[Construction and expression of vector with aroA-in gene and its transformation in tobacco]

Inteins are intervening protein sequences that undergo self-excision from precursor protein with concomitant joining of flanking sequences. Here, we demonstrated intein cis-splicing in Nicotiana tabacum nuclear genomes by using artificial cis Ssp DnaB and Rma DnaB intein. We want to test whether protein splicing can occur in higher eucaryotic cell,which would play an important role in transgene containment in transgenic plants. Glyphosate-resistant Salmonella typhimurium aroA gene was divided at position 235/236 aa within EPSPS by inverse PCR from pLEPSPS. Amplified gene products with artificial cis-Ssp DnaB/Rma DnaB intein and split-Ssp DnaB/Rma DnaB intein were inserted at position 235 of EPSPS respectively to construct plasmid pLEBC, pLERC, pLEBT and pLERT. Above four aroA-In gene fusions were ligated into pET-32 to obtain E. coli expression vectors termed pETLEBC, pETLEBT, pETLERC and pETLERT. E. coli DE3 cells containing individual recombinant plasmids described above were induced by IPTG to produce corresponding protein products. Detectable spliced EPSPS and unspliced precursor demonstrated that splicing occurred in bacteria. aroA-cis SSp DnaB and aroA-cis Rma DnaB were ligated into Agrobacterium tumefaciens binary vector pLYM. Then A. tumefaciens containing EPSPS-(cis) intein cassettes were used for leaf disk transformation in N. tabacum. Integration of aroA-In gene into plant genome was confirmed by genomic PCR analyses. To verify the expression of fusion genes at transcriptional level, RT-PCR analyses were performed and the expected products were identified. These results suggested that plant cells support expression of S. typhimurium aroA-In fusion gene in nulear genomes. Thus,we speculated the existence of protein-splicing activity in plant cells. This opens the possibility of applying intein trans-splicing technique to reduce/prevent gene transfer by way of pollen in transgenic plants.