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1998). Todd Wood from Clemson University provided the PERL script that creates the PlantEST databases as described above. The NR(aa) database is a nonredundant database of protein sequences from GenBank. Determination of Gymnosperm-Specific Genes All available plant ESTs were downloaded from GenBank and separated into three datasets consisting of angiosperms (monocots and dicots), gymnosperms, or lower plants (ferns, mosses and algae). , 1998). All matches with an expect value < 1e-5 were considered significant.
2005). , 2006). This is the first report of the upstream sequences of conifer defense-related genes and the putative cis-acting elements located in those regions. The upstream sequences of 3CAR and CYP720B4 each have more than five elements with sequence identity to cis-acting elements putatively involved in wound, stress, and defense responses in angiosperms. The promoter region of the CYP720B4 gene is 95–99% identical with the corresponding PCR-amplified regions across several genotypes of Sitka spruce, hybrid interior spruce, and white spruce (data not shown).
RESULTS Targeted Isolation of BAC Clones Containing TPS (3CAR) and P450 (CYP720B4) Genes Our first objective was to test if individual BAC clones containing conifer genes of large gene families could be isolated in a gene-specific manner. , 2005). , 2003), D. Hall, J. I. Keeling, J. Bohlmann, unpublished results). Primers used to screen for a specific target P450 gene were based on the functionally characterized diterpene oxidase CYP720B4 from Sitka spruce and its white spruce orthologue (B. Hamberger, T.