苦荞全生育期芦丁积累与其生物合成途径相关基因表达分析

doi:10.3864/j.issn.0578-1752.2017.18.004

摘要/Abstract

摘要： 【目的】探究苦荞全生育期芦丁含量变化与其合成途径关键酶基因和调控因子MYB基因表达量之间的相关性，以期进一步明确苦荞植株体内芦丁生物合成的分子机制。【方法】以九江苦荞为试验材料，整个生育期分别在萌发期、子叶期、真叶期、盛叶期、现蕾期、盛花期、灌浆期和籽粒成熟期共8个时期取材（S1—S8）。采用RT-PCR方法，克隆获得与黄酮类代谢相关的MYB类转录因子基因。采用T-coffee软件进行氨基酸同源序列比对及保守结构域分析。与拟南芥黄酮类代谢相关的MYB转录因子及荞麦同源MYB转录因子序列比对，基于邻近法构建系统进化树；实时荧光定量PCR技术分析芦丁合成途径中5个关键酶FtCHS、FtF3H、Ft4CL、FtFLS-like和FtUFGT及上述克隆到的MYB转录因子在不同组织中的表达模式。基于高效液相色谱法（HPLC）测定全生育期取材组织的芦丁含量。同时采用相关性分析方法分析不同组织中芦丁含量变化与基因表达模式的相关性。转换上述数据为矩阵，采用欧式距离法构建共表达层次聚类图谱。【结果】克隆到2个MYB类转录因子，即FtMYB7和FtMYB9，其核酸序列长度分别为876和912 bp，分别编码291和303个氨基酸残基。氨基酸序列同源性比对分析结果表明，二者均具有典型的R2R3保守结构域，为R2R3类型的MYB转录因子。结合Nr数据库中17个苦荞和3个拟南芥黄酮类代谢相关MYB转录因子同源氨基酸序列构建系统进化树，结果表明这22个基因分成6大类群，其中FtMYB7属于第II类群，FtMYB9属于第IV类群，二者分属于不同类群，暗示这2个MYB转录因子可能涉及调控植物生长发育过程中不同功能类型。荧光定量结果显示，FtMYB7在S3（真叶期）和S5（现蕾期）基因相对表达量最高，达到501和867倍；FtMYB9在S1（萌发期）和S2（子叶期）相对表达量最高，分别为34和72倍。上述基因表达量与芦丁含量变化模式相关性分析表明，8个生长时期中，Ft4CL、FtCHS、FtF3H、FtUFGT和FtMYB7相对表达模式与芦丁含量变化幅度呈正相关，其相关系数分别为0.748、0.683、0.704、0.890和0.862。而FtFLS-like和FtMYB9与芦丁含量的变化幅度呈负相关，其相关系数分别为-0.442和-0.501。【结论】FtMYB7和FtMYB9转录因子在整个生育时期中表达模式存在明显差异。其中FtMYB7可能在苦荞芦丁积累的过程中起到正调控作用，而FtMYB9则为负调控作用。

关键词: 苦荞, 芦丁, MYB基因, 基因表达

Abstract: 【Objective】 To explore the relationship among the rutin content at different developmental stages with rutin biosynthesis genes and MYB regulation genes, it will be very meaningful to understand the underlying molecular metabolism of rutin accumulation in tartary buckwheat. 【Method】 The whole developmental stages of F. tatarium Jiujiang were classified into 8 stages (germination, first pair of leaf formation, true leaf growth, vegetative growth, flowering, peak flowering, immature seeds , matured seeds),which were named S1-S8 stages. The whole seedlings from S1and S2 stage, leaves from S3 and S4 stage, flowers from S5 and S6 stage, seed formation organ from S7 and S8 stage were used as test materials. Two MYB genes, FtMYB7 and FtMYB9, were cloned and sequenced. The homologous amino acid sequence and conservative structure domain analysis were carried out by T-coffee software. The phylogenetic tree was constructed by the NJ method (neighbor-joining). The multiple sequence alignment of MYB transcription factors related with flavonoids among Arabidopsis and buckwheat were analyzed by MEGA 7.0 software. The rutin content of these tissues at S1-S8 stages was detected by HPLC. In the meantime, the expression level of rutin-biosynthesis-related genes (FtCHS, FtF3H, Ft4CL, FtFLS-like and FtUFGT) and regulator genes (FtMYB7 and FtMYB9) were investigated by qRT-PCR. The correlation of rutin content and the gene expression pattern during the whole developmental stage was estimated by Pearson correlation method. These data were transformed to matrix data, and constructed a Hierarchical cluster heat map by Euclidean distance method. 【Result】Cloned two MYB transcriptional factors, FtMYB7 and FtMYB9, their nucleic acid sequences were 876 and 912 bp, respectively, encoding 291 and 303 amino acid residues. The sequences of 17 MYB transcription factor from buckwheat, and 3 genes related to flavonoids metabolism in Arabidopsis were constructed for a phylogenetic tree. The phylogenetic tree showed that these genes were divided into six groups. Among them, FtMYB7 belongs to the group II, and FtMYB9 belongs to IV groups, the result indicated that the two MYB transcription factors involved in function diversity at regulation of plant growth and development process. The qRT-PCR results showed that the relative expression levelsof FtMYB7 at S3 and S5 stages were the highest at 501 and 867 times, respectively. To FtMYB9, the highest gene expression levels were detected at S1 and S2 stages, and 34 and 72 times, respectively. Correlation analysis showed that during 8 growth stages, Ft4CL, FtCHS, FtF3H, FtUFGT and FtMYB7 expression patterns were positively correlated with rutin content, and the correlation coefficient was 0.748, 0.683, 0.704, 0.890 and 0.862, respectively. However, FtFLS-like and FtMYB9 were negatively correlated with rutin content, and the correlation coefficients were -0.442 and -0.501, respectively. 【Conclusion】 The FtMYB7 and FtMYB9 were characterized by two R2R3 MYB genes. The different gene expression patterns and rutin content at the whole developmental stage of tartary buckwheat suggested that the FtMYB7 is positively regulated in rutin biosynthesis, but the FtMYB9 is negatively regulated.

Key words: Fagopyrum tataricum, rutin, MYB genes, gene expression

孙朝霞，侯思宇，令狐斌，刘荣华，王丽，杨武德，韩渊怀. 苦荞全生育期芦丁积累与其生物合成途径相关基因表达分析[J]. 中国农业科学, 2017, 50(18): 3473-3481.

SUN ZhaoXia, HOU SiYu, LINGHU Bin, LIU RongHua, WANG Li, YANG WuDe, HAN YuanHuai. Correlation Analysis on Rutin Accumulation and Gene Expression of Rutin Synthetic Enzymes and MYBs in the Whole Developmental Stage of Fagopyrum tataricum[J]. Scientia Agricultura Sinica, 2017, 50(18): 3473-3481.

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