[Cloning, prokaryotic expression and bioinformatics of Rspg1 gene of Rhizoctonia solani].

ABSTRACT

OBJECTIVE: The study was aimed at understanding the roles of polygalacturonases in the pathogenicity and the interaction between Rhizoctonia solani and rice. METHODS: According to the sequences of Rspg1 of R. solani deposited in GenBank, a pair of specific primers was designed. The gene Rspg1 was cloned and expressed using prokaryotic expression tool to elucidate its biological characteristics. The structures of the protein RsPG1 were predicted using bioinformatics tools. RESULTS: A 1395-bp fragment including an open reading frame (OFR) of Rspg1 was amplified from the genomic DNA of the pathogen. Compared with RT-PCR results, it was found that this sequence fragment contains five introns (positions 278-334, 545-601, 657-715, 1090-1155 and 1244-1304) and one 1095 bp ORF. The ORF was predicted to encode 364 amino acids. Bioinformatics analysis showed that RsPG1 contains an 18-amino acid signal peptide and 4 conserved sequence segments (180NTD, 202DD, 223GHG and 255RIK) characteristic of all the polygalacturonases. The main structural elements of the secondary structure are alpha-helix, beta-sheet and random coil. Six cysteines form three disulfide bonds (Cys24-Cys40, Cys204-Cys220 and Cys329-Cys333). Transmembrane prediction analysis suggested that RsPG1 could be secreted outside the cell. Tertiary structure is a right-handed helix which consisted of ten repeated beta-sheet, forming an opening activity cleft. CONCLUSION: RsPG1 is tentatively a 40 kDa protein with polygalacturonase enzyme activity at 277.78 U/mg. It is probably a secreted protein and has characteristics of all the polygalacturonases. The results can help to further understand the roles that R. solani polygalacturonases play during the pathogenicity and how the pathogen interacts with the host.