关键词: 马铃薯, 质体, 载体构建, GFP基因, 瞬时表达

Abstract: Plastid transformation in higher plants offers several advantages over nuclear transformation, including maternal inheritance of transgenic, lack of position effects and gene silencing, and high levels of transgenic expression, because the high ploidy level of the plastome in cells and the genes of interest are integrated into the plastome via homologous recombination. Based on the highly conservative features of trnI and trnA genes during the plastid genome evolution of higher plants, we described a distinct construction protocol of species-specific transformation vector of potato. We designed the primers and PCR-amplified the trnI-trnA targeting region from total genomic DNA of potato line of ‘Hui-2’. After sequencing and digesting the amplifed 2.7 kb fragment, which was used as homologous targeting sequences, we constructed the potato-specific plastid expression vector named pBMLSIA-GFP that carries the expression cassette of Prrn-gfp-aadA-TpsbA. Then, verified by digestion with restriction enzymes, the vector pBMLSIA-GFP was transformed into potato tubers using PDS-1000/He biolistic particle delivery system. The fluorescence upon excitation with 365 nm light 2 days after bombardment was observed, the results indicated that the transient expression of GFP gene was in high efficiency under the regulation of plastid promoter Prrn and signals TpsbA. Tubers bombarded twice at 1 100 psi pressure and a target distance of 6 cm emitted the most intensive fluorescence. The analysis results of SDS-PAGE electrophoresis showed that GFP protein in high-level expression was up to 15.4–30.2% of the total soluble protein in potato tubers. It indicated that this potato-specific plastid expression vector pBMLSIA-GFP is highly effective and desirable to be applied in traits improvement of potato via plastid genetic transformation.

Key words: Solanum tuberosum L., Plastid, Vector construction, GFP gene, Transient expression