Non-radioactive detection of β-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC

摘要: The use of transient gene expression assays for the study natural or engineered plant promoters is affected by a considerable degree inter-experiment variability. As means obtaining interpretable data from limited number experiments, we worked out conditions simultaneous determi nation activity two reporter genes, “sample” and “reference”, ona single extract co-transformed protoplasts. s-glucuronidase (GUS) chloramphenicol acetyl transferase (CAT) both under control CaMV 35S promoter, were transferred into tobacco (Nicotiana tabacum L.) protoplasts on independent plasmids. parallel genes in several co-transformation experiments was verified. Conditions protoplast extraction buffer assay activities set up. A HPLC method non-radioactive determination enzyme aliquot reaction mixture developed. resulting procedure tested using GUS as “reference” CAT gene, either wild type upstream-deleted (−90) “sample”. protocol simple allows fast analysis presence true internal standard which manipulations are reduced to minimum subjected same experimental treatments.