Ma H, Yanofsky M F, Klee H J, Bowman J L, Meyerowitz E M
Division of Biology, California Institute of Technology, Pasadena 91125.
Gene. 1992 Aug 15;117(2):161-7. doi: 10.1016/0378-1119(92)90725-5.
A series of vectors has been constructed for the purpose of introducing cloned DNAs into plant genomes, using Agrobacterium tumefaciens-mediated transformation methods. One of these vectors, pCIT20, is a plasmid that contains a multiple cloning site (MCS), and a marker (Hph) that confers hygromycin resistance to plant cells. The others are all cosmid vectors which allow insertion of up to 46 kb of plant genomic DNA, and which also contain all of the necessary sequences for A. tumefaciens-mediated plant transformation. The cosmid vectors either contain a Hph marker (pCIT30), or a kanamycin-resistance marker (pCIT101-104). Three of the cosmid vectors (pCIT30, pCIT101, and pCIT103) carry bacteriophage T7 and SP6 promoters flanking the cloning Bg/II site, for synthesis of end-specific RNAs. The end-specific RNAs may be used as probes when labeled with radioactive or biotinylated nucleotides, for example, in a chromosome-walking experiment. The other two cosmid vectors (pCIT102 and pCIT104) carry restriction sites flanking the insertion site (XhoI) for convenient release of the insert by restriction digests. These sites, in combination with sites internal to the insert, allow the generation of end fragments for subcloning or labeling probes. These vectors should be valuable for isolation and analysis of plant genes, using transformation, library screening, and chromosome-walking approaches.
为了利用根癌农杆菌介导的转化方法将克隆的DNA导入植物基因组，已经构建了一系列载体。其中一个载体pCIT20是一种质粒，它含有一个多克隆位点（MCS）和一个赋予植物细胞潮霉素抗性的标记（Hph）。其他载体都是黏粒载体，可插入长达46 kb的植物基因组DNA，并且还包含根癌农杆菌介导的植物转化所需的所有序列。黏粒载体要么含有Hph标记（pCIT30），要么含有卡那霉素抗性标记（pCIT101 - 104）。其中三个黏粒载体（pCIT30、pCIT101和pCIT103）在克隆Bg/II位点两侧带有噬菌体T7和SP6启动子，用于合成末端特异性RNA。例如，在染色体步移实验中，当用放射性或生物素化核苷酸标记时，末端特异性RNA可用作探针。另外两个黏粒载体（pCIT102和pCIT104）在插入位点（XhoI）两侧带有限制性酶切位点，以便通过限制性酶切方便地释放插入片段。这些位点与插入片段内部的位点相结合，可产生用于亚克隆或标记探针的末端片段。这些载体对于利用转化、文库筛选和染色体步移方法分离和分析植物基因应该是有价值的。
