Functional Analysis Of The Host Peanut Specific Virulence Gene Of Rastonia Solanacearum

A 12.8 kb DNA fragment (pGX1252) which carried specific virlence gene to peanut(host specific virulence,/m>) had been cloned in our laboratory. Later this hsv gene was located in a 4.5 kb DNA fragment (pGX1404).In this work, a 3.0 kb DNA fragment (pGX3418), which includes only ORF3 and its promoter sequence was obtained by exonucleaelll unidirectional digestion for limited time. Plasmid pGX3418 was transfered into T2014 by triparental mating. The resultant transconjugant T2418 could cause wilting on peanut GuihualV. This confirms that ORF3 is a host specific virulence gene.There were two ATGs(ATGl ^ ATG2) in the same reading frame at the 5' region of ORF3. The distance of the two ATGs is 189bp.In order to know where the ORF3 promoter is located, about 200bp upstream sequence of the two ATGs were fused to GusA report gene in two orientations by recombinant PCR, respectively. The recombinant fragments were cloned in pLAFR3 which is a broad host range vector .Recombinant plasmids was transfered into T2014> T2015. Plate test confirms that the upstream sequence of ATG1 has promoter activity, but that of ATG2 does not.A multi-copy indigenous plasmid of Ralstonia solanacearum strain T2015 was isolated -, sequenced and analysed. This plasmid may be developed into a new highly efficient vector for Ralstonia solanacearum.