Cloning And Functional Characterization Of Thga1Gene Of Trichoderma Harzianum Th-33

Trichoderma harzianum is an important biocontrol filamentous fungus which can antagonize avariety of plant pathogenic fungi and play an extremely important role in the biological control of plantdiseases. The mechanism of T. harzianum antagonize plant diseases contain competitive, antagonism,parasitism and induction of host plant defense reaction. The above mechanism are closely related to theprocess of T. harzianum cell signaling. So research of the signal transduction system how T. harzianumresponse pathogens, pass the signal and the regulation of cell reaction process is great significance forelucidate biocontrol mechanism of T. harzianum. GTP protein-coupled signal transduction system is animportant signaling pathway in cell. A lot of evidence show that G protein involved in growth,sporulation, development, pathogenicity, secondary metabolism activities of filamentous fungi. Gprotein is composed of α, β, and γ subunits that each subunit encoded by independent gene. The Gαsubunit is numerous in variety and generally considered to be a functional subunit of G protein. Soresearch of Gα gene and its functional properties is of great significance for reveal the mechanisms ofsignal transduction and regulation in T. harzianum. This article clone the heterotrimeric G protein alphasubunit gene of class I by PCR from T. harzianum, perform bioinformatics analysis, get the knockoutmutants by gene knock, observed phenotypic changes and explored its function. The results are asfollows:The Gα gene is cloned from T. harzianum Th-33genomic DNA and get a1203bp homologousfragment. Using genome walking technology to extend the fragment and get3673bp Gα gene and itsflanking sequences ultimately. The Gα gene is1283bp. The full-length cDNA sequence is amplifiedwith T. harzianum Th-33cDNA as a template. The Gα gene is named Thga1and is submitted toGenBank (accession number: JN874387).The result of comparative analysis of DNA and cDNA sequences show a total of four exons andthree introns sequence of Thga1gene which encode a polypeptide consisting of353amino acidsequence. The alignment and homology analysis show that Thga1belong to class I of Gα gene and andshares100%and99%acid amino identity with TgaA(AAK74191.1) of Trichoderma virens andTga1(AAO18659.1) of Trichoderma atroviride, respectively.In order to construction the binary vector pDHt/sk-Gα-Hyg, using hygromycin gene as a selectablemarker and the genes flanking sequences at both ends of Thga1as the homology arms. The resultingplasmids are subsequently transformed into the genome of T. harzianum by agrobacterium tumefaciensmediated-transformant (ATMT). According to the principle of homologous recombination, thehygromycin gene expression cassette replace Thga1gene and two Thga1gene knockout mutants1-1and10-1are got by the molecules confirmation.The Phenotype analysis of two mutants: colony morphology change significantly on PDA medium.The colony is thin and mycelium is uneven distribution. growth rate of1-1,10-1are significantly slowerthan the wild-type Th-33. Compared wild-type, the conidia yield of two mutants are decreasesignificantly. The conidia yield of1-1is least and less two orders of magnitude compared to the wild-type. Two mutants are delayed20hours compared to wild-type Th-33and the branch are less. Theconidiophore branch of two mutants are very sparse, and that secondary branch and phialide are less.Antagonistic effect of the two mutants are analyzed: The inhibition of two mutants of T. harzianumagainst two species plant pathogen of Rhizoctonia solani, Phytophthora capsici is reduced significantlyand the mycelia of two mutants can’t cover and parasitize pathogenic fungi’s mycelia in confrontationtest. The inhibition of two mutants’ volatile inhibitory substances against two species plant pathogen aresignificantly lower than wild-type which inhibition rates of mutant1-1are7.42%and11.95%,inhibition rates of mutant10-1are8.46%and8.80%. The inhibition of two mutants’ non-volatileinhibitory substances against two species plant pathogen compared with wild-type Th-33is also reducedin varying degree, in which inhibition rates of mutant1-1against R.. solani is not reducing significantthan wild-type. But inhibition rates of mutant1-1against P. capsici and mutant10-1against two speciesplant pathogen are reducing significant than wild-type. The parasitism between wild-type Th-33and R.solani is observed, but this phenomena can’t be observed in two mutants.