Computational protocol: Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits

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Protocol publication

[…] Total RNA was extracted from the flavedo of Satsuma mandarin fruits according to the method described by Ikoma et al. []. First-strand cDNA was synthesized from 2 μg of total RNA using TaqMan Reverse Transcription Reagents (Applied Biosystems). The full-length cDNAs of CitCYP97A, CitCYP97B, and CitCYP97C were amplified by RT-PCR using the primers designed within 5′ and 3′ UTRs according to the sequences obtained from the citrus genome database (Additional file : Table S1). The amplified cDNAs were cloned into the TOPO TA vector and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).The alignment of CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C was created using CLUSTAL W (http://www.clustal.org). The Neighbor-joining phylogenetic tree was constructed based on the alignment of the deduced amino acid sequences of carotene hydroxylases using MEGA6 software []. Accession numbers are: Arabidopsis AtBCH1, AY113923; Arabidopsis AtBCH2, AY117225; Brassica napus BnBCH, EF026098; Coffea canephora CcBCHY, DQ157165; Citrus CitHYb1, AB114653; Crocus sativus CsBCH2, AY579207; Lycopersicon esculentum SICrtR-b1, Y14809; Lycopersicon esculentum SICrtR-b2, Y14810; Marchantia polymorpha MpBHY, AB981062; Narcissus tazetta var. chinensis NcBCH, JN625263; Zea mays ZmBCH1, GQ131287; Zea mays ZmHYD3, AY844958; Zea mays ZmHYD4, AY844956; Arabidopsis AtCYP97A3, NM_102914; Chlamydomonas reinhardtii CrCYP97A5, EF587911; Citrus CitCYP97A, LC143646; Daucus carota DcCYP97A3, JQ655297; Haematococcus pluvialis HpCYP97A, JX308236; Lycopersicon esculentum SlCYP97A29, EU849605; Lycium ruthenicum LrCYP97A, KF957714; Marchantia polymorpha MpCYP97A, AB981063; Oryza sativa OsCYP97A4, AK068163; Vitis vinifera VvCYP97A, XP_002279984; Arabidopsis AtCYP97C1, AY424805; Citrus CitCYP97C, LC143648; Chlamydomonas reinhardtii CrCYP97C3, EF587910; Daucus carota DcCYP97C, ABB52076; Lycopersicon esculentum SlCYP97C11, EU849604; Medicago truncatula MtCYP97C10, ABC59096; Marchantia polymorpha MpCYP97C, AB981065; Oryza sativa OsCYP97C2, AK065689; Arabidopsis AtCYP97B3, NM_117600; Citrus CitCYP97B, LC143647; Haematococcus pluvialis HpCYP97B, JX272918; Marchantia polymorpha MpCYP97B, AB981064; Oryza sativa OsCYP97B2, XM_015771315; Ricinus communis RcCYP97B, XP_002520583; Sorghum bicolor SbCYP97B, XP_002451628; Vitis vinifera VvCYP97B, XP_002266883. Predictions of transit peptides of CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C were carried out using TargetP. […]

Pipeline specifications

Software tools Clustal W, MEGA, TargetP
Databases CGD
Applications Phylogenetics, Protein sequence analysis
Organisms Escherichia coli BL21(DE3)
Chemicals Carotenoids, Heme, Lutein