解析病原真菌特有毒力基因及其调控网络将为新药靶的筛选和利用提供依据。烟曲霉中存在人源缺乏的亮氨酸合成途径，本课题的前期研究从烟曲霉随机突变体库中筛选到该途径中的Cys(6)Zn(2)型转录因子LeuB1，信息学分析表明LeuB家族有三个成员LeuB1,2,3但仅有LeuB1缺失导致低铁敏感、亮氨酸合成受阻、全局性蛋白降解。本课题拟进一步通过烟曲霉CRISPR-Cas9基因编辑技术，构建基因缺失、位点突变和原位加标签的LeuB系列菌株，研究LeuB家族调控亮氨酸合成的分子机制；并结合染色质免疫沉淀(ChIP-seq)、凝胶迁移分析（EMSA）证实LeuB家族转录调控的靶基因、探析LeuB家族及其转录活性与产生动物毒性的关系，进而阐明LeuB家族与亮氨酸合成的网络LeuA和LeuC的调控，解析LeuB家族影响烟曲霉生长发育和毒力的分子功能特征，为新型抗真菌药物设计提供理论依据。

New broad-spectrum antifungals, which display high effectiveness and reduced toxicity, are required. Since the nutrition of a pathogen during infection is essential for growth and maintenance within the host, new antifungals may be derived from the inhibition of fungal metabolism during pathogenesis. Therefore, targeting of pathways required for nutrient acquisition and metabolism could lead to new antifungals. Since mammals do not have the capacity to produce branched-chain amino acids, the fungal leucine biosynthesis system may be useful as a novel antifungal drug target. .In this project, based on screening T-DNA mutagenesis library, we found that the Cys(6)Zn(2) transcription factor family LeuB (LeuB1, 2 and 3) plays important roles in both leucine biosynthesis and iron regulation. According to BLAST analysis, LeuB1 in A. fumigatus has a deduced 920 amino acids sequence in length and belongs to a Cys(6)Zn(2) transcription factor family, which exclusively exists in fungi with a DNA-binding domain and a specific domain for a transcription factor. Deletion of leuB1 greatly reduces the hyphal growth rates and sharply decreases conidiation in the absence of leucine and/or under the iron starvation condition. These defects could be completely recovered with the addition of both leucine and iron in media. To further address how the leuB family regulates the leucine synthesis pathway and whether leuB family is an important virulence determinant in A. fumigatus, we will construct different strains including single and/or multiple leuB deletions, site-mutated, precise and efficient in-frame integration with GFP and His-tag based on homology-direct integration and a highly efficient CRISPR mutagenesis system. Through phenotypic observation under both liquid and solid cultural conditions, we next further clarify what is the relationship among this putative LeuB family members and how LeuB shows a dual-function transcription factor for both of leucine biosynthesis and the iron acquisition. Thirdly, according to approaches of Chip-seq (Chromatin Immunoprecipitation sequencing) combined with electrophoretic mobility shift assay (EMSA), putative target genes regulated by leuB as a potential transcript factor could be identified. Leucine is not only acts as a substrate for protein metabolism, but can also serve as signaling molecules during signal transduction. In eukaryote, proteasome mediated protein degradation pathway is crucial for the protein metabolism. Thus, we hypothesize that LeuB-deficiency resulted in dramatic protein degradation under iron starvation may be due to impaired leucine biosynthesis which activates the proteasome mediated protein degradation pathway. To verify this hypothesis, SDS-PAGE for whole protein extraction, Western blotting and related biochemistry assays will be carried out under the treatment of the proteasome specific inhibitor MG132 in vivo and in vitro. Finally, virulence tests for different leuB mutants will be carried out in both intranasally and intravenously infected mice model. We propose that the identification of LeuB-targeted genes with a yet uncharacterized link to leucine biosynthesis with iron as well as protein metabolism will aid in finding for new antifungal drug targets required for fungal adaption to host niches and virulence traits.