草酸青霉纤维素酶基因的新关键调控基因POX01167的功能鉴定

甘蔗渣生物炼制可提高甘蔗糖业的经济效益，但微生物的纤维素酶产量低使其不能实现产业化。草酸青霉HP7-1能高效降解甘蔗渣纤维素，其突变体ΔPOX01167在含微晶纤维素Avicel培养基中几乎不产生纤维素酶，但在含麦麸加Avicel培养基中其正常产生纤维素酶。本项目拟通过比较分析ΔPOX01167和出发菌株ΔPoxKu70分别在含Avicel、和麦麸加Avicel培养基中的差异转录组，鉴定POX01167的调控元，筛选POX01167下游候选调控基因，通过在ΔPoxKu70中敲除和在ΔPOX01167中过量表达候选基因、突变体的酶活测定、凝胶迁移实验和体内报告基因检测，鉴定出下游关键调控基因。鉴定POX01167的DNA结合功能域的关键氨基酸残基和POX01167结合的DNA序列及关键碱基。对认识真菌纤维素酶基因的表达调控机理具有重要意义，为理性改造真菌、降低甘蔗渣生物炼制成本提供理论依据。

Biorefineries from sugarcane bagasse can improve economic performance of cane sugar industry, but it hasn’t been industrized because of low cellulase production from microorganisms. Penicillium oxalicum HP7-1 can efficiently degrade sugarcane baggase cellulose. A mutant ΔPOX01167 derived from P. oxalicum HP7-1 produces negligible cellulase in medium containing crystalline cellulose Avicel as the carbon source, while normally produces cellulase to the wild-type level in medium containing wheat bran and Avicel. In this proposed project, the regulon of the key regulatory gene POX01167 will be identified and the candidate regulatory genes acting downstream of POX01167 will be screened by differential transcriptomic analysis of ΔPOX01167 and background strain ΔPoxKu70 cultivated in media containing Avicel, and wheat bran plus Avicel as the carbon source. The screened candidate regulatory genes will be respectively knocked-out in ΔPoxKu70 and over-expressed in ΔPOX01167. The cellulase activity of the constructed mutants will be measured. The regulation of the found genes by POX01167 will be verified by electrophoretic mobility shift assay and reporter gene assay in vivo. Key regulatory genes acting downstream of POX01167 will be thus identified. Furthermore, key amino acid residues in the DNA-binding domain of POX01167, and bound DNA sequences and key bases by POX01167 will be characterized. .The expected outcome of this suggested project is of great theoretical significane in understanding regulation mechanism of cellulase gene expression in fungi, and thus provide a solid basis for rational genetic modification of fungal strains to reduce the cost of biorefinery from sugarcane bagasse.