| Saprophytic fungus Trichoderma reesei has a long history of important industrial production of different plant material hydrolyzing enzymes, especially cellulases and hemicellulase, due to its exceptional ability to secrete enzymes for substrate degradation to readily metabolizable breakdown products.Trichoderma reesei has the advantage of possessing a eukaryotic secretory machinery and, most likely, similar protein modification properties (e.g. high mannose type N-glycosylation) to mammalian systems. All the excellent characters improved the molecular biological research and genetic modification of Trichoderma reesei. There has few reports on construction of strong expressional transformation system and engineering strains reported in local literature. Therefore, eonstruction of high transformation and expression system is important for carrying out molecular biological research on Trichoderma reesei.The cbh1 gene in T. reesei is a single-copy gene in T. reesei. the promoter was considered as strong promoter. Therefore, it is significant to choose strong promoter Pcbh1 to construct expressional vector to control foreign protein expression in T. reesei.According to the reports by literature, the amount of heterogonous proteins can increase by many factors : genes copies , the intensity of promoter -multi-copies , protein stabilization.So we delete the glucose repressor-binding sites in the upstream region of T.reesei cellobilhydrolase I (cbhl) using recombinant PCR and then multi-copy the upsteam region of cbhl promoter containing CCAAT region , Ace2 binding sites and XlnR bindig sites.Fusing the reporter gene E. coli uidA gene with -1306~-16bp region and -868~-16bp region of the cbhl promoter and terminator resulted in the plasmid pLPTG and pCPTG respectively.The deletion of the -709~-661bp regioncontaining three glucose repressor CreA-binding sites in the promoter of cbhl on the basis of pCPTG was performered by PCR, through which the plasmid ApCPTG was constructed. -805~-606bp upstream of the translation intiation codon , a 200-bp DNA fragment containing Ace2 binding sites , XlnR bindig sites , CCAAT enhancer sequences located in the Pcbhl of the plasmid ApCPTG was amplied by PCR whose product was inserted into ApCPTG to create plasmid Ap2CPTG. Similarly, plasmid Ap4CPTG carrying four copies of - 805 to -606bp region and plasmid Ap6CPTG carrying 6 copies of -805 to -606bp region were obtainedFinally, the resulting plasmids were co-transformed with plasmid pAN7-l carrying a hygromycin resistance cassette into T. reesei Rut C30 U4, a protease deficient strain. 1200 hygromycin-resistant transformants were obtained. 50 transformants were isolated by PCR, and PCR emplification product was sequenced, demonstrating the uidA expression cassete has inserted into the chromosomal DNA. 6 of these transformants were verified further by RT-PCR. Then promoter activity was judged by using an assay for β-glucuronidase, which was expressed under the control of these promoters. An increase in β-glucuronidase activity, corresponding to the number of inserted -805 to -606bp region's copies, was observed. In transformant T.reesei A4-3, carrying 4 copies , the β-glucuronidase activity were shown to be 2.4 fold of transformant T.reesei L13, 2 fold of transformant T.reesei P12 . In transformant T.reesei △1-2, the promoter activity was shown to be 1.8 fold of T.reesei L13. But in transformant T.reesei △6-1, the activity was the same as that of T.reesei A4-3 . At the same time, the β-glucuronidase activity of T. reesei P12 grown in glucose medium is only 40% of that in the lactose medium, however it is similar for T.reesei △1-2, demonstrating that the CreI binding sites have been deleted. The multicopies plasmids constructed provided a tool for highly-expressing heterologous protein and function research in T. reesei and the basis on further application in industy0 .
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