Ambros P F, Matzke A J M, Matzke M A
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
EMBO J. 1986 Sep;5(9):2073-2077. doi: 10.1002/j.1460-2075.1986.tb04468.x.
We have used in situ hybridization to determine the sites of insertion of Agrobacterium rhizogenes Ri T-DNA in the chromosomes of Crepis capillaris (2n = 6) transformed roots. Four transformed root lines were obtained by infecting Crepis stem segments with A. rhizogenes. Southern hybridization analysis indicated that each root line was the result of one or more independent T-DNA insertion events. In two root lines, one copy of T-DNA was present; the other two root lines each contained two copies of T-DNA. To localize these T-DNA inserts on Crepis chromosomes, metaphase spreads were perpared from each root line, and hybridized in situ to a biotinlabeled T-DNA probe. The results indicated that T-DNA was present in a different chromosomal location in each root line, and that each chromosome had been a target for T-DNA insertion at least once. In the root lines containing two T-DNA inserts, two patterns of integration were observed: in one case the T-DNAs were present on separate chromosomes; in the other case the two T-DNAs were close together (but not tandemly arranged) on a single chromosome. A comparison of these results and those obtained previously for a fifth Crepis-transformed root line demostrated that Ri T-DNA does not insert preferentially into a particlar chromosomal location.
我们已使用原位杂交技术来确定发根农杆菌Ri T-DNA在毛状鸦葱（2n = 6）转化根染色体中的插入位点。通过用发根农杆菌感染毛状鸦葱茎段获得了四条转化根系。Southern杂交分析表明，每个根系都是一个或多个独立的T-DNA插入事件的结果。在两条根系中，存在一份T-DNA拷贝；另外两条根系各包含两份T-DNA拷贝。为了将这些T-DNA插入片段定位在毛状鸦葱染色体上，从每个根系制备中期染色体铺片，并与生物素标记的T-DNA探针进行原位杂交。结果表明，每个根系中T-DNA存在于不同的染色体位置，并且每条染色体至少有一次成为T-DNA插入的靶点。在含有两份T-DNA插入片段的根系中，观察到两种整合模式：一种情况下，T-DNA存在于不同的染色体上；另一种情况下，两份T-DNA在一条染色体上靠得很近（但不是串联排列）。将这些结果与之前从第五条毛状鸦葱转化根系获得的结果进行比较，表明Ri T-DNA不会优先插入特定的染色体位置。
