Molecular cloning and characterization of ClZE, a zeaxanthin epoxidase gene in watermelon (Citrullus lanatus)

Abstract

In this study, a watermelon (Citrullus lanatus) zeaxanthin epoxidase gene, ClZE, was isolated by reverse transcription-polymerase chain reaction (PCR) together with RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The full cDNA sequence of ClZE is 2535 bp in length containing a 1998 bp open reading frame (ORF) that encodes 665 amino acids. ClZE was shown to share high homology with the putative ZE genes in other plant species. Prediction analysis revealed that ClZE bears two large conservative domains, including Pyr_redox and FHA (forkheadassociated domain). Phylogenetic analysis suggested that ClZE shares high similarity (94.8%) with CsZE from Cucumis sativus, but is far from the ZE genes of other species. Prokaryotic expression indicated that ClZE possessed the apparent molecular mass consistent with its calculated molecular mass of 73.1 KDa. Based on quantitative real-time PCR (qRT-PCR), ClZE was shown to be down-regulated under chilling-low irradiance stress in watermelon leaves. Transgenic Arabidopsis lines harbouring ClZE were generated to test the gene function in mediating plant response to chilling stress. The results indicated that the transgenic lines exhibited decreased nonphotochemical quenching (NPQ) and maximum quantum efficiency of photosystem II (PSII) photochemistry (Fv/Fm), increased activities of peroxidase (POD), superoxide dismutase (SOD) and elevated content of malondialdehyde (MDA) relative to the wild type (WT) under chilling-low irradiance stress. In addition, overexpression of ClZE resulted in impaired xanthophyll cycle and aggravated PSII photoinhibition under chilling-low irradiance. All the results together suggested that ClZE plays major roles in regulating the photoinhibition behaviour.