Cloning Of Glyceraldehyde-3-phosphate Dehydrogenase Gene And Establishment Of Agrobacterium-Mediated Transformation System Of Rhizoctonia Solani

Rice sheath blight, which is caused by Rhizoctonia solani is considered to be the most destructive disease in rice all over the world. Studies of the genetic, pathology and ecology of this pathogen have been hampered by the lack of an efficient transformation system.In recent years, there were few reports showing that developing a transformation system for R. solani. Using strong promoters to express heterologous genes in appropriate hosts is a major strategy in biotechnological applications. GPD (glyceraldehyde-3-phosphate dehydrogenase) can represent up to 5% of the soluble cellular protein content in Saccharomyces cerevisiae and other eukaryotes. Furthermore, GPD mRNA accounts for 2-5% of the poly (A)+ RNA present in baker's yeast. These observations implied that the GPD gene was regulated by a highly active promoter. As a matter of fact, the homologous GPD promoters have been widely used in directing the expression of heterologous genes in yeast, basidiomycetous and filamentous fungi.In this study, a glyceraldehyde-3-phosphate dehydrogenase gene was isolated from Rhizoctonia solani using degenerate PCR and genome walking. The complete GPD sequence (from ATG to TAA) was 1,531 bp in length and contained ten introns. The locations of these ten introns were similar to those of other anastomosis group's Rhizoctonia solani, which might reflect the evolutionary divergence of these fungi. The corresponding protein of 343 amino acids shows high homology to the other known GPD genes and is 85.52% identical to the gyceraldehyde-3-phosphate dehydrogenase of AG-3 anastomosis group. Southern blot analysis suggested that there existed only one copy of the GPD gene in the genome of Rhizoctonia solani.The 5'-flanking region of sGPD was cloned by PCR. Analysis using TFSEARCH showed that there existed one core promoter region and many cis-acting regulatory elements. The result of PlantCARE and TFSitescan service analysis indicated that several transcription factors were in this region. It can be concluded that it's most probably a promoter sequence.Three special vectors were constructed with a modified hph gene for R. solani. This modified gene contains Laccase introns in the 5'and 3' untranslated regions, or several A-C conversions in the 5'part of the coding region and is driven by the R. solani GPD promoter. Blended mycelium and mycelium pellets were chosen as explants for Agrobacterium tumefaciens-mediated transformation (ATMT) of R. solani. The results showed ATMT was successfully applied to the mycelium pellets. However, these resistant colonies stopped growing after a short interval and failed to show any further growth even when transferred to fresh media. The results showed that the transgene had not integrated into the endogenome.