Characterization of a plant-transformation-ready large-insert BIBAC library of Arabidopsis and bombardment transformation of a large-insert BIBAC of the library into tobacco.

Plant-transformation-ready, large-insert binary bacterial artificial chromosome (BIBAC) libraries are of significance for functional and network analysis of large genomic regions, gene clusters, large-spanning genes, and complex loci in the post-genome era. Here, we report the characterization of a plant-transformation-ready BIBAC library of the sequenced Arabidopsis genome for which such a library is not available to the public, the transformation of a large-insert BIBAC of the library into tobacco by biolistic bombardment, and the expression analysis of its containing genes in transgenic plants. The BIBAC library was constructed from nuclear DNA partially digested with BamHI in the BIBAC vector pCLD04541. It contains 6144 clones and has a mean insert size of 108 kb, representing 5.2× equivalents of the Arabidopsis genome or a probability of greater than 99% of obtaining at least one positive clone from the library using a single-copy sequence as a probe. The transformation of the large-insert BIBAC and analyses of the transgenic plants showed that not only did transgenic plants have intact BIBAC DNA, but also could the BIBAC be transmitted stably into progenies and its containing genes be expressed actively. These results suggest that the large-insert BIBAC library, combined with the biolistic bombardment transformation method, could provide a useful tool for large-scale functional analysis of the Arabidopsis genome sequence and applications in plant-molecular breeding.

Transcriptome analysis of cadmium response in Ganoderma lucidum.

Ganoderma species are white-rot fungi widespread throughout the world. In this study, a wild isolate of Ganoderma lucidum was first collected and its tolerance was tested in a medium containing 3.0 mM CdCl(2). The cDNA-amplified fragment length polymorphism method was conducted to analyze the transcription profiling of this Ganoderma species in response to Cd treatment. In total, 12 925 transcript-derived fragments (TDFs) were amplified using 256 primer combinations. Forty-nine differentially expressed TDFs were confirmed by DNA dot-blot analysis. Northern blot analysis was used to verify the transcription levels of 34 Cd-inducible TDFs. Sequence analysis indicated that genes involved in reactive oxygen species generation, synthesis of sulfur-containing metabolites, translation machinery, DNA repair, transporting system, proteolysis pathway, mitochondria function, and cell wall biosynthesis were upregulated by Cd treatment. Our results provide a genome-wide transcriptome profiling of Cd response in Ganoderma species.

Transcriptome analysis of auxin-regulated genes of Arabidopsis thaliana.

Plant hormone auxin elicits diverse responses in plant growth and development. Accumulated data indicate that the ubiquitin-mediated proteolytic pathway plays a crucial role in transducing auxin signaling. To gain more understanding of the molecular mechanisms underlying auxin action, we performed a comparative transcriptome analysis of auxin responsive genes between Arabidopsis Columbia ecotype and the auxin insensitive mutant eta2 by cDNA-AFLP. Using 256 primer combinations, about 5900 transcript-derived fragments (TDFs) were amplified. Sixty-six differentially expressed TDFs were confirmed by DNA dot blot analysis. Sequence analysis indicated that, a large number of genes involved in transcription regulation or RNA metabolism were identified as auxin-regulated genes. Northern blot analyses confirmed transcription levels of 16 auxin-regulated genes. These genes include various forms of transcription regulators, defense related, RING-type ubiquitin ligases, and glycosyl hydrolase. This study demonstrates that auxin exerts its effect in complex transcriptional networks.

Bacterial artificial chromosome-based physical map of Gibberella zeae (Fusarium graminearum).

Fusarium graminearum is the primary causal pathogen of Fusarium head blight of wheat and barley. To accelerate genomic analysis of F. graminearum, we developed a bacterial artificial chromosome (BAC)-based physical map and integrated it with the genome sequence and genetic map. One BAC library, developed in the HindIII restriction enzyme site, consists of 4608 clones with an insert size of approximately 107 kb and covers about 13.5 genome equivalents. The other library, developed in the BamHI restriction enzyme site, consists of 3072 clones with an insert size of approximately 95 kb and covers about 8.0 genome equivalents. We fingerprinted 2688 clones from the HindIII library and 1536 clones from the BamHI library and developed a physical map of F. graminearum consisting of 26 contigs covering 39.2 Mb. Comparison of our map with the F. graminearum genome sequence showed that the size of our physical map is equivalent to the 36.1 Mb of the genome sequence. We used 31 sequence-based genetic markers, randomly spaced throughout the genome, to integrate the physical map with the genetic map. We also end-sequenced 17 BamHI BAC clones and identified 4 clones that spanned gaps in the genome sequence. Our new integrated map is highly reliable and useful for a variety of genomics studies.

The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization.

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.