The Construction Of CDNA Subtraction Libraries In Association With Mature Appressorium Of Magnaporthe Grisea

The appressorium is a specialized cellular structure formed by Magnaporthe grisea before penetrating plant issue. This appressorium stage is a key period for the rice blast fungus to infect rice. It is significant to find out the stage of gene expression of appressorium for us to understand the process of appressorium formation, development and penetration in the rice blast fungus. In this article, we reported the discovery of transcripts uniquely expressed during late appressorium stage in M. grisea by a suppression subtractive hybridization method, and tried to find the key gene related to appressorium formation, development and penetration. The results obtained are as following:The experiment was carried out with appressorium induced on projection membrane and mixture of mycelium and conidia control. As the conidial concentration was 1 × 10⁶ ml^-1, the ratio of appressorium formation on the hydrophobic surface of projection membrane was 96.5% 24 hours after conidia inoculated. Total RNA was extracted from appressorium and mixture of mycelium and conidia and the concentration was 1.42μg/μl and 5.09μg/μl respectively.The RNA of appressorium and mixture of mycelium and conidia was reverse-transcripted into cDNA by smart skill and long distance PCRskill.The length of two kinds of cDNA was between 200bp and 6Kb.The length of appressorium cDNA was mainly between 500bp and 5kb and that of driver cDNA was mainly between 400bp and 2kb.A subtractive suppressive library of mature appressorium cDNA subtracted by conidia and mycelium cDNA were constructed by SSH (Suppression subtractive hybridization). 250 EST sequences were obtained after the sequencing of recombinantcDNA clones. After contig assembly, 155 nonredundant ESTs were generated. Process sequences were subjected to similarity searches against 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 "C .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 SSH cDNA library was successful in the identification of the genes specifically expressed in appressoria.