ISOLATION AND CHARACTERIZATION OF THE 2S ALBUMIN GENE AND PROMOTER FROM GRAPEVINE

Abstract

High-throughput sequencing of cDNA libraries has resulted in millions of expressed sequence tags (ESTs) from plants. To exploit such a valuable molecular resource for functional analysis of genes and genetic elements, we developed an improved thermal asymmetric interlaced (TAIL) PCR technique. We demonstrated its usefulness by recovering the complete seed-specific 2S albumin gene and promoter using a partial EST and genomic DNA of Vitis vinifera grapevine. The 2S albumin VvAlbl (V. vinifera 2S albumin 1) gene obtained from different cultivars encompasses a coding region of 504 to 540 nucleotides corresponding to a deduced amino acid sequence of 167 to 179 residues. This deduced protein contains up to 30% glutamine residues and 8 cysteine residues arranged in a pattern highly conserved among 2S albumins for disulphide bond formation. DNA sequence alignment revealed that the same VvAlbl 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 V. vinifera 'Thompson Seedless'. DNA sequence analysis indicated that a number of highly conserved seed-specific regulatory motifs were present throughout a 2.2 kb region 5' upstream of the transcription start site of the VvAlbl gene. Comparative analysis of promoter activity using both EGFP and GUS genes directed by various artificially truncated promoter fragments established the function of several promoter regions in regulating seed-specific gene expression in both transiently and stably transformed grape SE. In particular, a 0.4 kbp promoter fragment (-1 to -404) supported the highest level of transient expression but failed to produce any detectable level of expression in stably transformed SE. However, an opposite situation was found when a 2.2 kbp promoter fragment was used. In addition, all transgenic SE lines harboring this long promoter fragment, but not other shorter fragments, showed arrested embryo development. Plants were regenerated successfully from all transgenic SE lines and are being evaluated for temporal and spatial regulation of promoter activity. These results exemplify TAIL-PCR as a cost-effective and target-specific method to utilize the vast amount of molecular information now available.