Comparative The Efficiency Of Obtaining Flanking Sequence Of T-DNA Tags Insertion In Rice Genome By Plasmid Rescue And TAIL-PCR

It is important to efficiently obtain flanking sequence of T-DNA tags insertion in genome a strategy for investigating functional genomics by using reverse genetics. In this study the two methods were compared for their efficiency to obtain the flanking sequences T-DNA tags insertion in rice (Oryza sative) genome. A plant binary vector pBsBar was constructed and transferred into rice immature embryo callus by Agrobacterium-mediated method. 85 percent of about 1000 TO transgenic plants were resistant to BASTA solution. The result from 150 TO T-DNA insertion lines showed that the efficiency of plasmid rescue was about 90%, and that of TAIL-PCR was 62%. About 80% of flanking sequence of T-DNA tags insertion in rice genome by plasmid rescue was rice genome, while about 89% by TAIL-PCR. As mentioned above, the efficiency of obtaining flanking sequence of T-DNA tags insertion from transgenic plants by plasmid rescue was about 72%, which was 18% higher than that of TAIL-PCR.We analysis the obtaining flanking sequences of T-DNA tags insertion in rice genome by two methods. Our results suggested that T-DNA insertions show a good correlation with the predicted size distribution of genie and intergenic regions in the rice genome and were evenly spread within genes, while the preference insertions in the 5'- and 3'-regulatory regions were observed. T-DNA tags also preferentially inserted into introns. The chromosome two arms had relatively more T-DNA integrations than the pericentrometic regions. The T-DNA tags were also seldom founded in repeat DNA region. It is suggested that T-DNA tags are uneasy to insert into the heterochromatin regions of rice chromosomes. The result showed that general AT content distribution (45%~65%) for the genomic DNA of T-DNA insertion sites showed a good match with the randomly selected genomic DNA. No obvious nucleotide composition preference close to the T-DNA insertion sites was observed.