Cloning and Functional Analysis of GjD27, a Carotenoid Isomerase Gene from Gardenia jasminoides

Purpose To clone and functionally characterize the carotene isomerase D27 gene (GjD27) from Gardenia jasminoides Ellis.

Methods Transcriptome data of the stem, leaves, fruits, seeds, and calyx of G. jasminoides were obtained using sequencing by synthesis (SBS) and single molecule real-time sequencing (SMRT). Bioinformatics tools were employed for gene annotation and identification of the GjD27 gene. The protein was expressed using the pET-32a(+) prokaryotic system. After co-transformation with a β-carotene-producing plasmid into Escherichia coli, the catalytic products were detected by high performance liquid chromatography (HPLC).

Results A total of 118.84 and 25.50 Gb of clean data were obtained from SBS and SMRT sequencing, respectively, and annotated 10535 unigenes. A carotenoid isomerase gene, GjD27, was identified and cloned. The GjD27 cDNA was 1116 bp in length and encoded 169 amino acids. The expression of GjD27 was highest in leaves. The pET-32a(+) plasmid harboring GjD27 produced high levels of soluble protein in E. coli BL21(DE3) when induced with 0.5 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) at 16 ℃ and 200 r/min. HPLC results showed that: compared with the control, the GjD27 protein significantly increased the content of all-trans-β-carotene and significantly reduced the content of 9-cis-β-carotene, demonstrating that GjD27 had the function of catalyzing the conversion of 9-cis-β-carotene to all-trans-β-carotene.

Conclusion The GjD27 gene encoding carotenoid isomerase is successfully cloned from G. jasminoides, and the functional analysis provides a theoretical foundation for elucidating and regulating the biosynthetic pathway of strigolactones in G. jasminoides.