Characteristics Of T-DNA Integration In Transgenic Cotton(gossypium Hirsutum L.)

The copy number of transgene is one of the factors which affect the transgene inheritance and it's expression. However, it is a common phenomenon in transgenic lines, transformed not only through pollen tube injection, but by gene-gun bombardment or Agrobacterium-mediated, that multiple copies were integrated into plant genome. Therefore, whether based on the perspective of breeding, or the study of the molecular characteristics of transgene integration, identification of it's copy number is the basic step. In this research, PfaffI method was, for the first time, applied to identify the transgene copy number in transgenic T0 transgenic cotton lines transformed through pollen tube injection, gene-gun bombardment or Agrobacterium-mediated, respectively.Foreign gene was randomly integrated into the different regions of each chromosomes. However, we prefer to think that the random distribution of transgene in the plant genome should not be regarded as a feature of arbitrary in the sequences and/or its structures of T-DNA integration sites. Therefore, a total of 135 flanking sequences of T-DNA in T0 transgenic cotton lines were cloned and sequenced to analyze the integration characteristics of T-DNA.All of the conclusions in this study were as follows:1) PfaffI method, which was usually used to study the gene expression, was, for the first time, applied to identify the copy number of transgene(T-DNA)in transgenic plants.2) The percentage of transgenic cotton T0 lines of 1-2 T-DNA copy(ies), transformed through pollen tube injection, gene-gun bombardment and Agrobacterium-mediated, respectively, were 60%, 42.8% and26.2%. In addition, as many as 27.3% of lines transformed by Agrobacterium-mediated were inserted more than 5 T-DNA copies.3) The backbone vector sequences outside both left and right border of T-DNA were integrated into cotton genome in some transgenic lines. In the left border, the nick position were mostly A or T(89.5%). On the contrary, in the right border, the nick position were mostly G(87%).4) There was not a preference for T-DNA integration in any regions in cotton genome. The cotton genome sequence , which has a 100% homology to sequence of T-DNA right border and it's adjacent region sequence is likely to be a hot sequence for T-DNA integration.