| Myceliophthora thermophila is a thermophilic filamentous fungus with a strong ability to produce a large number of thermostable enzymes to degrade lignocellulose during optimal growth at 45?.The genome of M.thermophila has been sequenced and annotated in 2011.Some hydrolytic enzymes from M.thermophila can maintain active over a temperature range from 70? to 80?.Therefore,M.hermophila represents a potential reservoir of thermostable enzymes and can be applied in various industrial bioprocessesEpigenetics is the study of heritable phenotypes that do not involve modifications in the DNA sequence.These changes do not alter DNA sequence but affect the expression of target genes,and thus it plays a significant role in organ development and individual growth.Broadly,epigenetic modifications include DNA methylation,histone modification,chromosome remodeling,and regulation of non-coding RNA.DNA methylation,promoted by DNA methyltransferases(DNMTs),is associated with down-regulation of gene expression.As one of the important epigenetic modification methods.In animals,DNA methylation can occur in promoters,transposons,enhancers,silencers,and gene bodies.DNMT1 is the most important methyltransferase in cells and is responsible for maintaining methylation patterns in animals.It was show in some paper that interference of the expression of dnmtl can reduce the methylation status of genes and increase the expression of some genes.In plants and filamentous fungi,DNA methylation is almost exclusively in association with repeat sequences and transposable elements.In addition,LaeA,a protein methyltransferase found in Aspergillus nidulans,is a wide-ranging regulator that modifies the structure of heterochromatin and regulates the expression of various genes.In order to investigate the effect of epigenetics on the cellulase activity of fungus and the regulation of corresponding cellulase genes of the Myceliophthora thermophila,we uses RNAi(RNA interference)technology to study the function of Mtdnmtl and Mtlae gene of Myceliophthora thermophilea respectivelyThe main research contents and results of this thesis are as follows(1)RNA interfering with the Mtdnmt1 gene:Mtdnmtl in Myceliophthora thermophila is an ortholog of DNA methyltransferase 1 of Neurospora crassa.In this study,the Mtdnmt1 gene interference expression vector pUC19-siRNA-Mtdnmt1 was constructed.pUC19-siRNA-Mtdnmtl and pAN7-1 vectors were co-introduced into Myceliophthora thermophila through protoplasts transformation.After screening with hygromycin and genomic PCR vertification,a positive transformant D1 with 75%interference efficiency was obtained successfully by real-time quantitative PCR and used for subsequent experimentsThe activities of paper(FPase),endo-1,4-?-D-glucannase(CMC),cellobiohydrolase(CBH)and ?-glucosidase(BG)in transformant D1 are determined under two different inducing cultures.When M.thermophila strains were incubated in medium containing 2%Avicel,FPA and CMC activities reached the highest on the fifth day,which were 1.17 and 1.04 fold than that of wild-type(WT),respectively,BG and CBH activities peaked on the sixth day,which were 1.13 and 1.16-fold higher than that of the WT respectively.When grown on 5%?-lactose,the activity of CMC showed relatively higher compared with the induction of 2%Avicel,and achieved the highest on the third day.The CMC activity of D1 was 1.18 fold compared of that of WT,while the CBH activity was extremely lowThe transcription levels of the major cellulase genes cbh1,cbh2,egl1,egl3,bgl1,and associated regulatory genes xyr1 and cre1 in WT and D1 strains cultured with 2%Avicel were performed by RT-qPCR analysis.The transcription levels of xyrland cre1 in D1 were lower than those of the wild type.In contrast,transcription quantities of the major cellulase genes was higher compared those of WT.However,the transcription levels of the main cellulase gene and related regulatory factors Dlwere were higher than that of WT when grown on medium including 5%?-lactose(2)RNA interfering with the Mtlae gene:The Mtlae gene in Myceliophthora thermophila share the highest homology with the protein methyltransferase LAE1 in Trichoderma reesei(Trire 2:41617)and the protein methyltransferase LAEA in Aspergillus nidulans respectively.Similarly,the Mtlae gene interference expression vector pUC19-siRNA-Mtlae was constructed and co-introduced into Myceliophthora thermophila with pAN7-1 by protoplasts transformation.S33,a positive transformant with 73%interference efficiency was obtained for subsequent observations selected with hygromycin B resistance and sequential identifcation via PCRThe activities of various enzymes in the supernatant of WT and S33 strains cultured with 2%Avicel or 5%?-lactose as the sole carbon source were determined,respectively,when incubated in medium containing 2%Avicel,the activities of FPA and CMC of transformant S33 reached the highest on the fifth day,which were 1.33 and 1.15 fold higher than that of WT.And the activities of BG and CBH peaked on the sixth day,which were 1.17 and 1.20 fold higher than those of WTIn contrast,almost all the enzyme activities of S33 were decreased as compared with those from WT when 5%?-lactose was used as carbon source.The activity of FPase of S33 declined from day 2,which was reduced by 20%compared with WT,and almost no enzyme activity can be detected on day 5.Similarly,the CMC activity of S33 showed a downward trend from the day 2 and was decreased by 36%compared to that of WT,while the WT strain reached the highest on the day 3Analysis of transcriptional levels of the main cellulase genes cbh1,cbh2,egl1,egl3,bgl1 and related regulatory genes xyr1 and cre1 of WT and S33 strains by RT-qPCR revealed that under the induction of 2%Avicel,the expression levels of cbh1,cbh2,egll,egl3,bgl1 and cre1 were improved in S33 compared with WT except xyr1 which was slightly down regulated.By contrast,the expression levels of the main cellulase gene cbh2,egl1,egl3 and bgl1 of S33 were all down regulated compared with WT on day 2 when grown on 5%?-lactose.However,the expression levels of xyrl and cre1 were slightly elevated in S33.These results was basically consistent with the enzyme activit ies that we observedIn summary,different carbon sources have distinct effects on the cellulase activities of M.thermophila.Although xyr1 has been regarded as a cellulase genes transcription activator in a variety of filamentous fungi,it barely get involved in regulation of cellulase gene expression of M.thermophila.After the Mtdnmt1 gene of M.thermophila was interfered,the cellulase activity of transformant D1 was significantly higher than that of wild-type,when both cultured in medium containing 2%Avicel or 5%?-lactose.The transcription levels of corresponding cellulases showed the same trend.This indicates that DNA methylation is an important mechanism that affects the expression of cellulase gene.And cellulase activities can be enhanced by interfering with DNA methylation to some extentInterfering with the Mtlae gene of M.thermophila,exhibited that when cultured in fermentation medium with 2%Avicel and 5%?-lactose,there were significant difference for four cellulase activities between WT strains and S33 strains.The four cellulase activities of S3 3 were higher than those of wild type on medium containing 2%Avicel.However showed opposite performance when 5%?-lactose was used as carbon source.This illustrates that the regulation of cellulase activities by protein methyl transferase is affected by different inducers. |
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