Study On The Function Of Regulatory Factor SaraC In Stereum Hirsutum

Recent years,transcriptional regulatory factors have been widely used in the research of fungal secondary metabolites.Among them,global regulatory factors with epigenetics modification functions are hotspots of current research.Lae A has successfully implemented genetic operations in many fungi,thus some new metabolites were discovered,and the yields of part active compounds(such as penicillin,lovastatin,and sterigmatocystin)were increased.Therefore,the discoveries of more similar global regulatory factors not only help us better understand mechanisms of fungal metabolism regulation,but also lay the molecular theoretical basis for the deep excavation of fungal secondary metabolites.This dissertation focus on Stereum hirsutum FP-91666: a gene,which postulates a transcriptional regulator from its genomic information,is selected from further research and named Sara C.After analysis of its protein sequence and conserved domain,we found that the Sara C contained zinc finger structure domain of methyltransferase,and Sara C showed very low homology with other genes.Further,according to the genetic family corresponding to the conservative sequence and the gene cluster analysis,Sara C shows individual clustering and has certain genus specificity.Due to the difficulty of genetic manipulation of S.hirsutum,we constructed two over-expressing strains by Agrobacterium-mediated genetic manipulation,and detect the colony morphology,growth rate,and resistance to stress.The function of Sara C gene was analyzed by a combination of methylationomics,mranscriptomics and metabonomics.In addition,we obtained Sara C protein by heterologous expression in Escherichia coli and assayed the enzyme activity of DNA demethylation by LC-MS detection.The main results are as follows:(1)Two transformants(SA4 and SA6)of over-expressing Sara C were obtained.After PCR verification and sequencing,the results showed that the Sara C were insertedinto SA4 and SA6 genome with GPDA promoter,and the transcriptional levels of two transformants were significantly up-regulated.Moreover,the transcriptional levels of SA4 and SA6 increased nearly triple at 15 d.(2)When evaluating its colony pattern and growth rate,the pigment production of SA4 was increased significantly and the growth rate was slightly faster than that of wild type after 5d,while the growth rate of SA6 was the slowest.(3)SA4 and SA6 showed very sensitive to cell wall synthesis interfering agent SDS,but generally sensitive to Congo red.And SA4 and SA6 grew better than the wild type on TG plates plus hypertonic reagents sorbitol and sodium chloride,and the pigment yield is significantly increased: On the TG plate containing 0.75 M sorbitol,SA4 and SA6 colonies also showed a significant increase in pigment yield.Therefore,Sara C may be involved in pigment synthesis under fungal hyperosmotic environment and oxidative stress,and Sara C may play a negative role in cell wall synthesis.(4)The data of the methylationomics revealed that SA4 and SA6 had higher levels of ultra-low methylation at the four sites of Cp G,CHH,CHG,and 6m A.And GO/KEGG enrichment analysis shows that the methylation of Cp G and 6m A loci was highly correlated with transcriptional regulatory genes.Transcriptomics data showed that many genes related to transcriptional regulation of SA6 were increase at 10 d,as well as those genes related with secondary metabolism(SM)biosynthesis.The expression of core SM genes in six gene clusters was increased by more than 1.5 times,and some were even more than triple.Metabolomics further conformed to the results of the methylationomics and transcriptomics.SA6 can produce more significantly different compounds,and these compounds were hardly produced or the yield was extremely low in wild type.Based on the above results,we presumed that Sara C is a transcriptional regulatory factor,which functioned by demethylation of DNA.(5)Sara C protein was obtained by heterologous expression in Escherichia coli and assayed the enzyme activity of DNA demethylation by LC-MS detection.In vitro enzyme activity experiments,the substrate peaks were detected in the in control groupand the substrate peak of demethylation could not be detected.But in the experiment group,the products of demethylation were detected.Thus the result indicated that Sara C protein can be demethylated on the ds DNA.