Abstract:

Soybean sharp eyespotis one of the most serious diseases in world. The protein encoded by G-protein β-subunit (Guanine nucleotide binding protein beta-subunit) gene plays an important role in pathogenesis mechanism. In this paper, the G-protein β-subunit from Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris)causing soybean sharp eyespot was identified. The genome of 1 864 and 1 047 bp open reading frame (ORF) were amplified by PCR and RT-PCR. The gene included 4 introns and 5 exons. Introns ranged in size from 54 to 65 bp, and their sequences complied with the rule of “5'-gt” and “ag-3'” (GenBank Accession No. EU663628). The ORF encoded 348-amino acid polypeptide with 38.24 kD of calculated molecular weight and 6.31 of pI. There were two alpha-helixes and seven beta-sheets including four beta-strands each in its amino acid secondary structure. Two alpha-helixes in its N-terminal and seven beta-sheets formed barrel structure by non-regular curl in the tertiary structure. The deduced amino acid sequence of β-subunit was identical to that from Rhizoctonia solani (GenBank Accession No. EU267677, AY884129), Lentinula edodes (GenBank Accession No. AAT74567), Coprinopsis cinerea (GenBank Accession Number EAU92269), Ustilago maydis (GenBank Accession Number AAN33051) and Filobasidiella neoformans (GenBank Accession No. AAD03596) with 99.43%, 89.19%, 87.97%, 83.66%, 80.23%, and 79.72%, respectively. The amplified ORF was ligated into the prokaryotic fusion expression vector pGEX-4T-2. E. coli BL21 was transformed with this recombinant vector and induced by IPTG for expression. The result indicated that the protein size of ORF matched the prediction. This cloning of this gene provides the evidence for controlling hyphal growth, development and virulence in R. solani.

Key words: Soybean, Rhizoctonia solani, Pathology, G-protein beta-subunit, Signal transduction