Vector DNA fragments integrating into transgenic carrot genome during Agrobacterium-mediated transformation
Insertion of foreign genes into plants using T-DNA is one of the major techniques for creating stable strains of transgenic plants. This technique, however, is prone to errors, in which vector backbone sequences are also inserted into the plant genome. In present work we assess the rate and diversity of such undesirable vector insertion using sampling of transgenic carrot (Daucus carota L.) plants. We have demonstrated that various fragments of vector DNA are transferred to the genome of transgenic carrot plants due to the errors in T-DNA processing. Totally, 17 types of vector DNA fragments were detected. We found vector sequences in 43 of the 119 analyzed plants (36.1%). The fragments can either be very small (300-400 bp) or contain the entire vector sequence (8600 bp in the case of plasmid pBi121). We have also demonstrated that both T-DNA borders can be erroneously determined and cut. Our data confirm the hypothesis that the error in identification of the left terminal repeat occurs during transfer of the entire vector sequence into the plant genome, as the number of the vector sequences adjacent to the left terminal repeat (22.7% 27 plants carrying such fragments) exceeds the number of those adjacent to the right terminal repeat (15.1% 18 plants carrying such fragments). Our data comply with the published data, stating that a precise cut in the region of the right terminal repeat is an important stage in T-DNA processing. Our work also showed that a vector sequence can be inserted independently of T-DNA into the same plant that carries an incorrectly excised T-DNA. We have found four plants (3.4%) which carry vector fragments that were inserted independently of T-DNA. Presumably, the transfer rate of the vector fragments independently of T-DNA during Agrobacterium-mediated transformation can be even higher. Some part of the transformed plants may contain only a vector fragment and lack T-DNA, such plants will be eliminated during selection. The regions of vector DNA from two carrot plants inserted adjacent to T-DNA into the genome of two carrot plants were cloned and sequenced. The search for homologous sequences in GenBank confirmed that they originated from the plasmid pBI121, used for producing these transgenic plants. Thus, the transfer of vector sequences that is an undesirable, but apparently at present unavoidable part of the mechanism of T-DNA transfer, should be kept in mind when producing transgenic plants.
Keywords
трансгенные растения,
краевые векторные последовательности,
встраивание Т-ДНК,
transgenic plants,
vector backbone sequences,
T-DNA integration,
Agrobacterium tumefaciens,
Agrobacterium tumefaciensAuthors
| Permyakova Natalya V. | Siberian Branch of the Russian Academy of Sciences (Novosibirsk) | puh@bionet.nsc.ru |
| Deineko Elena V. | Siberian Branch of the Russian Academy of Sciences (Novosibirsk); Tomsk State University | deineko@bionet.nsc.ru |
Всего: 2
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