Isolating And Identifying Plant MARs To Construct High Efficient Expression Vectors

Gene transfer technology is based to enhance agronomic performance or improve quality traits in a wide variety of crop species, and has become a fundamental tool for basic research in many subdisciplines of plant biology. But the applications of the technology in practical and basic research are sometimes handicapped by gene silencing and gene instable expression. Gene silencing was caused by many factors, such as DNA methylation, position effect, co-suppression, chromatin structure, and trans-inactivation. It is important to adopt efficient strategies to overcome gene silencing and increase transgene expression level for optimizing transgenic technology. Recently, MARs were proven to have the ability enhancing transgene expression level or reducing the variance between transgenic events when flanking transgene cassette. It is thought that the MARs anchor the introduced DNA to the nuclear matrix, after integration into the host genome, thereby isolating the new gene in a chromatin Loop which acts as an independent regulatory domain, permitting the transgene to transcript and express independently to enhance gene expression or reduce position effect. MARs not only enhance transgene expression or reduce the variance between transformants, but also stabilize such transgene expression in their progeny. Therefore, MAR is an efficient regulatory element to improve transgene expression and stabilize the expression level in the progeny. It is important to isolate efficient MARs with strong enhancing ability for constructing high efficient expression vector.In the present paper, four DNA fragments with MARs motifs were isolated from tobacco and Arabidopsis by PCR method. Considering that nuclear matrix-MAR interactions are evolutionarily conserved, we prepared rice nuclear matrix to bind with the fragments for identifying MARs and comparing their binding strength to nuclear matrix in vitro. In the binding assay, the new tobacco MARs and Arabidopsis MARs can bind to rice nuclear matrix in vitro, predicting MARs maybe have no specificity and can interact with nuclear matrix in vivo in different plants. High efficient expression vectors constructed with strong MARs can be widely used to increase gene expression in different kinds of plant species. The binding strength of the new MARs and the known MAR were distinguished in the assay by the ratios of binding MARs to the corresponding binding linear vectors. Two MARs (TM2, TM3) have greater binding strength than the known MAR, and sequence analysis shows the four new MARs have different numberand kind of MARs motifs. The primary relations between binding strength in vitro and MARs motifs can be observed from table.1and fig.2. TM3 has T-box, A-box, and its binding strength is stronger than that of TM2 with one T-box and AM1and AM2 which are absent of A-box and T-box, indicating that A-box and T-box are important for MARs binding strength to nuclear matrix and can be used as basic characteristics of MARs. The relations between binding strength and unbinding sites or topsomerase sites are not obvious in our study, suggesting that the two motifs possibly bind weakly to nuclear matrix. Moreover, although high AT content is a major feature of MARs, AT content is not proportional to binding strength in this experiment. AM1, the highest AT-rich MAR (81%), however, bound more weakly to nuclear matrix than TM3 did (69.1%). Taken together, the kind and number of MARs motifs may be the characteristics of MARs, but it is difficult to speculate MARs binding strength to nuclear matrix just based on the motifs. For further discovering the relations among MARs features, in vitro binding strength and in vivo enhancing ability, especially for isolating MARs which are capable of protecting plant transgene against gene silencing, the five different MARs were flanked the reporter gene uidA cassette in the vector pBI121, respectively. And the transgenic tobaccos were used to assay GUS stable expression. Our results show TM1, TM2, TM3 and AM1 can enhance gene expression 1.5-fold, 5-fo...