Gene cloning, sequence analysis, and expression profiles of a novel beta-ring carotenoid hydroxylase gene from the photoheterotrophic green alga Chlorella kessleri

摘要

In this study, a full-length complementary DNA (cDNA) sequence of beta-ring carotenoid hydroxylase (CHY), designated Ckecyp97a1, was isolated via reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends (RACE) methods. The cloned Ckecyp97a1 cDNA was 2,264-bp in length, and contained an open reading frame (ORF) of 1,944-bp with 5'-terminal untranslated region (UTR) of 66-bp and 3'-terminal UTR of 254-bp and encoded a beta-ring CHY protein of 647 amino acids. The deduced protein had a calculated molecular mass of 71.43 kDa with an estimated isoelectric point (pI) of 6.72. Multiple sequence alignment and phylogenetic analysis revealed that Ckecyp97a1 was homologs to known chloroplastic cytochrome P450 (P450) CHY. The typical catalytic motifs of the P450 were highly conserved in the protein sequences of CkeCYP97A1. The Ckecyp97a1 transcriptional expression and carotenoids accumulation were observed under high light (HL) of different wavelengths (white: 390-770 nm and blue: 420-500 nm). The results revealed that Ckecyp97a1 transcript increased strongly throughout the course of the HL illumination treatment (22-70 h) under white HL treatment, while decreased during 10-58 h under blue HL treatment. The concentrations of lutein, alpha-carotene, and beta-carotene were relatively steady and below the control level under both treatments. The zeaxanthin concentration was higher under white HL treatment than those under control and blue HL treatments. Ckecyp97a1 gene showed different expression patterns under different light wavelengths treatments. The data obtained in this study demonstrates that CkeCYP97A1 is the enzyme responsible for carotenoid hydroxylation involved in HL acclimation for photoheterotrophic green alga Chlorella kessleri CGMCC 4917.