Cloning And Functional Analysis Of Genes Encoding Heterotrimeric G Protein And Phospholipase C 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 play an important role in regulating the growth,development and pathogenicity of fungal pathogens.Five genes, which encoded different key components in signal transduction pathway,such as Gαprotein,Gβsubunit and phospholipase C,were cloned in this research and all of them shared high amino acid homology with the corresponding protein in other pathogenic fungi. Genes encoding G alpha subunit were named as Stga-1,Stga-2 and Stga-3,and genes encoding G protein beta subunit and phospholipase C were named as Stgb-1 and StPLC-1 respectively.Functional analysis of three genes was explored by constructing the gene replacement vector or anti-sense expression vector,which was used to create gene mutants. As a result,these genes regulated growth,conidiation,pathogenicity,osmitic stress response,laccase activity,mating,secondary metabolism,respectively.This work is helpful to study and understand the other conserved genes' function in signal transduction pathway of S.turcica.In addition,Stga-1 gene and Stgb-1 gene was respectively expressed in pET prokaryote gene expression system and it laid a solid fundation for analyzing the protein structure in vitro and exploring their expression model in vivo.Main results in this paper were as follows:1.Three Gαgenes(Stga-1,Stga-2,Stga-3),one Gβgene(Stgb-1)and one phospholipase C-encoding gene(StPLC-1)were cloned with the candidate gene cloning strategy.Among them,we had cloned the full length DNA and ORF of Stga-1,Stga-2 and Stgb-1 and also obtained partial coding region of Stga-3 and StPLC-1.The ORF of Stga-1 and Stgb-1 had been deposited in the GenBank/EBI Database under accession No. EF407554 and No.EF407555.2.Stga-1 included a 1249 bp DNA sequence with a 1062 bp coding region,4 exons and 3 introns and its predicted protein contained 353 aa.Stga-2 gene included 1318 bp and interrupted by 4 introns and its ORF of 1071bp encoded 356 aa.Stgb-1 gene of 1296 bp encoded a protein of 351 amino acid residues and was interrupted by four introns.All introns were accordance with GT-AG rules.3.Homology analysis had showed Stga-1,Stga-2 and Stga-3 possibly belonged to three different groups of G protein.4.Prokaryote gene expression vectors of Stga-1 and Stgb-1 was constructed, respectively,pET-28a(+)vector,E.coli BL21,and the target gene expression were confirmed by SDS-PAGE and Western blotting.5.Southern hybridization results have showed all these genes had single copy in the genome of S.turcica.6.The Stga-1 gene replacement vector was constructed based on the gene homologous combination theory and PEG-mediated gene transformation system. Transformants were screened by hygromycin B resistance and PCR with specific primers corresponding to hygromycin phosphotransferase or Stga-1 gene.Although both wild type and mutant A-23 of Stga-lgene were similar in vegetative growth rate,the mutant A-23 exhibited light grey colony,dense mycelia,cell lysis in the center colony,evident reduction of HT-toxin activity,sporulation and laccase activity,losing pathogenicity and mating ability.Moreover,the mutant A-23 was more tolerant than wild type under the osmotic stress.7.The sense and antisense Stgb-1 gene expression vectors were constructed based on the plasmid pSO-I.Spore germination rate was calculated after being induced by quinic acid for 12h.The spore germination rate of mutant strain anti-GB-4 was only 16%,while that of the wild type was about 90%.8.The StPLC-1 gene replacement vector was also constructed based on the same strategy with Stga-1.StPLC-1 mutant named as C-1 exhibited light grey colony,fluffy mycelia,significantly reduced HT-toxin activity,and reduced sporulation.It was similar with wild type in laccase activity and mating ability.Its pathogenicity was weakened significantly.The decreased growth rate of the mutant was recoverable after CaCl₂ was added into its medium.The above results can summarize the function of these genes:1)Stga-1 and StPLC-1 gene co-regulated the HT-toxin activity,pathogenicity and the osmotic response;2) StPLC-1 controlled the hypal growth by activating calcium signal pathway;3)Stga-1 gene regulated mating,pigmentation and laccase activity by activating other effectors except for StPLC-1;4)Stgb-1 gene was required for spore germination.