| The growth cycle of Coprinus cinerea is short,and the life history can be completed in two weeks under laboratory conditions.It is an ideal material for studying the growth and development process of Basidiomycetes.In the early stage of our laboratory,the transcription factors Nsd D1 and Nsd D2 were co-silenced by RNA interference technology.The resulting double-interfering strains grew slowly,and did not form the precursor of the fruit body after light induction,and there was no formation of fruit body.Transcriptome analysis and CHIP-seq analysis showed that CNR1 gene(a gene related to cell cycle control,a protein belonging to the Plac8 family)was down-regulated in the dual-interfering strain;SKN1 gene(a protein related to?-glucan synthesis,An important protein involved in the synthesis of fungal cell walls)was up-regulated in dual-interfering strains.The down-regulation and up-regulation of these two genes are caused by double interference,and CHIP-seq analysis shows that these two genes are downstream target genes of Nsd D2.Therefore,in this experiment,by silencing the CNR1 gene and overexpressing the SKN1 gene,observing the mycelial growth of Coprinus cinerea,the formation of the fruiting-body initials,the number of fruiting bodies and the elongation of the stem,etc.,to verify whether there will be a double The same phenotype of the interfering strain.We successfully constructed the CNR1-RNAi vector,and then transformed the interference vector into Amut Bmut using a PEG-Ca Cl2 mediated co-transformation system.Through colony PCR verification,we selected and saved 15 verified interference strains and 15 control strains.The CNR1 gene expression in the interfering strains was detected by real-time fluorescent quantitative PCR,and it was found that the CNR1 gene was silenced to varying degrees.It may be that different transformants have different silencing efficiencies due to the different copy numbers of the gene inserted.By culturing the interference strain and the control strain,it was observed that the growth rate of the hyphae of the interference strain was significantly slower than that of the control strain:the average growth rate of the hyphae in the control group was1.46 cm/d,while the average growth rate of the hyphae of the interference strain was1.26 cm/d;Observing the fruiting-body initials under a stereoscope,it was found that the number of the fruiting-body initials of the interfering strain was significantly less than that of the control group:the average number of the fruiting-body initials of the control group was 248.78/plate,and The average number of the fruiting-body initials of the interfering strains was 78.36 per plate;the growth of the fruiting bodies found that the number of fruiting bodies in the control group was significantly more than that of the interfering strains,and the elongation rate of the stalk was measured and the elongation rate of the interfering strains was lower Control strain:The average elongation rate of the control strain's stalk was 0.63 cm/h,while the average elongation rate of the interfering strain's stalk was 0.56 cm/h.In this experiment,the SKN1 overexpression vector was constructed.Similarly,the overexpression vector was transformed into Coprinus cinerea Amut Bmut using a PEG-Ca Cl2 mediated co-transformation system,and 15 SKN1 overexpression strains were verified.The expression of SKN1 gene in overexpression strains was detected by fluorescence quantitative analysis,and the results showed that they were all up-regulated.By culturing the overexpression strain and the control strain,it was found that the growth rate of the hyphae of the overexpression strain was significantly lower than that of the control strain:the average growth rate of the hyphae in the control group was 1.48 cm/d,while the average growth rate of the hyphae of the overexpression strain was 1.37 cm/d d;Observing the number of the fruiting-body initials under a stereoscope,it was found that the number of progenitors of the fruiting bodies of the overexpression strain was significantly less than that of the control group:the average number of the fruiting-body initials formed by the light-induced of the control group was 267.68Pcs/plate,and the average number of the fruiting-body initials of SKN1 overexpression was 76.36 pcs/plate;observing the number of fruiting bodies and measuring the stalk elongation,it was found that the number of fruiting bodies of the overexpression strain was significantly less than that of the control strain,and The stalk elongation rate of the overexpression strain was lower than that of the control strain:the average stalk elongation rate of the control strain was 0.64 cm/h,while the average elongation rate of the overexpression stalk was 0.59 cm/h.According to the experimental results,silencing the CNR1 gene and overexpression of the SKN1 gene both slowed down the growth rate of Coprinus crassipes hyphae(this is consistent with the phenotype after double interference),and both affected the fruit body of Coprinus coprinus.The formation of progenitors reduces the number of progenitors of fruiting bodies induced by light(this is similar to the phenotype after double interference),which in turn affects the growth of fruiting bodies and reduces the number of fruiting bodies and the rate of stipe elongation.CNR1 gene is involved in cell division and differentiation,is related to cell cycle regulation,and plays a positive regulatory role in the growth and development of Coprinus cinerea;SKN1 gene is a?-glucan synthesis-related protein,and?-glucan in turn It is an important component of the fungal cell wall,and it plays a negative regulatory role in the growth and development of Coprinus cinerea.In addition,studies have shown that?-glucosidase also plays an important role in the growth and development of Coprinus cinerea.We compared the amino acid sequence of the exo?-1,6-glucosidase of fission yeast with the gene of Coprinus cinerea and found that there is a gene that is 39%identical(XP?001829226).According to NCBI's conservative region analysis,we found that the protein belongs to the glycoside hydrolase GH1 family member in the Carbohydrate Active Enzyme(CAZy)database,which belongs to exo?-glucosidase.Using the Pichia pastoris eukaryotic expression system,heterologous recombinant expression of exo?-glucosidase 226,and the protein was purified by nickel column affinity chromatography.After identification by mass spectrometry,the purified protein is?-glucosidase 226 protein.We explored the enzymatic properties of?-glucosidase 226,including its optimum p H,optimum temperature,temperature stability,and p H stability.We found that the optimum temperature of Coprinus cinerea?-glucosidase 226 is 50°C,the enzyme activity is the highest under p H 6.0,and it has good thermal stability.We also explored the kinetic analysis of 226 pairs of p NPG,whose Km and Vmax were 2.18 m M and 254.98?mol mg-1 min-1,respectively;and the kinetic analysis of 226 pairs of laminarin showed Km and Vmax of 27.49m M and Vmax,respectively.501.82?mol mg-1 min-1.Therefore,the affinity substrate of 226 is p NPG,and 226 has a higher specific activity of 159.73U/mg to p NPG.226 has a wide range of substrate specificity,it can hydrolyze laminarin,p NPG,cellobiose,gentiobiose and lamina oligosaccharides.This study also found that?-glucosidase 226 cannot reverse synthesis of any disaccharides using glucose as a substrate,nor can it utilize cellobiose and gentiobiose for glycosylation,but it can use laminarin and laminarin Sugar is a substrate that undergoes glycosylation to form higher oligosaccharides. |
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