| Gene-stacking is one of the highlights in plant genetic engineering, of which linker peptide has overcome the drawbacks of traditional methods to some extent and become a new way to construct gene-stacking vectors. In addition, lots of proteins expressed in transgenic plants play their roles in subcompartments of cells, thus it's necessary to study where and how these'stacking genes'ahead to.In this study, reporter genes gfp and rfp were linked by linker peptide LP4/2A, with chloroplast transit peptide and endoplasmic reticulum transit peptide fusing to reporter genes respectively or simultaneously, to construct various vectors. After transformed into maize protoplasts by PEG-Ca2+, reporter genes were expressed and their sublocalization was analyzed by cellular and molecular methods.Firstly, 7 vectors with LP4/2A (pUgLr, pUCgLr, pUrLCg, pUEgLr, pUgLEr, pUCgLEr and pUErLCg) and 4 control vectors (pUg, pUr, pUEg and pSEb) were constructed. (U represented Ubi promoter, g for gfp, r for rfp, L for LP4/2A, C represented chloroplast transit peptide and E represented endoplasmic reticulum transit peptide)Secondly, based on protocol of Jen Sheen lab and after improved possible factors, the optimal way for maize protoplasts PEG transformation was as follows: digested maize leaves for 7～8 h, then added protoplasts/plasmid with 105/10μg and transformed by 30% PEG for 20 min, results showed that about 107 protoplasts could be obtained from 1 g leaves and transformation rate was 30～50%.Then maize protoplasts were transformed with above vectors, via laser scanning confocal microscope and western blot, analysis of sublocalization of stacking genes with LP4/2A indicated that, when there was only one kind of transit peptide, no matter where it was, it could lead the fused protein to its sub compartment, similarly, when there were two kinds of transit peptides, they also could make their proteins sublocalize accurately, while a small amount of uncleaved proteins existed in incorrect place for both situations. Furthermore, sublocaliztion was not disturbed by position, up stream or down stream of LP4/2A, of reporter genes.In conclusion, stacking genes with LP4/2A could be cleaved and sublocalized correctly in maize. |
