茄萼花色苷合成相关基因DFR和MYB克隆及表达分析

doi:10.3864/j.issn.0578-1752.2017.14.014

Abstract

Abstract: 【Objective】Anthocyanins are the water-soluble secondary metabolites synthesized via the flavonoid pathway responsible for red, violet, and blue in different kinds of plant organs. These pigments are of benefit to human health. Eggplant is rich in anthocyanins. The molecular mechanism of anthocyanin biosynthesis in eggplant calyx is still unclear. The primary objectives of this experiment are to clone DFR and MYB genes in the eggplant, detect their expression at different developmental stages and in calyx with different colors, explore the role of DFR and MYB in anthocyanin synthesis in calyx of eggplant. This work expanded our knowledge about the molecular mechanism of anthocyanin biosynthesis in eggplant peel and calyx. 【Method】Green calyx and purple calyx of eggplants were used as materials. The content of anthocyanins in the calyx was measured by spectrophotometry at different pH. cDNA sequence of the DFR and MYB were isolated and cloned by using RACE techniques. Analyze the conserved domains and characteristics of DFR protein were analyzed. Phylogenetic analysis of homologous protein sequences of other plants and DFR was conducted, the phylogenetic tree was constructed to further analyze and identify genes. To predict its secondary structure, SPOMA was used on-line which provided by the ExPAsy website. The bioinformatics analysis of MYB was the same as DFR. Expression of DFR and MYB genes in calyx with different colors were investigated through quantitative real-time PCR.【Result】In this study, two full-length cDNA sequences of DFR were successfully cloned and denoted as ouSmDFR and dongSmDFR (GenBank accession Nos KX224250 and KX224251). ouSmDFR is 1 285 bp in cDNA length with an open reading frame of 858 bp corresponding to 285 amino acid residues. dongSmDFR is 1 249 bp in cDNA length with an open reading frame of 864 bp, which encoding 287 amino acids. Two full-length cDNA sequences of MYB gene were cloned and denoted as ouSmMYB and dongSmMYB (GenBank accession Nos KX224253 and KX224254). The corresponding cDNAs are 969 bp and 959 bp in length and the deduced proteins contained 153 amino acids, respectively. Results of secondary structure analysis exhibited that α-Helix and random coil were primary secondary structural components of the two DFR genes and two MYB genes. Sequence analysis showed that ouSmDFR and dongSmDFR have a NADPH-binding domain and substrate specific binding domain. ouSmMYB and dongSmMYB belonged to the R2R3-MYB transcription factors, which have R2 and R3 conserved domains and a bHLH binding domain. Phylogenetic tree analysis indicated that ouSmDFR and dongSmDFR have a close relation with StDFR and SlDFR, ouSmMYB and dongSmMYB have the closest genetic relationship with EsMYB. Results of total anthocyanins measurement showed that high contents of anthocyanins were detected in purple calyx and increased gradually, while no anthocyanins were detected in green calyx. The qRT-PCR analysis indicated that the expression of DFR and MYB genes in the purple calyx were much higher than that in the green calyx. Moreover, from early budding to full flowering,the expression of dongSmDFR and dongSmMYB increased gradually. However, ouSmDFR and ouSmMYB almost had no change, which were consistented with the color variation of calyx.【Conclusion】ouSmDFR and dongSmDFR belong to the NADB-Rossmann superfamily, ouSmMYB and dongSmMYB are typical R2R3-MYB transcription factors. The expression of DFR and MYB in the purple calyx is significantly higher than that in the green calyx. Therefore, it is speculated that the DFR and MYB genes play a role in the color of eggplants’ calyx. Furthermore, DFR and MYB are involved in the process of anthocyanin biosynthesis.

Key words: calyx, anthocyanins, DFR, MYB, gene expression; Solanum melongena L.

WANG HaiZhu, QU HongYun, ZHOU TingTing, XU QiJiang. Cloning and Expression Analysis of Anthocyanin Biosynthesis-Associated DFR and MYB Genes in Calyx of Eggplant (Solanum melongena L.)[J].Scientia Agricultura Sinica, 2017, 50(14): 2781-2792.

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Cloning and Expression Analysis of Anthocyanin Biosynthesis-Associated DFR and MYB Genes in Calyx of Eggplant (Solanum melongena L.)

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