The Gypsy Insulator Of Drosophila Melanogaster Facilitates High And Precise Expression Of Transgenes In Arabidopsis Thaliana

To introduce foreign genes (sometimes modified upon requirements) into target organisms is popular for studying the function of corresponding genes and also important for the research upon gene regulation, thus useful to the investigation of growth and development. However, the expression level of these exotic genes varies greatly among independent transgenic lines, referred to as transgene variability. They may express ectopically or otherwise become inert and in turn hamper the further studies.The varying expression level of transgenes is caused by many possible factors including the transgene copy number, RNA silencing, and the transgene insertion site. Here, our work focused on the influences of the host genomic site of integration. Transgenes randomly integrate into the genome, and the influences of surrounding host chromatin should be responsible for the varying expression of transgenes, besides these, negative and positive regulatory elements in adjacent loci are also related to the unexpected results, these are usually referred to as "position effects" which troubled researchers for such a long time. Without exception, in our initial studies, we constructed various kinds of vectors for promoter analysis using a new cloning system mentioned as Gateway Technologytm. However, the expression levels of corresponding transgenic lines were so distinctive to each other that it brought great inconvenience to our further studies.We presented a strategy to counteract position effect in Arabidopsis thaliana by flanking the transgenes with the gypsy insulator from Drosophila melanogaster. The gypsy insulator, originally identified from the gypsy retrotransposon, is one of the best-characterized insulators. It has been reported that this insulator could regulate gene expression by establishing higher-order domains of chromatin structure and blocking the interference of nearby enhancers or repressors, thus became one of the best candidates for protection against position effect. In addition, Su(Hw) (Suppressor of Hairy-wing), the binding protein of the gypsy insulator was coexpressed. Results indicated that the gypsy insulators could efficiently improve the expression levels of GUS reporter genes driven by various kinds of promoters by 8-to 13-fold. Coexpression of the Su(Hw) protein led to a more uniform expression level of a transgene, as the coefficient of variation of expression levels was reduced further. Interestingly, the gypsy-Su(Hw) system enhanced expression levels, but did not alter the specificity of promoter activities, as experimentally evidenced by the promoters of the PIN and the AFB gene families. The gypsy insulator was also able to improve the expression of a selectable marker gene outside the insulated region, which facilitated the screen of transgenic plants. Our system will likely decrease the number of lines that experimenters need to create and examine for a given transgene by contributing to relatively high and precise expression of transgenes in Arabidopsis.