Large-scale Identification of Expressed Sequence Tags (ESTs) from Nicotianatabacum by Normalized cDNA Library Sequencing

An expressed sequence tags (EST) resource for tobacco plants (Nicotianatabacum) 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. All annotated ESTs were classified into 18 functional categories, unique transcripts involved in energy were the largest group accounting for 831 (32.32%) of the annotated ESTs. After excluding 2450 non-significant tentative unique transcripts (TUTs), 100 unique sequences (1.67% of total TUTs) were identified from the N. tabacum database. In the array result two genes strongly related to the tobacco mosaic virus (TMV) were obtained, one basic form of pathogenesis-related protein 1 precursor (TBT012G08) and ubiquitin (TBT087G01). Both of them were found in the variety Hongda, some other important genes were classified into two groups, one of these implicated in plant development like those genes related to a photosynthetic process (chlorophyll a-b binding protein, photosystem I, ferredoxin I and III, ATP synthase) and a further group including genes related to plant stress response (ubiquitin, ubiquitin-like protein SMT3, glycine-rich RNA binding protein, histones and methallothionein). The interesting finding in this study is that two of these genes have never been reported before in N. tabacum (ubiquitin-like protein SMT3 and methallothionein). The array results were confirmed using quantitative PCR.