茄萼花色苷合成相关基因DFR和MYB克隆及表达分析

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

【目的】花色苷是一类通过类黄酮途径合成的水溶性次生代谢产物,既能使植物的不同器官呈现红、紫、蓝等颜色,还有利于人体健康。紫茄富含花色苷,但是有关茄萼花色苷生物合成的分子机制还不是很清楚。本研究旨在通过克隆茄萼花色苷合成相关基因DFR和MYB,测定其在不同发育时期不同颜色茄萼中的表达量,探究DFR和MYB在茄萼花色苷合成中的作用。【方法】选用绿萼和紫萼长茄(Solanum melongena L.)果萼为试材,测定不同p H条件下茄萼花色苷含量;通过RACE方法分离克隆DFR和MYB cDNA全长序列,分析DFR和MYB的保守结构域及序列特征;分别对DFR和MYB及其同源蛋白序列进行系统进化分析,构建系统进化树来进一步分析鉴定基因;使用Ex PASy网站提供的在线分析软件SOPMA预测蛋白质二级结构;利用实时荧光定量PCR方法检测目的基因在不同发育阶段果萼中的表达情况。【结果】从绿萼和紫萼长茄果萼中克隆了DFR和MYB片段,分别命名为ouSmDFR、dongSmDFR和ouSmMYB、dongSmMYB,Gen Bank登录号分别为:KX224250、KX224251和KX224253、KX224254。ouSmDFR和dongSmDFR全长分别为1 285 bp和1 249 bp,开放阅读框为858 bp和864 bp,分别编码285个和287个氨基酸;ouSmMYB和dongSmMYB全长分别为969 bp和959 bp,开放阅读框均为462 bp,编码153个氨基酸。蛋白质二级结构分析表明α-螺旋和无规则卷曲均为两个DFR蛋白和两个MYB蛋白的主要二级结构元件。序列比对表明DFR蛋白具有NADPH结构域(NADPH binding domain)和底物特异性结合结构域(Substrate specific binding domain),属于NADB-Rossmann超基因家族;MYB蛋白属于R2R3-MYB转录因子,具有R2、R3两个MYB结构域和b HLH结合域。ouSmDFR和dongSmDFR与St DFR和Sl DFR具有相对较高的同源性;ouSmMYB和dongSmMYB与Es MYB同源性较高。花色苷含量测定显示,紫萼果茄萼花色苷含量较高且随着果实的发育成熟而逐渐增加;而绿萼茄萼几乎检测不到花色苷。荧光实时定量PCR分析表明,DFR和MYB在紫萼长茄果萼中表达量均远高于绿萼长茄;从初蕾期到盛花期,紫萼长茄果萼中DFR和MYB表达量逐渐升高,而绿萼长茄则几乎没有变化,与两个品种茄萼颜色变化相一致。【结论】ouSmDFR和dongSmDFR属于NADB-Rossmann超基因家族,ouSmMYB和dongSmMYB为典型R2R3-MYB转录因子,DFR和MYB在紫萼长茄果萼中表达明显高于绿萼长茄。推测DFR和MYB在茄萼呈色中发挥作用,并且参与花色苷生物合成。

【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.