Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice.

We have generated 47,932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41,234 lines, we obtained 27,621 flanking sequence tags (FSTs), among which 12,505 were integrated into genic regions and 15,116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http://www.postech.ac.kr/life/pfg/risd. On request, seeds of the T(1) or T(2) plants will be provided to the scientific community.

[1]  E. Ábrahám,et al.  Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. , 2002, The Plant journal : for cell and molecular biology.

[2]  Hong-Gyu Kang,et al.  Generation and Analysis of End Sequence Database for T-DNA Tagging Lines in Rice1 , 2003, Plant Physiology.

[3]  N. Chua,et al.  The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[4]  J. Kawai,et al.  Collection, Mapping, and Annotation of Over 28,000 cDNA Clones from japonica Rice , 2003, Science.

[5]  J. Chory,et al.  BAS1: A gene regulating brassinosteroid levels and light responsiveness in Arabidopsis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[6]  G. An,et al.  T-DNA Insertional Mutagenesis for Activation Tagging in Rice1 , 2002, Plant Physiology.

[7]  G. Angenent,et al.  Low frequency of T‐DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences , 2003, FEBS letters.

[8]  A. Oliphant,et al.  A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). , 2002, Science.

[9]  E. Zubko,et al.  Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants. , 2002, The Plant journal : for cell and molecular biology.

[10]  T. Gojobori,et al.  The genome sequence and structure of rice chromosome 1 , 2002, Nature.

[11]  J. Chory,et al.  A role for flavin monooxygenase-like enzymes in auxin biosynthesis. , 2001, Science.

[12]  Jia Li,et al.  BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[13]  F. Nagy,et al.  Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter , 1985, Nature.

[14]  A. Miyao,et al.  Target Site Specificity of the Tos17 Retrotransposon Shows a Preference for Insertion within Genes and against Insertion in Retrotransposon-Rich Regions of the Genome Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.012559. , 2003, The Plant Cell Online.

[15]  L. van der Fits,et al.  ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. , 2000, Science.

[16]  V. Sundaresan,et al.  Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. , 2000, Current opinion in biotechnology.

[17]  Srinivasan Ramachandran,et al.  Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences. , 2004, The Plant journal : for cell and molecular biology.

[18]  M. Schmid,et al.  Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana , 2003, Science.

[19]  Huanming Yang,et al.  A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. indica) , 2002, Science.

[20]  T. Takahashi,et al.  Heat-shock tagging: a simple method for expression and isolation of plant genome DNA flanked by T-DNA insertions. , 2000, The Plant journal : for cell and molecular biology.

[21]  Yujun Zhang,et al.  Sequence and analysis of rice chromosome 4 , 2002, Nature.

[22]  B. Bartel,et al.  Redundancy as a way of life - IAA metabolism. , 1999, Current opinion in plant biology.

[23]  R. Dixon,et al.  Activation tagging in Arabidopsis. , 2000, Plant physiology.

[24]  K. Shimamoto,et al.  Simple RNAi vectors for stable and transient suppression of gene function in rice. , 2004, Plant & cell physiology.

[25]  Jia Li,et al.  BAK1, an Arabidopsis LRR Receptor-like Protein Kinase, Interacts with BRI1 and Modulates Brassinosteroid Signaling , 2002, Cell.

[26]  Takuji Sasaki,et al.  From mapping to sequencing, post-sequencing and beyond. , 2005, Plant & cell physiology.

[27]  S. Strauss,et al.  Activation Tagging of a Dominant Gibberellin Catabolism Gene (GA 2-oxidase) from Poplar That Regulates Tree Stature1 , 2003, Plant Physiology.

[28]  G. Droc,et al.  High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. , 2004, The Plant journal : for cell and molecular biology.

[29]  Ping Wu,et al.  Distribution and characterization of over 1000 T-DNA tags in rice genome. , 2003, The Plant journal : for cell and molecular biology.

[30]  G. An,et al.  Molecular genetics using T-DNA in rice. , 2005, Plant & cell physiology.

[31]  Y. Yamazaki,et al.  Rice mutants and genes related to organ development, morphogenesis and physiological traits. , 2005, Plant & cell physiology.

[32]  H. Leung,et al.  Rice Mutant Resources for Gene Discovery , 2004, Plant Molecular Biology.

[33]  K. Jung,et al.  T-DNA insertional mutagenesis for functional genomics in rice. , 2000, The Plant journal : for cell and molecular biology.

[34]  D. R. Wagner,et al.  Activation Tagging in Tomato Identifies a Transcriptional Regulator of Anthocyanin Biosynthesis, Modification, and Transport Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.012963. , 2003, The Plant Cell Online.