敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

Effects of Knocking-out Histone H3 Lysine Methyltransferase on Cellulase Expression in Trichoderma reesei

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶(histone lysine methyltransferase,HKMT)参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T.reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reeseiΔhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活(filter paper enzyme activity,AFP)和羧甲基纤维素钠酶活(carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL-1、15.00 IU·mL-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。

Epigenetics is a heritable change that does not involve changes in DNA sequence,including DNA methylation,histone modification,and miRNA regulation.In the histone methylation modification,histone lysine methyl transferase(HKMT)is involved in regulation mainly.Some literature reported that the catalytic core of HKMT protein is the SET domain,which has the function of promoting or inhibiting gene expression.In Trichoderma reesei,the mechanism of HKMT regulates the expression of cellulase genes is not clear.In this paper,the gene hkmt knockout expression cassette was constructed by using Split-Maker technology,and T.reesei QM9414 was transformed.PCR and Southern blotting were used to verify the result.Microscopic observation showed that the T.reeseiΔhkmt strain had longer hyphae and more branches.The mutant strain was tested for 7 days of filter paper enzyme activity(AFP)and carboxymethyl cellulose sodium enzyme activity(CMCA).The results were 5.00 IU·mL-1 and 15.00 IU·mL-1higher than that of wild type strain,respectively.The expression of cellulase and related genes cbh1,egl1 and xyr1 in mutant strains were detected by RT-qPCR to be 4.51,3.87 and 2.51 times higher than that of wild type,respectively.Through the exploration of the morphological characteristics,cellulase activity,cellulase-related gene expression of wild-type strains and mutant strains,it provides a new idea and experimental data for further study on the influence of epigenetic regulation of T.reesei on cellulase expression.