Nagano Yukio, Takao Syoko, Kudo Takahiro, Iizasa Ei'ichi, Anai Toyoaki
Analytical Research Center for Experimental Sciences, Saga University, Saga, Japan.
Plant Cell Rep. 2007 Dec;26(12):2111-7. doi: 10.1007/s00299-007-0428-2. Epub 2007 Aug 7.
T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.
T-DNA双元载体常用于植物转化实验。由于它们通常非常大，且适合DNA连接反应的限制酶切位点很少，因此将DNA片段克隆到这些载体中很困难。我们在此提供一种将DNA片段克隆到非常大的载体中的替代方法。我们基于酵母的重组工程方法能够通过一步转化将DNA片段克隆到某些类型的T-DNA双元载体中，而无需特定的重组位点或精确定位的限制酶切末端，从而使克隆过程更加灵活。此外，该方法成本低廉，适用于多片段克隆。
