Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: Evidence for multiple complex patterns of integration
Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: Evidence for multiple complex patterns of integration
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DOI：
10.1046/j.1365-313x.1997.11050945.x
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发表时间：
1997-05-01
期刊：
影响因子：
7.2
通讯作者：
Gelvin, SB
中科院分区：
文献类型：
--
作者：
Kononov, ME;Bassuner, B;Gelvin, SB
在冠瘿肿瘤发生的过程中，根癌农杆菌将部分肿瘤诱导（Ti）质粒（T-DNA）转移到植物细胞中，在那里它最终稳定地整合到植物基因组中。直接重复的DNA序列，称为T-DNA边界，定义了T-DNA的左端和右端。T-DNA可以与Ti质粒的剩余部分物理分离，产生“二元载体”系统;该系统经常用于产生转基因植物。科学家们最初认为，只有那些位于T-DNA左右边界之间的序列转移到了植物中。然而，最近出现了一些报道，描述了非T-DNA二元载体“骨架”序列整合到转基因植物的基因组中。为了研究这一现象，我们构建了T-DNA双元载体，其含有T-DNA内的nosnptll基因和T-DNA边界外的mas 2 ′-gusA（β-葡萄糖醛酸酶）基因。我们再生卡那霉素抗性转基因烟草植物，并分析这些植物的载体定位的gosA基因的表达和二元载体骨架序列的存在。大约五分之一的植物表达可检测的GUS活性。PCR分析表明，约75%的植物含有gusA基因。Southern杂交分析表明，载体骨架序列可以整合到烟草基因组中，连接到左边或右边的T-DNA边界。载体骨架序列也可以独立于T-DNA（不与T-DNA连接）整合到植物基因组中。尽管我们可以容易地检测到细菌内含有T-DNA的T链，但我们不能检测到仅含有载体骨架序列或与T-DNA连接的这些载体序列的T链。
During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the plant genome. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant. More recently, however, several reports have appeared describing the integration of the non-T-DNA binary vector 'backbone' sequences into the genome of transgenic plants. In order to investigate this phenomenon, we constructed T-DNA binary vectors containing a nosnptll gene within the T-DNA and a mas2'-gusA (beta-glucuronidase) gene outside the T-DNA borders. We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gosA gene and for the presence of binary vector backbone sequences. Approximately one-fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border. The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T-DNA. Although we could readily detect T-strands containing the T-DNA within the bacterium, we could not detect T-strands containing only the vector backbone sequences or these vector sequences linked to the T-DNA.