Isolation And Functional Analysis Of MARs In Tobaccos And Poplars

Matrix attachment regions (MARs) are DNA sequences that bind to the nuclear matrix, which tend to be noncoding sequences and AT rich. MARs can regulate gene transcription and suppress position dependent transgene silencing by forming chromatin into independent loop domain througth their anchoring to the nuclear matrix. Previous studies have shown that, MARs play very important roles in suppressing transgene silencing, enhancing the level of transgene expression and decreasing the variability of transgene expression. MARs were isolated from tobaccos and Populus nigra by two different methods. Plant expression vectors containing MARs were constructed to investigate their effect on transgene expression regulation. Two DNA sequences (M14 and M17) were obtained from tobacco genome by PCR. Both of the sequences contained several typical consensus sequences of MARs, such as 90%AT-box, A-box, T-box, the base unpairing regions (BUR), autonomously replicating sequences (ARS), the consensus sequence for topoisomeraseâ…¡, MAR recognition sequence (MRS), origin of replication (ORI), curved DNA motifs and ATATTT and so on. To investigate the effects of these two sequences on foreign gene expression in transgenic plants, three plant expression vectors were constructed using uidA gene coding ?-glucuronidase (GUS) which were flanked on one side and both sides in plant expression vector pCAMBIA2301 by the MARs we obtained. These plant expression vectors were transformed into tobaccos via Agrobacterium-mediated transformation method, with the pCAMBIA2301 and wild type tobacco as controls. GUS histochemical staining showed that the uidA gene expressed stably in transgenic tobaccos. Compared to the controls, quantitative detection of GUS activity showed that MARs could increase GUS expression levels in vivo, wherever they were flanked on one side or both sides of uidA gene. Compared to the control vector of pCAMBIA2301, the vector cloned with double cis-copys of MARs increased the average GUS activity for 3.14 fold, while the two vectors cloned with a single MAR increased the average GUS activity for 1.56 fold and 2.43 fold respectively. Thus we concluded that the double cis-copys of MARs had a better effect on GUS expression than a single MAR. But the expression differences among individual transformants were still obvious. Therefore, the MARs obtained are two new MARs and could increase the gene expression in vivo. In the meanwhile, although the number of the typical MARs motifs in M14 was more than that in M17, especially the 90%AT-box considered to be the motif correlated highly with binding strength in vitro, the enhancement was lower yet, which implied no correlation between improvement of gene expression and binding strength of MARs and nuclear matrix in vitro. Two DNA sequences A7 and A23 were isolated from suspended cells of Populus nigra by high-salt method. Their seqence analysis suggested that both A7 and A23 contained the features of MARs, they both had the following motifs: 90%AT-box, A-box, T-box, the base unpairing regions (BUR), autonomously replicating sequences (ARS), the consensus sequence for topoisomeraseâ…¡, MAR recognition sequence (MRS), origin of replication (ORI), curved DNA motifs and ATATTT, thus they were confirmed as MARs elementarily according to these characters. To investigate the effect of these two sequences on foreign gene expression in transgenic plants , ?-glucuronidase(GUS) gene (uidA) in plant expression vector pCAMBIA1305.1 was flanked on one side or both sides by the MARs we obtained. The plant expression vectors with and without MARs were transferred into tobaccos via A grobacterium-mediated transformation procedure. The result of GUS histochemical staining showed that, the uidA gene was stably expressed in transgenic tobaccos, GUS activity of transformated tobaccos containing the single copy of A7 or A23 is lower than that of control. This demonstrated that the inserted MAR fragment decreased the expression of uidA gene instead of increasing its expression. The DNA sequences suppressed the transgene expression although they shared the sequence features of MARs. This study displayed the diversity of MARs.On the other hand, any MAR element may play various functions. A lack of reduction in variation that has been reported for some MARs illustrates that not all of these elements have boundary functions or the ability to eliminate transgene silencing. Some MARs have both general and specific properties. This heterogeneity can complicate functional analysis, because results obtained with different MAR sequences may not be comparable.