title: Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning.
abstract: 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.
authors.affiliation: Genetic Engineering Laboratory, College of Life Science, South China Agricultural University, Guangzhou 510642, PR China. ygliu@scau.edu.cn
authors.forename: Yao-Guang
authors.initials: YG
authors.lastname: Liu
authors.forename: Hongmei
authors.initials: H
authors.lastname: Liu
authors.forename: Letian
authors.initials: L
authors.lastname: Chen
authors.forename: Weihua
authors.initials: W
authors.lastname: Qiu
authors.forename: Qunyu
authors.initials: Q
authors.lastname: Zhang
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.forename: Chunyi
authors.initials: C
authors.lastname: Yang
authors.forename: Jing
authors.initials: J
authors.lastname: Su
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Wang
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Tian
authors.forename: Mantong
authors.initials: M
authors.lastname: Mei
mesh_terms: D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D015698:Genomic Library; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D018744:DNA, Plant; D015247:Deoxyribonuclease HindIII