| Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi, which produces and releases lytic enzymes in the presence of a suitable host. Various studies have provided evidence indicating that enzymatic activities enable T. harzianum to penetrate its hosts and use their biomass as a source of nutrients. Due to its mycoparasitic activity, T. harzianum been used for the biological control of a large variety of phytopathogenic fungi that are responsible for major plant diseases. In comparison to chitinases and glucanases, little is known about the proteases secreted by Trichoderma strains, despite the fact that it also plays a significant role in the biocontrol ability.Based on the induced cDNA library from T. harzianum mycelium, the DNA and cDNA sequences of subtilisin-like protease genes (sl41, ss10) and aspartic protease gene (sa76) were cloned using RACE approach. These cDNA sequences were submitted to GenBank. The numbers are DQ910533, EF063644 and EF063645, respectively. The full-length cDNA, encoding aspartic protease (asp83) has successfully been cloned from the cDNA library by combining the bioinformatics analysis and the molecular strategy. The sequence was submitted to GenBank under accession number EF063643.The expression patterns of genes (sl41, ss10, sa76 and asp83) in T. harzianum were analyzed under different starvation conditions or different pathogenic fungal cell walls. All of four genes were induced strongly by the presence of fungal cell walls, but the expression patterns were different. All five fungal cell walls employed strongly induced the expression of sl41. The expression of sl41 couldn't be affected under starvation conditions. ss10 showed a similar expression pattern. The expression of sa76 gene was induced by the presence of fungal cell walls and chitin. Starvation conditions could trigger the expression of sa76 gene. asp83 was subject to nitrogen catabolite repression and induced by the cell walls of Cytospora chrysosperma,Rhizoctonia solani,and Sclerotinia sclerotiorum, respectively. These results indicated that these genes might participate in the process of T. harzianum mycoparasitic.Genes of sl41, ss10, sa76 and asp83 from T. harzianum was respectively inserted into pYES2 vector, an expression vector of Saccharomyces cerevisiae with high efficiency promoter, and transformed them into the cells of S. cerevisiae H158. The results of Northern blotting analysis showed that all of genes were successfully expressed in the yeast. SDS-PAGE indicated that the heterologous protein was secreted successfully by S. cerevisiae.The optimum temperature of subtilisin-like protease SL41and SS10 was 40℃and 50℃, respectively. SL41and SS10 were optimally active in the pH8, and they were stable in a pH range 7.5~9.5. SL41 was sensitive to temperature in a range 25~30℃. The maximum enzyme activity of SL41 and SS10 were 19.4U/mL and 17.8U/mL, respectively. The maximum enzyme activity of SA76 was 11.8U/mL. The optimal enzyme reaction temperature was 45℃and optimal pH was 3.5. The maximum enzyme activity of ASP83 was 4.0U/mL with temperature 40℃, optimal pH 4.0. Both SA76 and ASP83 maintained higher activity in a pH range 3.0~5.0.In order to evaluate antagonistic activity of protease against pathogenic fungi, in vitro inhibition of mycelial growth of five pathogenic fungi by culture supernatant of protease were performed. The phytopathogenic fungi tested included Fusarium oxysporum, Alternaria alternate, C. chrysosperma, S. sclerotiorum and R. solani. The results showed that four protease were observed to possess different fungicidal activity against five pathogenic fungi. Both SL41 and SS10 showed strongly growth inhibitions against five pathogenic fungi, especially to F. oxysporum. The inhibition ratio of SL41 and SS10 against F. oxysporum was 58.22% and 47.38%, respectively. ASP83 could inhibit the mycelial growth inhibition of R. solani and C. chrysosperma. The mycelial growth of other three pathogenic fungi was less inhibited by ASP83. All pathogenic fungi were inhibited by SA76. SA76 was observed to possess obvious antagonistic activity against F. oxysporum at 42.11% of mycelial growth inhibition.
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