| Recently, thermostable enzymes have become one of an important directions in the field of enzyme engineering. Extracel ular enzymes produced by thermophilic fungi might be more active and stable under extreme conditions. Enzymes with thermal stability have economic advantages in the production of chemicals and biomass-based fuels. Myceliophthora thermophila is a mild thermophilic fungus which can grow between 25 ?and 55 ?. It belongs to Ascomycetous fungus. The genome of M.thermophila has been analyzed in 2011. Genomic data analysis reveals that this fungus has the potential ability to produce more than 200 kinds of glucoside hydrolases and a large number of accessory proteins related with cel ulase and hemicellulase degradation. We selected two factors, STL1 and AZF1, from transcription profile comparison of Myceliophthora thermophila that cultured in inducing and non-induc ing conditions, respectively. In the present study, methods of homologous expression and RNA interference were used in the researching of stl1 and azf1. The main contents of this study are as follows:In the present study, we focused on the influence of over-expression of sugar transporter protein STL1 in activity of fungus cel ulase. Myceliophthora. thermophila ATCC42464 is a kind of thermophilic fungus. Its genome has been sequenced. In this study, the gene sequence of sugar transporter protein STL1 was cloned. The recombinant plasmid was constructed by using the promoter of Mt Ppdc and terminator of Mt Tpdc. The stl1 gene was over-expressed in M. thermophile successfully. The expression amount of stl1 in transformant S02 was up to 1653-fold higher than that of the wild type. In the transformant, the filter paper hydrolyzing activity and ?-glycosidase activity were 18.6% and 49.9% higher, respectively, than those in the parental strain when the strains were cultured in inducing medium for 5 days. According the reasult of real time fluorescent quantitative Polymerase Chain Reaction, m RN A transcription level of the corresponding cel ulase gene cbh1, cbh2, egl1, egl3 and bgl1 in stain S02 were 3.28-, 2.70-, 1.66-, 1.79- and 1.55-fold higher than those in the parental strain. The expression of stl1 increased the activity of cellulase. The increase of beta-glycosidase activit y played a major role on the increment of total cel ulase activity.AZF1 is a kind of transcription factor with C2H2 doamin. The si RNA sequences were designed. The recombinant plasmid was constructed by using the promoter of Mt Ppdc and terminator of Mt Tpdc. By using specific primer PCR identification and sequencing comparison, three transformants have been obtained successfully. According to the results of real time fluorescent quantitative Polymerase Chain Reaction of azf1, A02 is the best one among three transformants. However, it the gene expression still has 70.8 % remaining. The ideal state should be under 20%. It indicated that these transformants have not achieved effective RNA interference in Myceliophthora thermophila. Cellulase enzyme activity and protein concentration of the transformant A02 were determined. Compare to parental strain, filter paper hydrolyzing activity, ?-1, 4- exoglucanase activity and ?-glycosidase activity of A02 had not stable and obvious difference when strains was cultured in inducing medium. Possible reasons are as follows: poor design of si RNA; low transformation efficiency; randomness of insertio n site inside of fungus; off-target effects, etc. |
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