150 POJ 6(2):150-156 (2013) ISSN:1836-3644 In-silico prediction of an uncharacterized protein generated from heat responsive SSH library in wheat (Triticum aestivum L.) Jasdeep Chatrath Padaria*, Deepesh Bhatt, Koushik Biswas, Gagandeep Singh, Rajkumar Raipuria Biotechnology and Climate Change Laboratory, National Research Centre on Plant Biotechnology, PUSA Campus, New Delhi- 110012, India *Corresponding author: jasdeep_kaur64@yahoo.co.in Abstract Wheat is exposed to various abiotic stresses at different stages of its life cycle leading to severe decline in productivity. With rapid climate changes, high temperature stress is a major limitation to wheat production. Certain cultivars of wheat display a tolerant response to heat stress. Studies on differential expression in response to heat stress leads to identification of genes involved in molecular mechanism of thermo tolerance. Large-scale differential display analysis generates a large number of transcripts, of which a few are stress responsive whereas, many are of unknown or uncharacterized functional identity. The present study was done to identify a transcript of uncharacterized function obtained from heat responsive subtractive library generated from anthesis stage of thermo-tolerant wheat cv. Raj3765. Real time PCR analysis showed a four-fold increase in expression of the identified transcript at a stress of 37°C at the anthesis stage, indicating its role in facilitating the plant to cope the deleterious effects of high temperature at anthesis stage. Protparam tool analysis revealed that the leucine (Leu) is dominant amino acid present in the sequence, involving 15.5% of total amino acids. In-silico analysis revealed the existence of conserved domain region similar to leucine rich repeat (LRR) motif, an important DNA-binding domain. The presence of LRR motif in the protein predicted from the transcript under study indicates that this protein has a role as a signaling molecule involved in stress responses. Functional validation of the identified transcript in a model plant system shall confirm its role in heat stress tolerance. Keywords: Wheat cv. Raj 3765; Heat stress; Suppressive Subtractive Hybridization; qPCR; Homology modeling; In-silico analysis. Abbreviations: BLAST-Basic Local Alignment Search Tool; CDD-Conserved Domain Database; cDNA-complimentary DNA; cv- cultivar; EST-Expressed Sequence Tag ; GFP- Green Fluorescent Protein; GO-Gene Ontology; LRR-Leucine rich repeat; NCBI- National Center for Biotechnology Information; PCR- Polymerase Chain Reaction; PDB-Protein Data Bank; qPCR- quantitative PCR; SAVES-Structural Analysis and Verification Server; SSH-suppressive subtraction hybridization; TIGR- The Institute for Genomic Research. Introduction Bread wheat (Triticum aestivum L.) is one of the most widely grown wheat species in India, occupying 37% of the total cultivated land. Gradual increase in temperature causes detrimental effect on physiological and biological processes resulting in drastic reductions in grain yield and quality (Asseng et al., 2011). Continued heat stress is a problem in about 7 mha while terminal heat is a problem in about 40% of the irrigated wheat growing areas of the world (Joshi et al., 2007). Heat stress occurs when the ambient temperature is higher than the threshold temperature. When the rate of evaporation exceeds the pace at which moisture is being replaced through water taken up by the plant, the plant becomes dehydrated. Genotypes tolerant to heat stress are known and their enhanced tolerance is due to the altered expression of stress-inducible genes that protect and maintain the function and structure of cellular components (Sanghera et al., 2011). As compared with the other methods for analyzing altered gene expression, such as mRNA differential display (Liang and Pardee, 1992), serial analysis of gene expression (Velculescu et al., 1997), and cDNA microarray (Chu et al., 1998); suppressive subtractive hybridization (SSH) (Diatchenko et al., 1996) is a productive and efficient approach for identifying and characterizing both known and unknown genes involved in complex developmental processes and stress responses (Fernandez et al., 2007; Namasivayam and Hanke, 2006; Tsuwamoto et al., 2007). In recent years, SSH method is widely employed as a method of differential expression of genes in corn (Zhang et al., 2004), rape (Wu et al., 2007), wheat (Li et al., 2008; Zhang et al., 2005), rice (Jiang et al., 2009), and cucumber (Terefe and Tatlioglu, 2005). The present study aimed at identifying heat responsive genes at anthesis stage in thermo tolerant wheat cv Raj3765 (Rane et al., 2007) by construction and sequence analysis of a subtractive cDNA library. Genes that were up- regulated under heat stress at anthesis stage were enriched in the SSH library. Sequencing and annotation analysis of the SSH library provided a basis for obtaining full-length genes related to anthesis stage under heat stress from cv. Raj3756 a thermo tolerant wheat genotype. A number of sequences identified as uncharacterized in the SSH library, prove to be very rare heat stress responsive transcripts (Gorantla el al., 2007). A 252bp EST sequence of uncharacterized protein from the generated library was selected for functional and structural analysis. qPCR studies confirmed the heat responsive stage specific expression of the transcript for uncharacterized protein. In-silico characterization of the raw sequence of the identified stress-inducible transcript was performed. The present investigation implies homology modeling to deduce a three dimensional structure of the uncharacterized protein under study, followed by validation