Effect Of Sec6 Gene Of Trichoderma Reesei On Mycelial Branching And Enzyme Production

Trichoderma reesei is widely used in industrial production of cellulase due to its high cellulase production and high enzyme production activity,but its cellulase production still needs to be improved.A large number of studies have shown that filamentous fungi secrete proteins to the outside of the cell through the mycelial tip.It is speculated that the increase in the mycelial branch can produce more mycelial tips,which can achieve the purpose of increasing protein yield.Many genes have been reported that affect the mycelial branch of fungi,such as ras1,ras2,rho A,spa2,cla4,cdc42 and rac A,etc.Most of these genes are involved in the establishment of cell polarity,intracellular material transport after editing these genes,the observed result is that the mycelial branch will have obvious changes.Saccharomyces cerevisiae Sec6 participates in the exocytosis of cells,and also plays an important role in the construction of cell walls and the establishment of cell polarity;Candida albicans Sec6 is involved in the transportation and delivery of intracellular substances,and the absence of the sec6 gene will cause Candida albicans mycelial branch reduction.In this study,a database search was performed to find genes homologous to Trichoderma reesei and Candida albicans sec6.It was planned to overexpress and knock out the Trichoderma reesei sec6 gene to confirm the gene's ability to branch and produce enzymes of Trichoderma reesei impact.This study mainly obtained the following results:(1)Calcium ion promotes the mycelial branch of Trichoderma reesei to a certain extent,but different strains are affected by calcium ions to different degrees.Among the four candidate Trichoderma reesei strains,340554 strain is affected by calcium ions The effect is the biggest.Under the culture conditions with and without calcium ions,the difference in mycelial branching is the most significant,indicating that this strain has a large difference in mycelial branching under different conditions,which is a good material for studying mycelial branching,so choose this Strains to study genes related to Trichoderma reesei mycelial branching.(2)Search for the gene homologous to Candida albicans sec6 in Trichoderma reesei in the NCBI database,and then design primers to amplify and sequence the gene in the Trichoderma reesei 340554 strain.The results of the amplified sequence sequencing and NCBI database The announcement sequence is consistent.Then,the transcription level of the sec6 gene under calcium ion culture conditions was measured,and as a result,the transcription level of the sec6 gene under calcium ion conditions was higher than that under calcium-free culture conditions.(3)Sec6 overexpression plasmid was successfully constructed and successfully transformed into T.reesei 340554 strain to obtain sec6 overexpression transformed strain.By comparing the physiological and biochemical characteristics of the overexpressing transformed strain and the starting strain,the mycelium growth rate of sec6 overexpressing transformed strain was slow,and the colony diameter of the solid culture was measured for 48 h.Compared with the starting strain,the growth rate of the overexpressing transformed strain Decreased by 31.29%.By measuring the total amount of extracellular protein in the enzyme liquid after fermentation for 5 days,it was found that the total amount of extracellular protein increased by 14.8% compared with the starting strain.Through fluorescent quantitative detection of sec6 gene,it was found that the sec6 gene transcription level of the overexpressing transformed strain was increased by 31.87% compared with the starting strain.(4)Through transformation,a T.reesei strain that can stably inherit and express Cas9 is obtained.The morphology and cellulase activity of the selected Cas9 transformed strain are not significantly different from the starting strain,which can ensure the introduction of the Cas9 encoding gene And expression does not interfere with the expression and function of other genes,and provides good materials for the study of Trichoderma reesei gene function;and successfully constructed g RNA and d DNA plasmids targeting the sec6 gene,which can be used for the knockout of the sec6 gene.Judging from the overexpression results,the sec6 gene does have a great influence on the mycelial branching and cellulase-producing ability of Trichoderma reesei.This result validates the initial conjecture in this study.