Gene expression in Vicia faba L. under regulation of the SV40 enhancer

Abstract

The similarities and differences between plants and other organisms can be studied by introducing non-plant regulatory sequences into the plant genome. Experiments were carried out to study gene expression under the regulation of an animal virus enhancer in faba bean (Vicia faba L. cv. Puma).

A binary vector, pGA643-GUS-SV, was constructed carrying the cauliflower mosaic virus 35S (CaMV 35S) plant promoter, the Simian virus 40 (SV40) animal enhancer and the β-D glucuronidase (GUS) reporter gene. This plasmid was transferred to hairy root-inducing Agrobacterium tumefaciens strain A4T. Vicia faba L. cv. Puma plants were inoculated with A. tumefaciens A4T carrying pGA643-GUS-SV. Control V.faba L. cv. Puma plants were inoculated with A4T carrying the enhancer-less binary vector pGA643-GUS. The hairy roots were assayed for GUS expression and DNA content. Southern blots were carried out on these hairy roots to confirm GUS gene transfer and integration into V.faba L. cv. Puma, and to assess GUS copy numbers.

The GUS activities showed high variability in both treatment pGA643-GUS and treatment pGA643-GUS-SV. This variability was reduced when the GUS activity values were adjusted by GUS copy numbers. The considerable amount of variability that remained after the adjustment was probably due to (i) differences in hairy root physiology and (ii) insert positioning effects in the plant genome.

The mean GUS activity was slightly higher in treatment pGA643-GUS-SV than that in treatment pGA643-GUS. This difference, however, was not statistically significant at the 5% level, probably partly due to the variability observed in GUS activity values. The inability of the SV40 enhancer to significantly stimulate GUS gene expression in V.faba L. cv. Puma might have also been due to (i) the lack of an appropriate transcription factor or factors to bind to the enhancer, (ii) the nature of the intervening DNA between the enhancer and the promoter and (iii) possibly, to an unknown inhibiting factor present in V. jaba L. cv. Puma.