Recombinant Protein for Biocontrol of Brown Rot Disease in Potato

In Egypt, potato has an important position among all vegetable crops. Potato crop is infected with the brown rot disease producing a major problem which caused by Ralstonia. Solanacearum. Soil samples were collected from Gharbia governorate, bacterial isolation was carried out using suitable media. Many bacterial isolates were obtained and used for bioagent against R. solanacearum. One isolate among all the examined isolate showed high antagonism activity against the R. solanacearum. This isolate was subjected to identification using the 16S rRNA gene and the sequence analysis revealed that the strain is Pantoea agglomerance. The bacterium was cultivated on PBG medium and the culture filtrate was fractionated, the entire fractions were used for the antagonism activity. One of these fractions showed high ability to control R. solanacearum (named Biocine). The Molecular weight of the purified Biocine was determined by SDS polyacrylamide gel electrophoresis revealing one band with a molecular weight of 29kDa. Differential display was used to study the Biocine regulating gene. Band in molecular weight 800bp were excised, cloned using TOPO TA cloning kit. The fragment was released using the EcoR1 restriction enzyme and subcloned into prokaryotic expression vector (Ptrac A). The recombinant bacteria were induced using the IPTG and the purified protein was obtained based on the His-tag technology. Bioassay was carried out for the purified recombinant protein compared with the wiled one, the same activity was shown. It could be concluded that production a high amount of biocine on semi-industrial scale available. To transfer the experiment from lab to the field are