Isolation And Characterization Of Promoters From Trametes Gallic

First of all, the sensitivity of Trametes gallic to hygromycin B was determined, the result showed that a hygroroycin B concentration of 100μg/mL can completely inhibit the growth of T. gallic at three representative culture media.Promoter-probe vector pSUPV8 was used to clone promoters from T. gallic. The chromosomal DNA of T. gallic digested by Sau3AI was ligated with BamHI-cut pSUPV8. The ligated mixture was used to transform E. coli XL-1 Blue. Eight hygromycin B -resistant clones (named pTP1-pTP8) were obtained. Restriction analysis indicated that all eight recombinants had inserts whose sizes ranged from 0. 6 to 1. 7kb. Their hygromycin B -resistant level was determined on different concentrations of hygromycin B-containing plates. The results demonstrated that the hygromycin B resistance level of these recombinants ranges from 1,50 to 350μg/mL. The recombinant pTP6 was chosed to be analysed via 10 different kinds of restriction enzymes. The result indicated that there are three restrition sites, KpnI, XhaI and Sad in the TP6 fragment with 1.7kb in size. Subcloning analysis of TP6 showed that deletion of the 0. 8kb or the 1. 2kb of 5'-terminal segment of pTP6, decreases the hygromycin B~resistant level from 2.50 to 1.50μg/mL It is deduced that there may be an upstream activator sequence in this 0.8kb segment. Southern hybridization using TP5 as probe showed that TP5 can hybrid with the total DNA of T. gallic but not with that of ?coli, indicating that TP5 fragment is from fromT. gallic. The 3'-terminal segment of TP5 fragment was a] so sequenced, The result indicates that it contains several sequences similar to both prokaryotic and eukaryotic gene promoter sequences. It also showed that the 5' -terminal segment of hygromycin phosphoric acid transferase can be changed without any influence on the activity of the expressed gene product. It is evident that this gene can be used as a reporter gene for the isolation of the promoters, determineation of the promoter efficiency and the study of the function of other DNA modulator element.