茄萼花色苷合成相关基因DFR和MYB克隆及表达分析

发布时间：2018-02-25 15:49

本文关键词： 萼片花色苷 DFR MYB 基因表达茄　出处：《中国农业科学》2017年14期 　论文类型：期刊论文

【摘要】：【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。
[Abstract]:[objective] anthocyanin is a kind of water-soluble secondary metabolites synthesized by flavonoid pathway, which can not only make different organs of plants appear red, purple and blue, but also benefit human health. However, the molecular mechanism of biosynthesis of anthocyanin is not clear. The aim of this study was to clone the genes DFR and MYB related to the biosynthesis of anthocyanin, and to determine the expression of these genes in different color calyx at different developmental stages. To explore the role of DFR and MYB in the synthesis of anthocyanins from eggplant calyx. [methods] Calyx and Solanum melongena L.) were selected as experimental materials to determine the content of anthocyanin in different pH conditions, and the full-length sequences of DFR and MYB cDNA were isolated and cloned by RACE. The conserved domain and sequence characteristics of DFR and MYB were analyzed, and the sequences of DFR and MYB and their homologous proteins were analyzed, respectively, and phylogenetic tree was constructed to further analyze and identify genes. Protein secondary structure was predicted by SOPMA, an online analysis software provided by Ex PASy website. The expression of target gene in Calyx was detected by real-time fluorescence quantitative PCR. [results] DFR and MYB fragments were cloned from green calyx and purple calyx. The full length of SmDFR and dongSmDFR are 1 285 BP and 1 249 BP, respectively. The open reading frame is 858 BP and 864 BP, encoding 285 amino acids, 287 amino acids, 969 BP and 959 BP, respectively, and the full length of open reading frame is 858 BP and 864 BP, respectively. The full length of Bank is 969 BP and 959 BP, respectively. The full length of open reading frame is 858 BP and 864 BP, respectively. The full length of open reading frame is 969 BP and 959 BP, respectively. The length of open reading frame is 858 BP and 864 BP, respectively. The full length of open reading frame is 969 BP and 959 BP, respectively. The full length of open reading frame is 858 BP and 864 BP, respectively. The reading frames were 462bp, encoding 153 amino acids. The analysis of protein secondary structure showed that both 伪 -helix and irregular curl were the main secondary structural elements of two DFR proteins and two MYB proteins. Sequence alignment indicated that DFR protein had NADPH. NADPH binding domain) and substrate specific binding domain substrate specific binding domain belong to NADB-Rossmann supergene family and belong to R2R3-MYB transcription factor. There are two MYB domains, two MYB domains, two MYB domains, two MYB domains, and two binding domains. OuSmDFR and dongSmDFR have relatively high homology with St DFR and SL DFR, and the homology between dongSmMYB and es MYB is relatively high. The anthocyanin content is determined by assays of anthocyanin content. The content of anthocyanin was higher and increased gradually with the fruit maturation, but the anthocyanin was almost not detected in the green calyx. Fluorescence real-time quantitative PCR analysis showed that the expression of MYB and DFR in the long purple calyx was much higher than that in the green calyx. The expression of DFR and MYB increased gradually from the first bud to the full flowering stage, while the expression of MYB in the long purple calyx increased, while the expression of MYB in the long green calyx was almost unchanged. [conclusion] ouSmDFR and dongSmDFR belong to the NADB-Rossmann supergene families, NADB-Rossmann SmMYB and dongSmMYB are the typical transcription factors of R2R3-MYB and MYB. The expression of DFR and MYB in the calyx of long purple calyx is obviously higher than that in the long calyx of green calyx. It is speculated that DFR and MYB play a role in the color of eggplant calyx. And participate in anthocyanin biosynthesis.
【作者单位】：东北林业大学生命科学学院/林木遗传育种国家重点实验室;黑龙江省农业科学院园艺分院;
【基金】：国家基础科学人才培养基金(J1210053) 中央高校基本科研业务费专项资金(DL12CA10,2572014EA03) 林木遗传育种国家重点实验室创新项目(2013A06,2013B010) 黑龙江省博士后科研启动金(LBH-Q14011)
【分类号】：S641.1

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