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The isolation and molecular characterization of a 2S albumin gene from Picea glauca McInnis, Stephanie Marie


2S albumins are a class of small seed storage proteins (SSPs) found widely among the Dicotyledonae and related to the prolamin SSPs of the Monocotyledonae. A genomic clone (PG2S) and related pseudogene with homology to the dicot 2S albumins were sequenced from the gymnosperm, Picea glauca. Northern blot analysis of developing spruce somatic embryos indicated embryo-specific expression of the conifer 2S albumin gene, in a pattern consistent with a seed storage protein. A translational fusion between 2.3 kb of the 5'-flanking region of PG2S and the uidA (β-glucuronidase) reporter gene was constructed to explore promoter function. Two distal deletions were also created, resulting in promoters deleted to a 5' position of -653 and -117, respectively, relative to the site of transcriptional initiation. The two larger constructs were used in the stable transformation of tobacco (Nicotiana tabacum cv. Xanthi) resulting in embryo-specific expression of the uidA gene in the developing tobacco embryo from long heart stage to embryo maturity with maximal expression in the torpedo stage embryo. Under control of the PG2S promoter GUS expression was not detected in any other tobacco tissues. There were no differences observed in pattern or strength of expression between the constructs in tobacco. The three PG2S promoter ; uidA reporter gene constructs were transiently expressed by microprojectile bombardment in a developmental series of interior spruce {Picea glauca/engelmannii) somatic embryos (proembryo, stage 2, early cotyledonary, mature and partially-dried mature), germinants and pollen. Transient expression of the 2.3 kb promoter construct mirrored 2S albumin mRNA levels in the proembryo, stage 2, early cotyledonary and mature embryo, as well as in germinants. In contrast, high levels of transient expression were observed in partially-dried mature embryos and in pollen, in which low and no 2S albumin mRNA was detected. The promoter deletions gave reduced levels of transient expression, but were not altered in seed-specificity. Putative regulatory motifs identified within the promoter were: ACGT, TGCA, CATG, CANNTG, and CCAC(C). Despite evolutionary distance, the 2S albumin promoter from spruce functions in a developmentally regulated, tissue-specific manner in an angiosperm; cis-elements and their co-ordinate transacting factors are conserved between gymnosperms and angiosperms.

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