Thellungiella HalophilaTable of Contents (Click on the topics below to go directly to them) - BAC (BIBAC) Library
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BAC (BIBAC) library. A BIBAC library has been constructed by Qi Xie (Institute of Genetics and Developmental Biology, CAS, Beijing) from a partial BamHI-digest of genomic DNA (Shandong ecotype) in the pBIBAC2 vector. In total, 23,040 clones have been arranged in 60 384-well plates that are stored at -80oC. With an average insert size of ~75kb (according to PFGE), and after subtracting empty vectors, the library is predicted to include approximately 4 equivalents of the T. halophila genome. We are presently generating a BAC library with a small-size (7.5 kb) vector and aiming for larger-sized inserts. We are aiming for 12,500 clones with an average of 130 kb or approximately 5-6-fold coverage of the genome. The library will be available by May 2005, and will be sorted (384-well format), and DNA printed on nitrocellulose sheets. cDNA libraries. One cDNA library was generated by the group at Shandong University (China). Seedlings of T.halophila were treated with 200 mmol/l NaCl for 48 h; RNA from aerial tissues was poly(A+) selected and clones (Wang et al., 2004). Approximately 1,800 sequences have been deposited in NCBI. The G+C content of cDNA inserts is 45.8%. Three libraries of cDNAs recovered from cold-, drought- or salinity-stressed plants have been generated for the Yukon ecotype of Thellungiella by the group of Barbara Moffatt (U. Waterloo, Canada). A combination of early and later time point tissues were used for the cold and salinity libraries; in the case of drought, the plants were sampled at the time of wilt. A total of 8,000 clones (average size 1.1 kb), sequenced form their 5' ends, resulting in 3,628 unigenes. The ESTs have an average nucleotide length of 535and an overall G+C content of 45.9%. Comparision of Thellungiella unigenes with the corresponding Arabidopsis cDNAs available at TAIR revealed that 6% of the Thellungiella unigenes displayed a precentage identity of 80% or less with the corresponding Arabidopsis transcripts. A cDNA library in a double 35S promoter vector (0.6 106 clones, ~80% with insert) has been made by Qi Xie (CAS, Beijing) and Arabidopsis transformed lines are being screened. Full-lengeth cDNA library. The Laboratory of K. Shinozaki (RIKEN, Japan) has generated Thellungiella full-length cDNAs following procedures established for the generation of Arabidopsis full-length cDNAs (biotinylated CAP trapper method) (Seki et al., 2002). As with Arabidopsis, plants at different developmental stages and treatment conditions have been used. Normalization/subtraction was chosen to increase frequency of unique sequences. ESTs. An analysis of ~1,800 ESTs from a library of seedlings treated with 200mM NaCl for 48h indicated generally strong sequence identity (90-95%) with transcripts from Arabidopsis. A smaller number of transcripts, approximately 15-20% showed lower sequence identity, as low as 60-70% with Arabidopsis. The presence of multiple transcripts for genes that in Arabidopsis are derived from single genes is indicated i.e., the Thellungiella genome could include paralogs for a number of genes (Wang et al., 2004). A comparison of the EST collections from Yukon and Shandong ecotypes reveals a bias in EST abundance for some sequences (e.g. chalcone synthase, mannitol dehydrogenase) that may contribute to deifferences in physiological responses between lines (M. Griffith, U. Waterloo, pers. comm.). Microarray Analyses. Taji et al. (2004) used Arabidopsis full-length cDNA array slides to probe transcript changes in Thellungiella in high salinity (RIKEN, Japan). Microarrays with long-oligo probes (50-70mers) designed to Arabidopsis genes also produce good levels of hybridization with Thellungiella cDNA (70-80% of Arabidopsis signal, Amtman and Bohnert labs). Homology of Arabidopsis 70-mer probe sequences with corresponding Thellungiella EST sequences has been documented (Inan et al., 2004). In contrast, very short targets as employed on Affymetric GeneChips proved to be less suitable for inter-species comparison (20% of Arabidopsis signal; Volkov et al., 2004). A Thellungiella cDNA microarray platform has been generated by the group of Barbara Moffatt (U. Waterloo). Unique cDNAs derived from the stress-induced libraries of Yukon ectotype were printed. These arrays have been used to evaluate the profile of the transcripts represented by the microarray in Thellungiella subjected to drought, long term cold or salinity treatments. A microarray experiment has also been performed to compare samples collected from the Yukon in comparison with chamber-grown Thellungiella plants. Manuscripts by our Canadian colleagues that analyze cDNA populations and transcript profiles have been submitted for publication (Griffith et al., 2005; Wong et al., 2005). In summary, microarray studies using Arabidopsis or Thellungiella platforms have indicated commonality as well as specificity of transcriptional stress responses between the species. Interestingly, Thellungiella shows constitutively high expression level of some genes that are stress-induced in Arabidopsis (Taji et al., 2004). The Examination of differential gene regulation, including, e.g., the identification of promoter elements, will be significantly enhanced by the availability of a genomic DNA sequence for Thellungiella. Proteomics and metabolomics analyses are underway in the labs of Anna Amtmann (U. Glasgow) and Elizabeth Weretilnyk (McMaster University) to identify components that accumulate or are being modified in stressed plants. A comprehensive metabolomics program is underway for the Yukon ecotype using both laboratory and field grown plants. Mutant Populations. For Theullungiella halophila, ecotype Shandong, the Purdue labs have generated T-DNA insertion mutant lines. The transformation vectors used include: SuperTag, At1190, and pSKI15, all including the BAR gene for selection on soil. As of February 2005, the collection includes 17,375 Bar-resistant lines that have been harvested in populations of 50 plants. The Xie lab (Beijing) added ~5,000 T-DNA tagged lines using the vector pSKI015. Salt cress seeds have also been mutagenized by soaking in 0.4% EMS for 14h (Zhu lab, UC Riverside). Plants (M1) from the resulting seeds were allowed to self-fertilize and M2 seeds were collected from 25 pools of M1 plants (1000-1500 Ml plants/pool). Several salt sensitive mutants have been selected from the M2 population using the root-bending assay developed for screening Arabidopsis sos mutants (Inan et al., 2004). Complementing these EMS mutants generated in the Zhu lab, the Xie lab generated 24, 000 lines (20 x 1,200 pools) of additional EMS mutants.
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