多基因协同调控里氏木霉高效分泌表达蛋白的研究

里氏木霉诱变菌纤维素酶高产性状来自于多个基因的共同作用，但基因间是否存在协同调控及作用机理并不清楚。辨识出能协同调控高效分泌内、外源蛋白的重要基因组合及表达方式可大大加快里氏木霉的基因工程育种。高产菌CL847中四个关键转录因子(cre1、ace1、ace2和xyr1)的表达发生了有规律的变化。据此选定这四个关键因子作为模型对基因间能否协同调控内源纤维素酶的高效分泌表达进行研究。对每个基因，构建突变启动子文库（插入能增强转录的gpd box）或RNAi质粒分别多水平上调或下调基因的表达。将不同数目或组合的突变启动子文库和/或RNAi质粒同时转化木霉，在转化子中引入这四个基因表达水平组合的丰富生物多样性，筛选纤维素酶分泌极大提高的转化子，从而揭示所研究的调控基因间是否存在协同作用及它们的作用方式。对外源基因的表达选定黑曲霉脂肪酶为模型、关键因子hac1、ftt1和ftt2为靶点同法进行研究。

The hyperproducing ability of mutated Trichoderma reesei strains are from the actions of multiple genes. However, whether there is synergistic regulation and how it works among multiple genes are not clear. Thus the identification of the combinations of important genes and their expression styles, which can synergistically regulate high efficiency production of endogenous or heterologous proteins, can largely speed up the breeding of T. reesei by genetic engineering. In the hyperproducing strain CL847, the expression styles of four key transcription regulators, that are cre1, ace1, ace2 and xyr1, are changed not by random. Based on this, we choose these four key transcription regulators as a model to explore the synergistic roles among multiple genes in regulating high efficiency endogenous gene expression and secretion. For each of these four genes, either a mutated promoter library (by inserting the gpd box which has a transcription-enhancing effect) or an RNAi plasmid will be constructed, which will allow up- or down-regulation of the expression of these genes at multiple levels. Then different numbers and combinations of the mutated promoter libraries and/or RNAi plasmids will be used to transform T. reesei simultaneously. This will introduce abundant biodiversity regarding the combinations of the expression of the four genes. By screening transformants for largely improved cellulase producing ability, the synergistic effect among special gene combinations and their expression styles will be revealed. In addition, for the heterologous genes, we will choose the Aspergillus niger lipase as a model and three key regulators hac1, ftt1 and ftt2, as the target genes and use the same method as described above.