Development and analysis of ESTs (expressed sequence tags ) in black pepper (piper nigrum L )
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Date
2008
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
Soil moisture is one of the major factors that influence plant growth and productivity. The stress is the second most important factor that affects crop production in black pepper. Various genotypes of black pepper are reported to vary in their response to water stress and the variety Kalluvally has been identified as a drought tolerant one among the cultivated genotypes.
Plants respond to stress by adaptation of the biochemical and physiological processes. These biochemical and physiological reactions are regulated by several genes that are induced during drought conditions. Thus the gene products directly or indirectly provide tolerance to the plants so that they can survive under water stressed conditions.
In the present study, an attempt was made to identify such water stress induced genes in variety Kalluvally using the molecular technique called Suppression Subtractive Hybridization (SSH) and finally to develop and analyze Expressed Sequence Tags (ESTs). Total RNA and mRNA were isolated from normal and water stressed plants and were used respectively as ‘driver’ and ‘tester’ in SSH reaction. The reactions were performed utilizing the PCR select cDNA subtraction kit provided by CLONTECH, USA.
Control subtraction was carried out first using PCR select™ cDNA subtraction kit, which gave satisfactory and expected results. For experimental subtraction, double stranded cDNAs were synthesized from 2µg mRNA from normal ‘driver’ and water stressed ‘tester’. Two tester populations were created and each ligated to two different adaptors. This was followed by two hybridization reactions and finally a selective PCR amplification. Only differentially expressed cDNAs were amplified exponentially. This was confirmed by analyzing the PCR products on agarose gel, which showed a smear ranging from 0.2 to1.5kb in the subtracted sample and was different from smear pattern of unsubtracted ones. The cDNA fragments from subtracted sample were cloned in pGEMT vector and sequenced. Total twenty clones were sequenced and analysed after vector and adaptor editing.
In silico analysis using bioinformatics tools revealed that some of the cloned sequences showed good homology with known sequences which play important role during water stress conditions directly or indirectly. These included Heat Shock Proteins (HSP-17 & 20), Secretory Carrier Membrane Protein (SCAMP), gamma thionins, MYB transcription factor, Ribonuclease enzyme, fatty acid desaturase, peptidylprolyl isomerase and NADH-ubiquinone oxidoreductase family protein. Also, these sequences had conserved domains for the above mentioned proteins. The rest of the clones did not show any good homology and therefore it was difficult to assign any reported role to these. In addition to this, all the sequences possessed Open Reading Frames (ORFs) many had transmembrane helices and some were found to have signal peptide.
The sequences were submitted to dbEST. For further exploitation of these sequences it is necessary to clone full length cDNA. ESTs thus generated in the present study will be of great use in future for further downstream applications.
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