| Background:Filamentous fungi is a important kind of microorganism owning abundant secondary metabolic spectrum, including antibiotic, anti-cancer substance, the cholesterol class, immunosuppressor and so on. Filamentous fungi is a treasury of natural product deserved to be lucubrated about its regulation of secondary metabolism and growth. According to the research of recent years, we know that the secondary metabolic regulation of fungi is very complex, which is also regulated by the global regulator except pathway-specific regulator.LaeA, a global regulator was isolated and identified from the Aspergillus nidulans and the recent research proved that LaeA could regulate the secondary metabolism and growth,meanwhile exists in most of filamentous fungi. There were many works centring on the LaeA in recent years and confirming that LaeA regulates the global network by interacting with VeA and VelB. The confirmation of the heterotrimer LaeA/VeA/VelB leads the researcher to expand the global network regulated by LaeA.Pennicillium citrinum is the strain producting Mevastatin. Our group had do many works about the pennicillium including the culture of strain, building the genetic transformation system and the colony of LaeA. Come so far, the research about the pennicillium citrinum is limited to the colony and the primary functional characterization of LaeA home and abroad. However the research about the proteic interaction of LaeA which related to the global regulation was barely reported.Obejective:To investigate the interaction between LaeA and VeA in vitro; Confirm the role of SAM Domain in the global regulation of LaeA; Excavate the protein interacting with LaeA and build the primary global network by the bioinformatics analysis.Method:1. Heterologous coexpression was used to verified the interaction relationship between LaeA and VeA in vitro.2. Building the genetic deficiency of SAM Domain in LaeA and confirming the role of SAM Domain in the interaction between LaeA and VeA by the method of heterologous coexpression.3. The overexpression of LaeA(ΔSAM) is used to verified the function of SAM domain in the LaeA’s regulation of secondary metabolism and growth of fungi4. The tandem affinity purification and Maladi-TOF-TOF were used to find and confirm the protein interacting with LaeA.Result:1. We builded the vector PET-Duet-VeA-LaeA successfully and purified the VeA and LaeA by Ni, proving that the LaeA interacts with VeA in vitro.2. We coloned the LaeA losing the SAM Domain and builded the vector PET-Duet-VeA-LaeA(ΔSAM); We just obtain VeA protein after nickel affinity purification detected by SDS-PAGE, confirming that the SAM Domain is the key domain of LaeA for the interaction with the other proteins.3. We builded the Vector PGiHTGi-LaeA(ΔSAM) succsesfully and sented it into Penicillium citrinum through ATMT; The yields of mevastatin of OE::LaeA(ΔSAM)are lower than OE::LaeA and similar to the wild type detected by HPLC; The spore output of OE::LaeA (ΔSAM) is lower than the wild type by31%.4. We builded the vector PGiHTGi-TAP-LaeA successfully, sented into Penicillium citrinum by ATMT; We obtained five proteins interacting with LaeA after SBP and nickel affinity purification.5. We identified five proteins by Maladi-TOF after gel enzyme solution; we speculate the three of them relating to LaeA:DST1-like protein, Sey-1protein and CBS; DST1-like protein is a kind of zinc finger protein relating to transcription elongation; sey-1protein is a kind of GTP-binding protein involving in signal channel; CBS protein has a CBS Domain in charge of binding to the other protein.Conclusion:There are interaction exsiting between LaeA and VeA in vitro and SAM Domain is the key domain for the global regulation function of LaeA; LaeA regulats the secondary metabolism and sporulation by interacting with sey-1, DST1-like protein, CBS, and the SAM is the key domain for the global regulation function of LaeA... |
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