Isolation and Characterization of the 2S Albumin Gene and Promoter from Grapevine

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 VvAlb1 (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 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 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 VvAlb1 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. INTRODUCTION Technological improvements for the genetic transformation of grape have significantly enhanced our ability to transfer foreign genes into the Vitis genome. This has facilitated efforts to genetically engineer grape in part by supporting genetic/genomic studies to determine gene functions and mechanisms of gene expression and regulation. International efforts to sequence various cDNA libraries in grapevine have produced a wealth of molecular information, e.g., over 183,000 expressed sequence tags (ESTs) of Vitis vinifera (http://www.ncbi.nlm.nih.gov/genomes/PLANTS/PlantList_sz.html#EST) have been deposited in the GenBank database of National Center for Biotechnology Information (NCBI). These sequence data are useful for the analysis of gene expression profiles, development of molecular markers, and the isolation and functional analysis of unknown genes and genetic elements. Proc. IS on Biotechnol. Temp. Fruit Crops & Trop. Species Eds. R.E. Litz and R. Scorza Acta Hort. 738, ISHS 2007 760 In order to isolate seed-specific promoters, we modified the thermal asymmetric interlaced PCR (TAIL-PCR) procedure originally described by Liu and Whittier (1995) and amplified several genomic DNA sequences utilizing the sequence of GenBank accession No. AY267256 an EST corresponding to a 2S albumin gene. Sequence analysis of the amplification products revealed the complete VvAlb1 gene encoding a 2S albumin precursor polypeptide and a 0.6 kb 5’ flanking sequence conferring seed-specific promoter activity (Li and Gray, 2005). In this study, we analyzed a 2.2 kb 5’ sequence flanking the VvAlb1 gene for promoter activity via progressive 5’ deletions. By using both transiently and stably transformed grape somatic embryos (SE) and plants, the importance of several regions of the extended VvAlb1 promoter in regulating temporal and spatial gene expression was revealed. The implication of genetic elements within promoter regions on efficacious DNA-protein interactions and tissue-specific gene activation is also discussed. MATERIALS AND METHODS Plant Materials and Agrobacterium-Mediated Transformation Somatic embryos were induced from in vitro-grown leaves of V. vinifera ‘Thompson Seedless’ and maintained as described previously (Gray, 1995; Li et al., 2001). Somatic embryos at the mid-cotyledonary stage of development were used in all transformation experiments. A modified Agrobacterium-mediated transformation procedure was used to introduce test constructs into grape (Li et al., 2005). After co-cultivation with Agrobacterium, SE were placed on selection medium to induce transgenic SE that were subsequently used for regeneration of transgenic plants (Li et al., 2001). Somatic embryos, 3 d post-co-cultivation, were collected, processed, and utilized in transient gene expression studies. Source of Promoter Sequences and Construction of Plant Transformation Vectors A 2.2 kb DNA fragment 5’ upstream of the VvAlb1 gene was previously amplified from V. vinifera ‘Merlot’ (Li and Gray, 2005) and separated into 5 fragments by progressive deletion at the 5’ end. These fragments were designated as -2145, -1711, -1071, -551, and -357 based on their 5’ end positions relative to the transcription initiation site. These promoter fragments were end-modified to incorporate a unique HindIII site at the 5’ end, a unique KpnI site at the 3’ end, and then cloned into a pBIN19-based binary vector. This resulted in a series of binary constructs referred to as A1 (-2145), A2 (-1711), A3 (-1071), SAG (-551) and A4 (-357) (Fig. 1). In each construct, a GUS expression unit under the control of the respective promoter fragment and a bi-functional EGFP/NPTII expression unit controlled by a doubly-enhanced CaMV 35S promoter (Mitsuhara et al., 1996) were arranged in tandem within the T-DNA region. A positive control vector (pd35G) containing a GUS gene controlled by a doublyenhanced CaMV 35S promoter (Kay et al., 1987), and a negative control vector (pD35S) lacking a GUS expression unit (Li et al., 2001, 2004) were included for comparison. All binary vectors were introduced into A. tumefaciens strain EHA105 via the freeze-thaw method (Burrow et al., 1990). Monitoring GFP Expression and GUS Assays GFP expression was monitored as described by Li et al. (2001). Multiple images of GFP expression in transformed tissues were recorded and representative images analyzed. Somatic embryos and leaf explants were collected and assayed for GUS activity according to Jefferson (1987). For abscisic acid (ABA) induction experiments, stable transgenic SE at the mid-cotyledonary stage of development were divided into two groups. One group was placed on medium containing 10 μM ABA and the other on identical medium lacking ABA for 5 d. These SE were then assayed for GUS activity. For quantitative expression analyses, three replicates of at least 30 SE were used for each treatment. Experiments were repeated two to three times depending on the availability of explant materials.