The litchi (Litchi chinesis Sonn.) is a tropical and subtropical fruit tree native to southern China, Taiwan, Bangladesh and Southeast Asia, and now cultivated in many parts of the world. The litchi has a history and cultivation going back as far as 2000 BC according to records in China. Cultivation began in the area of southern China, Malaysia and Vietnam. Wild trees still grow in parts of southern China and on Hainan Island. Litchi is extensively grown in China, and also elsewhere in Brazil, South-East Asia, India, Pakistan, Bangladesh, southern Japan and more recently in California, Jamaica and elsewhere in the Caribbean, Hawaii, Texas, Florida, the wetter areas of eastern Australia and sub-tropical regions of South Africa, Israel and also in the states of Sinaloa and San Luis Potosíin Mexico. A wide range of cultivars is available, with early and late maturing forms suited to warmer and cooler climates, respectively. They are also grown as an ornamental tree as well as for their fruit. Litchi was commonly sold fresh in Asian markets, and in recent years, also widely in supermarkets worldwide. The red rind turns dark brown when the fruit is refrigerated, but the taste is not affected. It is also sold canned year-round. Anthocyanin biosynthesis had been characterized in flowers of petunia, snapdragon, pericap of litchi, skins of grape and in kernels of maize, and the biosynthetic pathway is now one of the best known pathway in plants (Holton and Cornish, 1995). Anthocyanin was the predominant pigments of grape skins as well as other blue, red, or black fruits and flowers, and was biosynthesized through a flavonoid pathway. Regulation of the anthocyanin pathway had been shown to occur mainly at the transcriptional level in pigmented organs. Two classes of genes were required for anthocyanin biosynthesis. The structural genes encoded the enzymes that directly participate in the formation and storage of anthocyanins and other flavonoids. The UGFT enzyme of plants was the final gene in the anthocyanin pathway. The transfer of the glucosyl moiety from UDP-glucose to the 3-hydroxyl group of anthocyanidins by UFGT was shown to be the key for anthocyanidin stability and water solubility (Yoshihara et al., 2005). In grapevines, the main control point for anthocyanin quantitative variation was downstream in the pathway at theUFGT level, in agreement with the early observations (Boss et al., 1996a, b, c). In the grape berry, anthocyanin biosynthesis pathway, some cDNAs encoding structural genes had been cloned in Vitis vinifera (Sparvoli et al., 1994). It was showed that only the expression of the gene coding for UFGT was consistently associated with the berry color, depending on developmental stage and cultivar (Boss et al., 1996a, b, c). Expression analysis of UFGT genes in white and red-skinned cultivars revealed that the UFGT gene was present, but not expressed in the white cultivars (Boss et al., 1996a, b, c; Kobayashi et al., 2001). The UFGT sequences were identical in white and red-skinned sports including the promoter region, suggesting that the phenotypic change from white to red could be the result of a mutation in a regulatory gene controlling the expression of UFGT (Kobayashi et al., 2002). Commercial litchi cultivation had focused on fruit crops that develop red color within a month time on the tree. Therefore, this fruit  could  provide  a  model for investigating regulation of anthocyanin synthesis during fruit maturation. Here, we reported the isolation of the promoter of UFGT gene in the anthocyanin biosynthetic pathway. Expression of the GUS gene was examined in a series of nested UFGT promoter. The evidence presented revealed that GUS activity was the highest in one element of about 360 bp upstream of the transcription start site.

Plant material and bacterial strains

Litchi (L. chinesis Sonn.) cv. Nuomici was grown at College of Horticulture, South China Agricultural University in Guangzhou, China. Fresh leaves and fruit samples were taken to the laboratory and immediately frozen in liquid nitrogen and stored at -80°C. Escherichia coli strain DH5a was cultivated in LB medium for vector constructs and DNA manipulation. Agrobacterium tumefaciens strain EHA105 was cultivated in LB medium for onions transformation. White skin onion cultivar was used for transformation.

PCR cloning of the UFGT promoter region

Total genomic DNA was isolated from fresh leaves samples by CTAB method (Stewart and Via, 1993). The promoter region was cloned with high-efficiency thermal asymmetric interlaced PCR method described previously (Liu and Chen, 2007). The corresponding produce was subjected to pre-amplification reactions of TAIL-PCR with inner gene-specific primer (GSP1) (5'-TTC CTT CTT TGT TCT CTC AGC AGA CC-3') and the primary or secondary TAIL-PCRs with outer gene-specific primer (GSP2) (5'-ATT GTG TGTAGC AAC AGC AGG GC-3'). Major bands were isolated from using TaKaRa Agarose Gel DNA Purification kit (TaKaRa, Japan), and the isolated fragments were cloned into a pMD-19T vector (TaKaRa, Japan). The inserted sequence in recombinant plasmid DNA was analyzed using a BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, USA) on an ABI PRISMTM 377 DNA Sequencer. In order to find out motifs regulation UFGT gene expression, functional motifs in UFGT gene promoter were predicted by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Construction of vectors

With the genomic as a template, a fragment upstream of the translational start codon of UFGT gene and its 5' deletion derivatives were generated by PCR with five primers: (Pf1:5'-CAAGCTTGGCCGCCCTGTTGGTTAAATAGTAT-3’; Pr1:5'-CGGATCCAGTGGTGGTGTTGTGGTGGTGGATA-3'; Pf2:5'-CAAGCTTTTAGTAAGGGCTGGATTAAGTGAAT-3'; Pr2:5'-CGGATCGAGGTTCTTAGTGCGTATCGGTCTTCG -3';Pf3:5'- CAAGCTTGACTGATATTTCGGTTTGAACTGCA-3'; Pr3: 5'-CGGATCCCCCCACGCTCACTACGAGACTACCC-3';), the introduced HindIII sites in the forward primers and the introduced BamHI site in the reverse primers. The amplified fragments were respectively inserted into the plasmid pBI121 (Clontech) as a HindIII-BamHI fragment at the corresponding restriction sites in place of the cauliflower mosaic virus (CaMV) 35S promoter region, resulting in a series of pBI-pUFGT::GUS vectors.

Onions transformation

Individual   binary   vectors,   including   pBI121   and   a   series   of pBI-pUFGT::GUS were introduced into A. tumefaciens EHA105 by the freeze-thaw method (Walkerpeach and Velten, 1994).

Isolation and characterization of litchi UFGT promoter

With the genomic as a template, a 1487 bp fragment upstream of the translational start codon of UFGT gene was gotten (Figure 1). The three expression of vector were named P(-1487/-18), as the full-length promoter construct in this study, P1(-1487/-857), P2(-910/-344) and P3(-477/-18) (Figure 2).

The transcriptional start sites of UFGT were determined and located 85 bp upstream from the ATG codon. The AE-box, and ELI-box in anthocyanin biosynthesis of angiosperm were found in UFGT promoter. MRE which was MYB binding site involved in light responsiveness, control the transcription of genes in anthocyanin biosynthesis (Grotewold et al., 1994; Jin et al., 2000; Moyano et al., 1996; Sablowski et al., 1994; Sainz et al., 1997; Sugimoto et al., 2000; Tamagnone et al., 1998; Yang, 2001). The UFGT gene promoter contained TCA elements and a HSE homologous sequence, UFGT promoter also contained WUN-motif that was wound-responsive element (Figure 3).

Expression pattern of the UFGT promoter in onions (Allium cepa Linn.)

In order to identify promoter regions that confer the spatial and developmental expression of litchi genes, the 1.48 kbp promoter fragment and three promoter deletions of UFGT were fused to a GUS reporter gene (Figure 4). All constructs were transferred into onions plants by Agrobacterium-mediated transformation system. Ten to 15 independent onions transformants were obtained for each construct. Figure 5 showed the GUS activities in onions transformants with the UFGT promoter-GUS construct. The GUS expression levels of the the P1promoter-GUS construct (-1487/-857) and the P3promoter (-477/-18) fragments were very low in onions plants. The highest level of GUS activity was detected in transformants carrying UFGT full length (-1,487 bp from the transcriptional start site). We then analyzed the histochemical localization for every individuals obtained from each UFGT promoter-GUS construct. In onions tissue, the expression levels of the P1promoter (-1487/-857) and the P3 promoter (-477/-18) deletions were very low (Figure 5) and CK no histochemical localization was detected. These results suggest that the region–910 to–344 of the UFGT promoter, in which reside HSE, ABRE, MRE and TCA-element (Table 1) responsible for the strong heat, light, and ABA expression in the transgenic onions. The UFGT promoter construct, P2 produced significant GUS activity in response to bending in onions (Figure 5), whereas other constructs did not show high activity. This indicates that the UFGT promoter region between -910 and -344 bp also contains possible bending stress responsive elements in litchi.