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应用农杆菌介导的方法获得了有Ds因子插入的3000多株水稻转化群体,用Inverse PCR方法,从部分独立转化植株中分离了590条含Ds因子的T-DNA插入位点处的右侧旁邻水稻染色体序列.根据旁邻序列中T-DNA右边界与侧翼水稻序列之间的插入序列的特征可分成6个主要类别,其中类型Ⅰ是主要类型,为通常的T-DNA整合,即T-DNA右边界序列与水稻染色体序列相连,或者其间插入小于50 bp的序列片段;类型Ⅱ为T-DNA右边界旁先接T-DNA载体序列,再与水稻序列相接的重组类型.340个类型Ⅰ和Ⅱ的旁邻序列通过与已知的水稻染色体序列数据库一致性比较分析,确定了它们在水稻染色体上的分布位置,构建了一个Ds因子在水稻12条染色体插入的框架结构.这340个有Ds因子插入的位点在整个染色体上平均相距0.8 Mb.分析在第1条染色体上T-DNA(Ds)插入情况显示有21％的频率插入到预测基因的外显子中.T-DNA(Ds)在染色体上分布位置的确定,使我们可以选择合适的Ds因子插入株作为起始株系,导入Ac转座酶基因后,使Ds发生转座,从而获得新的Ds插入突变株,为进一步利用Ds转座标签法分离水稻基因创造了条件.
More than 3000 rice transformed individuals with Ds factor insertion were obtained by Agrobacterium tumefaciens-mediated transformation. 590 pairs of Ds factor-containing T-DNA insertion sites were isolated from some independently transformed plants by Inverse PCR The adjacent rice chromosome sequence can be divided into six major categories based on the characteristics of the insert sequence between the right border of T-DNA and the flanking rice sequence in the adjacent sequence, of which type I is the predominant type and is the usual T-DNA integration, ie The right border sequence of T-DNA is linked to the rice chromosome sequence or a sequence fragment of less than 50 bp is inserted in it. Type II is a recombination type in which the T-DNA vector sequence precedes the T-DNA right border and then is connected with the rice sequence. The adjacent sequences of type I and II were compared with known rice chromosome sequence databases to determine their distribution on chromosomes of rice and a frame structure of Ds inserted in 12 chromosomes of rice was constructed. 340 sites with Ds insertion averaged 0.8 Mb across the entire chromosome Analysis of T-DNA (Ds) insertion on chromosome 1 showed 21% insertion into exons of the predicted gene T -DNA Ds) on the chromosome, we can select the appropriate Ds factor insertion strain as the starting strain. After the introduction of the Ac transposase gene, Ds can be transposed to obtain a new Ds insertion mutant. Further use of Ds transposon tagging method to create a condition for the isolation of rice genes.