Functional Analysis Of Matrix Attachment Region Of Pea In Transgenic Rice

Gene transfer technology is being used to enhance agronomic performance or improve quality traits in a wide variety of crop species. However , it is sometimes severely handicapped by difficulty in obtaining material in which transgene expression is predictable and stable over many generations. Transgene silencing is a common phenomenon in plant genetic engineering researching, while gene silencing caused by DNA methylation and multiple copies integrating is more common. Transgene silencing is a great disadvantage for the application of transgenic crops culturing. It is important to study mechanism of transgene silencing and adopt effective strategies to overcome transgene silencing. Matrix attachment region (MAR) are DNA sequences that bind to the nuclear matrix. MAR can regulate gene transcription and expression , and suppress position dependent transgene silencing by forming chromatin into independent loop domain through their anchoring to the nuclear matrix. Studies have shown that MAR play very important roles in enhancing the level of transgene expression, decreasing the variability of transgene expression and suppressing transgene silencing.In this research, The pea MAR was used to construct T-DNA constructions, in which the pea MAR flanked the sides of gene gus and hpt (-MAR-gus-MAR-hpt, -gus-MAR-hpt-MAR-) respectively, the pea MAR had no influence on gene expression in transient expression; GUS quantitative activity showed that MAR could increase gene expression level in MAR-gus-MAR-hpt constructions mediated by Agrobacterium. GUS activity increased two times compared with gus-MAR-hpt-MAR constructions, and stable expression in T₁ generation, but, we found gene silencing in contral construction. The TaqMan primers and probes for real-time PCR were designed according to the sequence of gus and sps gene, the gene intergration copy number were estimated by two absolute quantitative and one relative quantitative. Real-time PCR and hpt segregation analysis showed that gus genes were most integrated into rice genome at one copies.