A Putative Stress Regulatory Gene Agsebl Involved In Asexual Development,Salt Stress Response And Secondary Metabolisms In Aspergillus Glaucus

Fungi possess sophisticated regulatory systems for responding to a vast array of environmental signals.Their developmental program and secondary metabolites always change synchronously for rapidly adapting to various ecological niches and satisfying reproduction and survival needs.The numerous environmental signals including light,ambient pH,nutrient,temperature,osmolality and oxidative stress.Aspergillus glaucus HB1-19,a marine filamentous fungi,produces a novel anti-tumor anthraquinone derivant named aspergiolide A.We found that aspergiolide A biosynthesis was related to the asexual stage which somehow induced by salt stress.The conidiation and production of aspergiolide A were both enhanced in proportion to increased concentration of artificial seawater(0?100%).These responses to stressful stimuli usually result from transcriptional regulation of specific genes in mutil-layers.msn2/4 of Saccharomyces cerevisiae was reported to regulate a wide variety of genes which associated with multi-stress response through binding the STRE(stress response elements)of their promoter.Thus,it possibly exist the same functional gene in A.glaucus,and participate in stress response regulation,then influence the asexual development and secondary metabolisms of A.glaucus as well.In order to verify this speculation,Agsebl as the msn2/4 homologous gene was identified from A.glaucus based on amino acid alignment,degenerate PCR and Hi-tail PCR.The Agsebl ORF is 1855 bp with an intron at 545-659 bp site,which encodes a protein of 579 amino acids.Sequence alignment of protein shows that Agsebl shares high identity with the homologs in other Aspergilli:A.ruber 94%,A.oryzea 61%,A.niger 60%,A.nidulans 55%.Additionally,it only shares 59%and 54%identity with Msn2p and Msn4p of S.cerevisiae.However,two highly conserved zinc-finger DNA-binding domains was found at 480-503 aa and 509-531aa sites which shows 74%and 62.5%identity with Msn2p.On the basis of the full length and flanking sequence of Agsebl,?Agseb1 strain was constructed through homologous integration of double crossover.Analysis of ?Agseb1 strain was carried out by three aspects,macroscopic and ultrastructual morphology,product biosynthesis and developmental state under salt stress.The results showed that,the Agsebl deletion strain of A.glaucus exhibited retarded colony growth with a smaller-size and unusual reduced peripheral edge with a uniform length.This morphology presented in contrast to wild type strain of A.glaucus for which the peripheral hyphae diverge as radial extention and differ in length.Meanwhile,the ?Agsebl produced abnormal hyphal cells with hyperbranching at new sites.The colonies of ?Agsebl strain also produced reduced conidiation instead of copious cleistothecium when the wild type strain formed a amount of conidiophores.It indicates that the deletion mutant switched its former asexual development to sexual development.Meanwhile,defects in conidial heads were found in ?Agsebl strain through scanning electron microscopy(SEM)detection.Apparently,the conidiospores inclined to separate from the phialides as long chains instead of individual part which probably resulted from AgabaA down-regulating.Besides,cell structure observation revealed that the deletion mutant was more electron dense than equivalent layer in wild type strain using trainsmission electron microscopy(TEM).Outer surface of cell wall in the deletion strain was more smooth,as opposed to wild type strain where presents a rough surface.All these phenotypes indicates that Agseb1 affects hyphal growth and possibly participates in later regulation of asexual development.In addition,the disfunction of Agsebl also drastically reduced aspergiolide A production of A.glaucus through severely down-regulating the expression of the key biosynthetic gene Agpks1.Compared to wild type strain,the transcription of Agbr1A,a crucial gene in central regulatory pathway of conidiogenesis in ?Agsebl was increased implying its derepression from Agsebl.But AgabaA,AgwetA were both deceased which caused the overall weakening of asexual development as well as the formation of abundant cleistothecium.Moreover,the deletion mutant showed decreased sensitivity to salt stress(both sorbitol and NaCl).Taken together,these data strongly suggested that Agseb1 is not only playing a direct role in response to high salt stress but also involved in the conidiation and aspergiolide A synthesis.