Matrix Attachment Region Increased Reporter Gene Expression in Stably Transformed Dunaliella salina

摘要

Nuclear matrix attachment regions (MARs) are known to bind specifically to components of the nuclear scaffold and thought to influence the expression of the flanking genes. In our previous studies, a new DSM2 DNA fragment was isolated from Dunaliellasalina, can bind to the nuclear matrix in vitro and has the MAR typical characteristics. In this study, we investigated its effect on transgene expression in D. salina under the control of RbsC promoter in stably transformed D. salina, expression vectorscontaining chloramphenicol acetyltransferase (CAT) reporter gene with and without MARs were constructed and delivered into D.salina cells by electroporation. Twenty stably transformed D.salina strains were randomly picked and CAT enzyme activities wereassayed, the results showed that CAT enzyme activities of the strains transformed with the construct containing MARs were averaged 4.5-fold higher than the controls. The transgene expression variation between individual transformants was also reduced tosome extent. Southern blotting demonstrated that CAT gene was integrated into the genome DNA, and the gene copy number was not significantly increased in transformed lines with the construct containing MARs, suggesting that MARs might have no influence on the copy number of transgene. Northern blotting results showed that CAT gene could transcribe and express at RNA level, and CAT enzyme activity was positively associated with stable mRNA in the transgenic D.salina, supporting that MAR could increase transgene expression at transcriptional stage.