Identification Of ARS Function Of Plant MARs And Its Influence In Gus Gene Expression

Gene transfer technology enhances agronomic performance orimproves quality traits in a wide variety of crop species, and has become afundamental tool for basic research in many subdiscriplines of plant biology.However, the applications of the technology in practical and basic research aresometimes handicapped by transgene silencing and gene instable expression. Itis important to find efficient strategies to overcome gene silencing andincrease transgene expression level for optimizing transgenic technology. MARs were proven to have the ability enhancing transgene expressionlevel or reducing the variance between individual transformants when flankingtransgene cassette. MARs not only enhance transgene expression or reducevariance between transformants, but also stabilize such transgene expressionin their progeny. Therefore, MAR is an efficient regulatory element to improvetransgene expression and stabilize the expression level in the progeny. In this paper, six DNA fragments with MARs motifs are isolated fromtobacco and Arabidopsis are investigated for their ARS activities. The resultsshows that among the six plant MARs, only TM1 and AM4 had strong ARSactivity which is almost the same as that of ARS from yeast chromosome. Inorder to further identify the core region of the two MARs for the ARSactivity,a series of subclones were created by PCR strategy, and the correspondingsubclones were designated as TM1-1, TM1-2, TM1-3, AM4-1, AM4-2,andAM4-3, respectively. Our studies revealed that TM1-3 and AM4-3 not only 3李宏:植物 MARs 的 ARS 功能鉴定以及对 gus 基因表达活性的研究had higher ARS activity, but also displayed higher transformation frequency,plasmid stability and growth rate compared to their intact MARs, TM1 andAM4. These data present an important clue for further elucidating therelationship between MAR and ARS. For further studying the influence of MARs having ARS activities on thegus gene expression in transgenic plants, TM1, AM4, TM1-3 and AM4-3 wereflanked the gus gene cassette in the vector pB121, respectively. Then thetransgenic tobaccos were used to assay GUS stable expression. The resultsshow that TM1, AM4, TM1-3 and AM4-3 can enhance gene expression1.38-fold, 1.23-fold, 2.41-fold and 1.81-fold compared to control plants,respectively, but they had no obvious effect on decreasing the variation ofexpression between transformants.