Olszewski N E, Martin F B, Ausubel F M
Department of Genetics, Harvard Medical School, Massachusetts General Hospital, Boston 02114.
Nucleic Acids Res. 1988 Nov 25;16(22):10765-82. doi: 10.1093/nar/16.22.10765.
We constructed a cosmid vector, pOCA18, designed for transferring plant genomic libraries from Agrobacterium tumefaciens to plants. Clones from a genomic library of Arabidopsis thaliana DNA in pOCA 18 were propagated stably in both Escherichia coli and A. tumefaciens. Clones from the pOCA18 A. thaliana library were used to construct transgenic Nicotiana tabacum plants; the DNA inserts were transferred intact in 10 out of 16 transgenic N. tabacum plants examined but were partially deleted in six others. Transgenic N. tabacum plants constructed with a mutant A. thaliana acetohydroxy acid synthase gene (from the pOCA18 library) that encodes an enzyme resistant to the herbicide chlorsulfuron were resistant to chlorsulfuron. A statistical analysis indicated that if the A. thaliana library contains 10(7) members and if 10(7) A. tumefaciens transconjugants containing the library were used to transform plant cells, then 2 x 10(4) transformed plant cells must be generated to have a 95% probability of constructing a transgenic plant carrying a specific DNA sequence from the A. thaliana library.
我们构建了一种黏粒载体pOCA18，其设计目的是将植物基因组文库从根癌农杆菌转移至植物中。来自拟南芥DNA基因组文库的pOCA 18克隆在大肠杆菌和根癌农杆菌中均能稳定繁殖。来自pOCA18拟南芥文库的克隆用于构建转基因烟草植株；在所检测的16株转基因烟草植株中，有10株的DNA插入片段完整转移，但另外6株中出现了部分缺失。用一个突变的拟南芥乙酰羟酸合酶基因（来自pOCA18文库）构建的转基因烟草植株对除草剂氯磺隆具有抗性，该基因编码一种对氯磺隆有抗性的酶。统计分析表明，如果拟南芥文库包含10⁷个成员，并且如果用10⁷个含有该文库的根癌农杆菌转接合子来转化植物细胞，那么必须产生2×10⁴个转化植物细胞，才有95%的概率构建出携带拟南芥文库中特定DNA序列的转基因植物。
