敲除组蛋白去乙酰化酶基因hdac对里氏木霉纤维素酶表达的调控作用

Effects of knocking-out histone deacetylase gene hdac on cellulase expression in Trichoderma reesei

摘要:

【背景】里氏木霉(Trichoderma reesei)是木霉属中产纤维素酶最具代表性的真菌之一，表观遗传调控是不涉及DNA序列变化的可遗传变化，组蛋白去乙酰化是其中一种。组蛋白去乙酰化酶(histone deacetylase，HDAC)负责脱乙酰化，敲除去乙酰化酶基因可引起菌株孢子、菌丝及纤维素酶活性等的一系列改变。【目的】通过敲除里氏木霉组蛋白去乙酰化酶基因(histone deacetylase，hdac)建立了里氏木霉hdac缺失突变株(T. reesei Δhdac)，以研究对纤维素酶基因表达的调控作用。【方法】利用Split-Maker技术构建了组蛋白去乙酰化酶基因敲除表达盒，并转化了里氏木霉T. reesei QM9414。经PCR及Southern blotting验证正确后，对突变体T. reesei Δhdac连续7 d检测滤纸酶活(filter paper activity，AFP)、羧甲基纤维素钠酶活(carboxymethyl cellulase activity，CMCA)，利用RT-qPCR检测纤维素酶及其相关基因cbh1、egl1和xyr1的表达。【结果】突变体T. reesei Δhdac两种酶活力均显著高于出发菌株，分别高出8.00、30.00 IU/mL。突变体T. reesei Δhdac纤维素酶及其相关基因cbh1、egl1和xyr1的转录水平分别为出发菌株T. reesei QM9414的6.50、6.01和4.51倍。【结论】里氏木霉中纤维素酶的基因表达明显受到组蛋白去乙酰化酶基因(hdac)的调控，这为研究里氏木霉表观遗传调控对纤维素酶的影响提供了新的证据。

Abstract:

[Background] Trichoderma reesei is the most representative fungus to produce cellulase. Epigenetic regulation is a heritable change that does not involve changing DNA sequence. Histone deacetylation is one of epigenetic regulation, and histone deacetylase (HDAC) is responsible for deacetylation. Knocking out the acetylase gene can cause a series of changes in spores, hyphae and cellulase activities. [Objective] A mutant of T. reesei Δhdac was developed by knocking out the histone deacetylase (hdac) gene to investigate the effects of hdac on regulation of cellulase expression. [Methods] The hdac knocking-out expression boxes were constructed by the Split-Maker technique, and were transformed into Trichodermium reesei QM9414. After being verified by PCR and Southern blotting, filter paper enzyme activity and carboxymethyl cellulose natriuretic enzyme activity of the mutant were continuously detected for 7 days, and the related gene expressions were detected by RT-qPCR. [Results] The two enzyme activities in mutant strains were 8.00 IU/mL and 30.00 IU/mL higher than those of the start strain Trichodermium reesei QM9414 respectively. The transcription level of cbh1, egl1 and xyr1 in the T. reesei Δhdac were 6.50 times, 6.01 times and 4.51 times higher than those of the start strain, respectively. [Conclusion] The cellulase gene expression in Trichoderma reesei QM9414 was significantly regulated by the histone deacetylase, this provides a new way to study the effects of epigenetic regulation of Trichoderma reesei on cellulase.