Cloning And Functional Analysis Of Genes Encoding Heterotrimeric G Protein Gamma Subunit In Setosphaeria Turcica

Setosphaeria turcica is a destructive pathogen of maize all over the world and causes significant yield losses. The universal signal tranduction pathways mediated by heterotrimeric GTP binding protein (G protein) in fungi plays an important role in regulating the growth, development and pathogenicity of fungal pathogens.Homologous fragment of G-proteinγsubunits gene in S. turcica was obtained by candidate gene method and Genome walking technology. The fragment length was 1129 bp and had 82% homology with G-proteinγsubunit of Phaeosphaeria nodorum and Pyrenophora tritici-repentis by BLAST analysis.The completed cDNA sequence of Stgγ-1 gene was obtained by PCR with specific primers designed on the full-length genomic DNA. There was one open reading frame composed of 97 codons and three deduced introns of 9, 50 and 104 nucleotides in the Stgγ-1 gene. Phylogenetic analysis showed that the fragments had the nearest relationship with Phaeosphaeria nodorum G proteinγsubunit gene. The gene encoding G gamma subunit was named as Stgγ-1.SignalP soft predicted that there was no signal peptide, and through TMPRED analysis, it was known that there was not a transmembrane domian in the deduced amino acid sequence of Stgγ-1 gene. The study to the second structure predicted that there are 43.3% alpha helix, 10.31% extended strand, 1.03% beta turn and 45.36% random coil.DNA Southern blot analysis indicated that there was a single copy of the Stgγ-1 gene in the genome of Setosphaeria turcica. Functional analysis of the gene was explored by constructing RNAi vector of the gene, which was used to create gene mutants.The RNAi vector was transformed into S. turcica protoplast and 20 transformants were obtained.