里氏木霉翻转酶DRS2对木质纤维素降解酶基因表达及分泌的影响

Effects of flippase DRS2 on expression and secretion of lignocellulases in Trichoderma reesei

摘要:

【目的】本研究以工业生产菌株里氏木霉为研究对象,鉴定翻转酶基因drs2对其纤维素酶表达及分泌的影响。【方法】首先通过BLAST序列比对,从里氏木霉中鉴定出翻转酶基因drs2,并通过同源重组的方法在里氏木霉中构建了drs2基因的敲除菌株∆drs2。对∆drs2菌株及其对照株在不同碳源上的生长发育、蛋白分泌、纤维素酶及半纤维素酶的表达水平等进行比较分析,并对DSR2蛋白进行了亚细胞定位。【结果】与对照菌株Cpyr4相比,∆drs2菌株在葡萄糖、乳糖、微晶纤维素(avicel)等条件下生长速率都明显降低。∆drs2菌株在纤维素诱导条件下的总蛋白分泌量、纤维素酶活力、半纤维素酶活力均显著提高,但drs2基因的缺失并不影响关键纤维素酶基因的转录水平。DRS2蛋白位于里氏木霉菌丝近顶端的顶体位置。【结论】在纤维素为唯一碳源的条件下,drs2基因的缺失导致纤维素酶的产量显著提高,drs2基因不调控纤维素酶基因的转录,而是在其分泌过程中发挥作用。

Abstract:

[Objective] To study the effects of flippase DRS2 on the expression and secretion of lignocellulases in Trichoderma reesei.[Methods] The homologous gene drs2 in T. reesei was firstly identified through sequence alignment by BLAST and then deleted via homologous recombination strategy from T. reesei.The growth,protein secretion,and activities of cellulase and hemicellulase were compared between the drs2-deleted strain ∆drs2 and the control strain Cpyr4.Furthermore,the subcellular localization of DRS2 was predicted.[Results] Compared with Cpyr4,∆drs2 showed significantly decreased growth rate in the medium supplemented with glucose,lactose or microcrystalline cellulose (avicel).The total protein secretion and the activities of cellulase and hemicellulase of ∆drs2 were significantly higher than those of Cpyr4.The transcriptional levels of the key cellulase genes were similar between ∆drs2 and Cpyr4. Subcellular localization prediction revealed that DRS2 was located in spitzenkörper of the hypha tip.[Conclusion] The deletion of drs2 gene can significantly improve the production of lignocellulases in T. reesei cultured with cellulose as the sole carbon source. DRS2 may play a role in the secretion rather than the transcription of cellulases.