Liu Yao-Guang, Liu Hongmei, Chen Letian, Qiu Weihua, Zhang Qunyu, Wu Hao, Yang Chunyi, Su Jing, Wang Zhonghua, Tian Dongsheng, Mei Mantong
Genetic Engineering Laboratory, College of Life Science, South China Agricultural University, Guangzhou 510642, PR China.
Gene. 2002 Jan 9;282(1-2):247-55. doi: 10.1016/s0378-1119(01)00849-6.
The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.
已证明具备转化能力的人工染色体载体（TAC）系统在拟南芥中高效分离基因方面非常有用（《美国国家科学院院刊》96 (1998) 6535）。为使该载体系统适用于作物基因分离，开发了两种新的TAC载体和水稻基因组文库。新载体pYLTAC17和pYLTAC27分别使用由水稻Act1启动子驱动的Bar基因和Hpt基因作为植物选择标记，适用于水稻和其他禾本科植物的转化。利用pYLTAC17，使用不同大小类别的部分消化DNA片段构建了杂交水稻恢复系明恢63的两个代表性基因组文库（I和II）。文库I和文库II分别由34,560个和1.2×10⁵个克隆组成，平均插入片段大小分别约为77 kb和39 kb。文库I和文库II的基因组覆盖率估计分别约为5个和11个单倍体基因组当量。文库I的克隆单独保存在90个384孔板中，文库II的克隆则收集为每个包含30 - 50个克隆的混合池，并保存在8个384孔板中。使用多种探针与通过改进的复制方法制备的文库I的高密度菌落滤膜杂交，每个探针检测到2 - 9个阳性克隆。开发了一种通过混合菌落杂交快速筛选文库II的方法。一个具有80 kb水稻DNA插入片段的TAC克隆通过农杆菌介导的转化成功转入水稻基因组。这些新载体和基因组文库应有助于水稻和其他作物的基因克隆和基因工程。
