Molecular cloning and identification of a flavanone 3-hydroxylase gene from Lycium chinense, and its overexpression enhances drought stress in tobacco

Abstract

Flavonoids, as plant secondary metabolites, are widespread throughout the plant kingdom and involved in many physiological and biochemical processes. Drought resistance is attributed to flavonoids with respect to protective functions in the cell wall and membranes. The flavanone 3-hydroxylase (F3H) gene which encodes flavanone 3-hydroxylase, is essential in flavonoids biosynthetic pathway. Lycium chinense (L. chinense) is a deciduous woody perennial halophyte that grows under a large variety of environmental conditions and survives under extreme drought stress. A novel cDNA sequence coding a F3H gene in Lycium chinense (LcF3H, GenBank: KJ636468.1) was isolated. The open reading frame of LcF3H comprised 1101 bp encoding a polypeptide of 366 amino acids with a molecular weight of about 42 kDa and an isoelectric point of 5.32. The deduced LcF3H protein showed high identities with other plant F3Hs, and the conserved motifs were found in LcF3H at similar positions like other F3Hs. The recombinant protein converted naringen into dihydrokaempferol in vitro. Since studies have shown that amongst flavonoids, flavan-3-ols (catechin and epicatechin) have direct free radical scavenging activity to maintain the normal physiological function of cells in vivo, these data support the possible relationship between the oxidative damage and the regulation of LcF3H gene expression in L. chinense under drought stress. In order to better understand the biotechnological potential of LcF3H, gene overexpression was conducted in tobacco. The content of flavan-3-ols and the tolerance to drought stress were increased in LcF3H overexpressing tobacco. Analysis of transgenic tobacco lines also showed that antioxidant enzyme activities were increased meanwhile the malondialdehyde (MDA) content and the content of H2O2 were reduced comparing to nontransformed tobacco plants. Furthermore, the photosynthesis rate was less decreased in the transgenetic plants. These results suggest that LcF3H plays a role in enhancing drought tolerance in L. chinense, and its overexpression increases tolerance to drought stress by improving the antioxidant system in tobacco.