Molecular Cloning And Character Description Of The Global Regulate Gene Pci-veA In Penicillium Citrinum

Background:Filamentous fungi is an important class of microorganisms, some industrial strains can produce a lot of antibiotics for human benefit such as, organic acids, cholesterol-lowering substances, anti-viral substances, immunosuppressants anti-cancer substances and substances,it will also produce some secondary metabolites harmful to humans, such as handle music adriamycin, aflatoxins and other mycotoxins.Fungal secondary metabolite biosynthetic pathways mainly included the PKS and NRPS this two ways. secondary metabolite biosynthesis regulate genes are clustered exist, the control system of fungal secondary metabolite is a complex network. Regulation of fungal secondary metabolites of regulatory factors consist of:specific transcription factors which is located on the way to biosynthesis gene cluster, and the signaling pathways regulatory factors that not directly related to the biosynthetic gene cluster,the last one is the global regulative factor VeA and LaeA discovered and researches in recent years.Global regulatory factor VeA,was firstly found by E. Kfer et al in 1965 in Aspergillus nidulans with the effects of the proportion of sexual and asexual reproduction, a further study in recent years revealed that it not only effected the microbe morphological development of filamentous fungi, but also control in the production of secondary metabolites such as antibiotics and mycotoxins as a very important global regulator in filamentous fungi.its positive regulatory role of secondary metabolite and relative regulators some extent provids a important new channel to rational genetic reconstruction of fungi; it also contributed to activate the expression of cryptic gene clusters and resulted in generating novel products.it makes the genetic manipulation based on global regulatory factors searching for new natural products become a new direction.So far, veA homologous genes have been respectively cloned in Aspergillus fumigatus, Aspergillus parasiticus, Penicillium chrysogenum and Neurospora crassa.Statin drugs can not be compared because of the cholesterol-lowering effect, is the first choice of lipid-lowering drugs. Since 1987, lovastatin was approved for clinical by FDA, and became the first market using statins, a number of semi-synthetic and fully synthetic statins was developed, and many of them was approved by FDA. In addition to commonly used functions, the most important lipid-lowering statin drugs also discovered in many new therapeutic areas. Mevastatin was the first statin and was concerned as the main premise of other statins.Aim:In our study, a mevastatin producing strain M2, was cloned veA homologue Pci-veA using TELL-PCR and RACE technology, design different fragments of the size of the primer,and then analysis its bioinformatics. Analysis of its expression at different stages, the changes in biomass,growth rate changes, while observing the light and dark conditions, the impact on its morphology, and then infer the regulation of their relationship may exist between them.Methods:Based on highly conserved sequence regions, synthesized degenerate primers to amplify the specific fragment of veA with template first-stranded cDNA from P. citrinum mycelial samples. Using"Rapid Amplification of cDNA Ends" (RACE) technique amplification of the veA 3'cDNA ends and isolate the 5'-end of the veA gene fragement by TAIL-PCR (thermal asymmetric interlaced PCR) technique.Then Amplification of the full-length gene.Analyze the sequence character and its physiological functions in Penicillium citrinum.Result:This article successfully cloned a global regulatory veA homologue Pci-veA from Penicillium citrinum, the producer of extremely successful drug for the treatment of hypercholesterolemia mevastatin. The full-length Pci-veA DNA sequence is 1,774 bp in size, which contains an open reading frame (ORF) of 1704 bp encoding a peptide of 567 residues and the deduced amino acid sequence of Pci-veA shows 91% identity with that of Penicillium chrysogenum and 61%with that of Aspergillus clavatus respectively.The predicted molecular mass of Pci-veA is 62762.5 Daltons and theoretical isoelectric point is 9.10. The N-terminal region of the Pci-VeA also contains a putative classical bipartite nuclear localization signal (NLS) motif, NLSI and a pat7 motif as well as Aspergillus nidulans. Studies have shown that the expression of the gene in the growth of Penicillium citrinum expressed in the entire period, and inhibit its conidial production in lighe which contrast to in the darkness conditions.Conclusion:This article successfully cloned a global regulatory veA homologue Pci-veA from Penicillium citrinum,which may possible positive regulate mevastatin biosynthesis and effeces the growth morphology of Penicillium citrinum.Further characterize and fucnction analysis of Pci-VeA will be useful for understanding of molecular regulation mechanism of mevastatin biosynthesis.