基于URA3基因快速构建表达载体及其在里氏木霉的应用

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

摘要:

【背景】表达载体是基因工程必不可少的工具，对于真菌如里氏木霉(Trichoderma reesei)等，由于缺乏商品化的表达载体，使其基因工程蛋白表达既复杂又费时而难以开展。【目的】建立一种利用URA3序列快速构建表达载体的方法，解决真菌研究中难以简单、高效和快速构建表达载体的难题。【方法】基于URA3基因的序列，利用其启动子上游5′端序列和终止子下游3′端序列构建同源重组臂，通过同源重组臂定向同源重组到宿主基因组上，利用强启动子和终止子替换URA3基因，从而实现外源基因的表达。根据此方法构建pTRUC表达载体，将红色荧光蛋白基因mCherry克隆到该表达载体上，转化里氏木霉中并验证mCherry的表达。【结果】阳性转化子在荧光显微镜下观察到强的红色荧光信号，在基因组PCR中检测到mCherry，Western blotting结果表明红色荧光蛋白mCherry能在里氏木霉中表达，以上结果说明该载体构建成功，使外源基因mCherry在里氏木霉中正确表达。【结论】基于URA3基因的快速构建表达载体及其构建方法切实可行，将成为推动真核表达系统表达异源蛋白的有力工具。

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.