干扰MHR1对嗜热毁丝霉纤维素酶活性的影响

The effect of RNA interference of MHR1 on cellulase activity in Myceliophthora thermophila

利用RNA-Seq技术,筛选到潜在的负调控因子MHR1(fungal_TF_MHR1).为研究MHR1与纤维素酶基因表达之间的关联,设计基因mhr1的RNA干扰序列,并使用丙酮酸脱羧酶强启动子pdc成功构建mhr1基因的RNA干扰表达载体,经原生质体转化,获得一株干扰效率较高的阳性转化子MtR5.通过RT-q PCR、酶活测定和胞外蛋白浓度测定等分析发现,在诱导和非诱导培养条件下,转化子MtR5的胞外蛋白浓度、纤维素酶活性以及主要纤维素酶基因的表达量均高于野生型的.干扰转录因子MHR1可增加纤维素酶基因的表达量,研究表明,MHR1是一种与纤维素酶基因表达调控相关的转录因子.

The potential negative regulatory factor MHR1 was screened by RNA-Seq technique. A positive transformant MtR5 was successfully obtained by constructing the RNAi expression vector for mhr1 through the designing of the RNA interference sequence aimed at mhr1 and the use of the strong promoter of pdc gene. Real-time quantitative PCR,enzymatic and protein concentration determination show that the cellulase activities,extracellular protein concentration,and main cellulase genes expression of the transformant MtR5 are higher than those of the wild type under both inducing and non-inducing conditions. The above results elucidate the relationship between MHR1 and cellulase gene regulation for the first time and the association between the protein MHR1 and carbon catabolite repression. MHR1 works as a novel transcription factor associated with the cellulase gene expression and it is a repressor in carbon catabolite repression.