Preparation and biological application of antibodies against leucoanthocyanidin reductase and anthocyanidin reductase from grape berry
DOI:
https://doi.org/10.5073/vitis.2009.48.69-75Keywords:
LAR, ANR, polyclonal antibody, translational expression, immunohistochemical localization, grape berryAbstract
Proanthocyanidins (PAs) endow wine with the flavor of bitterness and astringency. Both leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are two key enzymes of PA biosynthesis in grape berries, but the previous studies on these enzymes only focused on the transcriptional expression of these genes. Here, the full-length cDNAs of VvLAR1, VvLAR2 and VvANR, respectively, were cloned from wine grape berries and were then introduced into the pGEX-4T-1 expression vectors, which were highly expressed in Escherichia coli DH5α cells with the induction of the isopropyl-β-D-thiogalactoside (IPTG). The purified fusion proteins were used as the antigens to immunize rabbits, separately. The obtained antiserums were further purified to obtain the immunoglobulin G (Ig G) fractions, which were demonstrated to be capable of specifically immuno-recognizing the VvLAR1, VvLAR2 and VvANR from the crude protein extracts from grape berries with weight masses of approximately 43 kD. The analyses of translational expression of these enzyme genes during berry development and immunohistochemical localization of these proteins, by using the obtained antibodies, showed that a high amount of VvLAR1, VvLAR2 or VvANR was present at the pre-veraison stage and these enzyme proteins were all localized on the outer layer of the berry skin and the vascular bundle, as well as in the inner layer of the seed coat. This work provides an important basis for further studies on PA biosynthesis in grape berries.
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