MEDICAL HYPOTHESES AND RESEARCH
VOL. 2, No. 3, July 2005



H.-T. Dong, et al. [2005] Med Hypotheses Res 2: 481-496.


Large-Scale Identification of ESTs from Nicotiana
Tabacum by Normalized cDNA Library Sequencing


Hai-Tao Dong, Sandra Pérez Alvarez, Jack Chen and De-Bao Li*

Bioinformatics and gene network research group, Zhejiang University, Hangzhou, 310029,
China (H.-T.D., S.P.A., D.B.L.), and The Unit of Biocomputing, NICHD/NIH, Bethesda, MD
20892, USA (J.C.)


Abstract. An expressed sequence tags (EST) resource for tobacco plants (Nicotiana
tabacum) was established using high-throughput sequencing of randomly selected clones
from 1 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. 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,
seven EST from this analysis may have a relation to TMV resistance in tobacco plants
particularly to the Hongda variety. The array results were confirmed using a Real-time
quantitative RT-PCR.



*Address all correspondence to: Dr. De-Bao Li, Biotechnology Institute of Zhejiang
University, No. 268, Kaixuan Road, Zhejiang Province, China 310029.
Telephone: 86-571-86971183. Fax: 86-571-86961525. E-mail address:
xchen@helix.nih.gov



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