Isolation by improved thermal asymmetric interlaced PCR and characterization of a seed-specific 2S albumin gene and its promoter from grape (Vitis vinifera L.).

A seed-specific 2S albumin gene and its promoter region of grape (Vitis vinifera L.) were isolated using an improved thermal asymmetric interlaced PCR that allowed efficient amplification of target sequence of up to 3 kbp in length directly from genomic DNA. The 2S albumin VvAlb1 (for V. vinifera 2S albumin 1) gene from different grape cultivars encompasses a coding region of 504-540 nucleotides corresponding to a deduced amino acid sequence of 167-179 residues. This deduced protein contains up to 30% glutamine residues and eight cysteine residues arranged in a pattern highly conserved among 2S albumins for disulfide bond formation. DNA sequence alignment revealed that the same VvAlb1 gene among different grape cultivars varied greatly, including an insertion of up to 36 bp near the 3' end of the gene sequence isolated from 'Thompson Seedless'. DNA sequence analysis indicated that several conserved seed-specific regulatory motifs were clustered within a 0.6-kbp region 5' upstream of the transcription start site. To further test promoter activity, the sequence of this region was used to drive a bifunctional EGFP/NPTII fusion gene in Agrobacterium-mediated transformation of grape somatic embryos and leaf discs of grape and tobacco (Nicotiana tabacum L.). A high level of GFP expression, comparable with that derived from an enhanced double CsVMV promoter, was observed in the cotyledonary but not hypocotyl and vegetative tissues of grape and tobacco. These results suggest that the VvAlb1 gene promoter isolated is capable of conferring seed-specific gene expression.