Cloning and Expression Analysis of MYB Transcription Factor in Blood Orange (Citrus sinensis)
XIA Rong-Na1, LI Lin1, YANG Xin1, WANG Jian-Hui2,*
1 College of Ecology and Environment, Chengdu University of Technology, Chengdu 610059, China; 2 Key Laboratory of Horticultural Crops Biology and Germplasm Enhancement in Southwest, Ministry of Agriculture and Rural Affairs, Horticulture Research Institute of Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
Abstract：Anthocyanins belong to a kind of flavonoid and are widely found in different plants. The biosynthesis of anthocyanin is transcriptionally regulated by MYB transcription factor. In this study, the full length of MYB type transcription factor was cloned from flesh of blood orange (Citrus sinensis), then bioinformatic analysis and differential expression of the gene were performed. Furthermore, the prokaryotic expression vector PET28-CsMYB was developed and used to induce expression of MYB recombinant protein, which was used to immune rabbit (Oryctolagus cuniculus) to prepare polyclonal antibody. Then Western blot was used to detect MYB expression between different samples. The results showed that the full-length of MYB nucleotide sequence was 789 bp, which encoded a protein with 262 amino acids. The protein had R2 and R3 MYB conserved domains at the position of 1~56 and 57~111 of amino acids residues, so that it was designated by CsMYB (GenBank No. KT757348). Multiple alignments were performed using different homologous MYB-type proteins. The phylogenetic tree showed that CsMYB protein was clustered with Litchi chinensis, an anthocyanin biosynthesis regulator (LcMYB1), and the amino acid identity between the two proteins was 56.1%. Comparison of differential gene expression in different tissues of blood orange showed that higher transcription level was found in the flesh only. In addition, chalcone synthase (CHS) and glutathione S-transferase (GST) also exhibited higher expression levels in the flesh. The relative expression of CsMYB significantly increased during fruit development. Compared with a mutant fruit with less anthocyanin contents, the relative expression of CsMYB in the red-fleshed fruit with normal anthocyanin contents was significantly higher, and the expression change of CHS was consistent with CsMYB. Western blot results showed that a weak hybridization band at 36 kD was occurred only in the red-fleshed fruit. Taken together, CsMYB might be involved in the anthocyanin regulation of blood orange. The present study could provide a reference for further studies on fruit coloring mechanism and genetic breeding of blood orange.
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