Afolabi A S, Worland B, Snape J W, Vain P
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Theor Appl Genet. 2004 Aug;109(4):815-26. doi: 10.1007/s00122-004-1692-y. Epub 2004 May 15.
Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70-78% of the plant lines causing 14-21% of the loci to contain only the mid to right border part of a T-DNA. In 53-66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60-80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53-66% of loci) were generally a greater limitation to the production of marker-free T(1) plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).
在使用双元载体系统pGreen（含bar和gus基因的T-DNA）/pSoup（含aphIV和gfp基因的T-DNA）转化的水稻植株群体中，评估了转基因位点组成和T-DNA连锁构型。在62个独立转化的株系和大约4000个后代植株中分析了转基因结构、表达和遗传情况。这些株系表现出多种转基因位点数和组成。最常见的整合形式是两个T-DNA都整合在同一位置（占位点的56%）。当发生单类型T-DNA整合时（占位点的44%），pGreen T-DNA优先整合。在大约一半的株系（52%）中，T-DNA整合在两个或更多独立位点。在这些植株中，在转化过程中，混合和单类型T-DNA整合常常在不同位点同时发生。70 - 78%的株系中存在不完整的T-DNA，导致14 - 21%的位点仅包含T-DNA的中右边界部分。在53 - 66%的位点中，T-DNA与载体骨架序列整合。后代植株中转基因存在情况和表达的比较表明，转基因表型的分离既不是转基因遗传的可靠指标，也不是T-DNA连锁的可靠指标，因为通过表达研究仅检测到60 - 80%的转基因位点。与共转化（71%的株系）和非连锁整合（44%的位点）相比，共表达（28%的株系）和骨架转移（53 - 66%的位点）通常对产生表达目的基因的无标记T(1)植株的限制更大。
