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Evaluation Of Phosphorus Dissolving Effect Of Aspergillus Niger And Expression Of Key Enzyme Genes In Pichia Pastoris

Posted on:2019-07-10Degree:MasterType:Thesis
Country:ChinaCandidate:B B WuFull Text:PDF
GTID:2370330542994615Subject:Biochemistry and Molecular Biology
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Phosphorus?P?is a major and most important nutrient in plants.whereas organophosphorus in soil solutions is much lower,ranging from 0.001 to 1 mg/L.Even though organophosphorus is phosphorus?Up to 50%of the soil?but it cannot be used directly for nutrients unless it is degraded by soil enzymes.Given the high cost of chemical phosphate and the ability of phosphorus to form complexes with soil components,finding an inexpensive and viable alternative to chemical phosphate has become a top priority.This study mainly includes the growth promoting effect of Aspergillus niger on Maize and the expression of the key enzyme gene in Pichia pastoris,which provides the theoretical basis and technical support for the preparation of microbial phosphate fertilizer.Five endophytic fungi of Kosteletzkya pentacarpos were assayed for phosphate-solubilizingactivity,includingAspergillusniger,Penicillium aurantiogriseum,Penicillium funiculosum,Trichoderma asperellum and Trichoderma viride.Among them,Aspergillus niger,Penicillium citricum and Penicillium funiculosum dissolved phosphorus circle obviously.At the same time,1ml spore suspension?1×106cfu/ml?was added to PVK liquid medium containing Ca3?PO4?2.The pH of A.Niger solution was the fastest and stable.The pH value of A.Niger solution was 2.68 at the 7th day.Soluble phosphorus in the solution reached a maximum value of 5.264 mg/L on the 7th day.Aspergillus Niger had the best phosphorus solubilization effect.In the laboratory,corn was planted and the A.niger spore suspension?1×106 cfu/ml?was infested into the roots of maize seedlings when it grew into two leaves.After 21 days of treatment,the results showed that Aspergillus Niger significantly increased the dry weight and chlorophyll content of maize,and the effect of increasing available phosphorus content in soil was very obvious.The key enzyme gene of organic acid metabolism pathway of A.Niger,malate dehydrogenase?MDH?gene,was cloned and its length was 1303bp.The recombinant plasmid pPIC9K-MDH was constructed and electrotransformed into Pichia pastoris GS115.2.0 mg/ml G418 was used to screen the transformant B22.The transformants were centrifuged in methanol-induced BMMY medium to remove the supernatant.After 1×SDS-PAGE Protein Loading Buffer was added,the cells were ruptured by repeated freeze-thaw cycles.A 37 KDa band was obtained,corresponding to the size of its monomer protein.The phosphorus solubilization of transformant B22 under Ca3?PO4?2 stress was studied.A preliminary study on phosphorus solubilization of transformant B22 under Ca3?PO4?2 stress was carried out.Yeast transformant B22 was incubated in 250ml capacity flask at 28?and inoculated with pH of YPD medium pH.The maximum available phosphorus content in the solution was 0.449 mg/L at48h.The key enzyme gene of organic acid metabolism pathway of A.niger,citrate synthase?CS?gene,was cloned and its length was 1812bp.The recombinant plasmid pPIC9K-CS was constructed and electrotransformed into Pichia pastoris GS115.2.0mg/ml G418 was used to screen the transformant B4.After being induced by methanol in BMMY medium and condensed with TCA in the supernatant,a 134KDa band was obtained,which was in line with the size of its dimer.The phosphorus solubilization of transformant B4 under Ca3?PO4?2 stress was studied.Yeast transformant B4 was incubated in 250ml capacity flask at 28?and inoculated with pH of YPD medium pH.The maximum available phosphorus content in the solution is 0.462 mg/L at 60h.
Keywords/Search Tags:Aspergillus niger, Malate dehydrogenase gene, Citrate synthase gene, Phosphate-solubilizing
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