Cloning And Functional Verification Of Phosphate-solubilizing Related Genes From Fungi

Phosphorus is an essential macronutrient for plant growth and development,the use of phosphate?P?fertilizer is a usful method to increase crop yields.However,the mass use of P fertilizer and the accummulation of P in soil has caused serious environmental pollution.Phosphate-dissolving strains play an important role in phosphorous cycling in soils,convert insoluble phosphate into soluble phosphate.The aims were to screen efficient and stable P-solubilizing strains,to study the mechanism of the P-solubilization,to clone related P-solubable gene has important value and significance in agricultural production.In this stud y,5 fungi with high efficient P-solubilization were isolated from saline-alkali soils in Inner Mongolia province.2 of the 5 strains,M1?Aspergillus japonicus?and M2?Penicillium oxalicum?,were further studied on their P-solubilizing ability,the secretion of organic acids and phytohormones,the plant growth-promoting ability.The results showed that the concentration of available phosphorus in Ca₃?PO₄?₂ was 1020.89 mg/L at 6 d shaking incubation with isolate M1,isolate M2 was 971.87 mg/L.Soluble phosphorus in AlPO₄ liquid culture by M1was lower than in Ca₃?PO₄?₂ added medium,the content concentration of available phosphorus was 995.69 mg/L,isolate M2 was 987.74 mg/L mg/L.The greatest concentration of available phosphorus dissolved by isolate M1 from Jinning rock phosphate was 363.64 mg/L after 6 d shaking incubation.In greenhouse pot experiment,isolate M1 had a significant growth-promoting effect on corn in four kinds of soils?paddy soil,viscous fluvo-aquic soil,salinized fluvo-aquic soil and calcareous fluvo-aqui soil?under three kinds of insoluble phosphates treatment such as Ca₃?PO₄?₂,AlPO₄ and Kaiyang rock phosphate with 4 replicates per treatment.Compared with control?CK?,inoculation with isolate M1 or isolate M1 increased the fresh weight of corn biomass by 2.14%-90.91%and dry weight of corn biomass by 22.15%-268.28%,and soil available phosphorus content increased 21.81 mg/kg-24.24 mg/kg.The adaptability of isolate M1,M2 with four kinds of soils is greater than strain DSM 821,P83.Out of seven acids,significant increases in the concentration of oxalic acid?616.16 mg/L?and citric acid?413.69 mg/L?were recorded in 3 liquid cultures by isolate M1,respectively.Inoculation with isolate M2 the concentration of oxalic acid?623.34 mg/L?and citric acid?437.21 mg/L?were detected,respectively.The concentration of indole acetic acid?IAA?was 15.45-77.58 mg/L,zeatin was 0.06-0.11 mg/L.The cDNA libraries of isolate M1 and M2 were constructed.The DNA fragments of inserting cDNA library were 1000-1500 bp mostly.The recombination rate of isolate M1 and M2 was 99.91%and 99.93%with blue and white dots screening.The dilution of cDNA libraries from isolate M1 and M2was 6.19×10⁶ cfu/mL and 6.43×10⁶ cfu/mL,respectively.Four P-solubilizing related genes named as psf-p1,psf-p2,psf-p3,psf-p4 were isolated from the cDNA library of isolate M2.The four P-solubilizing related genes were 2427 bp,1846 bp,1161 bp,1035 bp.The four DNA strands translated protein containing 681,483,251,80 amino acid residues,respectively.gene psf-p1 had 99%identity with Penicillium decumbens strain 114-2?KC121314.1?,gene psf-p2 had 99%identity with Penicillium decumbens strain 114-2?KC121314.1?,gene psf-p3 had 78%identity with Penicillium expansum strain HAD Super hydrolytic enzyme family?XM₀16744571.1?,gene psf-p4 had 74%identity with Penicillium chrysogenum strain Wisconsin 54-1255?XM₀02559341.1?.PSF-p1 had 99%identity with Penicillium oxalicum hypothesis protein?EPS30995.1?,its function is unknown.PSF-p2 had 99%identity with Penicillium decumbens Intracellular?-glycosidase enzymes?AGC10824?.PSF-p3 had 99%identity with Penicillium oxalicum hypothesis protein PDE₀9609?EPS34645.1?.At present,PSF-p4 was no similar protein at all.The four P-solubilizing related genes were new genes.Plasmid pBluescript SK?+?-Sfi I which inserted into gene psf-p1,psf-p2,psf-p3 and psf-p4respectively was transformed into E.coli HD5a.4 transformants could reduce the pH value of medium significantly,the lowest value was 4.8.Out of seven acids,significant increases in the concentration of oxalic acid,malic acid andsuccinic acid were recorded in 3 liquid cultures by transformant.Phosphate solubilizing gene psf-p1 recombinated into vector pET-32a?+?-Sfi I in strain BI21?DE3?was obtained the function of dissolving insoluble phosphorus.