| The appressorium is a specialized cellular structure formed by Magnaporthe grisea before penetrating plant tissues. The appressorium stage is a key period for the rice blast fungus to infect rice. It is significant to find out the state of gene expression of appressorium for us to understand the process of appressorium formation, development and penetration in the rice blast fungus. In the first part of this article, we reported the construction of a cDNA library during late appressorium stage and discovery of transcripts uniquely expressed during late appressorium stage in M. grisea by a suppression subtractive hybridization method, and tried to find the key genes related to appressorium formation, development and penetration. Chaetomium globosum and Trichoderma viride are important fungi with roles in the biological control. Cell wall degrading enzymes (CWDEs), such as endochitinase and glucanase, have important affects in the biocontrol process of Trichoderma. But the roles of CWDEs in Chaetomium's biocontrol process have not been known until now. In the second part of this article, we reported the results of the variances in endochitinase expression and biocontrol effectiveness of C. globosum CG12, introduced with an endochitinase gene (chbi) cloned from T. viride, and tried to find out the possible roles of endochitinase in the biocontrol process of Trichoderma.The results obtained are as following:A mature appressorium cDNA Library of rice blast fungus, M. grisea, was constructed in a λTriplEx2 vector by smart skill and long distance PCR skill. The cDNA library contained 2.37 × 10~6 independent clones nearly about 100% of which harbored foreign cDNA inserts with average size of 660 bp. Of 12 randomly selectedclones to sequence, nine ESTs having an average length with 470bp were obtained. After contig assembly, eight nonredundant EST sequences were generated. All 8 unique EST sequences were subjected to similarity searches against GenBank, and two sequences were found to be no homologous EST sequences in GenBank. On the whole, the appressorium cDNA library could stand for the state of gene expression in late appressorium stage and is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M. grisea.A subtractive suppressive library of mature appressorium cDNA subtracted by conidium and mycelium cDNA were constructed by SSH (Suppression subtractive hybridization). 250 EST sequences were obtained after the sequencing of recombinant cDNA clones. After contig assembly, 155 nonredundant ESTs were generated. Process sequences were subjected to similarity searches agaist Magnaporthe grisea Database (Broad Institute). After every matching record of all homology searches were checked and counted, 142 genes were identified. Among 142 genes examined with RT-PCR, 71 genes were expressed only in appressoria incubating for 23.5-24.5h at 25℃. The homology searches of ESTs in GenBank using blastn revealed that fifty-eight per cent of the 142 ESTs had homologous EST sequences at the time of submission, and the rest ESTs had not homologous EST sequences with expect value less than e-3. The above results show the construction of SHH cDNA library was successful in the identification of the genes specifically expressed in appressoria.After the endochitinase gene (chbi) cloned from the genome DNA of T. viride was introduced into C. globosum CG12, thirty transformants were obtained. It was indicated that the endochitinase activities of one-fifth transformants were enhanced significantly. And it was found the antifungual abilities of four C. globosum CG12 transformants against several pathogen fungi were promoted on PDA plate as well. But there are no relations between endochitinase activity and antifungual abilities. |
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