| Targeted integration in Trichoderma reesei is typically hampered by very low rate of homologous recombination and the homologous recombination rate is usually less than10%,so it is difficult to gene modifify in Trichoderma reesei.The previous solution for improving the targeted integration efficiency has been deleting non-homologous end joining DNA repair genes,such as ku70 gene.Presently,there are few reports on RNAi silencing the T.reesei NHEJ pathway-related genes to increase the rate of homologous recombination.However,deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to accumulation of spontaneous mutations.So it is important to obtain a high homologous recombination rate without destroying the NHEJ pathway,using the RNAi tool to silence the T.reesei NHEJ pathway-related genes.In this study,we takes the strain Rut-C30 as the parental strain,and constructed the NHEJ pathway silenced strain with the RNAi method to silence the gene ku70.And then we constructed a Proinsulin expression vector and transformed it into the silenced strain genome with the Agrobacterium tumefaciens-mediated transformation method.This way,it can save time and labor to screen transformants and avoid strain instability caused by NHEJ pathway deletion.In summary,it is important for genetic engineering in Trichoderma reesei,and it could be an important and solid theoretical and technical foundation for some important enzyme preparations to apply in T.reesei.The main results of this research including:(1)we constructed two vectors pCB1300-Pku70 T and pCB1300-dual,containing two different RNAi elements.Then the vectors were transformed into T.reesei with the Agrobacterium tumefaciens-mediated transformation method.The NHEJ Pathway silenced strains were screened with hygromycin B and acetamide marker respectively.It has been demonstrated that the acetamide screening method is feasible for screening of T.reesei.Moreover,the transformants obtained by the acetamide screening method showed significantly fewer transformants than hygromycin screening.Our results demonstrate that the vectors pCB1300-Pku70 T and pCB1300-dual for RNAi silencing could be used as a powerful genetic tool in T.reesei.The ku70 gene expression level decreased 70%approximately.It was more efficient for RNAi silencing with the double – reverse promoters.(2)The vectors pCB1300-INS was constructed based on the pCAMBIA1300 vector,and transformed into silenced strain T4 and the wild type strain Rut-C30,the final homologous recombination rate was 32.15% in silence strain T4,6.6% in wild type strain Rut-C30,and the homologous recombination rate was obviously increased.(3)We compared the difference between the directed integration transformants and randomly inserted transformants at the transcriptional level,protein expression,and fermentation production.The transcriptional level of cbh1 gene in the targeted integrated transformant T-7 was approximately 67% of the randomly inserted transformant T-1,measured by quantitative RT-PCR and statistical analysis shows significant differences.However,the expression level of Prosoinlin gene did not change significantly.The protein expression level,CBHI expression of T-7 decreased most,and almost no expression,while CBHI of T-1 still showed a small amount of expression.The fermentation enzyme production level,the INS activity of T-7 was 35.6 mIU/L,it was significantly higher than T-1which INS activity was 16.1 mIU/L.The filter paper activity of T-7 was 0.36 IU/mL,and the filer paper activity of T-1 was 0.98 IU/mL.The filter paper activity of T-7 was greatly reduced,which was the similar to the upward trend of INS activity.This indicates that the decrease in the secretion of CBHI can enhance heterologous expression in T.reesei. |
PDF Full Text Request