Screening And Application Of Phosphate-dissolving Strains And Its Gene Cloning And Identification

Phosphate solubilizing microorganisms which can be suitable for colonization were isolated from soils. And One isolated wae screened, which can be suitable for corn production. Its colonization and phosphate-dissolving characteristics in soil were conducted. Cloning and identification of P-dissolving gene were studied in this article. The main conclusions were as follows:Twenty-three bacteria strains with high performance of phosphate dissolving were screened. Sixteen fungi were isolated, including 9 strains of Penicillium oxalicum, 2 strain of Aspergillus niger, 2 strains of Penicillium variabile, 1 strain of Penicillium aculeatum, 1 strain of Aspergillus nidulans ,1 strain of Trichoderma viride.The P-dissolving capbility of strains on different insoluble phosphorus(Ca₃(PO₄)₂, Zn₃(PO₄)₂, FePO₄, AlPO₄) were conducted. The P-dissolving ability of fungi were better than bacteria. 16 strains reduced the pH value from 7.0 to lower 2.0 in 18 h.They could dissolve all phosphate in solid culture.Colonization and P-dissolving characteristics in soil results showed fungi presented high colonization ability in soil. However, colonization ability of fungi in different soils was significant differences. The P-dissolving ability of isolated were significantly correlated with their colonization. 6 strains of Z33,Zh,ZC,Zh3,ZI1 and Z30 presented high colonization ability in soil. The number of 6 strains increased 9.83-48 times than initial number in 28 days and the content of available phosphorous was increased 44%¹60% than initial content.The plate experiment and soil experiment of the matching between strains and crop in laboratory indicated that the 6 strains of Z15+,Zh,ZQ3,ZI1,ZJ1 and Z30 could propagate in plates and soil useing the root secretion of corn as sole carbon source and dissolve insoluble phosphorus. Six strains of Z33,Zh,ZC,Zh3,ZI1 and Z30 presented high colonization ability in soil, the number of 6 strains increased 9.60-48.03 times and the content of available phosphorous was increased 62.08%-147.55% in 21 days than initial content. The pot experiment inoculating with 5 strains was conducted in greenhouse by planting corn. The result demonstrated that the content of available phosphorous was increased remarkably with strains ZI1 and Zh treatment. The dry matter yield of corn significantly increased than CK, Then strains ZI1 and Zh were used in field experiment as microbial inoculums. The yield of corn treated with strain Zh increased remarkably in two years. However, the yield of corn treated with strain ZI1 presented yield increased in one year. The application of P-solubilizing microorganisms had a significant impact on microbial community structure . The numbers of bacteria, fungi, actinomycetes were increased than CK. There were positive correlation between the biodiversity and the number of bacteria.Aspergillus nidulans Zh and Penicillium oxalicum ZI1 secrete lactic acid and acetic acid, the content of acid at 49th day was 372.5μg/mLand 593.9μg/mL , respectively. The strains ZI1 and Zh could colonizate in soil at 15,20,30℃and remarkably decreased the pH of soil. There were negative correlation between pH value decreased of soil and the number of fungi. The strain ZI1 and Zh promoted the transformation of insoluble phosphate which was the same law in different temperature. The content of Ca₂-P,Ca₈-P,Al-P,Fe-P and Ca₁₀-P were changed with ZI1 and Zh treatment. The transformation efficiency of Ca8-P of the strain ZI1was higher, but transformation efficiency of Al-P and Fe-P of the strain Zh was higher in soil. The mechanism of P-dissolving of the strain ZI1 and Zh was that they secrete organic acids in the progress of propagation chelating Ca²⁺, Fe³⁺, Al³⁺ for dissolving phosphate.Sixty clone were screened from cDNA library of Aspergillus niger Zh3 according to the transparent zone of P-dissolving in plate. And 72 clone were screened from cDNA library of Penicillium oxalicum ZI1. The function of these clone were stable at the second generation. The gene from A.niger Zh3 was named pag T, sequence analysis showed that it was 1407bp in length, and shared 98% identity at the nucleotide level to Aspergillus niger contig An07c0220, and shared less than 80% identity to other isolated. The gene from Penicillium oxalicum ZI1 was named ppg T, Sequence analysis showed that it was 1160bp in length, and shared only 68% identity at the nucleotide level to Penicillium chrysogenum Wisconsin 54-1255, it showed ppg T was a novel gene.Sequence analysis showed that the reading box of gene ppg T has 702bp, and its deduced 233 amino acid, this protein shares 59% homology with the Pc21g23580 in Penicillium chrysogenum Wisconsin 54-1255—the highest homology. And there was no report about the P-dissolving function of this protein, so the function of this protein PPG– T is a novel one. Sequence analysis showed that the reading box of gene pag T in Zh3 has 819bp, and its deduced 333 amino acids, this protein shared 100% homology with the bax Inhibitor family protein in Aspergillus niger CBS 513.88, but there is no report about the P-dissolving function of this protein. So it's a novel function of the protein.Connecting ppqT and paqT to expression vectors pET28 and pGEX ,we constructed four engineering strains, which mainly produce acetic acid and formic acid.They could reduce the pH of the inorganic undissolved phosphorus medium to 3.6 or so .The 4 engineering strains was inducted by undissolved phosphorus, because they couldn't reduce the pH value of medium without undissolved phosphorus. The P-dissolving mechanism of ppq T in E. coli. DH5αis the oxidation of glucose into acetic acid to dissolve phosphorus ; The phosphorus-dissolving mechanism of ppq T in E. coli. DH5αis the oxidation of glucose into acetic acid and formic acid to dissolve phosphorus.