里氏木霉组成型表达siRNA干扰cre1基因对纤维素酶表达的调控作用

使用组成型siRNA干扰载体对里氏木霉碳阻遏抑制因子CRE1进行siRNA干扰以研究其对里氏木霉纤维素酶基因表达的调控作用。根据里氏木霉cre1基因序列设计siRNA干扰片段。利用里氏木霉组成型表达载体将干扰片段分别构建至里氏木霉cre1干扰载体并将其转化里氏木霉QM9414。分别在48和144 h对各转化子进行纤维素酶酶活力测试(CMC酶活力测试和滤纸酶活力测试)及利用qPCR检测相关基因的表达。在诱导144 h时转化子的两种酶活力平均约比出发菌株高出1倍。qPCR检测cre1基因的表达结果表明,转化子的cre1表达量比出发菌株平均降低约50%,而ace1基因表达量变化不大。其他纤维素酶相关基因的表达水平也均高于出发菌株。通过组成型表达siRNA干扰里氏木霉cre1基因可以明显调控纤维素酶基因的表达,为研究纤维素酶的基因表达与调控提供参考。

Trichoderma reseei Constitutive Type of Expression Vector siRNA Interfering cre1 Gene on Cellulases Expression Regulation

The constitutive type of siRNA interference vector was employed on carbon repressor (cre1) gene to carry out siRNA interference in order to study the expression regulation on cellulase gene of Trichoderma reseei. The siRNA interfering fragments were designed according to T. reseei cre1 gene sequence. And the constitutive type expression vector of T. reseei was used to construct T. reseei interfering vectors respectively with interfering fragments and converted to T. reseei QM9414. The cellulase activity of each convertor was tested at 48 h and 144 h respectively (CMC and filter paper enzyme activities tests) as well as the expression of correlative genes using qPCR determination. Two enzymatic activities of the transformants were about 2 times higher than the starting strain. The expression results of cre1 by qPCR test showed that the expression of transformant cre1 reduced by 50% in average as compared with the starting strain, while the expression of ace1 gene had little change. The expression levels of the other cellulase correlative genes were all also higher than the starting strain. Adopting constitutive type expression of siRNA to interfere T. reseei cre1 gene could significantly regulate the expression of cellulase gene; these had laid experimental foundation to study the cellulase gene expression and regulation.