Cloning, expression analysis, and transformation vector construction of DAM homologs in peach and poplar

Genetic fine mapping and sequencing of the EVG locus in peach [ Prunus persica (L.) Batsch] identified six tandem arrayed Dormancy-Associated MADS-box (DAM) genes as candidates for regulating growth cessation and terminal bud formation in the non-dormant evergrowing (evg) mutant. Since the mutant is lacking expression of six genes in the mapped locus, further functional analysis is needed to narrow the list of gene candidates for the non-dormant evg phenotype. Here I report three sets of experiments designed to functionally test DAM genes in peach and their homologs in a model tree, hybrid poplar.;First I constructed overexpression and knockdown vectors for the reverse genetic experiments needed to assign DAM gene function in peach via Agrobacterium-mediated transformation of plants. Peach DAM full-length open reading frame (ORF) cDNAs were cloned for construction of overexpression vectors. The 3' UTR sequences of DAM genes, which are gene-specific, were cloned and used for the construction of hairpin-forming inverted repeat cassettes which are known to reduce endogenous gene expression via RNA interference (RNAi).;Second, I report results from experiments where I have constructed overexpression and knockdown vectors for putative DAM homologs from a hybrid poplar clone (Populus tremulaxalba, INRA 717-1B4). The six peach DAMs are members of the SVP/StMADS11 clade of type-II MADS-box genes and the sequenced P. trichocarpa genome contains eight genes in this clade. However, only six DAM homologs were isolated from the INRA 717-1B4 clone. Full-length ORF cDNAs of these six hybrid poplar homologs of the peach DAM genes were cloned. Overexpression and knockdown vectors were constructed using full-length ORF cDNAs and 3' UTR sequences, respectively, as described above for peach. These vectors were used for Agrobacterium-mediated transformation of the hybrid poplar clone INRA 717-1B4. Putative transgenic plants were obtained for three of the six poplar DAM homologs: PtMADS7, PtMADS26 and PtMADS48.;Finally, I measured gene expression of the six PtMADS genes over a six month period from summer to winter solstice in order to find correspondences between the expression patterns of these genes in peach and hybrid poplar. The expression pattern was notably different from that observed in the peach DAMs. This suggests the function of PtMADS in poplar may be different from the DAMs in peach. Additionally, the expression patterns of all of the six PtMADSs genes were very similar, which opens the possibility that these highly similar genes may be functionally redundant.;This work is the first to specifically monitor hybrid poplar DAM homolog gene expression through a seasonal transition from active growth through growth cessation, bud set, and endodormancy entrance. The seasonal behavior of the hybrid poplar genes differs dramatically from that of the peach DAMs. The role of these genes in hybrid poplar is possibly divergent from that of peach. The function of the hybrid poplar DAM homologs is currently not known; the overexpression and gene silencing vectors construction in this work is a necessary first step to understand the function of the DAM homologs in hybrid poplar.