Cloning and expression analysis of the FvNCED3 gene and its promoter from ash (Fraxinus velutina)

Abstract

The 9-cis-epoxycarotenoid dioxygenase (NCED) gene is rate-limiting in abscisic acid (ABA) biosynthesis. In this study, an NCED gene, designated FvNCED3 (KY008746), was cloned from velvet ash (Fraxinus velutina Torr.) with a RACE method. The full length cDNA of FvNCED3 encodes a 573-amino acid polypeptide. Sequencing analysis showed that the FvNCED3 protein was highly homologous to other NCED proteins. The expression patterns of FvNCED3 in different ash organs were analyzed by real-time PCR which revealed that FvNCED3 expression levels were highest in leaves and lowest in roots. The gene expression patterns of FvNCED3 under abiotic stress indicated that its expression increased under drought, salt and ABA stress and decreased due to high and low temperatures. There were no obvious changes under ultraviolet light. The 1094-bp upstream sequence 5′ flank regulation region of the FvNCED3 gene was also cloned from ash using the Genome Walking method. To assess the activity of the FvNCED3 promoter, a pFvNCED3p::GUS plant expression vector was constructed for tobacco transformation. GUS expression of the FvNCED3 GUS enzyme activity was detected in almost all transgenic tobacco tissues, especially in the young leaves, stigma, anther, ovule and ovary. After treating the transgenic tobacco with NaCl and placing it under drought stress, GUS staining of tobacco leaves increased compared with that under normal growth conditions. This result indicates that gene expression driven by the FvNCED3 promoter can be induced by salt and drought stress.

Keywords

Ash / Function analysis / NCED gene / Promoter / Tobacco

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