Functional Mechanism And Application Of TM2 MAR Sequence From Tobacco

Matrix Attachment Regions (MARs) are the DNA sequences with rich A/T nucleotides that may be involved in anchoring DNA/chromatin to the nuclear matrix. By the interaction with the binding proteins, MARs play an important role in the maintance and modification of the DNA/chromatin structure and the regulation of the gene expression.TM2 is a matrix attachment region isolated from the genomic DNA of tobacco, which can strongly bind to the nuclear matrix and significantly enhance the transgene expression in transgenic plants of rice and tobacco. To gain insights into the regulatory mechanism of TM2 by which transcription enhancement of transgene occurs, we give the detailed analysis of the expression variation of flanking transgenes drived by different promoters in different expression systems. We present the main results as follows:1.The tandem repeats of TM2 sequence can enhance the transgene expression activation mediated from the flanking MAR in transformants, although this effect does not correspond with the copy number of the MARs. The average level of transgene expression in tobacco calli is markedly lower than that in plants, however, this difference from the cell development does not influnce the function of TM2.2 . The TM2 bidirectionally regulated the flanking transgene expression without dependence on the sequence orientation on the integrating site. The 5′-TM2 and 3′-TM2 can both enhance the transgene expression, although the increasing distance of the MAR relative to the promoter might decrease the activation to a certain extent. This characterization indicates TM2 enhance the transgene expression in a bidirectional manner. The effect of location suggests both the 5′-TM2 and 3′-TM2 flanking the transgene in the construct are desirable for maximal enhancement of transgene expression. In addition, when the individual TM2 was constructed in the opposite direction relative to the transgene, either in the upstream or in the downstream of the cassette, the GUS assay does not show a significant difference between the constructs. These results indicate there is no direction effect of the TM2 sequence on transcription enhancement of the target gene expression.3.The TM2 can change the expression lelvel of the flanking transgene rather than the expression pattern mediated from the promoter. The effect of TM2 on GUS expression in transgenic cells that harbored the CaMV 35S or PNZIP minipromoters suggests that transcription enhancement from the tobacco MAR requires the basic expression of reporter gene. Without the upstream enhancer elements of minipromoter the MAR present to be invalid.4.The effect of the site-specific deletion of two unwinding sites, one topoisomerase II binding site and one T-box element in TM2 indicated that they are all functional elements. Deletion of any kind of elements always results in the decreasement of the flanking transgene expression. These four sites perform the vast majority of the enhancement mediated from TM2, although there is some functional redundancy in their contribution.5.According to the micrococcal nuclease accessibility analysis, the CaMV 35S promoter adjacent to the TM2 was degraded more rapidly than the control without MAR. By contrast, the time-course digestion of the promoter linked to mutant MAR was similar with that of the control. Considering the increasing accessibility to micrococcal nuclease would result in the decrease of PCR products for the regions of interest, the difference revealed that TM2 plays a role in nucleosome remodeling of the promoter region. The deletion of the four sites determined the effect of TM2 on the micrococcal nuclease accessibility.6.The transcription activation of flanking gene expression from the tobacco TM2 does not simply depend on the nuclear matrix but the condensed DNA structure. The MAR does not present any regulation ability in agrobacterium-mediated tobacco transient expression of the transgene expression. However, it can enhance the transcription efficiency so as to increase the his3 gene expression on plasmid expression vector out of the yeast nuclear. The above elements in TM2 also play important role in out-of-nuclear expression enhancement.7.Several potential genes encoding TM2 sequence-binding proteins were screened from the cDNA pool by yeast one-hybrid method. These proteins show high affinity with the TM2 fragment by electrophoresis mobility shift assay. These potential multiple targets implicate the functional complexity of the TM2 MAR. 8.With TM2 and other regulation sequences, we construct the plant overexpression and RNAi vectors. These constructs can be used in high-efficient transformation and regulating gene expressions. The recombination between the high-efficient plant expression vector and the cDNA pool by the In-Fusion Smart method bring an important perspective to large-scale screening of genes. These constructs will provide efficient tools for function study of the plant genes.