Cloning And Functional Characterization Of The Global Regulator VeA In An Anti-tumor Polyketide Deacetylmycoepoxydiene(DNA)Producing Fungus

Filamentous fungi are renowned microorganism which are able to synthesize a wide variety of secondary metabolites ranging from threatening mycotoxins such as sterigmatocystin to benign antibiotics such as penicillin. The study has found that fungal secondary metabolism is closely related to their morphological development under control of G-protein signal transduction pathway. In addition, the global regulator VeA play an important role in coordinating secondary metabolism with morphological development in this pathway.Phomopsis sp.G818, a rare marine endophytic fungi, has the specific metabolic pathways that can produce a range of novel secondary metabolites. DAM and MED are a group of the rare metabolites which have good anti-tumor activity and activation of AMPK activity. In order to further the development and use of the value of DAM, our group screened out a DNA fragment from the gencmic library of its producing strain based on the transcriptome differences library between DAM high-producing strain G818and DAM low-producing strain A123, and hypothesized the possible synthesis gene cluster of DAM.To further investigate and confirm the biosynthesis and regulation pathway of DAM, the present work explored the regulation of VeA on the morphological development and secondary metabolism in Phomopsis sp., especially DAM.Firstly, according to the known VeA homologous protein sequences, the veA fragment of Phomopsis sp. was successfully amplified by PCR using degenerate primers designed by CODEHOP programme. Then the whole veA gene sequences were obtained by the gene-specific primer using the LA-PCR technology and confirmed the ORF by means of softberry software and NCBI database. Sequence analysis showed that the veA ORF of Phomopsis sp. has a intron with length of64bp and can encode381amino acids.Secondly, the colony morphology, DAM production, veA and related genes expression of the two stains under different conditions be analyzed. The results showed that the expression levels of veA down-regulated, aerial mycelium significantly reduced, color deepened and mycelia tuberculosis formed under illumination. But the yield of DAM was no significant change. In addition, We found that veA as a positive regulator in the biosynthesis of the DAM. With the increased expression of veA in Phomopsis sp. G818, the expression of pks gene was detected and the yield of DAM is increasing uninterruptedly under darkness.Furthermore, the veA RNAi strains were constructed by means of pSilent-1plasmid vector. Results showed that the yield of DAM of RNAi mutants is decreased with the reduced expression of veA by detecting the expression levels of veA and the yield of DAM, which indicated veA genes promote the biosynthesis of DAM to some extent.The characterize and expression analysis of veA can be a foundation for the functional analysis of veA in Phomopsis sp.and the exploration of DAM biosynthetic pathway.