Large-scale Identification Of ESTs From Nicotiana Tabacum By Normalized CDNA Library Sequencing

An expressed sequence tags (EST) resource for tobacco plants {Nicotiana tabacum) was established using high-throughput sequencing of randomly selected clones from one cDNA library representing a range of plant organs (leaf, stem, root and root base). Over 5000 ESTs were generated from the 3' ends of 8000 clones, analyzed by BLAST searches and categorized functionally. Clustering of these ESTs identified a unigene set of 3600 genes that are represented in the EST collection. A number of 696 unique sequences (19.33 % in TUTs) were identified highly significant matches (E< 10-20) to the known gene sequences and was regarded as representation of known genes according to result of BLASTX research for available database. All annotation ESTs were classified in 18 functional categories, unique transcripts involved in energy were largest group accounting for 831 (32.32 %) in all annotation ESTs. After excluding 2450 non-significant TUTs in sequence similarity, we summarized that 100 unique sequences (1.67 % in total TUTs) were identified from N. tabacum database and the remaining 1050 TUTs showed partial homologous to the genes of the other organisms. To identify genes among of 5927 clones the amplified cDNAs were arrayed and hybridized. After comparing the hybridization data 359 high quality ESTs from four tobacco varieties were generated and analyzed using the Cyber-T statistic program. In the array result two genes strongly related to tobacco mosaic virus (TMV) were obtained, basic form of pathogenesis-related protein 1 precursor (TBT012G08) and ubiquitin (TBT087G01) both of them were found in the variety Hongda, some others important genes were grouped into two group genes related with plant development like those genes related with a photosynthetic process (Chlorophyll a-b binding protein, Photosystem I, Ferredoxin I and III, ATP synthase) and the other group that include genes related with plant stress response (Ubiquitin, Glycine-rich RNA binding protein, Histones and Methallothionein). The interesting finding in this study is that approximately 98 % of these genes have never been reported before in N. tabacum. The array results were confirmed using a Quantitative PCR.