Cloning, antibody production, expression and cellular localization of universal stress protein gene (USP1-GFP) in transgenic cotton
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This study was aimed to clone the universal stress protein (GUSP1) gene isolated from Gossypium arboreum in E. coli expression vector pET30(a) and to raise the specific antibody in rabbit to devise a system that could be used for localization and expression of this gene under drought stress. The amplification of GUSP1 transgene revealed a fragment of 500 bp via PCR in genomic DNA of transgenic cotton plants and expression was confirmed through ELISA and Western blot by using the GUSP1 specific polyclonal antibodies. ELISA showed the expression of GUSP1 protein in roots, stem and leaves of transgenic plants at seedling, vegetative and mature plant developmental stages. Total protein isolated from drought stressed transgenic plants revealed a fragment of 47 kDa (GUSP1-GFP fusion protein) in Western blot which confirmed the expression of transgene. Confocal microscopy detected the GFP fluorescence as localization of GUSP1 in the midrib, guard cells of stomata, trichome and globular trichome of intact leaf of transgenic plants. The co-localization was observed within cytoplasm, palisade, spongy mesophyll, guard cells of stomata, vascular bundle, trichome and globular trichome of transgenic plants by using the GUSP1 specific primary antibodies and Alexa fluor conjugated secondary antibodies. This study of GUSP1 gene will advance the mechanism of abiotic stress tolerance in plants.
KeywordsUniversal stress protein gene GFP Antibody production Protein purification Confocal microscopy Transgenic cotton
Universal stress protein gene1
Enzyme linked immunosorbent assay
Green fluorescent protein
Reactive oxygen scavengers
Reactive nitrogen species
Polymerase chain reaction
Sodium dodecyl sulphate
Polyacrylamide gel electrophoresis
Authors are thankful to Higher Education Commission (HEC) Pakistan for financial support to complete this study.
SH conducted the main experiments of this study, TRS performed the antibody production experiments while MRA performed the confocal microscopy analyses and BR supervised the experiments and drafted the manuscript. TH won the grant from funding source and BR and TH designed and managed the project. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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