| Coprinopsis cinerea is a model basidiomycete.Its growth and development process mainly includes the formation of initial mycelium and fruiting body,the elongation of stipe,the maturation of basidiospore,the expansion of canopy and the autolysis of canopy.It is estimated that 17%of genes are differentially expressed in the process of fruiting body formation,and the role of these genes is still unclear.In our previous study,we found that after double interference of Nsd D1 and Nsd D2 genes,the two interfering strains nsd D1/nsd D2 could not produce the primordial and fruiting bodies.Through transcriptome analysis,it was found that the relative expression of sty8was significantly up-regulated in the double interference strain,while the relative expression of sty17 was also significantly down regulated.Subsequently,CHIP-sec experiment showed that two genes,sty8 and sty17,were the target genes of transcription factor Nsd D2.It is inferred that sty8 plays a negative role in the growth and development of fruit bodies,while sty17 plays a positive role.Both STY8 and STY17are tyrosine like kinases.Some studies have shown that the growth of Arabidopsis plants is retarded after the deletion of sty8 and sty17 genes.However,the specific functions,related signaling pathways and molecular regulatory mechanisms of STY8and STY17 have not been studied in fungi.The purpose of this experiment is to explore the function of sty8 and sty17 in the development of fruit body.Because of the existence of genes that prevent foreign genes from being inserted into the genome by homologous recombination,the efficiency of gene knockout strains obtained by homologous recombination method is very low,we decided to overexpress sty8 and silence sty17 by RNA interference,and observed the phenotype of the corresponding strains to speculate their functions.In this study,we constructed a double stranded RNA interference vector of tyrosine like kinase STY17.The interference vector was transferred into Pholiota cinerea by PEG/Ca Cl2 mediated transformation system.After the interference strain(sty17i strain)of sty17 was obtained,the expression level of sty17 gene in sty17i strain was detected by fluorescence quantitative PCR,and the silencing efficiency was found to be between20%and 90%.Then,the phenotypes of 15 strains of sty17i were observed and compared with the control strain(CK strain).It was found that the mycelial growth rate of 6 strains of sty17i was 51.3%slower than that of CK strain,and the mycelia could not grow on the plate after dark culture for 7 days in the incubator.The mycelial growth rate of the other 9 strains of sty17i was 15%faster than that of CK strain,and the number of fruiting-body initial was 43.3%less than that of CK strain.The results showed that the average growth rate of mycelia was 16%slower than that of CK strain,the average number of fruiting-body initial was 59.6%less than that of CK strain,and the average number of fruiting body was 36.8%less than that of CK strain.T-test showed that the above results were significantly different from that of CK strain.The above results showed that,firstly,by comparing 15 strains of sty17i with CK,it was found that STY17played a positive role in the growth and development of fruiting body.Interference with this gene reduced the number of fruiting body initiation and fruiting body of Coprinopsis cinerea,but the related signaling pathways and molecular mechanism of its regulation need to be further studied.At the same time,the experimental results showed a serious heterogeneity,which may be because the interference vector was randomly inserted into the genome of Coprinopsis cinerea,and some genes in Coprinopsis cinerea were destroyed,which led to the uneven effect of interference sty17 on it.Therefore,the mycelia growth rate of 6 strains of sty17i was slow and could not grow fruiting bodies.Secondly,comparing the phenotypes of 9 strains of sty17i with that of CK,it was found that the number of fruiting bodies of CK was more than that of 9 strains of sty17i,but there was no significant difference in the number of fruiting bodies between them.The reason may be that not all of the fruiting bodies could eventually develop into fruiting bodies during the development of fruiting bodies;it is also possible that there is a gene homologous to sty17 in the Coprinopsis cinerea.When sty17 is down-regulated,the expression of the homologous gene changes correspondingly,resulting in the number of fruiting bodies not decreasing,but this does not mean that sty17 does not play a role in the development of fruiting bodies.In addition,because the expression of sty8 was up-regulated in the double interfering strain,it was speculated that sty8 played a negative regulatory role in the growth and development of fruit bodies.In this study,we further constructed the overexpression vector of tyrosine kinase sty8 by using the previously constructed pccproter1 skeleton vector.After the overexpression vector was transferred into the body of fimbria huigai by transformation system,fifteen strains of sty8-OE were obtained.The relative expression of the target gene sty8 at m RNA level was detected by real-time PCR.It was found that the relative expression of sty8-OE was up-regulated in different strains.Then,the mycelial growth rate,the number and morphology of fruiting-body initial and the number of fruiting bodies were observed and compared with CK strain.The results showed that the mycelia growth rate of strain sty8-OE was not significantly different from that of CK strain;the number of fruiting-body initial of sty8-OE was 22%less than that of CK,and the difference was significant by T-test;The number of fruiting bodies of sty8-OE was 40%less than that of CK,and there was significant difference between them by T-test.These results indicate that STY8 plays a negative regulatory role in the growth and development of fruiting body,and the overexpression of sty8 affects the number of fruiting-body initial and fruiting body.However,the related regulatory mechanisms and signaling pathways need to be further explored and clarified.The results showed that STY8 played a negative role and STY17 played a positive role in the growth and development of Coprinopsis cinerea.Both genes could affect the mycelia growth rate,the initiation and formation of the fruiting body of Coprinopsis cinerea,therefore,this study is of great significance for the study of the role of tyrosine-like-kinases in the growth and development of basidiomycetes and the molecular regulation mechanism.In addition,this paper studied bgl1 gene was transformed into Pichia pastoris,and BGL1 protein was successfully induced and expressed by Pichia pastoris.In our follow-up experiments,we found that BGL1 has a unique glycosylation ability at low concentration,which shows that it plays an indispensable role in the process of synergetic recombinant cell wall elongation and growth.The relevant experimental results have been published in the form of papers. |
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