| Lignocellulose is an abundant and important component of renewable resources in nature,but its structure is strong and difficult to degrade,resulting in low utilization efficiency.At present,the use of filamentous fungi to secrete cellulase to degrade cellulose is a mild,high-yield,non-polluting method.Trichoderma reesei as a filamentous fungus that mainly produces cellulase in the industry,can secrete a complete set of enzymes that degrade cellulose,but its cellulase production still needs to be improved.Studies have shown that the secretion of cellulase is positively correlated with the number of hyphae at the tip of its hyphae,so it is speculated that cellulase secretion can be affected by modifying the morphology of Trichoderma reesei hyphae.The cell wall of filamentous fungi has a certain influence on the morphology of their hyphae.Previous studies have found that the absence of chitin synthase III(CHSⅢ),the main enzyme involved in the cell wall of filamentous fungi,can cause the change of hyphal morphology.In this project,the overexpression of CHSⅢ gene and RNA interference were verified to determine the function of the gene in Trichoderma reesei.The following results were obtained in this study:(1)The overexpression plasmid of CHSⅢ gene was constructed and transformed into the protoplast of Trichoderma reesei QM6 a,and two overexpressed strains of CHSⅢ gene were successfully obtained,OE-3 and OE-7.The fluorescence quantitative results showed that the relative expression of CHSⅢ in OE-3 was about35 and that of CHSⅢ in OE-7 was about 29.(2)The determination of the cellulase total enzyme activity of CHSⅢ overexpressed strain and QM6 a strain found that the enzyme activity of CHSⅢ overexpressed strain was lower than QM6 a,the total cellulose enzyme activity of OE-3 was reduced by 24% compared with QM6 a on day 7,and the cellulose total enzyme activity of OE-7 and QM6 a was the largest difference on day 5,and OE-7decreased by 56.5% compared with QM6 a.It was found that the extracellular protein content of overexpressed strains was consistently lower than QM6 a,and the extracellular protein secretion of OE-7 strains was 13% lower than QM6 a and 7.6%lower than QM6 a when the exocytoprotein secretion was highest on day 7.However,intracellular protein content showed the opposite result,with OE-3 being 4% higher than QM6 a and OE-7 5.3% higher than QM6 a at day 5.(3)The determination of CHSⅢ overexpression strains and the properties of QM6 a strains found that the colony diameter of OE-3 and OE-7 was consistently smaller than QM6 a,but in liquid culture,when the biomass reached the maximum on the third day,the biomass of OE-3 increased by 20.63% compared with QM6 a,and OE-7 increased by 14.02% compared with QM6 a.Observing the branching of hyphae,OE-3 and OE-7 form fewer hyphae than QM6 a.OE-3 and OE-7 had 18.88% and13.06% higher chitin content than QM6 a,respectively.Secondly,the analysis of transcriptome data found that a batch of protein gene expression related to membrane synthesis and related transport showed upregulation.These results allow preliminary conclusions to be concluded that the hyphae of Trichoderma reesei by the CHSⅢ gene are associated with protein secretion.(4)The RNA interference plasmid of CHSⅢ gene was constructed to transform the interfering plasmid into the protoplast of Trichoderma reesei QM6 a,and successfully obtained a stable interfering transformant,which was named interference-10.The identification of interfering transformants and starter strain QM6 a found that the transcription level of CHSⅢ gene in scrambled-10 was not significantly different from QM6 a,and there was little difference in colony growth and biomass. |
