Mycoparasitism-associated Genes Of Gliocladium Catenulatum HL-1-1Associating With Sclerotia Of Sclerotinia Sclerotiorum

Gliocladium catenulatum, which distributes worldwide, is an important mycoparasite and has showed great potential for biocontrol of many plant fungal pathogens. In order to investigate mycoparasitism-related genes, a subtractive cDNA library of G catenulatum HL-1-1associating with sclerotia of Sclerotinia sclerotiorum was constructed in our lab and differentially expressed sequences were analyzed.504positive clones from the library were sequenced by randomLy, and303valid sequences of ESTs with the average size of423bp were gained after removing vector sequence and discarding fragments less than100bp. After sequence assembly,128unigenes, including23contigs and105singletons, were obtained. Sequence alignment of the unigenes was performed using Blastx in a non-redundant protein database in NCBI, and32deduced amino acid sequences were similar to some known functional proteins, including cytochrome P450, ribosomal protein, heme oxygenase, perilipin and endoglucanase which related to organism response under certain stress conditions.85unigenes had homology with hypothetical proteins derived from various genus and species; however their biological functions were not clear. No any homologous sequences were found for the other11unigenes in NCBI, indicating that these sequences might be fragments of novel genes.Two genes coding hypothetical proteins6-61and8-13and endoglucanase gene8-20were chosen from128unigenes, and their function during parasiting on sclerotia of S. sclerotiorum were investigated. Expressions of the three gene fragments were quantitatively monitored by real-time PCR using SYBR Green I. The results showed that expression level of6-61decreased when HL-1-1was cultured in sclerotia powder broth, and the lowest value was achieved at96h. The trend of gene8-13expression decreased firstly and then increased, and the level was higher than that without induction. The expression level of endoglucanase gene8-20increased as the incubation continued, and it was9times higher at96h than that at0h. The determination of relative expression levels indicated that the three genes might involve in the mycoparasitism of G catenulatum to sclerotia.The full-length cDNA of genes of6-61,8-13and8-20were completely cloned by RACE technique. Bioinformatics analysis demonstrated that cDNA of6-61was1247bp in length (GenBank accession number JQ811207) and encoded129amino acids. The function of the deduced protein which had high homology to hypothetical protein from Metarhizium acridum CQMa was still not known. The cDNA sequence of8-13included1308bp (GenBank accession number JQ811208) and encoded176amino acids, whose sequence was high homology to hypothetical protein from Penicillium marneffei ATCC18224. The sequence of gene8-20, named eg8-20, was1479bp in length (GenBank accession number JQ728996), including1269bp of open reading frame which encoded426amino acids. The deduced amino acid sequence of eg8-20was94%identical to endoglucanase from Trichoderma sp. SSL.An eg8-20gene-deficient mutant ED-3and a8-13gene-deficient mutant UD-6were constructed by inserting resistant gene G418into HL-1-1genome using homologous recombination technique. The wild and gene-deleted strains were similar in growth rate and sporulation. However, mycoparasitism of ED-3to S. sclerotiorum was greatly decreased to grade2from grade4by wild type, and UD-6decreased to grade3. The results demonstrated that endoglucanase8-20and hypothetical protein8-13secreted by G catenulatum HL-1-1played an important role in mycoparasitism to S. sclerotiorum.