RESUMO
BACKGROUND: Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. RESULTS: A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. CONCLUSIONS: Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.
RESUMO
In the fully sequenced Arabidopsis (Arabidopsis thaliana) genome, many gene models are annotated as "hypothetical protein," whose gene structures are predicted solely by computer algorithms with no support from either expressed sequence matches from Arabidopsis, or nucleic acid or protein homologs from other species. In order to confirm their existence and predicted gene structures, a high-throughput method of rapid amplification of cDNA ends (RACE) was used to obtain their cDNA sequences from 11 cDNA populations. Primers from all of the 797 hypothetical genes on chromosome 2 were designed, and, through 5' and 3' RACE, clones from 506 genes were sequenced and cDNA sequences from 399 target genes were recovered. The cDNA sequences were obtained by assembling their 5' and 3' RACE polymerase chain reaction products. These sequences revealed that (1) the structures of 151 hypothetical genes were different from their predictions; (2) 116 hypothetical genes had alternatively spliced transcripts and 187 genes displayed polyadenylation sites; and (3) there were transcripts arising from both strands, from the strand opposite to that of the prediction and possible dicistronic transcripts. Promoters from five randomly chosen hypothetical genes (At2g02540, At2g31270, At2g33640, At2g35550, and At2g36340) were cloned into report constructs, and their expressions are tissue or development stage specific. Our results indicate at least 50% of hypothetical genes on chromosome 2 are expressed in the cDNA populations with about 38% of the gene structures differing from their predictions. Thus, by using this targeted approach, high-throughput RACE, we revealed numerous transcripts including many uncharacterized variants from these hypothetical genes.