Isolation And Identification Of Mycoparasitism-associated Genes Of Gliocladium Catenulatum HL-1-1 Parasitizing On Sclerotia Of Sclerotinia Sclerotiorum

Gliocladium catenulatum is widely distributed in the soil and is of great potential for biological control of crop pathogenic fungus as a mycoparasite on Sclerotinia sclerotiorum and other plant pathogenic fungi. To reveal the molecular mechanism for G. catenulatum HL-1-1 parasitizing on sclerotia of S. sclerotiorum, my laboratory constructed a library of differentially expressed sequences. This paper reports the results on differentially expressed genes that related to mycoparaitism .420 positive clones were sequenced by randomly selecting from the constructed library. The length of the inserted fragments was between 200 and 600bp in all clones. 391 valid sequences were generated with average size of 409bp after vector trimming and discarding of the sequences less than 100 bp. 181 unigenes, including 27 contigs and 154 singlets, were obtained after initial assembly. Blastx was performed against a non-redundant protein sequence database in NCBI. Results showed that 38 unigenes were functional genes, among them, some sequences encoded proteins similar to endoglucanase, perilipin-like protein, MFS transporter, peroxidase etc. Another 39 unigenes had no homology with predicted proteins deposited in database and therefore were supposed to be novel genes. Other genes encode proteins with homology with hypothetical proteins deposited in database, but the actual biological functions are unclear.2-99 and 8-3 are two genes supposed to be related with appressorium formation and appressorium turgo formation, repectively. In order to clarify the role of these two genes during mycoparasitic process, the author used SYBR Green I relative quantitative Real-time PCR to monitor the expression of the above two genes. Result showed that no change was observed for the expression of gene 2-99 after 24h induction by cell wall preparation, while the expression quantity was significantly enhanced after 48h treatment, and elevated almost 8 times after 96h. As for Gene 8-3, after 48h inducing treatment, the expression quantity was much lower than the original, but after 72h, the expression quantity was began increasing, and after 96h, the expression quantity elevated approximately 2.5 times. Quantitative results indicated that these two genes were involve in the process of mycoparasitism.We have also cloned the full-length cDNA of gene 8-3 and analysed it by using bioinformatics network resources. We predicted that 8-3 cDNA had a 555bp open reading frame (ORF) from the 314bp site to the 868bp site which codes a 184-amino acid (aa) residues without signal peptide, typical hydrophobic regions and transmembrane region. A perilipin superfamily box was also supposed to exist in the residues as main functional region which contained 72-amino acid (aa) residues from 40 aa to 111 aa of proteide sequence.We constructed a homologous recombination gene-knockout vector of 8-3 of G. catenulatum HL-1-1. This preliminary experiment laid the foundation for success of genes knockout of Gliocladium and their functional verification in near future.