Expression pattern of bacterial polycistronic genes in tobacco cells

Abstract

Various plasmid constructs containing bacterial genes of arylsulfatase ( ats) and β-glucuronidase ( gus) were introduced into tobacco cells (BY-2) using a pneumatic particle gun device. A 3.6-kb atsB-atsA operon or a 1.8-kb atsA gene was inserted between the cauliflower mosaic virus (CaMV) 35S promoter and the gus gene in the pBI121 vectors. The frequency of transient GUS expression of the vector containing the atsA-gus genes was comparable to that of pBI121 without the atsA gene. The gus gene was also expressed in BY-2 cells with the vector containing the atsB-atsA operon and gus gene, although the degree of transient GUS expression was less than that of the vector with atsA. However, no Ats activity was observed in these transformants. Stable integration of the gus and atsA-gus genes in transformed callus lines was confirmed by Southern hybridization analysis. Northern blot analysis showed 1.9-kb and 3.7-kb transcripts that were identical in size to the gus and atsA-gus genes, respectively. These results suggest that the atsA-gus genes are transcribed polycistronically under the control of the CaMV 35S promoter. Protein gel assay showed two bands with GUS activity in extracts from transformed cells with the vector containing atsA-gus. These results and deletion analysis suggest that the polycistronic transcripts are translated from at least two initiation codons upstream of the gus gene.