Cloning, antibody production, expression and cellular localization of universal stress protein gene (USP1-GFP) in transgenic cotton

  • Sameera Hassan
  • Tahir Rehman Samiullah
  • Mahmood ur Rahman Ansari
  • Bushra Rashid
  • Tayyab Husnain
Original Article
  • 107 Downloads

Abstract

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.

Keywords

Universal stress protein gene GFP Antibody production Protein purification Confocal microscopy Transgenic cotton 

Abbreviations

USP1

Universal stress protein gene1

ELISA

Enzyme linked immunosorbent assay

GFP

Green fluorescent protein

ROS

Reactive oxygen scavengers

RNS

Reactive nitrogen species

SOD

Superoxide dismutase

CAT

Catalase

PCR

Polymerase chain reaction

IPTG

Isopropyl β-d-1-thiogalactopyranoside

PMSF

Phenylmethylsulfonyl fluoride

SDS

Sodium dodecyl sulphate

PAGE

Polyacrylamide gel electrophoresis

Notes

Funding

Authors are thankful to Higher Education Commission (HEC) Pakistan for financial support to complete this study.

Author contributions

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.

Supplementary material

13562_2017_427_MOESM1_ESM.docx (26 kb)
Supplementary material 1 (DOCX 26 kb)
13562_2017_427_MOESM2_ESM.docx (13 kb)
Supplementary material 2 (DOCX 13 kb)

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Copyright information

© Society for Plant Biochemistry and Biotechnology 2017

Authors and Affiliations

  1. 1.Centre of Excellence in Molecular BiologyUniversity of the Punjab LahoreLahorePakistan
  2. 2.Government College University FaisalabadFaisalabadPakistan

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