Document Type: Research Paper
Centre of Excellence in Molecular Biology, University of the Punjab Lahore, 87 W Canal Bank Road, Thokar Niaz Baig, Lahore-53700, Pakistan
centre of excellence in molecular biology, university of the punjab lahore, pakistan
Background: Drought stress seriously affects the cotton fiber development. Universal stress protein cDNA isolated from native species Gossypium arboreum has the promising tolerance role against these stresses.
Objectives: This study aimed to clone, characterize, and genetically transform the GUSP1 gene in local cotton and to observe its expression in transgenic plants under drought stress.
Materials and Methods: Universal Stress Protein (GUSP1) cDNA from Gossypium arboreum was cloned in pCEMBIA (-) 1301plant expression vector by replacing Hygromycin and GUS exon with GUSP1-GFP fusion fragment. The construct was transformed into Agrobacterium tumefaciens and transient expression assay was confirmed by agro-infiltration of Nicotiana benthamiana leaves and green fluorescence under a confocal microscope. Gene integration and expression in transgenic plants was observed through Southern blot and real-time PCR analyses. Cellular localization was observed through a confocal microscope and the copy number of the transgene was observed in progeny plants.
Results: Transformation efficiency was 1.9%. Developmental and spatial expression of GUSP1 was observed through Real-time PCR in stem, root, leaf, inflorescence, and seeds of transgenic plants at the vegetative and flowering stage. Integration of GUSP1 revealed a fragment of approximately 500 bp in Southern Blot analyses. Localization of GUSP1 was detected in the intact leaf of transgenic plants through GFP fluorescence in midrib, guard cells of stomata, and trichomes. Single gene copy was detected in the chromosome of transgenic seeds.
Conclusion: GUSP1 has cloned from native species of local cotton and its integration and expression in transgenic plants confirmed that the role of GUSP1 will provide direction to breed economically important cotton varieties.