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

发布时间：2018-06-07 19:00

本文选题：苦荞 + 芦丁　；参考：《中国农业科学》2017年18期

【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Ft CHS、Ft F3H、Ft4CL、Ft FLS-like和Ft UFGT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Ft MYB7和Ft MYB9,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Ft MYB7属于第II类群,Ft MYB9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,Ft MYB7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;Ft MYB9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、Ft CHS、Ft F3H、Ft UFGT和Ft MYB7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Ft FLS-like和Ft MYB9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】Ft MYB7和Ft MYB9转录因子在整个生育时期中表达模式存在明显差异。其中Ft MYB7可能在苦荞芦丁积累的过程中起到正调控作用,而Ft MYB9则为负调控作用。
[Abstract]:[objective] to explore the relationship between the changes of rutin content and the expression of key enzyme genes and MYB gene in the whole growth period of Tartary buckwheat. In order to further clarify the molecular mechanism of rutin biosynthesis in Tartary buckwheat plants. [methods] with Jiujiang Tartary Buckwheat as experimental material, the whole growth period was at germination stage, cotyledon stage, true leaf stage, full leaf stage, budding stage, full flowering stage, respectively. There were 8 stages of grain filling and grain maturation. The MYB transcription factor gene related to flavonoids metabolism was cloned by RT-PCR. Amino acid sequence alignment and conserved domain analysis were carried out by T-coffee software. The MYB transcription factors associated with flavonoid metabolism in Arabidopsis thaliana and the homologous MYB transcription factor sequences of buckwheat were compared to construct phylogenetic tree based on proximity method. The expression patterns of five key enzymes in rutin synthesis pathway, Ft CHSN, Ft F3H, Ft 4CLC, Ft FLS-like and Ft UFGT, and the cloned MYB transcription factors in different tissues were analyzed by real-time fluorescence quantitative PCR. The content of rutin was determined by high performance liquid chromatography (HPLC). Correlation analysis was used to analyze the correlation between rutin content and gene expression patterns in different tissues. Using Euclidean distance method to construct coexpression hierarchical cluster map. [results] two MYB transcription factors were cloned. The nucleic acid sequences of Ft MYB7 and Ft MYB9 were 876 and 912 BP, respectively, encoding 291 and 303 amino acid residues, respectively. The results of amino acid sequence homology analysis showed that both of them had a typical R2R3 conserved domain, which was a MYB transcription factor of R2R3 type. Phylogenetic tree was constructed by combining 17 Tartary buckwheat and 3 Arabidopsis thaliana flavonoid metabolism-related MYB transcription factor homologous amino acid sequences in Nr database. The results showed that the 22 genes were divided into 6 groups. FtMYB7 belongs to Group II and FtMYB9 belongs to Group IV, and they belong to different groups, suggesting that these two MYB transcription factors may be involved in regulating different functional types of plant growth and development. The results of fluorescence quantitative analysis showed that Ft MYB7 had the highest relative expression in S3 (true leaf stage) and S5 (budding stage), reaching 501 and 867 times FtMYB9 in S1 (germinating stage) and S2 (cotyledon stage), which were 34 and 72 times higher than those in S1 (germinating stage) and S2 (cotyledon stage), respectively. The correlation analysis between the expression of these genes and rutin content showed that the relative expression patterns of Ft4CLA Ft CHSN Ft F3Hfft UFGT and FtMYB7 were positively correlated with the range of rutin content, and the correlation coefficients were 0.7480.6830.7040.890 and 0.862respectively. Ft FLS-like and FtMYB9 were negatively correlated with rutin content, and the correlation coefficients were -0.442 and -0.501 respectively. [conclusion] there were significant differences in the expression patterns of Ft MYB7 and FtMYB9 transcription factors during the whole growth period. Ft MYB7 may play a positive role in the accumulation of Tartary buckwheat rutin, while Ft MYB9 plays a negative role.
【作者单位】：山西农业大学农学院;
【基金】：国家自然科学基金(31301385) 山西省科技攻关项目(20150311007-1) 山西省回国留学人员科研项目(2017-069) 山西省主要农作物种质创新与分子育种重点科技创新平台(2016-246)
【分类号】：S517

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