| Expressed sequence tags (EST) and gene chip are commonly high throughout research methods in functional genomics. Based on EST sequencing on a large scale and cDNA chip, and at the same time combining with some bioinformatics analysis concerned, the gene expression related to resistance to black spot disease in poplar has been analyzed in this study. The plant material of this study is the leaf of poplar induced by pathogen Marssonina brunnea f. sp. brunnea after 24h, 72h and 120h. Using the two high throughout technologies simultaneously, the mechanism of resistant black spot disease in poplar could be studied on the whole gene expression level and it would provide theoretical basis for the breeding of poplar resistant to black spot disease in poplar. Total 21657 EST have been sequenced from 5'ends based on clones randomly selected from two cDNA libraries in which the leaf of poplar has been induced by pathogen Marssonina brunnea f. sp. Brunnea for 72h. After removing the vector sequences and sequences with low quality, the final 20023 effective ESTs with the length greater than 100bp have been obtained and the average effective length was 615.9bp. The 20023 EST have been submitted to GenBank database and their accepted accession number is form CX167465 to CX187487 . Using the software Phrap, 20023 effective ESTs have been cluster analyzed and assembled into 10816 UniGene including 3734 contigs and 7082 singletons. According to the accession in Swissprot database, the unigene has been functionally classified based on Gene Ontology. The 8853 genes with homology alignment have been annotated 21100 kinds function according to GO three classification levels. The result showed that there were two kinds of gene abundant according to the functional annotation, which one kind of expressed genes were special to leaf tissue of poplar, another kind of expressed genes were about the resistance to stress. The mining single nucleotide polymorphisms (SNP) marker and simple sequence repeat(SSR) marker has been done based on the ESTs which we sequenced. According to the screening conditions , 94 substitution type of candidate SNPs and 39 insert/missing type of candidate SNPs have been selected from 3734 contigs. Using the software SSR Finder and according to the demands, 713 candidate SSR have been found from 10816 assembled sequences and there were 191 kinds of SSR type. 　　ESTs is good gene resource for cDNA microarray. In this study, based on the functional annotation of EST, 3000 clone have been selected and amplified by PCR and used to cDNA microarray ( repeated 3 times).The expression profiling of gene in poplar leaf induced by pathogen Marssonina brunnea f. sp. brunnea after 24h, 72h and 120h have been analyzed by the cDNA microarray. According to the screening standard of differential expressed gene(Ratio>=2 或<=0.5,S/N>2,p-value<0.05), the expression analysis showed that differentially expressed genes mainly took part in protein synthesis, cell defense reaction and signal transduction. Differentially expressed genes related to disease resistance for plant in this study mainly concluded such kinds of as pathogenesis-related protein,phenylalanine ammonia-lyase,chitinase,β-1,3-glucanase,ascorbate peroxidase,peroxidase,hydroxyproline-rich glycoprotein family protein,leucine-rich repeat family protein and so on. The others kinds such as metallothionein,zinc finger family protein,cytochrome P450 oxydoreductase,polyubiquitin were differentially expressed in the leaf of poplar induced by pathogen Marssonina brunnea f. sp. Brunnea, it showed that these kinds of genes played an important role in defense reaction. Differentially expressed genes related to replication, transcription and translation were translation initiation factor,elongation factor 1-α,transcription factor,histone,ribosomal protein and so on. There were several kinds of protein kinase related to signal transduction differentially expressed in this study.
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