Ustilagionidea virens is a common rice pathogen, which causes rice false smut. G protein may involve in the pathogenicity of U. virens. To study the role of G protein of U. virens in the pathogenicity, a gene that encodes G protein βsubunit had been isolated and characterized. The degenerate primers based on conserved domains of other reported genes were designed to obtain a homologous fragment. Thermal asymmetric interlaced PCR (TAILPCR) was used to isolate flank sequence of the homologous fragment. The gene was obtained by jointing all the fragments and named UvGβ1. Full length of UvGβ1 was 2037 bp, which contained 4 introns, 5 extrons, and an open reading frame (ORF) encoding 359 amino acids. The cDNA sequence containing entire ORF of UvGβ1 was cloned through RTPCR by primers based on the sequence of UvGβ1. The DNA and cDNA sequences of UvGβ1 have been registered in GenBank with accession numbers GU014921 and GU065745. Phylogenetic analysis showed that UvGβ1 had the nearest relationship with the G protein βsubunit gene of Cryphonectria parasitica. The ORF of UvGβ1 was subcloned into pET30a and was expressed in E. coli BL21. The recombinant protein was obtained by IPTG inducing.
Liu Y G, Huang N. Efficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR. Plant Mol Biol Rep, 1998, 16: 175-181.
Gilman A G. G proteins: Transducers of receptor-generated signals. Annu Rev Biochem, 1987, 56: 615-649.
Nishimura M, Park G, Xu G R. The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea. Mol Microbiol, 2003, 50: 231-243.
Ganem S, Lu S W, Lee B N, et al. The G protein β subunit of Cochliobolus heterostrophus involved in virulence, asexual and sexual reproductive ability andmorphogenesis. Eukaryot Cell, 2004, 3: 1652-1663.
Jain S, Akiyama K, Kan T, et al. The G protein β subunit FGB1 regulates development and pathogenicity in Fusarium oxysporum. Curr Genet, 2003, 43: 79-86.
Yu H Y, Seo J A, Kim J E, et al. Functional analyses of heterotrimeric G protein Gα and Gβ subunits in Gibberella zeae. Microbiology, 2008, 154: 392-401.
Kasahara S, Nuss D L. Targeted disruption of a fungal G protein β subunit gene results in increased vegetative growth but reduced virulence. Mol Plant Microbe Interact, 1997, 10(8): 984-993.
Muller P, Leibbrandt A, Teuissen S, et al. The G β-subunit-encoding gene bpp1 controls cyclic-AMP signaling in Ustilago maydis. Eukaryot Cell, 2004, 3: 806-814.
Sagaram U S, Shim W B. Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein β subunit, is associated with fumonisin B1 biosynthesis and hyphal development but not with fungal virulence. Mol Plant Pathol, 2007, 8: 375-384.