해충저항성 GM 배추에서 T-DNA와 식물체 게놈의 인접 부위 분석
Analysis of junction between T-DNA and plant genome in insect resistance GM Chinese cabbage
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The Agrobacterium-mediated transformation has been successfully used method to introduce foreign genes into some monocotyledonous as well as a large number of dicotyledonous plants genome. We developed transgenic Chinese cabbage plants with insect-resistance gene, modified CryIAc, by Agrobacterium- transformation and confirmed transgene copy number by Southern blot analysis. We confirmed that twenty-nine out of 46 transgenic Chinese cabbage plants have single copy of CryIAc. To obtain the sequences information on the transferred DNA (T-DNA) integration into plant genome, we analyzed left border (LB) flanking sequences by genome walking (GW) PCR method. Out of 46 transgenic Chinese cabbage plants examined, 37 carried the vector backbone sequences. This result indicates that the transfer of the vector backbone from the binary vectors resulted mainly from inefficient termination of LB site. Analysis of T-DNA LB flanking region of 9 transgenic Chinese cabbage plants without vector backbone revealed that all LB ends were not conserved and nucleotides up to 36bp from the LB cleavage site were deleted.
Abstract
The Agrobacterium-mediated transformation has been successfully used method to introduce foreign genes into some monocotyledonous as well as a large number of dicotyledonous plants genome. We developed transgenic Chinese cabbage plants with insect-resistance gene, modified CryIAc, by Agrobacterium- transformation and confirmed transgene copy number by Southern blot analysis. We confirmed that twenty-nine out of 46 transgenic Chinese cabbage plants have single copy of CryIAc. To obtain the sequences information on the transferred DNA (T-DNA) integration into plant genome, we analyzed left border (LB) flanking sequences by genome walking (GW) PCR method. Out of 46 transgenic Chinese cabbage plants examined, 37 carried the vector backbone sequences. This result indicates that the transfer of the vector backbone from the binary vectors resulted mainly from inefficient termination of LB site. Analysis of T-DNA LB flanking region of 9 transgenic Chinese cabbage plants without vector backbone revealed that all LB ends were not conserved and nucleotides up to 36bp from the LB cleavage site were deleted.
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