Transient expression of electroporated gene in leaf protoplasts of indica rice and influence of template topology and vector sequences |
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Authors: | A Chaudhury S C Maheshwari A K Tyagi |
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Institution: | Dept of Plant Molecular Biology, Univ. of Delhi South Campus, New Delhi 110021, India. |
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Abstract: | Electroporation was used for gene delivery and evaluation of various parameters affecting transient expression of a gene for β-glucuronidase ( gus ) in leaf protoplasts of Oryza sativa var. Basmati-370. Transient expression was found to be dependent on voltage, capacitance, amount of plasmid and carrier DNA as well as period of culture. Maximum GUS activity was obtained when a 150 ms pulse at 300 V cm-1 and 200 μF was applied to the protoplasts (l–2×106ml−1) in an electroporation buffer containing 20 μg of plasmid and 30 μg of calf thymus DNA, assayed 48 h after electroporation. DNA topology did not influence expression of the gene as both linear and supercoiled templates resulted in similar activities, but a 4-fold decrease in expression was observed if the gene was excised, reflecting the positive influence of vector sequences on gene expression. |
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Keywords: | Electroporation GUS assay indica rice Oryza sativa protoplasts transient gene expression template topology vector sequences |
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