Distribution And Characterization Of T-DNA Tags In Rice Genome

In the present study, transgenic rice plants were generated using T-DNA tag. The flanking sequences of T-DNA insertions in rice genome were rescued by TAIL-PCR. One thousand and nine flanking sequences were precisely mapped on 12 rice chromosomes by bioinformatics analysis. The distribution and characterization of one thousand and nine T-DNA tags integration in rice genome were analyzed in detail.The results were summarized as follows:1. Two thousand transgenic rice plants of Nipponbare and Zhonghua No. 11 were obtained by using T-DNA tag. TAIL-PCR was performed with three arbitrary degenerate primers to rescue the flanking sequences of T-DNA insertions in the rice genome. One thousand and six hundred thirty-one flanking sequences were successfully rescued and the ratio was approximately up to 80%.2. 1009 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T-DNA tags were unevenly spread on rice chromosomes and preferentially inserted in gene-rich regions. The distal regions of most of the chromosome arms had relatively high levels of T-DNA integration, while the pericentromeric regions had lower levels. On chromosome 4, the short arm and the pericentromeric region of the long arm had a low density of T-DNA insertions. Chromosomes 1, 2, and 3 had relatively high levels of T-DNA integration, over twice those of chromosomes 11 and 12, and contained 48% of the total mapped T-DNA tags.3. The T-DNA insertions were analysed in genie, intergenic and repeat DNA regions. Only 2.4% T-DNA insertions were identified in repeat DNA regions. The frequencies of T-DNA insertions in genie and intergenic regions (58.1% and 41.9%) showed a good correlation with the predicted size distribution of genie and intergenic regions in the rice genome (57.8% and 42.2%). The density of T-DNAinsertions in 5'- and 3'- regulatory regions of 500bp around the ATG and stop codons, respectively, was approximately 2-3.5 times those in genie and intergenic regions, suggesting that T-DNA is preferentially inserted in the 5'- and 3'-regulatory regions of the genes. This is confirmed by analysis of chromosome 1 and also found that T-DNA insertions had a relatively high density in introns, (more than twice that in exons), which indicates that T-DNA was preferentially inserted into introns compared to exons. The results also suggested that T-DNA were evenly spread within genes, without any obvious bias toward the 5' or 3' ends of the genes.4. Analysis of AT content of the rice genome sequences near T-DNA insertion sites indicates that no obvious bias was seen in the AT nucleotide composition close to T-DNA insertion sites. The junction sequences between T-DNA borders and the rice genome were determined for the left borders of 233 T-DNA insertions and the right borders of 190 T-DNA insertions. The results showed that truncations of T-DNA borders, insertions of filler DNA and the formation of inverted and direct repeat occurred during T-DNA integration. From the cleavage site of the T-DNA borders, no truncations occurred for nine left borders, most of the left borders deleted less than 35bp and most of the right borders truncated to 21bp. The size of filler DNA ranged from several bp to approximately 300bp and originated from T-DNA or genome sequences. The insertions of filler DNA occurred for both the single copy of T-DNA borders and the T-DNA repeats. The complex configuration of T-DNA integrations could be explained by DSB repair model.