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Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

ABSTRACT: BACKGROUND: Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage was cloned, characterized and its role was validated under heat stress by proteomics and in-silico studies. In the present study we report the characterization at molecular and in-silico level of peroxisomal TapAPX gene isolated from heat tolerant wheat cultivar of India. RESULTS: qPCR studies of TapAPX gene displayed up to 203 fold level of expression at 42 °C heat stress exposure. A full length cDNA of 876 bp obtained by RACE deduced a protein of 292 amino acid residues which gives a complete 3D structure of pAPX by homology modeling. TapAPX cDNA was cloned in expression vector pET28 (a+) and the recombinant protein over-expressed in E. coli BL21 showed highest homology with APX protein as deduced by peptide mass fingerprinting. CONCLUSIONS: TapAPX gene from wheat cv Raj3765 has a distinct role in conferring thermo tolerance to the plants and thus can be used in crop improvement programmes for development of crops tolerant to high temperature.

SUBMITTER: Padaria JC

PROVIDER: S-EPMC4209082 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

Padaria Jasdeep Chatrath JC Vishwakarma Harinder H Biswas Koushik K Jasrotia Rahul Singh RS Singh Gyanendra Pratap GP

BMC research notes 20141010

<h4>Background</h4>Heat stress leads to accelerated production of reactive oxygen species (ROS) which causes a huge amount of oxidative damage to the cellular components of plants. A large number of heat stress related genes as HSPs, catalases, peroxidases are overexpressed at the time of stress. A potent stress responsive gene peroxisomal ascorbate peroxidase (TapAPX) obtained from heat stress (42 °C) responsive subtractive cDNA library from a thermo tolerant wheat cv. Raj3765 at anthesis stage ...[more]

PMID: 25304397

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Project description:BackgroundHead formation of broccoli (Brassica oleracea var. italica) is greatly reduced under high temperature (22 °C and 27 °C). Broccoli inbred lines that are capable of producing heads at high temperatures in summer are varieties that are unique to Taiwan. However, knowledge of the early-activated pathways of broccoli head formation under high temperature is limited.ResultsWe compared heat-tolerant (HT) and heat-sensitive (HS) transcriptome of broccoli under different temperatures. Weighted gene correlation network analysis (WGCNA) revealed that genes involved in calcium signaling pathways, mitogen-activated protein kinase (MAPK) cascades, leucine-rich repeat receptor-like kinases (LRR-RLKs), and genes coding for heat-shock proteins and reactive oxygen species homeostasis shared a similar expression pattern to BoFLC1, which was highly expressed at high temperature (27 °C). Of note, these genes were less expressed in HT than HS broccoli at 22 °C. Co-expression analysis identified a model for LRR-RLKs in survival-reproduction tradeoffs by modulating MAPK- versus phytohormones-signaling during head formation. The difference in head-forming ability in response to heat stress between HT and HS broccoli may result from their differential transcriptome profiles of LRR-RLK genes. High temperature induced JA- as well as suppressed auxin- and cytokinin-related pathways may facilitate a balancing act to ensure fitness at 27 °C. BoFLC1 was less expressed in HT than HS at 22 °C, whereas other FLC homologues were not. Promoter analysis of BoFLC1 showed fewer AT dinucleotide repeats in HT broccoli. These results provide insight into the early activation of stress- or development-related pathways during head formation in broccoli. The identification of the BoFLC1 DNA biomarker may facilitate breeding of HT broccoli.ConclusionsIn this study, HT and HS broccoli genotypes were used to determine the effect of temperature on head formation by transcriptome profiling. On the basis of the expression pattern of high temperature-associated signaling genes, the HS transcriptome may be involved in stress defense instead of transition to the reproductive phase in response to heat stress. Transcriptome profiling of HT and HS broccoli helps in understanding the molecular mechanisms underlying head-forming capacity and in promoting functional marker-assisted breeding.

Project description:Terminal heat stress during reproductive stage in wheat (Triticum aestivum L.) causes pollen grain sterility and has a drastic impact on wheat crop production. Finding genotypes with high pollen viability under heat stress is crucial to cope with the impact of climate change through developing heat-tolerant cultivars. To assess the effect of terminal heat stress on pollen viability in a panel of spring wheat genotypes (N = 200), RCBD (randomized complete block design) field trials were conducted under normal and heat stress conditions for two consecutive years (2020-2021 and 2021-2022). Analysis of variance showed significant variation in genotypes, treatments, and genotype × treatment interaction. Fifty and 46 genotypes were categorized as heat tolerant (HSI pv < 0.5) in the first and second year, respectively. Twelve genotypes, namely, Chenab-70, Pari-73, Pak-81, MH-21, Punjab-76, NIFA-Aman, NUWYT-63, Swabi-1, Nisnan-21, Frontana, Amin-2000, and Pirsabak-2004, were found to be heat tolerant across the years. The violin plot displayed a trend of improvement in heat tolerance (HSI pv < 0.5) over the period of time in many modern wheat varieties. However, some modern wheat varieties released after 2001 such as Janbaz-09 (57%), Ghazi-2019 (57%), and Sindhu-16 (43%) had very low pollen viability under heat stress conditions. The results of phenotypic coefficient of variance (PCV%), genotypic coefficient of variance (GCV%), broad sense heritability (h2 bs), and genetic advance (GA) suggested the major contribution of genetic factors in controlling pollen viability trait. Higher values of h2 bs and GA under heat stress conditions suggested pollen viability as a heat tolerance trait controlled by additive genetic effects. Taken together, these results suggested pollen viability as a useful trait for selection in early generations under elevated temperatures. The genotypes identified as heat tolerant in both years can be used as genetic resources for breeding cultivars with higher pollen viability under elevated temperature conditions.

Dataset Information

Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

Publications

Molecular cloning and in-silico characterization of high temperature stress responsive pAPX gene isolated from heat tolerant Indian wheat cv. Raj 3765.

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