Plant & Animal Genome IX Conference

Poster: Sequencing & EST
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A FUNCTIONAL GENOMIC APPROACH TO IMPROVE DROUGHT TOLERANCE IN RICE: EST ANALYSIS USING NORMALIZED cDNA LIBRARIES

¹ Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad-500 046, India

² Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA

The drought stress response in rice is a complex phenomenon involving multiple but functionally related genes dispersed throughout the genome. In rice information on drought stress-responsive changes in gene expression is mostly confined to protein profiles. Very little is known about the drought induced changes in global gene expression in rice. We have taken a genomic approach to define the genetic determinants that control drought stress responses in different tissues at different stages of plant adaptation and tolerance to drought. We have made cDNA libraries from different tissues of indica rice (Oryza sativa cv. Nagina 22) seedlings grown under controlled growth conditions, and subjected to drought stress at 70% through 40% field capacity. Primary cDNA libraries made from Poly A+ mRNA preps from leaf, leaf sheath and root tissues at different stages of drought stress were normalized by reassociation of PCR products of ssDNA primary libraries with ssDNA followed by separation of ssDNA from dsDNA on hydroxyapatite columns. The cDNA clones from the normalized library were sequenced from the 3' end and the ESTs were analyzed using BLAST, PhredPhrap and other programs. Of the one thousand cDNAs sequenced so far, nearly 85% are non-redundant ESTs. Further analysis revealed that about 40% of these ESTs do not show significant homology to the published sequence databases. A broad based functional categorization of database-matched ESTs showed that about 15% are stress/defense related genes, about 35% are primary metabolic pathway genes, and about 10% are transcription and translation control genes. This library includes several ESTs that are highly homologous to known drought responsive genes such as Arabidopsis DREB1A, AP2, RD22, and catalases, peroxidases, kinases, HSPs, LEA and others. These EST clones are being used for investigation of stress induced gene expression in rice. Further, this EST library will serve as a resource for the discovery of genes associated with drought stress tolerance and their functional organization in rice. (This project is supported by the Rockefeller Foundation, New York. Dr. N.P. Saxena of ICRISAT, Hyderabad, is thanked for facilitating the work).