Department of Plant Pathology and Crop Physiology, 302 Life Sciences Building, Louisiana State University and LSU Agricultural Center, Baton Rouge, LA 70803-1720, USA.
Plant Sci. 2012 May;187:49-58. doi: 10.1016/j.plantsci.2012.01.012. Epub 2012 Feb 2.
Small high-yielding binary Ti vectors of Agrobacterium tumefaciens were constructed to increase the cloning efficiency and plasmid yield in Escherichia coli and A. tumefaciens for transformation of higher plants. We reduced the size of the binary vector backbone to 4566bp with ColE1 replicon (715bp) for E. coli and VS1 replicon (2654bp) for A. tumefaciens, a bacterial kanamycin resistance gene (999bp), and the T-DNA region (152bp). The binary Ti vectors with the truncated VS1 replicon were stably maintained with more than 98% efficiency in A. tumefaciens without antibiotic selection for 4 days of successive transfers. The transcriptional direction of VS1 replicon can be the same as that of ColE1 replicon (co-directional transcription), or opposite (head-on transcription) as in the case of widely used vectors (pPZP or pCambia). New binary vectors with co-directional transcription yielded in E. coli up to four-fold higher transformation frequency than those with the head-on transcription. In A. tumefaciens the effect of co-directional transcription is still positive in up to 1.8-fold higher transformation frequency than that of head-on transcription. Transformation frequencies of new vectors are over six-fold higher than those of pCambia vector in A. tumefaciens. DNA yields of new vectors were three to five-fold greater than pCambia in E. coli. The proper functions of the new T-DNA borders and new plant selection marker genes were confirmed after A. tumefaciens-mediated transformation of tobacco leaf discs, resulting in virtually all treated leaf discs transformed and induced calli. Genetic analysis of kanamycin resistance trait among the progeny showed that the kanamycin resistance and sensitivity traits were segregated into the 3:1 ratio, indicating that the kanamycin resistance genes were integrated stably into a locus or closely linked loci of the nuclear chromosomal DNA of the primary transgenic tobacco plants and inherited to the second generation.
为提高农杆菌转化高等植物的克隆效率和质粒产量，构建了小而高效的农杆菌二元 Ti 载体。我们将二元载体骨架缩小至 4566bp，其中包含大肠杆菌的 ColE1 复制子（715bp）和农杆菌的 VS1 复制子（2654bp）、一个细菌卡那霉素抗性基因（999bp）和 T-DNA 区（152bp）。带有截短 VS1 复制子的二元 Ti 载体在没有抗生素选择的情况下，在农杆菌中稳定维持，连续转接 4 天的效率超过 98%。VS1 复制子的转录方向可以与 ColE1 复制子相同（同向转录），也可以与广泛使用的载体（pPZP 或 pCambia）相反（头对头转录）。在大肠杆菌中，具有同向转录的新型二元载体的转化频率比具有头对头转录的载体高 4 倍。在农杆菌中，同向转录的效果仍然是正向的，转化频率比头对头转录高 1.8 倍。新型载体在农杆菌中的转化频率比 pCambia 载体高 6 倍以上。在大肠杆菌中，新型载体的 DNA 产量比 pCambia 高 3 到 5 倍。通过农杆菌介导的烟草叶片转化，证实了新型 T-DNA 边界和新的植物选择标记基因的正确功能，导致几乎所有处理的叶片都发生转化并诱导愈伤组织。对后代中卡那霉素抗性性状的遗传分析表明，卡那霉素抗性和敏感性性状按 3:1 的比例分离，表明卡那霉素抗性基因稳定地整合到初级转基因烟草植株核染色体 DNA 的一个或紧密连锁的位点上，并遗传到第二代。
