粗糙脉孢菌丝氨酸/苏氨酸激酶家族基因敲除库纤维素酶表达分泌研究

【目的】蛋白磷酸化在丝状真菌细胞对外界纤维素酶诱导信号感应以及信号胞内的传导过程中有着重要的作用,而蛋白磷酸化是由蛋白激酶来完成的。为了挖掘在丝状真菌纤维素酶表达过程中发挥重要作用的激酶基因,对粗糙脉孢菌丝氨酸/苏氨酸家族的61株蛋白激酶单基因突变体的纤维素酶表达分泌情况进行了分析测定。【方法】在以微晶纤维素为唯一碳源的条件下,7株单基因突变体胞外分泌蛋白产量有显著变化,随后,对这7株突变体胞外蛋白进行了详细的SDS-PAGE分析和内切-β-1,4-葡聚糖酶酶活、β-葡萄糖苷酶酶活、外切纤维素酶酶活以及木聚糖酶酶活的测定。【结果】突变株W14、W38、W87和W40胞外分泌蛋白含量提高了30%以上,除了突变株W14外,其它突变体的内切-β-1,4-葡聚糖酶酶活分别显著提高了62%、42%和42%。而突变株W85、W26和W46胞外分泌蛋白含量降低了50%以上,相对应的内切-β-1,4-葡聚糖酶酶活也分别下降了86%、75%和84%。【结论】这些关于粗糙脉孢菌丝氨酸/苏氨酸家族蛋白激酶基因的挖掘,为进一步深入研究蛋白激酶在纤维素酶诱导表达调控中的分子机理奠定了基础。

Cellulase expression analysis of serine/threonine kinase gene deletion mutants of Neurospora crassa

Objective] Protein phosphorylation plays an important role in many important cellular processes, such as the extracellular cellulase induction signal sensing and intracellular signal transduction processes in filamentous fungi. The protein phosphorylation is controlled by protein kinases. Methods] to discover the roles of protein kinases in cellulase induction signaling pathway, we analyzed cellulase expression levels of 61 kinase mutants using 2% crystalline cellulose as the sole carbon source. Compared with the wild type, cellulase productions in 7 mutants were significantly changed. Then, we did the SDS-PAGE analysis and measured the endo-beta-1,4-glucanase activity, beta-glucosidase activity, cellobiohydrolase activity, xylansae activity of these 7 mutants. Results] We found that extracellular proteins of mutants W14, W38, W87 and W40 increased more than 30%, and their endo-beta-1,4-glucanase activities increased by 62%, 42% and 42% respectively, except for mutant W14. Moreover, the extracellular proteins of mutants W85, W26 and W46 decreased over 50%, and their endo-beta-1,4-glucanase activities were also reduced by 86%, 75% and 84% respectively. Conclusion] These observations about serine/threonine protein kinase genes in Neurospora crassa could be helpful to better understand the roles of protein kinases in cellulase induction pathway.