Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures

Abstract

The Orange (Or) gene is responsible for the accumulation of carotenoids in plants. We isolated the Or gene (IbOr) from storage roots of orange-fleshed sweetpotato (Ipomoea batatas L. Lam. cv. Sinhwangmi), and analyzed its function in transgenic sweetpotato calli. The IbOr gene has an open reading frame in the 942 bp cDNA, which encodes a 313-amino acid protein containing a cysteine-rich zinc finger domain. IbOr was strongly expressed in storage roots of orange-fleshed sweetpotato cultivars; it also was expressed in leaves, stems, and roots of cultivars with alternatively colored storage roots. IbOr transcription increased in response to abiotic stress, with gene expression reaching maximum at 2 h after treatment. Two different overexpression vectors of IbOr (IbOr-Wt and IbOr-Ins, which contained seven extra amino acids) were transformed into calli of white-fleshed sweetpotato [cv. Yulmi (Ym)] using Agrobacterium. The transgenic calli were easily selected because they developed a fine orange color. The expression levels of the IbOr transgene and genes involved in carotenoid biosynthesis in IbOr-Wt and IbOr-Ins transgenic calli were similar, and both transformants displayed higher expression levels than those in Ym calli. The contents of β-carotene, lutein, and total carotenoids in IbOr-Ins transgenic lines were approximately 10, 6, and 14 times higher than those in Ym calli, respectively. The transgenic IbOr calli exhibited increased antioxidant activity and increased tolerance to salt stress. Our work shows that the IbOr gene may be useful for the biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands.

Keywords: 2,2-diphenyl-1-picrylhydrazyl; 9-cis-epoxycarotenoid dioxygenase; ABA; CHY-β; CRTISO; CaMV; Carotenoid; DPPH; GFP; HPLC; Hm; LCY-β; Metabolic engineering; NCED; NT; Orange gene (IbOr); PSY; Pftf; ROS; Salt stress; Shinhwangmi; Shinzami; Sweetpotato; Ym; Yulmi; Zm; abscisic acid; carotenoid isomerase; cauliflower mosaic virus; green fluorescent protein; high-performance liquid chromatography; lycopene β-cyclase; non-transgenic; phytoene synthase; plastid fusion/translocation factor; reactive oxygen species; β-carotene hydroxylases.