Rapid construction of an expression vector based on URA3 gene for application in Trichodermium reesei

Abstract:

[Background] The expression vector is an indispensable tool for genetic engineering. However, some of the fungus such as Trichoderma reesei lacks commercial expression vector and the traditional vector construction method is complicated and time-consuming, which makes protein expression difficult to carry out. [Objective] To generate a fast and novel method to construct the expression vectors based on the sequences of URA3 gene to generate a fungus’s universal expression system for research and development. [Methods] Based on the sequence of URA3 gene, we used the upstream gene fragment of URA3 promoter and downstream gene fragment of URA3 terminator to form homologous recombination arms. The promoter and terminator of URA3 was replaced with the promoter of CBH1 and the terminator of PDC during the construction process, respectively. The elements of the vector were linked seamlessly in one reaction using DNA topoisomerase I. Pcbh1-MCS-Tpcd, flanked by the homologous recombination arm of URA3, was assembled with other element fragments to form vector pTRUC. Red fluorescence protein, mCherry, was inserted into pTRUC which later was transformed into T. reesei to generate the final RUT-C30. Such expression system proves its feasibility via observation and determination of mCherry protein expression. [Results] After culturing the positive transformants on the PDA medium with 5-FOA, red fluorescence appeared on the hyphae tips and the septum. PCR result proved the expression of mCherry in the host. Western blotting analysis also showed the expression of mCherry. This method of vector construction is feasible as the constructed expression vector could express the recombinant protein. [Conclusion] The results demonstrate that the rapid construction of the expression system based on homologous recombination with the URA3 gene and the construction method is feasible. This will become a powerful tool to promote the expression of recombinant proteins in many eukaryotic expressing systems.