Song J, Bradeen J M, Naess S K, Helgeson J P, Jiang J
Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA.
Theor Appl Genet. 2003 Sep;107(5):958-64. doi: 10.1007/s00122-003-1334-9. Epub 2003 Jul 26.
Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium. We tested the possible factors that may cause instability, including the insert sizes of the BIBAC and TAC constructs, potato DNA fragments consisting of highly repetitive or largely single-copy DNA sequences, different Agrobacterium transformation methods and different Agrobacterium strains. The insert sizes of the potato BIBAC and TAC constructs were found to be critical to their stability in Agrobacterium. All constructs containing a potato DNA fragment larger than 100 kb were not stable in any of the four tested Agrobacterium strains, including two recA deficient strains. We developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem.
开发将大DNA片段导入植物的高效方法将极大地促进基于图谱的基因克隆。最近开发的BIBAC和TAC载体已显示出通过农杆菌介导的转化将大DNA片段导入植物的潜力。在此我们报告，含有大于100 kb的马铃薯基因组DNA片段的BIBAC和TAC克隆在农杆菌中不稳定。我们测试了可能导致不稳定的因素，包括BIBAC和TAC构建体的插入片段大小、由高度重复或主要为单拷贝DNA序列组成的马铃薯DNA片段、不同的农杆菌转化方法以及不同的农杆菌菌株。发现马铃薯BIBAC和TAC构建体的插入片段大小对其在农杆菌中的稳定性至关重要。所有含有大于100 kb马铃薯DNA片段的构建体在四种测试的农杆菌菌株（包括两种recA缺陷菌株）中的任何一种中都不稳定。我们开发了一种基于转座子的技术，可用于将BAC插入片段高效亚克隆到两到三个BIBAC/TAC构建体中，以规避不稳定性问题。
