Isolation and characterization of expressed sequence tags (ESTs) from subtracted cDNA libraries of Pennisetum glaucum seedlings

Abstract

Pearl millet (Pennisetum glaucum), used as forage and grain crop is a stress tolerant species. Here we identify differentially regulated transcripts in response to abiotic (salinity, drought and cold) stresses from subtracted cDNA libraries by single-pass sequencing of cDNA clones. A total of 2,494 EST sequences were clustered and assembled into a collection of 1,850 unique sequences with 224 contigs and 1,626 singleton sequences. By sequence comparisons the putative functions of many ESTs could be assigned. Genes with stress related functions include those involved in cellular defense against abiotic stresses and transcripts for proteins involved in stress response signaling and transcription in addition to ESTs encoding unknown functions. These provide new candidate genes for investigation to elucidate their role in abiotic stress. The relative mRNA abundance of 38 selected genes, quantified using real time quantitative RT-PCR, demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a kinetics-specific manner in response to different abiotic stresses. Notably, housekeeping and non-target genes were effectively reduced in these subtracted cDNA libraries constructed. These EST sequences are a rich source of stress-related genes and reveal a major part of the stress-response transcriptome that will provide the foundation for further studies into understanding Pennisetum's adaptability to harsh environmental conditions.

Transcriptional profiling in pearl millet (Pennisetum glaucum L.R. Br.) for identification of differentially expressed drought responsive genes

“…HG516611-HG517355. These EST sequences from the subtracted cDNA library are rich sources of drought stress-related genes that can help in understanding the molecular basis of drought tolerance in pearl millet by revealing a major part of the stress-responsive transcriptome (Mishra et al 2007). More than 25 % of the ESTs generated were classified into response to abiotic stimulus category.…”

“…Among the various methods for differential transcriptome analysis, Suppression Subtractive Hybridization (SSH) proves to be an efficient approach to isolate and identify cDNAs of differentially expressed genes in the absence of sequence information (Mishra et al 2007;Almeida et al 2013;Ding et al 2014;Khan et al 2014). In this technique differentially expressed genes can be normalized and enriched over 1000-fold in a single round of hybridization (Diatchenko et al 1996).…”

“…SSH has successfully been used to identify genes responsive to various biotic and abiotic stresses in various plant species such as Saccharum (Watt 2003); Festuca (Zhang et al 2005); Pennisetum (Mishra et al 2007); Agrostis (Xu et al 2009); Triticum (Chauhan et al 2011). Thus in the current study we focus on identifying, validating and establishing the putative functions of the major differentially expressed genes in P. glaucum during drought stress response using SSH, real-time quantitative qRT-PCR and homology searches.…”

Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses

“…Isolation and cloning of PgHsc70 cDNA clone One of the EST clones (CD726670) from P. glaucum heat stress-responsive EST database (Mishra et al 2007) that showed maximum homology to PgHsc70 gene was used as a DNA probe for screening the Pennisetum subtractive heat stress cDNA library by the plaque hybridization method. The positive plaques were separated and DNA samples prepared for sequencing analysis (Reddy et al 2008a).…”

Identification of differential expressed transcripts of almond (Prunus dulcis ‘Sefied’) in response to water-deficit stress by cDNA-AFLP

“…other genes in response to stress (Mishra et al 2007 12 Table 3. Relationships in predicted gene interaction network of drought resistance in almond.…”