| Trichosporon mucoides as a kind of Oleaginous Microorganisms, It can be able to convert carbohydrates and ordinary fats into microbial oils stored in vivo. But it can not be directly use lignocellulose for the synthesis of SCO(single cell oil). P-glucosidase as a member of the family of glycosides,it belongs to the cellulose enzyme system, Mainly involved in the hydrolysis at the end of unreducible, namely glycosyl atoms with aryl or alkyl glycoside bond formation between beta -D-glucose. Such as beta glucosidase and beta galactosidase, also has a certain effect on xylose and fructoside. Hydrolysis of cellulose produced sugars can be fermented by trichisporon synthesis microbial oil. This experiment aims to study the p-glucosidase fermentation trichisporon efficient utilization of microbial oil production of lignocellulose to lay a solid foundation. Select the β-glucosidase as the research object. We extracted Trichosporon mucoides total RNA and reverse transcription for get cDNA. According to the sequence of the different strains of the different strains which have been reported in NCBI design primers. PCR amplification was obtained in some CDs areas and the use of bioinformatics software was analyzed. The changes of PCR expression in different culture time, temperature, C/N ratio and pH were detected by fluorescence quantitative PCR technique. The CDS region was introduced into expression vector and expressed in Escherichia coli (Top10 E.coil).The results are as follows(1) After grinding in liquid nitrogen, freezing and thawing, ultrasonic crushing, ball and ball together with shock enzyme pretreatment and using Trizol method to extract total RNA of Trichosporon mucoides. The results showed that the RNA extracted from grinding in liquid nitrogen and ball together with shock enzyme pretreatment was relatively intact and the quality of the non degradation was better and followed by repeated freezing and thawing. There was an obvious degradation after ultrasonic pretreatment. Electrophoresis bands was not detected after the steel ball shock pretreatment. It is showed that liquid nitrogen grinding is an effective pretreatment method for broken cell wall to extract total RNA from the Trichosporon mucoides.(2) We get partial CDs sequences of glucosidase gene through the TA clone, the CDs is part of P-glucosidase gene sequence contains 570 bp, encoding 189 amino acids. The polypeptide is a hydrophilic protein, secondary structure in helix accounted for 25.9%, extension chain accounted for 22.89% and random coil accounted for 51.2%, no beta folding structure.(3) The changes of mRNA expression in different culture time, temperature, C/N ratio and pH expression were analyzed by fluorescence quantitative PCR technique combined with SPSS software. The results showed that BG gene was analyzed in different pH, carbon and nitrogen ratio(C/N), temperature and time by RT-PCR. Temperature, pH and C/N had no significant effect on the expression of BG gene in Trichosporon mucoides. whereas time was different from them. The expression level in 3d was significantly higher than that in other culture phase, and was decreased after increasing.(4) The pET28a plasmid expression vector and beta glucosidase CDs gene constructed prokaryotic expression vector, expression of the encoding part by Escherichia coli. The bacteria PCR and agarose gel electrophoresis test, expression vector exists in E.coli. SDS-PAGE gel electrophoresis showed that the molecular weight of the target with HisTaq was in accordance with the expected. Due to the expression of target protein with HisTaq and confirmed by Western blot results showed his tag radioactive reaction, indicating that the His tag antibody and combined demonstrate and HisTaq expression of fusion protein is also expressed in E.coli, is further evidence of the accuracy of the results of SDS-PAGE, through the above experiments prove that protein is part of the beta glucosidase from glycosidase gene coding protein and lay a foundation for the subsequent experiments. |
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