不同条件下两株溶磷菌溶磷量及葡萄糖脱氢酶基因表达与酶活分析

Phosphorus dissolving capability,glucose dehydrogenase gene expression and activity of two phosphate solubilizing bacteria

【目的】获得大豆根际土壤中溶磷能力较强的菌株,明确在菌株溶磷过程中葡萄糖脱氢酶(GDH)的作用特点及其基因的表达水平。【方法】利用溶磷圈方法分离与纯化溶磷菌株,采用Vitek 2系统和16S r RNA序列分析菌株的分类地位;测定2菌株的溶磷量、GDH活性,并根据GDH基因的保守区序列设计引物,克隆GDH基因,利用实时荧光定量PCR测定不同条件下基因的相对表达量。【结果】筛选出2株具有较强溶磷能力的溶磷菌,分别鉴定为Pseudomonas sp.和Enterobacter sp.,2菌株最高溶磷量分别为558μg/m L和478μg/m L;成功地克隆了2株溶磷菌的GDH基因,片段大小分别为2007 bp和2066 bp;2菌株在不同磷源、不同p H值培养基中GDH活性及基因表达量不同,菌株wj1在高磷条件下基因表达量最高,磷胁迫条件下基因表达量较低,而wj3在不同磷源条件下GDH基因表达量都较低。且GDH基因表达量及酶活的变化与wj3菌株溶磷量没有直接的关系。【结论】从大豆根际土壤中分离获得溶磷能力较强的菌株Pseudomonas sp.wj1和Enterobacter sp.Wj3,GDH活性及基因表达在2株菌溶磷过程中具有不同的作用特点,2菌株溶磷机制不完全相同。

[Objective] To identify the function of glucose dehydrogenase（GDH） and gene expression level in the process of solubilizing phosphorus.[Methods] Phosphate solubilizing bacteria（PSB） were isolated and purified by soluble phosphorus circle method,and identified by Vitek 2 system and 16 S r RNA sequence.The phosphate solubilization capacity and GDH activity of PSB were determined.GDH genes were cloned by PCR and the relative expression level of both genes under different conditions were determined by real-time quantitative PCR.[Results]Two PSB were identified as Pseudomonas sp.and Enterobacter sp.and the highest phosphorus solubilizing capability was 558 μg/m L for the former and 478 μg/m L for the latter.GDH genes of the two bacteria were cloned and the fragments were 2007 bp and 2066 bp.Different GDH activity and GDH gene expression were cultivated under the condition of different phosphorus sources and p H value.GDH gene expression of strain wj1 was higher than the other under high phosphorus,and the result was opposite under phosphorus stress.However,GDH gene expression of strain wj3 was lower in all phosphorus levels.The expression of GDH gene and the change of the enzyme activity were not obviously related with phosphorus solubilizing capability for strain wj3.[Conclusion] There were different characteristics of GDH activity and GDH gene expression in two isolated strains that have different phosphate solubilizing mechanisms.