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SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat
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 clo...
Autores principales: | , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988357/ https://www.ncbi.nlm.nih.gov/pubmed/27582756 http://dx.doi.org/10.3389/fpls.2016.01230 |
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author | Goswami, Suneha Kumar, Ranjeet R. Dubey, Kavita Singh, Jyoti P. Tiwari, Sachidanand Kumar, Ashok Smita, Shuchi Mishra, Dwijesh C. Kumar, Sanjeev Grover, Monendra Padaria, Jasdeep C. Kala, Yugal K. Singh, Gyanendra P. Pathak, Himanshu Chinnusamy, Viswanathan Rai, Anil Praveen, Shelly Rai, Raj D. |
author_facet | Goswami, Suneha Kumar, Ranjeet R. Dubey, Kavita Singh, Jyoti P. Tiwari, Sachidanand Kumar, Ashok Smita, Shuchi Mishra, Dwijesh C. Kumar, Sanjeev Grover, Monendra Padaria, Jasdeep C. Kala, Yugal K. Singh, Gyanendra P. Pathak, Himanshu Chinnusamy, Viswanathan Rai, Anil Praveen, Shelly Rai, Raj D. |
author_sort | Goswami, Suneha |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4988357 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-49883572016-08-31 SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat Goswami, Suneha Kumar, Ranjeet R. Dubey, Kavita Singh, Jyoti P. Tiwari, Sachidanand Kumar, Ashok Smita, Shuchi Mishra, Dwijesh C. Kumar, Sanjeev Grover, Monendra Padaria, Jasdeep C. Kala, Yugal K. Singh, Gyanendra P. Pathak, Himanshu Chinnusamy, Viswanathan Rai, Anil Praveen, Shelly Rai, Raj D. Front Plant Sci Plant Science 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. Frontiers Media S.A. 2016-08-17 /pmc/articles/PMC4988357/ /pubmed/27582756 http://dx.doi.org/10.3389/fpls.2016.01230 Text en Copyright © 2016 Goswami, Kumar, Dubey, Singh, Tiwari, Kumar, Smita, Mishra, Kumar, Grover, Padaria, Kala, Singh, Pathak, Chinnusamy, Rai, Praveen and Rai. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Plant Science Goswami, Suneha Kumar, Ranjeet R. Dubey, Kavita Singh, Jyoti P. Tiwari, Sachidanand Kumar, Ashok Smita, Shuchi Mishra, Dwijesh C. Kumar, Sanjeev Grover, Monendra Padaria, Jasdeep C. Kala, Yugal K. Singh, Gyanendra P. Pathak, Himanshu Chinnusamy, Viswanathan Rai, Anil Praveen, Shelly Rai, Raj D. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat |
title | SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat |
title_full | SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat |
title_fullStr | SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat |
title_full_unstemmed | SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat |
title_short | SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat |
title_sort | ssh analysis of endosperm transcripts and characterization of heat stress regulated expressed sequence tags in bread wheat |
topic | Plant Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988357/ https://www.ncbi.nlm.nih.gov/pubmed/27582756 http://dx.doi.org/10.3389/fpls.2016.01230 |
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