title: SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.
abstract: Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat-a novel step toward the development of "climate-smart" wheat.
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Suneha
authors: S
authors: Goswami
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Ranjeet R
authors: RR
authors: Kumar
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Kavita
authors: K
authors: Dubey
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Jyoti P
authors: JP
authors: Singh
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Sachidanand
authors: S
authors: Tiwari
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Ashok
authors: A
authors: Kumar
authors: Centre for Agricultural Bio-Informatics, Indian Agricultural Statistics Research Institute New Delhi, India.
authors: Shuchi
authors: S
authors: Smita
authors: Centre for Agricultural Bio-Informatics, Indian Agricultural Statistics Research Institute New Delhi, India.
authors: Dwijesh C
authors: DC
authors: Mishra
authors: Centre for Agricultural Bio-Informatics, Indian Agricultural Statistics Research Institute New Delhi, India.
authors: Sanjeev
authors: S
authors: Kumar
authors: Centre for Agricultural Bio-Informatics, Indian Agricultural Statistics Research Institute New Delhi, India.
authors: Monendra
authors: M
authors: Grover
authors: National Research Center on Plant Biotechnology New Delhi, India.
authors: Jasdeep C
authors: JC
authors: Padaria
authors: Division of Genetics, Indian Agricultural Research Institute New Delhi, India.
authors: Yugal K
authors: YK
authors: Kala
authors: Division of Genetics, Indian Agricultural Research Institute New Delhi, India.
authors: Gyanendra P
authors: GP
authors: Singh
authors: Centre for Environment Science and Climate Resilient Agriculture, Indian Agricultural Research Institute New Delhi, India.
authors: Himanshu
authors: H
authors: Pathak
authors: Division of Plant Physiology, Indian agricultural Research Institute New Delhi, India.
authors: Viswanathan
authors: V
authors: Chinnusamy
authors: Centre for Agricultural Bio-Informatics, Indian Agricultural Statistics Research Institute New Delhi, India.
authors: Anil
authors: A
authors: Rai
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Shelly
authors: S
authors: Praveen
authors: Division of Biochemistry, Indian Agricultural Research Institute New Delhi, India.
authors: Raj D
authors: RD
authors: Rai
keywords: DEGs; SAGs; SSH library; Triticum aestivum; abiotic stress; differential expression; differentially expressed proteins (DEPs); terminal heat stress