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首页> 外文期刊>Journal of Plant Physiology >Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA :: barley genomic DNA junctions
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Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA :: barley genomic DNA junctions

机译:农杆菌介导的大麦(Hordeum vulgare L.)转化,使用绿色荧光蛋白作为可视标记,并对T-DNA ::大麦基因组DNA连接进行序列分析

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摘要

Agrobacterium-mediated barley transformation promises many advantages compared to alternative gene transfer methods, but has so far been established in only a few laboratories. We describe a protocol that facilitates rapid establishment and optimisation of Agrobacterium-mediated transformation for barley by instant monitoring of the transformation success. The synthetic green fluorescent protein (sgfpS65T) reporter gene was introduced in combination with the hpt selectable marker gene into immature embryos of barley (Hordeum vulgare L.) by cocultivation with Agrobacterium tumefaciens strain AGLO harboring binary vector pYF133. Using green fluorescent protein (GFP) as a nondestructive visual marker allowed us to identify single-cell recipients of T-DNA at an early stage, track their fate and evaluate factors that affect T-DNA delivery. GFP screening was combined with a low level hygromycin selection. Consequently, transgenic plantlets ready to transfer to soil were obtained within 50 days of explant culture. Southern blot- and progeny segregation analyses revealed a single copy T-DNA insert in more than half of the transgenic barley plants. T-DNA/barley genomic DNA junctions were amplified and sequenced. The right T-DNA ends were highly conserved and clustered around the first 4 nucleotides of the right 25 bp border repeat, while the left T-DNA ends were more variable, located either in the left 25 bp border repeat or within 13 bp from the left repeat. T-DNAs were transferred from Agrobacterium to barley with exclusion of vector sequence suggesting a similar molecular T-DNA transfer mechanism as in dicotyledonous plants.
机译:与替代的基因转移方法相比,农杆菌介导的大麦转化有望带来许多优势,但迄今为止,仅在少数几个实验室中得到了证实。我们描述了一种协议,可通过对转化成功的即时监测来促进快速建立和优化大麦农杆菌介导的转化。通过与携带二元载体pYF133的根癌农杆菌菌株AGLO共培养,将合成的绿色荧光蛋白(sgfpS65T)报告基因与hpt选择标记基因组合导入大麦(Hordeum vulgare L.)的未成熟胚。使用绿色荧光蛋白(GFP)作为非破坏性视觉标记,使我们能够在早期识别T-DNA的单细胞受体,追踪其命运并评估影响T-DNA传递的因素。 GFP筛选与低水平潮霉素选择相结合。因此,在外植体培养的50天内获得了准备转移到土壤中的转基因苗。 Southern印迹和后代分离分析显示,在一半以上的转基因大麦植物中有一个单拷贝的T-DNA插入片段。 T-DNA /大麦基因组DNA连接被扩增和测序。右边的T-DNA末端高度保守,并聚集在右边25 bp边界重复序列的前4个核苷酸周围,而左边的T-DNA末端更具可变性,位于左边25 bp边界重复序列中或距离碱基25 bp以内左重复。 T-DNAs从农杆菌转移到大麦,排除了载体序列,这暗示了与双子叶植物相似的分子T-DNA转移机制。

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