Molecular Cloning And Function Analysis Of Matrix Attachment Regions From Tobacco And Its Application In Poplar

Transgene scilencing (or transgene inactivation) is a common phenomenon in transgenic plants studies, which has frustrated attempts at crop improvement through genetic engineering. The problem was solved using removing methylation and modifying promoter, but the effect was very little. Use of matrix attachment regions (MARs) to minimize transgene scilencing and breed transgenic crops with higher expression of foreign gene provides an effective approach. In the studies, three MARs were cloned and better MARs were selected and applied to poplars. The results acquired from the studies were followed:1. Two pairs of primers were degined basing on the characteristic of MARs. Three MARs were acquired using PCR from tobacco genemic DMA, respectively named TM1 (1165bp), TM2 (1002bp), TM3 (475bp). TM1 was thought to be the same MARs sequence to the Rb7 in GenBank. TM2 and TM3 were enrolled in GenBank as two new MARs.2. Sequences analysis showed, TM1, TM2 and TM3 were rich with A/T base composition and had a number of motifs commonly occuring in MARs, including the A-box,T-box,TATAAA,Drosophila toposiomerase II consensus cleavage site and DNA unwinding sequence.3. The T-DNA constructions containing MARs were constructed, in which GUS gene was flanked with MARs and the distance between two MARs was 2.9 kb. Three expression vectors were acquired, respectively named pBITMlGUSTMl, pBITM2GUSTM2 and pBITM3GUSTM34. The plant expression vectors with and without MARs were transferred into tobaccos via Agrobacterium-mediated transformation procedure. PCR analysis showed the presence of the GUS gene in all PCR positive transformants, suggesting the integration of the gene into tobacco genome.5. Effects of different MARs on the average GUS expression activity in transgenic plants were compared by fluorimetric assay. The result showed that all the three MARs had increased GUS gene expression levels, but had no obvious effect on decreasing th variation of expression between transformants. In the transgenic plants, the average GUS expression level in plants with TM1, TM2 and TM3 were respectively 1.4-fold, 5.1-fold and 1.3-fold greater than in control plants without MARs.6. MARs had no effect on the GUS gene expression levels in transient expression.7. A chitinase cDNA sequence from soybean was cloned and The T-DNA construction containing TM2 and chitinase gene was constructed, in which chitinase gene was flanked by TM2. The plant expression vectors with and without MARs were transferred into poplar via Agrobacterium-mediated transformation procedure. We had acquired kanamycin-resistant poplar shoots.