T‐DNA Integration Category and Mechanism in Rice Genome

Abstract

Abstract: T‐DNA integration is a key step in the process of plant transformation, which is proven to be important for analyzing T‐DNA integration mechanism. The structures of T‐DNA right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the T‐DNA right border occurred mainly on five nucleotides “TGACA” in inverse repeat (IR) sequence of 25 bp, especially on the third base “A”. However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the T‐DNA right border, which made the T‐DNA, carrying vector sequences, integrated into the rice genome. These results seemingly suggest that the IR sequence of the right border plays an important role in the process of T‐DNA integration into the rice genome, but is not an essential element. The appearance of vector sequences neighboring the T‐DNA right border suggested that before being transferred into the plant cell from Agrobacterium, the entire T‐DNA possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the T‐DNA right border homologous with plant DNA and filler DNAs were frequently discovered in the integrated position of T‐DNA. Some small regions in the right border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler DNA, through mutual twisting, and then the T‐DNA was integrated into plant chromosome through a partially homologous recombination mechanism. The appearance of filler DNA would facilitate T‐DNA integration. The fragments flanking the T‐DNA right border in transformed rice plants could derive from different parts of the inner T‐DNA region; that is, disruption and recombination could occur at arbitrary positions in the entire T‐DNA, in which the homologous area was comparatively easier to be disrupted. The structure of flanking sequences of T‐DNA integrated in the rice chromosome presented various complexities. These complexities were probably a result of different patterns of recombination in the integrating process. Some types of possible integrating mechanism are detailed.(Managing editor: Li‐Hui ZHAO)