沉默里氏木霉组蛋白赖氨酸甲基转移酶基因对纤维素酶表达的影响

Effects of silencing histone lysine methyltransferase gene on cellulase expression in Trichoderma reesei

【背景】里氏木霉(Trichoderma reesei)是一种比其他真菌小很多的多细胞真核微生物,在工业上受到广泛应用,而里氏木霉QM9414是目前研究最多基因产纤维素酶丰富的突变菌株。【目的】构建里氏木霉中组蛋白赖氨酸甲基化酶(Histone Lysine Methyltransferase)基因hkmt的siRNA沉默载体和过表达载体来降低或者增强hkmt在里氏木霉QM9414中的表达量,以分析其对里氏木霉纤维素代谢的调控作用。【方法】根据里氏木霉hkmt序列设计siRNA沉默片段并用反转录的方法获得过表达hkmt片段。将沉默片段和过表达片段克隆至里氏木霉组成型表达载体中,构建沉默hkmt的载体和过表达hkmt的载体,并将其转化里氏木霉QM9414。通过荧光显微镜观察重组菌的菌丝生长情况,此外对各重组菌进行纤维素酶的滤纸酶活性(Filter Paper Enzyme Activity,FPA)和羧甲基纤维素钠酶活性(Carboxymethyl Cellulose Enzyme Activity,CMCA)的测试;利用荧光定量PCR的方法检测hkmt、纤维素酶基因cbh1、egl1及木聚糖酶激活因子xyr1的表达量变化。【结果】通过使用荧光显微镜观察,发现沉默、过表达hkmt重组菌的菌丝形态均与出发菌株无明显差异。荧光定量PCR测定结果表明,沉默载体和过表达载体可以分别沉默和促进hkmt的表达。沉默hkmt重组菌株中FPA和CMC酶活力相比出发菌平均升高2.5倍。此外,纤维素酶相关基因和激活因子在沉默hkmt重组菌中的表达量均有所增加,但是在过表达hkmt重组菌株中以上相应指标均呈现相反的趋势。【结论】组蛋白赖氨酸甲基转移酶基因表达产物负调控里氏木霉产纤维素酶基因的表达,这为提高里氏木霉产纤维素酶水平提供了参考,并为里氏木霉产纤维素酶的表观遗传调控研究提供了新的证据。

[Background]The filamentous fungus Trichoderma reesei is a eukaryotic microorganism and smaller than any other multicellular,which is widely used in industry,and T.reesei QM9414 were one of the most abundant cellulase producing mutant.[Objective]In order to study cellulase activities and expression regulation in T.reesei,we constructed siRNA interfered vectors and overexpressed vectors to inhibit and enhance HKMT expression in T.reesei QM9414,respectively.[Methods]The siRNA interference fragment was designed according to the hkmt gene sequence of T.reesei and the overexpressed hkmt gene fragment was obtained by reverse transcription.The two fragments were cloned into the constitutive expression vector and the two vectors were transformed the protoplasm of T.reesei QM9414.The hyphae growth of the recombinant strains was observed through a fluorescence microscope.In addition,cellulase activities(CMC enzyme activity and filter paper enzyme activity)of recombinant strains were detected and related gene expressions were also detected by quantitative PCR.[Results]The mycelial morphology of the interfered and the overexpressed recombinant strains were not significantly different from the original strain.Quantitative PCR results showed that the interfered vector and the overexpressed vector can silence and promote the expression of hkmt gene,respectively.The enzyme activities of FPA and CMC in the interfered recombinant strains were increased by an average of 2.5 times compared with the original strain.In addition,the expression levels of cellulase-related genes and activators in interfered recombinant strains increased.However,the recombinant strains for overexpressing HKMT showed the opposite patterns in the genes’expression levels and enzymatic activities.[Conclusion]These findings indicate that HKMT in T.reesei is involved in the negative regulation of cellulase expression and production.