Barton K A, Binns A N, Matzke A J, Chilton M D
Cell. 1983 Apr;32(4):1033-43. doi: 10.1016/0092-8674(83)90288-x.
Cloned DNA sequences encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase have been inserted into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the "rooty" locus. Transformation of tobacco stem segments with the engineered bacterial strains produced attenuated crown gall tumors that were capable of regeneration into intact, normal tobacco plants. The yeast gene and entire transferred DNA (T-DNA) were present in the regenerated plants in multiple copies, and nopaline was found in all tissues. The plants were fertile, and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA. Expression of nopaline in the germinated seedlings derived from one regenerated plant was variable and did not correlate with the levels of T-DNA present in the seedlings. Preliminary evidence indicates that nopaline in progeny of other similarly engineered plants is more uniform. The disarming of pTiT37 by insertions at the "rooty" locus thus appears to produce a useful gene vector for higher plants.
编码酵母乙醇脱氢酶和细菌新霉素磷酸转移酶的克隆DNA序列已插入到根癌农杆菌质粒pTiT37的T-DNA的“根瘤”位点。用工程改造的菌株转化烟草茎段产生了弱化的冠瘿瘤，这些瘤能够再生为完整的正常烟草植株。酵母基因和整个转移DNA（T-DNA）在再生植株中以多拷贝形式存在，并且在所有组织中都发现了胭脂碱。这些植株可育，自花授粉产生的幼苗也含有完整且多拷贝的工程改造T-DNA。来自一株再生植株的萌发幼苗中胭脂碱的表达是可变的，并且与幼苗中T-DNA的水平无关。初步证据表明，其他类似工程改造植株后代中的胭脂碱更为一致。因此，通过在“根瘤”位点插入而使pTiT37解除武装，似乎产生了一种用于高等植物的有用基因载体。
