Cloning, antibody production, expression and cellular localization of universal stress protein gene (USP1-GFP) in transgenic cotton
- 107 Downloads
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.
- Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to abiotic stresses. In: Gruissem W, Buchannan B, Jones R (eds) Biochemistry and molecular biology of plants. Amer Soc Plant Physiol, Rockville, pp 1158–1249Google Scholar
- Galuszka P, Frébortová J, Luhová L, Bilyeu KD, English JT, Frébort I (2005) Tissue localization of cytokinin dehydrogenase in maize: possible involvement of Quinone species generated from plant phenolics by other enzymatic systems in the catalytic reaction. Plant Cell Physiol 46(5):716–728CrossRefPubMedGoogle Scholar
- Gury J, Seraut H, Tran NP, Barthelmebs L, Weidmann S (2009) Inactivation of PadR, the repressor of the phenolic acid stress response, by molecular interaction with USP1, a universal stress protein from Lactobacillus plantarum, in Escherichia coli. Appl Environ Microbiol 75:5273–5283CrossRefPubMedPubMedCentralGoogle Scholar
- He L, Yang X, Wang L, Zhu L, Zhou T, Deng J, Zhang X (2013) Molecular cloning and functional characterization of a novel cotton CBL-interacting protein kinase gene (GhCIPK6) reveals its involvement in multiple abiotic stress tolerance in transgenic plants. Biochem Biophys Res Comm 435(2):209–215CrossRefPubMedGoogle Scholar
- Hongzhong C (2015) Subcellular localization of CAX proteins in plants. Mol Soil Biol 6(3):1–5Google Scholar
- Isokpehi RD, Mahmud O, Mbah AN, Simmons SS, Avelar L, Rajnarayanan RV, Udensi UK, Ayensu WK, Cohly HH, Brown SD, Dates CR, Hentz SD, Hughes SJ, Smith-McInnis DR, Patterson CO, Sims JN, Turner KT, Williams BS, Johnson MO, Adubi T, Mbuh JV, Anumudu CI, Adeoye GO, Thomas BN, Nashiru O, Oliveira G (2011) Developmental regulation of genes encoding universal stress proteins in Schistosoma mansoni. Gene Regul Syst Biol 5:61–74Google Scholar
- Kuppu S, Mishra N, Hu R, Sun L, Zhu X, Shen G, Blumwald E, Payton P, Zhang H (2013) Water-deficit inducible expression of a cytokinin biosynthetic gene IPT improves drought tolerance in cotton. PLoS ONE 8(5):e64190. doi: 10.1371/journal.pone.0064190
- Saha S, Callahan FE, Dollar DA, Creech JB (1997) Effect of lyophilisation of cotton tissue on quality of extractable DNA, RNA, and protein. J Cotton Sci 1:10–14Google Scholar
- Sambrook J, Fritschi EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Hassan S, Ansari MR, Aslam A, Sarwar, MB, Rashid B, Ali Q, Husnain T (2016) Cloning, genetic transformation and cellular localization of universal stress protein gene (GUSP1) in Gossypium hirsutum. PLoS ONE (revision submitted)Google Scholar
- Wilhite DA (2009) Drought monitoring as a component of drought preparedness planning. Drought management and policy development in the Mediterranean. Springer, Berlin, pp 3–20Google Scholar