利用红色荧光蛋白分析里氏木霉合成纤维素酶的机理

Analysis of cellulase synthesis mechanism in Trichoderma reesei using red fluorescent protein

摘要:

以红色荧光蛋白作为报告蛋白研究了里氏木霉的纤维素酶合成机理。构建了里氏木霉的表达盒，通过该表达盒使红色荧光蛋白的基因整合到里氏木霉的基因组DNA上，并受纤维二糖水解酶基因启动子的调控，得到重组菌株T. reesei TR2。在不同的条件下培养T. reesei TR2，红色荧光蛋白的表达情况可以反映在不同条件下里氏木霉合成纤维素酶的情况。在诱导的情况下，红色荧光蛋白随时间变化的情况与培养液中纤维素酶活性的变化相似，培养至36h后可以观察到荧光，并且不断增强，到菌丝自溶时荧光减弱。另一方面，诱导后里氏木霉菌丝的各个部位均可以观察到荧光，而且分布均匀，表明菌丝的各个部位在纤维素酶合成过程中所起的作用相同。在非诱导的情况下，培养时间较长时也可以观察到较弱的荧光，表明在此条件下里氏木霉仍可以合成少量的纤维素酶，这一结果为解释纤维素诱导里氏木霉合成纤维素酶的机理提供了另一个试验依据。

Abstract:

Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2.Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.