| Potassium is one of the three major elements essential to plants grow,and the most part of soil potassium(90%-98%)are maintained in the structure of primary potassium-bearing minerals in soil.Microbial transformation of potassium-bearing silicate minerals can accelerate releasing of the structural potassium ion,which help to alleviate soil soluble potassium lack and also is a trial developping a new type of mineral potassium fertilizer.Aspergillus niger has been used for the weathering of potassium-bearing silicates by our research team,potassium feldspar weathering by the fungus is a integrative effect including elongated hypha biomechanical role,organic acid dissolution,chelation of ligands and the mycelium-mineral aggregates micro-environment formation.However the above conclusions are not involved in regulation of gene expression.How to understand the process and the microscopic mechanism of microbial weathering mineral at the molecular level?For example,what is the molecular mechanism of interaction between microbes and minerals and how genes participate the mineral weathering and so on?There are many unknown secrets about the molecular mechnism of microbial weathering mineral.In soluble potassium lacking of adversity,A.niger needs to regulate gene expression to adapt to the lack of potassium environment,and then attack the mineral by secreted metabolites,which leads to potassium releasing from the potassium-bearing silicates and the fungal growth can be maintained.RNA-seq can quantify gene expression within the scope of the whole genome in a single base resolution.We can analyze comprehensively the differentially expressed genes of the fungus in process of potassium feldspar weathering by RNA-seq and expound the molecular mechanism of fungal weathering potassium feldspar.Differentially expressed genes were be annotated and found that polysaccharide,protein synthesis,organic acid biosynthesis and carbohydrate metabolic pathways were be significantly enrichment.Meanwhile,a series of differentially expressed genes related to transportation of protein(enzyme)and organic acid,oxidation reduction also be found,they are closely related to the fungal weathering of potassium mineral and K+-releasing.Specially,individual gene differentially expressed significantly,such as Na+,K+-ATPase up-regulated 447.60 times which plays an important role of K+transportation in the fungal weathering of mineral.From another angle,understanding differentially expressed genes and metabolic pathways involved in A.niger weathering of potassium feldspar,is equal to obtain the key genes which is a linchpin of mineral weathering,which provides important information for the fungal genetically modification and superbug construction.Intersecting the genes of Aspergillus(A.niger and A.nidulans)related to the potassium-bearing silicates weathering,laccase(mcoA)was acted as an object of genetically modification.The expression vector commonly used in filamentous fungi pAN7-1 and A.niger glucose amylase gene promoter(pglaA)were used for constructing a mcoA over-expression vector,and then the vector was introducted into protoplast to obtain a strain of mcoA over-expressed A.niger.Phenotype,the growth and acid production of the genetically modification A.niger strain were no difference compared with the wild type of A.niger.Comparing with wild type of A.niger,the genetically modification fungus has a better effect of promoting potassium silicate mineral weathering and the release of potassium ions in shake flask culture conditions.In order to further study the direct interaction between laccase and potassium-bearing silicates,we performed the heterologous expression of laccase gene,and then obtained a single protein size of 66 KDa by using DEAE-Sephedra anion column purification,its oxidation substrate N,N-dimethyl-p-phenylenediamine(DMPPDA)of Km 0.27 mM,Vm is 0.45 mM/min.The recombinated MCOA was used to the weathering of potassium feldspar for 30 min and potassium ion dissolution has more 2.55?g than that of heat inactive laccase.The direct electrochemistry of recombinated MCOA showed that the reduction potential is 302mV,and there is electron transfer between the enzyme and potassium-bearing silicates through taking off the electron of-OH on the surface of the mineral,which disbalance the charge of mineral and offered help to other mineral weathering factor to attack the mineral.The weathering products of potassium feldspar are usually clay mineral including illite,kaolinite etc.,which have certain adsorption of heavy metal ions due to their larger specific surface area and ion exchange capacity.The genetically modification fungal weathering of potassium was conducted for 5d and then the secondary mineral formation was deteced.The results show that there may be a small amount of natrolite and illite.The weathering products of potassium feldspar can absorb common metal ions pollutions(Cu2+,Zn2+,Pb2+,Cd2+).To sum up,in this paper,the following conclusions are drawed:1.In the process of weathering potassium mineral,A.niger is able to actively overcome the difficulties of soluble potassium ions lacking,regulate the gene expression to maintain homeostasis to adapt to the environment lacking of potassium and K+-releasing from the mineral.The fungus improve cellular energy metabolism and material synthesis,and produce more mineral weathering agents such as protein(enzyme),organic acids,which promotes potassium-bearing silicates weathering and K+-releasing.2.PglaA can improve the downstream gene expression,it make expression of laccase in the superbug is 1439.55 times higher than that in wild type A.niger.Moreover,the superbug can improve the potassium feldspar weathering and K+-releasing.3.We obtained a recombinated laccase through eukaryotic expression and purification,the molecular weight is 66 KDa and it can promote the potassium feldspar weathering.4.There is electron transfer between laccase and potassium feldspar.5.The mineral residues have a good adsorption of Cu2+,Zn2+,Pb2+,Cd2+,especially the maxium adsorption capacity of Pb2+is 5.68mg/g. |
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