草酸青霉新调控基因POX01907调控生淀粉糖化酶基因表达的分子机理研究

淀粉加工中用生淀粉糖化酶可降低生产成本。前期研究发现草酸青霉突变体ΔPOX01907的生淀粉酶产量下降77.6%、一个特性优良的生淀粉糖化酶PoxGA15A的基因表达下调93.5%。POX01907是未知功能的新蛋白质。本项目拟通过凝胶迁移电泳、定点突变等证实POX01907对PoxGA15A基因表达的直接调控和POX01907的DNA结合结构域及关键氨基酸。构建POX01907不同序列删除的突变菌株，鉴定POX01907结构域的功能。通过DNA亲和纯化测序、定点突变、ΔPOX01907的转录组分析和突变体构建及分析等鉴定POX01907的直接靶标基因、结合的DNA序列和关键碱基及下游关键调控基因。通过串联亲和纯化-质谱、酵母单杂交、Pull down、Co-IP分析等鉴定POX01907的靶蛋白及调控POX01907基因表达的上游调控蛋白，对认识真菌的基因表达调控机理有重要的科学意义。

Employment of raw starch-digesting glucoamylase (RSDG) in the processing of starch can greatly reduce production cost. In our previous work, a Penicillium oxalicum mutant ΔPOX01907 was found to show 77.6% reduction in raw starch-digesting amylase (RSDA) production compared with the parental strain ΔPoxKu70, and 93.5% downregulation of the transcript of gene PoxGA15A encoding a highly active RSDG. POX01907 is a novel hypothetical protein with unknown function. In this proposal, electrophoretic mobility shift assay (EMSA) and site-directed mutagenesis will be employed to confirm the direct regulation of expression of the gene PoxGA15A by POX01907, DNA-binding domain of POX01907 and the contained key amino acids. The function of each domain contained in POX01907 will be determined through the construction of a series of POX01907 mutants with nested deletions in P. oxalicum parental strain ΔPoxKu70 and RSDA activity measurement of the resultant mutants. The direct targets and DNA motif bound by POX01907, the key bases in the motif and the key regulatory genes acting downstream of POX01907 will be identified through DNA affinity purification sequencing, site-directed mutagenesis, transcriptomic sequencing of mutant ΔPOX01907 in comparison with that of the parental strain ΔPoxKu70, mutant construction and analysis. Moreover, target binding protein(s) of POX01907 and protein(s) regulating the expression of gene POX01907 will be identified through tandem affinity purification-mass spectrometry and yeast one-hybrid assays, Pull down and Co-IP experiments. The expected outcome of this suggested project will help to understand molecular regulatory mechanism of gene expression in fungi.