Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo
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Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspensioncultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was discussed.
KeywordsMAR isolation nuclear matrix in vitro binding assay β-glucuronidase foreign gene expression high efficient expression vector construction
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