芋花青素合成相关R2R3-MYB基因家族鉴定与表达分析

Identification and expression analysis of the anthocyanin synthesis-related R2R3-MYB gene family in Colocasia esculenta (L.) Schott

  • 摘要: 目的 鉴定并分析芋R2R3-MYB基因家族成员,为揭示R2R3-MYB基因家族在芋球茎花青素生物合成途径中的分子作用机制,选育高花青素芋品种提供参考依据。方法 以赣芋2号白肉芋和赣芋3号红肉芋为试验材料,通过pH示差法测定2种芋3个球茎发育期(膨大前期、膨大中期和膨大后期)芋肉和皮层组织中的总花青苷含量。利用生物信息学方法从芋全基因组数据库中鉴定芋R2R3-MYB基因家族成员,并分析预测其蛋白理化性质、保守结构域、染色体定位、基因结构特征,使用MEGA 7.0构建R2R3-MYB家族成员系统发育树,结合芋球茎膨大后期芋R2R3-MYB家族基因的表达量筛选与花青素生物合成相关的基因,通过相关分析和实时荧光定量PCR检测进一步鉴定出重要芋花青素合成基因。结果 在3个芋球茎发育期,同一组织赣芋3号红肉芋中总花青苷含量均显著高于赣芋2号白肉芋(P<0.05,下同)。球茎膨大后期赣芋3号红肉芋皮层组织中总花青苷含量在所有样品中最高,达6.23 mg/100 g。芋全基因组中共筛选出97个芋R2R3-MYB基因家族成员,均定位于细胞核,不均等地分布在芋13条染色体及9个重叠群上。大多数的蛋白编码200~400个氨基酸残基,蛋白呈酸性,稳定性较差。MYB结构域的R2和R3重复序列虽然表现出不同的特征氨基酸模式,但在芋和拟南芥间高度保守。芋的96个R2R3-MYB家族基因均包含Motif 1、Motif 2和Motif 3以及1~5个内含子。不同作物R2R3-MYB家族成员系统发育分析结果显示,芋R2R3-MYB蛋白划分为29个亚家族,其中有8个芋R2R3-MYB家族蛋白与参与花青素合成相关的拟南芥蛋白亲缘关系较近。转录组测序及相关分析结果表明,基因CeMYB63CeMYB85CeMYB89在赣芋3号红肉芋球茎膨大后期表达量较高,而在赣芋2号白肉芋球茎中表达量较低;上述3个基因与总花青苷含量呈显著或极显著(P<0.01)正相关。芋花青苷合成相关基因CeMYB63CeMYB85CeMYB89的相对表达量与转录组测序结果基本一致。结论 在芋中共鉴定出97个R2R3-MYB基因家族成员,该家族基因在进化过程中具有一定保守性和多样性。筛选到3个与红肉芋花青素生物合成相关的重要调控基因,分别是CeMYB63CeMYB85CeMYB89

     

    Abstract: Objective This study aimed to identify and analyze the members of the R2R3-MYB gene family in taro Colocasia esculenta (L.) Schott, and to reveal the molecular mechanisms of the R2R3-MYB gene family in anthocyanin biosynthesis pathway in taro corm, thereby providing a theoretical foundation for breeding new taro varieties with high anthocyanin content.Method Ganyu 2 (white-fleshed) and Ganyu 3 (red-fleshed) were taken as experimental materials. The total anthocyanin content in flesh and cortex tissues of the two taro varieties during three corm development stages (early enlargement stage, middle enlargement stage, and late enlargement stage) were determined using the pH differential method. The members of taro R2R3-MYB gene family were identified using bioinformatic methods from the whole genome database of taro. Protein physicochemical properties, conserved domains, chromosomal localization information, and gene structural characteristics were analyzed and predicted. A phylogenetic tree of the R2R3-MYB gene fa-mily was constructed using MEGA 7.0. Anthocyanin biosynthesis-related genes were screened according to expression of R2R3-MYB family genes at the late corm enlargement stage. Key anthocyanin synthesis-related genes were further identified using correlation analysis and real-time fluorescence quantitative PCR.Result At the three corm development stages, the total anthocyanin content in the same tissue of Ganyu 3 (red-fleshed) was significantly higher than that in Ganyu 2 (white-fleshed) (P<0.05, the same below). The highest total anthocyanin content among all samples was observed in the cortex tissue of Ganyu 3 (red-fleshed) at the late enlargement stage (6.23 mg/100 g). A total of 97 members of the taro R2R3-MYB gene family were screened from the whole genome, which were all localized in the nucleus and unevenly distributed on 13 chromosomes and 9 contigs. Most proteins encoded 200~400 amino acid residues, which were acidic, and exhibited poor stability. Although the R2 and R3 repeat sequences of the MYB domain showed different cha-racteristic amino acid patterns, they were highly conserved between taro and Arabidopsis thaliana. All 96 taro R2R3-MYB family genes contained Motif 1, Motif 2, and Motif 3, and 1-5 introns. Phylogenetic analysis of different R2R3-MYB family members showed that taro R2R3-MYB proteins could be divided into 29 subfamilies, among which eight taro R2R3-MYB family proteins were closely related to Arabidopsis thaliana proteins involving in anthocyanin synthesis. Transcriptome sequencing and correlation analysis indicated that CeMYB63CeMYB85, and CeMYB89 exhibited high expression in Ganyu 3 (red-fleshed) at the late corm enlargement stage but showed low expression in Ganyu 2 (white-fleshed) corms. Transcriptome sequencing and correlation analysis showed that genes CeMYB63CeMYB85, and CeMYB89 in Ganyu 3 (red-fleshed) exhibited high expression at late corm enlargement stage, yet the three genes exhibited low expression in Ganyu 2 (white-fleshed); the three genes were positively or highly positively correlated (P<0.01) with total anthocyanin content. Relative expression and transcriptome sequencing were consistent for anthocyanin synthesis-related genes CeMYB63CeMYB85, and CeMYB89 in taro.Conclusion A total of 97 R2R3-MYB gene family members are identified in taro, and these family genes show conservation and diversity during evolution. Three key genes regulating anthocyanin synthesis in red-fleshed taro are identified as CeMYB63CeMYB85, and CeMYB89.

     

/

返回文章
返回