Hsp70参与调控真菌漆酶基因转录研究

The 70 kDa heat-shock proteins (Hsp70s) are central components, which are mainly expressed in response to different cellular and environmental stresses in fungal cells. Hsp70s assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, etc. However, little information is gained about its role as a DNA binding transcriptional activator of fungal genes in response to extracellular stresses. Previously, we found that two of the five Hsp70s (designated as ThHspA1 and ThHspA7) in Tramets sp. AH28-2 may act as DNA binding proteins and are involved in regulation laccase (lacA) expression during induction with o-toluidine and guaiacol, respectively. In order to know more about Hsp70s, in this proposal, we plan to induce lacA production with more types of phenolic compounds, to elucidate the relationship between Hsp70 expression and the types/characteristics of phenolic compounds. For example, whether there is always a type of Hsp70 acts as a DNA binding regulator to regulate lacA expression after phenolic compound exposure? Then, we will study on the interaction between the Hsp70s and their binding sequences through DNA footprinting, EMSA, and protein crystallography technique, etc, to elucidate the mechanism by which the Hsp70s regulate the expression of lacA. The results are supposed to improve our understanding of Hsp70 that acts as a DNA binding protein, and will provide some clues on exploring the mechanism of differencial laccase regulation.

70kDa热休克蛋白（Hsp70）是真菌细胞响应压力的关键蛋白。随着Hsp70新的亚型被发现，Hsp70的新功能也被逐渐发掘，但对其作为与DNA结合的转录因子功能目前知之甚少。栓菌AH28-2基因组编码5个Hsp70，其中ThHspA1和ThHspA7可能作为DNA结合蛋白，分别参与邻甲苯胺和愈创木酚压力下的漆酶lacA表达调控。本项目拟以栓菌AH28-2的5个Hsp70为研究对象，选择多种类芳香化合物，诱导漆酶lacA表达，并通过分子生物学等技术，探明在所选化合物暴露下是否均有Hsp70调控lacA表达，不同条件下参与调控的Hsp70类型与化合物类型/结构之间是否存在联系；明晰Hsp70通过何种方式、识别何种DNA序列进而参与调控漆酶lacA表达。研究结果有助于深入理解Hsp70参与调控基因表达的功能，并为揭示芳香化合物压力下漆酶基因差异表达调控机制研究提供新线索。

热休克蛋白70（Hsp70）是真菌细胞响应压力的关键蛋白，但其作为与DNA结合的转录因子功能知之甚少。前期，我们发现栓菌AH28-2基因组编码的2个Hsp70可参与芳香化合物压力下的漆酶LacA表达调控，本项目综合使用分子生物学、生物化学和细胞生物学的多种技术方法，获得可与DNA直接结合的Hsp70并研究其调控lacA转录的机制。AH28-2基因组中共有5个Hsp70，多种芳香化合物暴露下的lacA转录模式分析显示，不同结构芳香化合物对应不同的Hsp70表达；进一步，通过基因干扰和过表达实验，确定ThhspA1可响应芳香化合物特别是邻甲苯胺，进而调控lacA的转录和细胞壁抵抗芳香化合物的能力；制备获得重组ThhspA1和ThhspA5纯蛋白，结合EMSA和荧光偏振分析确定ThhspA1的核心DNA结合序列为lacA启动子区的一段含XRE元件的GC富集区；ThhspA5不直接与lacA启动子区结合，但可与ThhspA1相互作用；ThhspA1的核苷酸结合结构域和多肽结合结构域均参与其与DNA的结合，而C端无序区不参与；ThhspA1与DNA结合的亲和力与蛋白的聚合形式相关，且其与DNA的结合部位不同于多肽结合部位；进一步的多组学研究和酵母单杂交筛选结果表明，磷脂酰肌醇信号通路和多种蛋白共同参与了邻甲苯胺暴露下的LacA的表达调控过程。我们的结果有助于理解Hsp70参与调控基因表达的功能，并为揭示芳香化合物压力下漆酶基因表达调控机制研究提供新线索。