Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respecti...Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.展开更多
基金supported by the National High Tech R&D Program(863 Program)of China(2001AA212161)the National Natural Science Foundation of China(30170747).
文摘Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.
