内生细菌GZJR-8溶磷相关基因鉴定及LTYR-11和LTJR-57的生理生化特性研究

发布时间：2018-03-08 16:05

  本文选题：溶磷能力　切入点：pqqE　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：在农业生产中磷的需求量只低于氮,在自然条件下,可被吸收利用的可溶性磷经常是限制农业生产的一类矿质营养。内生细菌Pseudomonas sp.GZJR-8分离于拐枣七的茎部,初步鉴定其具有显著溶磷作用。本研究鉴定了GZJR-8的pqqE和GDH基因在溶磷方面的功能;利用同源重组技术分别获得pqqE和GDH基因的缺失突变体,采用电转法获得它们的互补菌株;采用难溶性无机磷培养基(PKO)和抗坏血酸-钼蓝显色定磷法分别定性和定量检测了野生型、突变体、互补菌株是否产生溶磷圈以及产生有效磷的总量和培养液的pH;结果表明:pqqE和GDH基因是GZJR-8的溶磷相关基因,其中突变体的溶磷能力下降,但并未完全丧失溶磷能力,而互补菌株可以恢复溶磷能力;该菌株通过产生酸性物质来溶磷;能够促进植物生长;该菌株在农业生产方面具有潜在应用价值。本研究从骆驼刺的叶和茎部分离得到两个菌株LTYR-11和LTJR-57,16S rRNA基因序列测定与系统发育分析,发现这两株菌分别隶属Bosea属和Blastococcus属,亲缘关系最近的模式菌株分别为Bosea vaviloviae Vaf-18T(95%)和Blastococcus saxobsidens BC448T(98.8%)。与已知模式菌株较低的序列相似性暗示这两株菌可能是潜在新分类单元。随后,本研究检测了这2株内生菌的培养条件、生长温度、生长pH、耐盐性、抑菌活性、多种酶活、产铁载体等生理生化指标;结果表明:Bosea sp.LTYR-11的最适生长条件是0.1TSA和NA培养基;30-37℃生长良好,44℃不能生长;最适pH为7.5-8;在NaCl浓度0-1%时生长良好、在1.5%NaCl不生长;不能在厌氧环境下生长;可以泳动;具有产铁载体能力;具有过氧化氢酶、脲酶活性。Blastococcus sp.LTJR-57的最适生长条件是MA培养基;37-44℃生长良好,25℃以下和50℃以上停止生长;能在pH9的碱性条件下生长;在NaCl含量为0%时生长良好、当NaCl浓度大于4%时不能生长;有产铁载体能力;能降解脱脂奶(酪蛋白)和酪氨酸;具有过氧化氢酶和脲酶活性。
[Abstract]:The demand for phosphorus in agricultural production is only lower than that of nitrogen. Under natural conditions, soluble phosphorus that can be absorbed and utilized is often a mineral nutrient that restricts agricultural production. Pseudomonas sp.GZJR-8, an endophytic bacterium, is isolated from the stem of Jujube. In this study, the functions of pqqE and GDH genes of GZJR-8 in phosphorus solubilization were identified, the deletion mutants of pqqE and GDH genes were obtained by homologous recombination technique, and their complementary strains were obtained by electroporation. Wild type and mutants were detected qualitatively and quantitatively by insoluble inorganic phosphorus medium (PKO) and ascorbic acid-molybdenum blue colorimetric method. The results showed that the pqqE and GDH genes were the phosphorus-soluble related genes of GZJR-8, and the phosphorus-soluble ability of the mutants was decreased, but the phosphorus-soluble ability of the mutants was not completely lost. The complementary strain can restore phosphorus solubilizing ability, it can dissolve phosphorus by producing acid substance, it can promote plant growth. In this study, two strains of LTYR-11 and LTJR-57716S rRNA gene were isolated from the leaves and stems of Camellia spp. The results showed that the two strains belonged to Bosea genus and Blastococcus genus, respectively. The most closely related model strains were Bosea vaviloviae Vaf-18Tf95) and Blastococcus saxobsidens BC448T988, respectively. The low sequence similarity with known model strains suggested that the two strains might be potential new taxa. Subsequently, the culture conditions of the two endophytic bacteria were examined. Growth temperature, pH, salt tolerance, bacteriostatic activity, enzyme activity, iron production carrier and other physiological and biochemical indexes, the results showed that the optimum growth conditions of sp.LTYR-11 were 0.1TSA and Na medium 30-37 鈩，

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