| Iron is an important cofactor for organisms to carry out various metabolic activities.The resistance of fungi to oxidative stress and the improvement of the tolerance of fungi themselves need iron metabolism related proteins.In this study,the major iron metabolism related genes of G.pseudoferreum were cloned by homologous cloning technology..By using bioinformatics technology to predict and analyze the function of iron metabolism related proteins.The toxicity of hexaconidol and artesunate on G.pseudoferreum was measured,and the inhibitory rate and the expression of iron metabolism-related proteins were measured to provide a theoretical basis for elucidating the molecular mechanism of drug resistance of G.pseudoferreum.In order to explore the effects of pesticide mixtures the rapamycin,sodium foscamet,hexaconazole,and cinemycin and vemurafenib targeted inhibition on FECH pesticides were used to co-treat G.pseudoferreum to determine the co-toxicity coefficient.The experimental toxicity of artesunate on zebrafish embryo development will help us understand its dose effect and environmental compatibility,and lay the foundation for field trials.The main results as follows:1.Three iron metabolism related genes have been cloned and the physicochemical properties of three iron metabolism related proteins have been predicted and analyzed.Based on the GP020 transcriptome sequencing of G.pseudoferreum of rubber tree,through NCBI comparison and the method of homologous cloning,and with the ues of Primer 5.0,three iron metabolism related genes have been cloned and named--iron permease encoding gene Gpftrl,multiple copper oxidases encoding gene Gpfet3 and ferrochelatase encoding gene Gpfech.The length of three coding gene region was 1191 bp,1887 bp,and 1071 bp,respectively.Bioinformatics analysis technology was used to analyze the physicochemical properties of three iron metabolism related proteins.The results showed that,The Gpftrl gene encoded 396 amino acids.its molecular weight was 43.23 kDa,and its isoelectric point was 6.92.The GpFTR1 was a hydrophobic protein.It had a conserved group REXLE,seven transmembrane domains,and no signal peptide.The genetic relationship between Gpftrl and Ganoderma sinense iron permease gene(accession number:PIL27756.1)was the closest,and the amino acid homology was 89%;The Gpfet3 gene encodes 628 amino acids,its molecular was 39.21 KDa,and its isoelectric point was 7.73.It had a hydrophilic protein.It had a transmembrane domain,and a signal peptide.The genetic relationship between Gpfet3 and Ganoderma lucidum polyoxon copper oxidase gene(Accession number:AHA83598.1)was the closest,and the amino acid homology was 96%;Gpfech gene encodes 356 amino acids.Its molecular weight was 68.38 KDa and its isoelectric point is 5.02.It was a hydrophilic protein with no transmembrane domain and no signal peptide.Gpfech was closely related to the Ganoderma sinense ferrochelatase gene(accession number:PIL34031.1)and the amino acid homology was 96%.2.Subcellular localization detection of iron transporter GpFTR1 and GpFET3Through the instant expression of Agrobacterium tumefaciens,using the plant expression vector pBin-GFP,the target gene FTR1 and FET3 were connected to the plasmid pBin-GFP respectively.The plant expression vector pBin-GpFTR1-GFP and pBin-GpFET3-GFP were both constructed.By the way of the electric shock of Agrobacterium tumefaciens GV3101,G V 3101-pB in-GpFTRI and GV3010-pBin-GpFET3 bacteria solution were obtained.The location of green fluorescence signal could be observed by laser confocal microscope.The green fluorescence signal of GpFTR1 protein showed that the protein was located on the cell membrane.The green fluorescence signal of GpFET3 protein showed that the protein was also located on the cell membrane.3.Toxicity test of hexaconazole and artesunate against G.pseudoferreum and detection of iron metabolism protein expressionThe growth rate method for the determination of EC50 was 0.192 mg/L and artesunate EC50 was 26.288 mg/L.In this experiment,hexaconazole and artesunate were used to treat G.pseudoferreum at different time and different concentration,and the expression of iron metabolic gene was detected.The results of real-time fluorescence quantitative PCR showed that all three proteins were the highest expression of 6 h when treated with hexaconazole,and all three proteins were the highest expression of 4 h when treated with artesunate.In the treatment of G.pseudoferreum with different concentrations of hexaconazole at 0.1,0.4,1.6,6.4,and 25.6 mg/L,the Gpftrl gene showed a tendency of first up-regulation and then down-regulation.And It was up-regulated by 1.21 times at 0.1 mg/L;Gpfet3 gene showed down-regulation;Gpfech gene showed a trend of first down-regulation and up-regulation.And it was up-regulated by 1.81 times at 25.6 mg/L.Different concentrations of artesunate at 2.8,8.6,26,78,and 234 mg/L treated G.pseudoferreum.The Gpftrl gene showed a tendency of first up-regulation and then down-regulation.At 2.8 mg/L,the expression was significantly up-regulated by 1.21 times.Gpfet3 Gene and Gpfech gene were down-regulated at different concentrations.4.Combined effects of several agents used on G.pseudoferreumThe use of Vertical filter strip method for hexaconazole and griseofulvin,hexaconazole and rapamycin,hexaconazole and Phosphachomycotic sodium combined against G.pseudoferreum.The results showed that all had a additive effect.Hexaconazole,the single pharmacy was still the best effective.The bacteriostasis zone was 3.5 mm.5.Acute toxicity test of artesunate on zebrafish embryosAccording to the OECD criteria,the acute toxicity of artesunate to zebrafish embryos was detected.Five concentrations were set at 31 mg/L,42 mg/L,56 mg/L,75 mg/L and 100 mg/L.With the increase of concentration,the toxicity is increasing.At 24 h,artesunate had no effect on zebrafish embryos.48 h zebrafish began to appear yolk cysts and pericardial cysts.The acute lethal concentration of 96 h was 9.435 mg/L,which had an influence on the heart toxicity of zebrafish,which belonged to poisoning. |
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