光假单胞菌CLW17菌株的pqqE和GDH基因的生理学功能。【方法】利用生物在线软件对2个基因编码蛋白进行生物信息学分析。利用同源重组技术分别获得pqqE和GDH基因缺失突变株(CLW17ΔpqqE，CLW17ΔGDH)，并使用接合转移的方式获得回补菌株(ΔpqqE/pqqE，ΔGDH/GDH)。分别采用NBRIP培养基、钼锑抗比色法及高压液相色谱法(HPLC)对野生型、突变株及互补株的溶磷及产有机酸能力进行检测。【结果】pqqE和GDH基因编码氨基酸数目分别为390和803，均无信号肽。pqqE无跨膜结构域，而GDH预测有5个跨膜结构域。pqqE和GDH基因是CLW17菌株的溶磷相关基因，2个基因的缺失均使该菌株的溶磷能力显著下降，而回补株可以恢复溶磷能力。CLW17野生株能分泌多种有机酸，其中葡萄糖酸(gluconic acid，GA)含量最多，其次是乙酸；但敲除株产有机酸的能力明显降低，尤其是产GA能力与空白培养基对照相差甚微，而且无乙酸产生。【结论】pqqE和GDH基因是CLW17溶磷能力发挥的关键基因，该菌株溶磷能力与产有机酸尤其是产GA和乙酸密切相关。本研究为进一步研究该菌株与南方红豆杉的互作和溶磷机制奠定了基础。

关键词:荧光假单胞菌;溶磷基因;基因敲除;同源重组;南方红豆杉

Abstract:

[Background] Phosphorus is a macronutrient for plant growth, but most of it cannot be absorbed by plants. Phosphate-solubilizing microorganism (PSM) can secrete organic acids to dissolve the insoluble soil phosphates, improve plant growth, crop yield and quality by increasing phosphorus utilization.[Objective] The research aims to study the physiological functions of pqqE and GDH genes in the Pseudomonas fluorescens CLW17 strain. [Methods] Bioinformatics analyses have been done for two gene encoding proteins using bioinformatics software. We obtain two deletion mutant strains (CLW17ΔpqqE and CLW17ΔGDH) of pqqE and GDH gene by homologous recombination technology, and the corresponding supplementary strains (ΔpqqE/pqqE and ΔGDH/GDH) were obtained using the combined transfer method. The phosphate solubilizing abilities and organic acid production abilities of wild-type, mutant strains, and complementary strains were detected by NBRIP medium, molybdenum anti colorimetry method, and high-pressure liquid chromatography (HPLC) method, respectively. [Results] The numbers of amino acids encoded by pqqE and GDH genes were 390 and 803, respectively. There is no signal peptide for both genes. pqqE had no transmembrane domain, while GDH predicted five transmembrane domains. The pqqE and GDH genes are the phosphate-solubilizing genes of the CLW17 strain, and the deletion of the two genes greatly reduces the phosphate-solubilizing ability of this strain, moreover, the complementary strains of these two genes can restore the phosphate-solubilizing ability. Wild strain CLW17 can secrete a variety of organic acids, among which gluconic acid content is the highest, followed by acetic acid. The ability to produce organic acids in the knocked-out strain was greatly reduced, especially the ability to produce gluconic acid and acetic acid. [Conclusion] The pqqE and GDH genes are the key genes for the phosphate solubilization of the CLW17 strain, the phosphate solubilization ability of the strain is closely related to the production of organic acids, especially gluconic acid and acetic acid. This study laid the foundation for further study on the interaction and phosphorus solubilization mechanism between the strain and Taxus chinensis var. mairei.

Key words:Pseudomonas fluorescens;phosphate-solubilizing gene;gene knockout;homologous recombination;Taxus chinensis var. mairei