里氏木霉分泌型表达载体的构建及绿色荧光蛋白的表达

【目的】构建里氏木霉分泌型表达载体,通过表达绿色荧光蛋白论证载体的可行性并初步观察绿色荧光蛋白在里氏木霉中的分泌过程。【方法】应用PCR及分子克隆技术将里氏木霉(Trichoderma reesei)纤维二糖水解酶(CBH1)的启动子及CBH1自身信号肽、终止子和潮霉素筛选基因依次插入骨架质粒pUC19中,构建出T.reesei表达载体Ppth15。将增强型绿色荧光蛋白(eGFP)基因装载入Ppth15中,获得eGFP表达载体Ppth15-eGFP。再将Ppth15-eGFP转化进T.reesei原生质体,通过潮霉素抗性筛选、基因组PCR检测等方法鉴定,获得阳性重组转化子。【结果】用PDA培养基培养阳性转化子2-3 d后,可在菌丝顶端、隔膜及培养基中清晰地观察到大量绿色荧光。【结论】表达载体构建成功且能够用于eGFP的表达,实验为进一步研究T.reesei表达其他基因提供了有效工具,同时为T.reesei胞外蛋白分泌的研究提供了参考。

Construction of secretory expression vector of Trichoderma reesei and expression of enhanced GFP

[Objective] To construct a secretory expression vector of Trichoderma reesei and prove its feasibility by recombinant expression of enhanced green fluorescence protein (eGFP), and observe the process of the expression of eGFP, spontaneously. [Methods] Construct the expression vector pPth15 by successively ligate the promoter of the CBH1 gene of T. reesei, the signal peptide sequence, the terminator and the hygromycin resistance gene into the backbone plasmid pUC19. Insert eGFP gene into pPth15 to aquire the recombinant plasmid pPth15-eGFP, and transformate it into the T. reesei protoplasts. Screening positive transformants via hygromycin resistance and PCR amplification. [Results] After culturing the positive transformants on the PDA medium for 2 to 3 days, green fluorescence distribution was emerged on the hyphae tips, the septum and the extracellular medium. [Conclusion] The constructed expression vector could be used for eGFP expression, and this work makes contribution to the follow-up study of heterogeneous protein expression in T. reesei and provides a reference in the research of extracellular protein expression of T. reesei.