Functional Identification Of Phosphate Specific Transport System Related Genes In Phosphate-solubilizing Bacteria Strain Wj1

Inorganic phosphate is an essential nutrient for all organisms,and its utilization begins with transmembrane transport of inorganic phosphate.Inorganic phosphate is taken up in bacteria mainly by two different transport systems,the inorganic phosphate transport?Pit?system and phosphate specific transport system?Pst?.Pit is a low-affinity,high-velocity phosphate-uptake and is dependent on the proton motive force for energy.Pst is an ABC transporter that has an ATP-driven high-affinity inorganic phosphate uptake.Through the Pit and Pst system,the bacteria can transfer the inorganic phosphate under the condition of high phosphorus and low phosphorus,which can meet the demand of the bacteria for phosphorus nutrition.Through the transcriptome sequencing of phosphate-solubilizing bacteria wj1 cultured in the condition of high phosphorus and low phosphorus,we found the genes related to Pst system and carried out a series of basic studies on it.The main results are as follows:By compared the Pst systems of 500 strains of common bacteria in soil,it was found that the genes encoding Pst in different strains were different and could be divided into three classes.The first class is encoded by five genes,including pstSCAB-phoU.The second class is also encoded by five genes,which is different from the first class.It contains two pstB genes,and there is no phoU gene.The third class is only encoded by the pstSCAB gene,the phoU gene was not found in this.The intergenic spacer regions pstCA and pstAB of Pst system were obtained by PCR,and it was found that the genes of each component encoding Pst system were successively arranged in the phosphate-solubilizing bacteria wj1,with a total length of 7120 bp,including pstS2CAB-phoU.Therefore,the Pst system of phosphate-solubilizing bacteria strains wj1 belongs to the first class.The physicochemical properties,secondary structure,signal peptide,transmembrane region,conserved domain and tertiary structure of pstS1,pstS2,pstC,pstA,pstB and phoU genes were analyzed by bioinformatics on-line analysis software.To further study the function of the gene to provide a theoretical basis.The nucleic acid sequences of pstS1 and pstS2 of phosphate binding protein gene were cloned.The length of open reading frame was 1335 bp and 999 bp,respectively.The prokaryotic expression vector pET-28a-pstS1 and pET-28a-pstS2 were successfully constructed,in which the pstS2 gene was induced to express at about 37.0KDa,it indicated that the target protein was expressed successfully.The phosphate binding capacity of recombinant strain was detected,and the phosphate binding ability of recombinant strain was enhanced.When the phosphate concentration was 0.2-0.5 mg·mL-1,the recombinant strain had a high ability to bind phosphate.When the concentration of phosphate was greater than 1 mg·mL-1,the ability to bind phosphate decreased significantly.With the increase of cell density,the ability of recombinant strain to bind phosphate was enhanced,and reached the maximum at cell density of 2.0 OD₆₀₀.When the cell density of OD₆₀₀ was 2.0 and the phosphate concentration was 0.4 mg·mL-1,the phosphate binding capacity was the highest,up to 79.54%.The results showed that the affinity of phosphate binding protein PstS2 to phosphate was high,and the Pst system played a role in the low phosphorus condition.