| Sclerotinia sclerotiorum (Lib.)de Bary is a worldwide plant pathogen causing destructive Sclerotinia stem rot disease in many oil crops, vegetables,etc. Sclerotinia stem rot disease is particularly important in production of soybean and greenhouse vegetables in China, and can cause destructive yield loss when the disease is severe. There is no evidence to reveal that pathogen and host interaction follows the typical gene-for-gene relationship. This has brought great difficulty to study the pathogenicity and resistancing to the toxin which is secreted by theirselives, So we construct the Library of Sclerotinia sclerotiorum to solve this problem.The generation of expressed sequence tags (ESTs) has been proven to be a rapid and efficient approach by which to identify the novel genes and analyze the function of genes. In order to studing the pathogen and toxin resistence cDNA library of Sclerotinia sclerotiorum was constructed, and it was subjected to high-throughout 5?-EST sequencing and bioinformatical analysis. The main results were as follow:1. The cDNA library of Sclerotinia sclerotiorum was constructed. The sink size of the primary cDNA is 4.01×10~5, in which 97.3% phages were recombinant, insert sizes ranging from 0.5～3.0kb were obtained. The results indicated that the library was integrated and efficacious, which is suited for ESTs sequencing.2. Totally 2150 clones were randomly selected from cDNA library of Sclerotinia sclerotiorum and partially sequenced, 1800 readable sequences that are more than 100bp were produced by analysis with Phred software .And the average length of the 1800ESTs was 554bp.3. Using the Phrap software, the 1800 effective ESTs of Sclerotinia sclerotiorum has been cluster analyzed and assembleed into 844 UniGene including 236 contigs and 606 contigs were composed of only one EST which was called singlet.4. B1astX analysis of significant homology with the non-redundant protein database suggested that about 4.15% of the total sequences were high expression abundance genes (expression frequency≥5), 23.82%were moderate abundance genes (expression frequency scope 2～5), and 71.80% were low abundance genes (expression frequency<2). The results indicated that most genes in Sclerotinia sclerotiorum were low or moderate expression abundance.5. The 3584 ESTs were blasted against the non-redundant nucleotide and protein database of NCBI. On the basis of NCBI database searches, 3.44% of the total 1800ESTs showed significant homology with the nucleotide database and 10.00% with the protein database.6. According to the accession in Swissprot database, the unigene has been functionally classified based on Gene Ontology. The 242 genes with homology alignment have been annotated 681 different functions according to GO three classification levels ( the molecular function, biological process and cell component). Genes with function annotation or putative function annotation have displayed that there are several genes related to the pathogenicity which should be studied further7.The conditions for ATMT in the Sclerotinia sclerotiorum fungus was further optimized based on the basic procedures established in Fusarium by Mullins and co-workers,including the optimal concentration of hydromycin B in transformant selection,the ratio of cefotaxime to carbenicillin for Agrobacterium inhibition,the efficient medium in the different stage of transformation,etc.
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