大豆根际土壤溶无机磷细菌的溶磷特性研究

发布时间：2018-06-23 11:31

  本文选题：溶磷菌 + 溶磷特性　； 参考：《吉林农业大学》2015年硕士论文

【摘要】：溶磷微生物能将土壤中难溶性磷酸盐转化为能被植物吸收利用的可溶性磷形态,从而提高土壤磷素利用效率,在农业磷素循环利用方面有着重要作用。本论文针对大豆根际土壤中溶无机磷细菌溶磷特性进行一系列的研究,主要取得以下成果:本试验从东北大豆根际土壤中分离和筛选出4株具有较强溶磷能力的细菌,其最大溶磷量分别为558μg/mL、478μg/mL、596μg/mL和586μg/mL。经过Vitek2生理生化系统分析和16S rDNA序列测定,鉴定4株菌株分别为假单胞菌、肠杆菌、苍白杆菌、克雷伯氏菌,并命名为wj1、wj3、wj5和wj6。本文以NBRIP分离培养基为基础,利用L25(53)正交设计试验,从碳源、氮源及pH值三个方面对菌株溶磷培养基进行优化。结果表明:最佳碳源为葡萄糖,氮源为蛋白胨,初始pH值为8。对wj6溶解Ca3(PO4)2、FePO4和AlPO4特性的研究发现,该菌株能不同程度的降解Ca3(PO4)2、FePO4和AlPO4三种磷酸盐,最高溶磷量分别为409.28μg/mL、60.59μg/mL、32.90μg/mL。但是,在溶解FePO4和AlPO4时受培养环境pH的影响较大,将wj6接种到pH 8的溶磷培养基中,该菌株能溶解FePO4和AlPO4,在培养基原液(pH 3)中不具有溶磷能力。从溶磷菌产酸角度对溶磷机制进行了研究。在以磷酸钙为唯一磷源的条件下,菌株溶磷过程中伴随着pH值的降低。采用GC-MS对菌株在溶磷过程中产生的有机酸分析:4株溶磷菌主要产生的有机酸有:2,2-二甲氧基丙酸、α-酮戊二酸、4-hexadienedioicacid、3-hydroxydecanoic Acid、苹果酸、hepta-2,4-dienoic acid、乙酸、琥珀酸、柠檬酸等。酶活性检测结果表明,在溶磷过程中菌液磷酸酶活性显著增强。从wj1、wj3、wj5和wj6溶磷菌中克隆GDH基因,分别获得大小为2007bp、2066bp、1727bp、2391bp的基因片段。序列分别与GenBank中Pseudomonas brassicacearum subsp.brassicacearum NFM421、Enterobacter asburiae L1、Ochrobactrum anthropi ATCC 49188、Klebsiella pneumoniae subsp.pneumoniae KP5-1中的GDH基因相似性达到92.30%、96.51%、85.12%、99.29%。综合12h和48h时间下wj1和wj3GDH基因在不同磷源以及不同pH值的NBRIP培养基中GDH基因表达量的变化结果表明,菌株在溶磷过程中GDH基因都有表达,同种菌在不同磷源的NBRIP培养基中表达量差异较大,且随着培养时间的延长表达量也在变化。培养相同时间,wj1和wj3两种菌株GDH基因在pH值为5~9的NBRIP培养基中表达量变化趋势一致。12h时,在pH 5的培养基中表达量最大,48h时在pH 9的培养基中表达量最大。综上所述,本研究可以推测4株溶磷菌在溶磷过程中pH值下降以及产酸都是在适宜的环境下诱导出来的。本试验成功克隆了产葡萄糖酸的GDH基因,GDH基因的表达量也受磷源和菌液pH值的影响。
[Abstract]:Phosphorus soluble microorganisms can transform insoluble phosphate in soil into soluble phosphorus forms which can be absorbed and utilized by plants, thus improving the utilization efficiency of phosphorus in soil, which plays an important role in the recycling of phosphorus in agriculture. In this paper, a series of studies were carried out on the phosphorus dissolving characteristics of inorganic phosphorus bacteria in soybean rhizosphere soil. The main results were as follows: in this experiment, four strains of bacteria with strong phosphorus solubilizing ability were isolated and screened from soybean rhizosphere soil in Northeast China. The maximum phosphorus solubilization was 558 渭 g / mL and 586 渭 g / mL, respectively, and 596 渭 g / mL and 586 渭 g / mL, respectively. The four strains were identified as Pseudomonas pseudomonas, Enterobacter pallidus, Klebsiella klebsiella, and named wj1wj3wj5 and wj6 by physiological and biochemical analysis of Vitek2 and 16s rDNA sequencing. On the basis of NBRIP separation medium, using L25 (53) orthogonal design experiment, the phosphorus solubilizing medium of the strain was optimized from three aspects: carbon source, nitrogen source and pH value. The results showed that the optimum carbon source was glucose, the nitrogen source was peptone, and the initial pH was 8. The characteristics of dissolving Ca _ 3 (PO _ 4) _ 2 FePO _ 4 and AlPO _ 4 by wj6 were studied. It was found that the strain could degrade three kinds of phosphate, Ca _ 3 (PO _ 4) _ 2 FePO _ 4 and AlPO _ 4 to varying degrees, and the highest dissolved phosphorus content was 409.28 渭 g 路mL ~ (-1) ~ 60.59 渭 g 路mL ~ (-1) ~ (32. 90) 渭 g 路mL ~ (-1), respectively. However, the pH of the culture environment affected the dissolution of FePO4 and AlPO4. The strain could dissolve FePO4 and AlPO4 by inoculating wj6 into pH 8 medium, but had no ability to dissolve phosphorus in the medium solution (pH 3). The mechanism of phosphorus dissolving was studied from the point of acid production of phosphorus-soluble bacteria. Under the condition of calcium phosphate as the sole phosphorus source, the pH value of the strain decreased in the process of phosphorus dissolution. GC-MS Analysis of Organic acids produced in the process of phosphorus dissolution by GC-MS the main organic acids produced by the four strains were: 1: 2 (2-dimethoxypropionic acid), 4-hexadienedioicacididine (4-hexadienedioic acid) 3-hydroxydecanoic Acid-malic acid hepta-24-dienoic acidic acid, acetic acid, succinic acid, citric acid and so on. The results of enzyme activity test showed that phosphatase activity of bacteria liquid increased significantly in the process of phosphorus dissolving. The GDH gene was cloned from wj1wj3wj5 and wj6, and the gene fragment of 2007bpn2066bp1 1727bpmc2391bp was obtained. The sequence was similar to that of Pseudomonas brassicacearum subsp.brassicacearum NFM421 Enterobacter asburiae L1 Ochrobactrum anthropi ATCC 49188 and Klebsiella pneumoniae subsp.pneumoniae KP5-1, respectively. The similarity of the GDH gene in Pseudomonas brassicacearum subsp.brassicacearum NFM421 and Klebsiella pneumoniae subsp.pneumoniae KP5-1 was 92.3096.51and 85.120.99.29. The expression of wj1 gene and wj3GDH gene in different phosphorous sources and different pH value NBRIP medium under 12h and 48h time were analyzed. The results showed that the GDH gene was expressed in the process of phosphorus solubilization. The expression of the same strain was different in NBRIP medium with different phosphorus sources, and the expression was also changed with the extension of culture time. At the same time, the expression of GDH gene was the same in NBRIP medium with pH 5 ~ 9, and the highest in pH 5 medium for 48 h, and the maximum expression level in pH 5 medium for 48 h. The expression level of GDH gene was the same in NBRIP medium with pH 5 and pH 5 at 48 h. The expression of GDH gene was the highest in pH 9 medium. To sum up, it can be inferred that the pH value decreased and the acid production induced by the four phosphorus soluble bacteria in the suitable environment. The expression of GDH gene was also influenced by phosphorus source and pH value.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S565.1;S154.3

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