Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number

Plant Molecular Biology
E H GendloffW G Buchholz

Abstract

A monoclonal antibody to chloramphenicol acetyl transferase (CAT) was used in an indirect competitive enzyme immunoassay (ELISA) for the quantitation of CAT in leaf extracts of eighteen transgenic tobacco plants containing the CAT gene fused to the cauliflower mosaic virus 35S promoter. The ELISA could be used to quantify CAT when present in extracts at 20 ng/ml. Enzymatic activity and electrophoretic mobility of CAT in these extracts was not different from CAT from Escherichia coli. Concentrations of CAT in these transgenic plants ranged from 79 to 732 ng CAT/mg protein. The average coefficient of variation among three replicate samples was 15%. All plants were sampled on two separate occasions. The CAT concentrations often varied between the two sampling dates. We determined the CAT gene copy number and the number of independently segregating loci in each plant by Southern blot analysis and progeny testing. We found no significant differences in CAT expression among all ten plants with a single CAT gene. We also found a significant correlation between CAT gene copy number and the level of CAT expressed in each plant, although plants with one gene copy sometimes had more CAT than plants with more than one gene copy. In this po...Continue Reading

References

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Citations

Apr 2, 1996·Proceedings of the National Academy of Sciences of the United States of America·N S LinY H Hsu
Nov 1, 1992·Molecular & General Genetics : MGG·P BreyneA Depicker
Oct 9, 1998·The Journal of Biological Chemistry·M S ResnickC S Hahn
Sep 3, 2018·Transgenic Research·Parimala ChinnaduraiLuis A Burzio
Oct 3, 2018·Planta·Dmytro P Yevtushenko, Santosh Misra

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