多靶向沉默里氏木霉碳代谢阻遏物对纤维素酶活性和表达的调控研究

Cellulase activities and expression regulation by multiple targeted silencing carbon catabolic repressors in Trichoderma reesei

摘要:

[目的]构建多靶向siRNA表达载体对里氏木霉碳阻遏抑制因子CRE1、CRE2、CRE3和CRE4进行同时多靶向siRNA干扰，以研究其对里氏木霉纤维素酶基因表达的调控作用。[方法]根据此前研究筛选出沉默cre1、cre2、cre3和cre4基因的4个最佳siRNA序列，设计并构建了A多靶向表达载体，另根据cre1、cre2、cre3和cre4基因中所含有的5个共有序列设计并构建了B多靶向表达载体，将两者转化至里氏木霉QM9414。经筛选后分别在48 h和120 h对各转化子进行纤维素酶酶活力测试（CMC活力测试和滤纸酶酶活力测试）及利用qPCR检测相关基因的表达。[结果]通过RT-qPCR测定结果表明，两种表达载体均可同时抑制里氏木霉的分解代谢物阻遏基因cre1、cre2、cre3和cre4的表达，纤维素酶活力比出发菌株明显升高，多靶向抑制菌株的CMC酶活和滤纸酶活比出发菌株平均提高了1.95倍和2.66倍。纤维素酶基因cbh1和egl1的表达水平比出发菌株也有明显提升，平均提高了3.83倍和3.95倍。纤维素酶相关基因xyr1的表达水平与出发菌株相比也明显上升，平均提高了2.78倍。[结论]多靶向沉默里氏木霉的碳代谢阻遏蛋白有利于解除葡萄糖效应，提高非还原糖的利用，从而提高纤维素酶的产量，使纤维素酶的表达得到更大的提升，为里氏木霉表达纤维素酶在分解代谢物阻遏基因调控方面提供了实验依据和新的技术思路。

Abstract:

[Objective] In order to study carbon metabolic repressors on cellulase activities and expression regulation in Trichoderma reesei, we constructed multi-targeting siRNA expression vectors to perform silencing carbon metabolic repressor cre1, cre2, cre3 and cre4 by simultaneous producing multi-targeting siRNAs. [Methods] According to previous studies and screening, the four best siRNAs targeting the cre1, cre2, cre3 and cre4 were selected and they were constructed a multi-targeting siRNA expression vector A. Additionally, according to 5 overlap common sequence in the cre1, cre2, cre3 and cre4, we designed and constructed the vector B. The two vectors were transformed the protoplasm of Trichoderma reesei QM9414 and selected on the hygromycin selection medium. Cellulase activities (CMC activity and filter paper activity) of transformants were detected and related gene expressions were also detected by RT-qPCR after incubation for 48 and 120 hours respectively. [Results] RT-qPCR results showed that the cre1, cre2, cre3 and cre4 expression levels in Trichoderma reesei were simultaneously silenced, and the cellulase activities were much higher than that of the staring strain. The CMC activity and filter paper activity of the multi-targeted inhibitor strains were 1.95-fold and 2.66-fold higher than those of the original strains. The cellulase-related genes expression levels were also increased significantly. The expression levels of cbh1, egl1 and xyr1 were 3.83-fold, 3.95-fold and 2.78-fold higher than those of the original strain. [Conclusion] Our results indicated that simultaneous silencing multi-targets of the carbon metabolic repressors in Trichoderma reesei is beneficial to release the glucose repressor effects and increase the expression and production of cellulase. These results provide evidence and techniques for study of regulation of carbon catabolite repressor genes on cellulase expression in Trichoderma reesei.