组蛋白修饰对里氏木霉纤维素酶表达的初步研究
发布时间：2018-08-22 16:55
【摘要】：为探讨组蛋白修饰对丝状真菌里氏木霉(Trichoderma reesei,T.reesei)表达纤维素酶的表观遗传调控机制,本文通过基因敲除方法使组蛋白H3赖氨酸甲基转移酶(histone H3 lysine methyltransferase,HKMT)和组蛋白去乙酰化酶(histone deacetylase,HDAC)的基因缺失,成功筛选获得突变菌株Δhkmt与Δhdac;通过siRNA技术干扰组蛋白乙酰化酶(histone acetylase,HAT)的基因gcn5并成功筛选出一株干扰效果较好的菌株T.reesei-gcn5-T10。通过显微镜观察突变菌株Δhkmt与Δhdac以及干扰重组菌株T.reesei-gcn5-T10的菌丝的变化;通过滤纸酶活(Filter paper enzymatic activities,FPA)和羧甲基纤维素钠酶活(Carboxymethyl Cellulose-Na enzymatic activities,CMCA)检测纤维素酶的活性变化;实时荧光定量PCR(Real-time fluorescent quantitative PCR,RT-qPCR)检测了突变菌株Δhkmt与Δhdac以及干扰重组菌株T.reesei-gcn5-T10中纤维素酶基因cbh1、egl1与酶激活因子xyr1的mRNA表达水平,以及hkmt、hdac与gcn5的mRNA水平的变化,探讨他们与纤维素酶表达之间的关系。结果显示,(1)在相同倍数的物镜下观察到突变菌株Δhkmt与Δhdac的菌丝均长于出发菌株T.reesei QM9414,并且分支较多而长。而干扰重组菌株T.reesei-gcn5-T10的菌丝相较于出发菌株T.reesei QM9414,变短而粗,并且末端膨大,内部变浑浊。(2)突变菌株Δhkmt与Δhdac的FPA和CMCA明显高于出发菌株T.reesei QM9414。突变菌株Δhkmt各时段比出发菌株T.reesei QM9414分别平均高出5.00 IU/m L和15.00 IU/mL;突变菌株Δhdac各时段比出发菌株T.reesei QM9414分别平均高出6.50 IU/m L和15.00IU/mL。而干扰重组菌株T.reesei-gcn5-T10的FPA和CMCA明显低于出发菌株T.reesei QM9414,分别平均低出10.00 IU/mL和5.20 IU/mL。(3)RT-qPCR结果显示,突变菌株Δhkmt中纤维素酶基因cbh1、egl1与酶激活因子xyr1基因的相对表达量均高于出发菌株T.reesei QM9414,分别高出4.51倍、3.87倍和2.51倍,突变菌株Δhdac中cbh1、egl1与xyr1基因表达量也均高出发菌株T.reesei QM9414,分别高出6.50倍、6.01倍和4.51倍,而干扰重组菌株T.reesei-gcn5-T10中cbh1、egl1与xyr1基因表达量均低于出发菌株T.reesei QM9414,分别为原菌的0.34倍、0.84倍以及0.44倍。(4)RT-qPCR对hkmt、hdac与gcn5的分析结果显示,干扰gcn5,即HAT表达量下降,导致hdac表达量上升,hkmt的表达量也随之升高。结论:hkmt基因与hdac基因被敲除后,组蛋白的甲基化修饰与去乙酰化修饰被抑制,纤维素酶基因cbh1,egl1与激活因子xyr1基因的表达水平显著增加,最终激活纤维素酶的表达。而gcn5被干扰后,组蛋白乙酰化修饰被抑制,使得相关基因表达显著下降,从而导致纤维素酶活下降。
[Abstract]:To investigate the epigenetic regulation mechanism of histone modification on the expression of cellulase in filamentous fungus Trichoderma reei. The gene deletion of histone H3 lysine methyltransferase and histone deacetyltransferase (HDAC) was studied by gene knockout. The mutant strain 螖 hkmt and 螖 hdac were successfully screened, and a strain T.reesei-gcn5-T10 was successfully screened by siRNA technique which interfered with the gene gcn5 of histone acetylase. The changes of 螖 hkmt and 螖 hdac and mycelium interfering with recombinant strain T.reesei-gcn5-T10 were observed by microscope, and cellulase activity was detected by (Filter paper enzymatic actives and Carboxymethyl Cellulose-Na enzymatic actives. Real time fluorescence quantitative PCR (PCR) was used to detect the mRNA expression levels of cellulase gene cbh1negl1 and activator xyr1 in mutant strains 螖 hkmt and 螖 hdac, and the levels of mRNA of hkmttHDAC and gcn5 in T.reesei-gcn5-T10. To explore the relationship between them and cellulase expression. The results showed that (1) the hypha of 螖 hkmt and 螖 hdac were longer than that of the original strain T.reesei QM9414 under the same objective lens, and the branches were more and longer than those of the original strain T.reesei QM9414. Compared with the original strain T.reesei QM9414, the mycelium that interfered with the recombinant strain T.reesei-gcn5-T10 became shorter and thicker, and the ends expanded and the interior became turbid. (2) the FPA and CMCA of the mutant 螖 hkmt and 螖 hdac were significantly higher than those of the original strain T.reesei QM9414. The 螖 hkmt of mutant strain was 5.00 IU/m L and 15.00 IU/m / mL higher than that of the original strain T.reesei QM9414, and the 螖 hdac of the mutant was 6.50 IU/m L and 15.00 IU / mL higher than that of the original strain T.reesei QM9414 in each stage, respectively. The FPA and CMCA of the interfering recombinant strain T.reesei-gcn5-T10 were significantly lower than that of the original strain T.reesei QM9414, which were 10.00 IU/mL and 5.20 IU / mL 路(3) RT-qPCR, respectively. The relative expression of cellulase gene cbh1negl1 and enzyme activator xyr1 in 螖 hkmt was higher than that of original strain T.reesei QM9414, 4.51 times higher than that of T.reesei QM9414, 3.87 times and 2.51 times higher than that of original strain T.reesei QM9414, respectively. The expression of cbh1negl1 and xyr1 genes in 螖 hdac was also higher than that of T.reesei QM9414, which was 6.50 times higher than that of T.reesei QM9414 and 4.51 times higher than that of T.reesei QM9414, respectively. However, the expression of cbh1egl1 and xyr1 genes in T.reesei-gcn5-T10 was lower than that of the original strain T.reesei QM9414, which was 0.34 times and 0.44 times of the original strain, respectively. (4) the results of RT-qPCR analysis of hkmttn5 and gcn5 showed that the expression of HAT decreased. As a result, the expression of hdac increased and the expression of hkmt increased. Conclusion the methylation modification and deacetylation modification of histone were inhibited after the knockout of the two genes, and the expression level of cellulase gene cbh1egl1 and activator xyr1 increased significantly, and finally the expression of cellulase was activated. After gcn5 was interfered, histone acetylation modification was inhibited, and the expression of related genes was significantly decreased, which resulted in the decrease of cellulase activity.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55
本文编号：2197742
[Abstract]:To investigate the epigenetic regulation mechanism of histone modification on the expression of cellulase in filamentous fungus Trichoderma reei. The gene deletion of histone H3 lysine methyltransferase and histone deacetyltransferase (HDAC) was studied by gene knockout. The mutant strain 螖 hkmt and 螖 hdac were successfully screened, and a strain T.reesei-gcn5-T10 was successfully screened by siRNA technique which interfered with the gene gcn5 of histone acetylase. The changes of 螖 hkmt and 螖 hdac and mycelium interfering with recombinant strain T.reesei-gcn5-T10 were observed by microscope, and cellulase activity was detected by (Filter paper enzymatic actives and Carboxymethyl Cellulose-Na enzymatic actives. Real time fluorescence quantitative PCR (PCR) was used to detect the mRNA expression levels of cellulase gene cbh1negl1 and activator xyr1 in mutant strains 螖 hkmt and 螖 hdac, and the levels of mRNA of hkmttHDAC and gcn5 in T.reesei-gcn5-T10. To explore the relationship between them and cellulase expression. The results showed that (1) the hypha of 螖 hkmt and 螖 hdac were longer than that of the original strain T.reesei QM9414 under the same objective lens, and the branches were more and longer than those of the original strain T.reesei QM9414. Compared with the original strain T.reesei QM9414, the mycelium that interfered with the recombinant strain T.reesei-gcn5-T10 became shorter and thicker, and the ends expanded and the interior became turbid. (2) the FPA and CMCA of the mutant 螖 hkmt and 螖 hdac were significantly higher than those of the original strain T.reesei QM9414. The 螖 hkmt of mutant strain was 5.00 IU/m L and 15.00 IU/m / mL higher than that of the original strain T.reesei QM9414, and the 螖 hdac of the mutant was 6.50 IU/m L and 15.00 IU / mL higher than that of the original strain T.reesei QM9414 in each stage, respectively. The FPA and CMCA of the interfering recombinant strain T.reesei-gcn5-T10 were significantly lower than that of the original strain T.reesei QM9414, which were 10.00 IU/mL and 5.20 IU / mL 路(3) RT-qPCR, respectively. The relative expression of cellulase gene cbh1negl1 and enzyme activator xyr1 in 螖 hkmt was higher than that of original strain T.reesei QM9414, 4.51 times higher than that of T.reesei QM9414, 3.87 times and 2.51 times higher than that of original strain T.reesei QM9414, respectively. The expression of cbh1negl1 and xyr1 genes in 螖 hdac was also higher than that of T.reesei QM9414, which was 6.50 times higher than that of T.reesei QM9414 and 4.51 times higher than that of T.reesei QM9414, respectively. However, the expression of cbh1egl1 and xyr1 genes in T.reesei-gcn5-T10 was lower than that of the original strain T.reesei QM9414, which was 0.34 times and 0.44 times of the original strain, respectively. (4) the results of RT-qPCR analysis of hkmttn5 and gcn5 showed that the expression of HAT decreased. As a result, the expression of hdac increased and the expression of hkmt increased. Conclusion the methylation modification and deacetylation modification of histone were inhibited after the knockout of the two genes, and the expression level of cellulase gene cbh1egl1 and activator xyr1 increased significantly, and finally the expression of cellulase was activated. After gcn5 was interfered, histone acetylation modification was inhibited, and the expression of related genes was significantly decreased, which resulted in the decrease of cellulase activity.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55
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