苦荞转录因子FtMYBF的克隆、亚细胞定位及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0861

摘要/Abstract

摘要：

R2R3-MYB转录因子SG7亚家族成员在植物黄酮醇生物合成中具有极其重要的调控作用。探究苦荞中SG7 R2R3-MYB转录因子在黄酮醇生物合成中的功能,为苦荞黄酮醇生物合成的分子调控机制研究奠定基础。采用RT-PCR技术从苦荞中克隆到一个前期经转录组与代谢组联合分析鉴定到的SG7 R2R3-MYB转录因子,命名为FtMYBF。利用生物信息学、亚细胞定位、基因共表达、基因表达量与总黄酮含量相关性对该基因进行了分析。结果表明,FtMYBF全长CDS序列为1 119 bp,编码372个氨基酸。蛋白多序列比对和系统进化树分析表明,FtMYBF属于R2R3-MYB SG7亚家族成员。亚细胞定位显示,FtMYBF蛋白定位于细胞核。基因共表达分析表明,FtMYBF与多个苦荞黄酮醇生物合成结构基因共表达。基因表达量与总黄酮含量间相关性分析表明,FtMYBF在不同组织部位表达量与总黄酮含量高度正相关。FtMYBF属于R2R3-MYB SG7亚家族成员,是一个核定位转录因子,其可能通过正调控苦荞黄酮醇生物合成结构基因的表达来正调控苦荞黄酮和黄酮醇的生物合成。

关键词: 苦荞, FtMYBF, R2R3-MYB转录因子, 基因克隆, 亚细胞定位, 黄酮

Abstract:

The SG7 subfamily members of R2R3-MYB transcription factors play a critical role in the biosynthesis of flavonols in plants. Exploring the function of SG7 R2R3-MYB transcription factors in flavonol biosynthesis in Fagopyrum tataricum would lay a foundation for the molecular regulation mechanism of flavonol biosynthesis in F. tataricum. RT-PCR was used to clone 1 SG7 R2R3-MYB transcription factor identified by the combined analysis of transcriptome and metabolome in previous study,and named as FtMYBF. This gene then was analyzed by bioinformatics,subcellular localization,gene coexpression and the correlation between gene expression level and total flavonoid content. As a result,the CDS length of FtMYBF was 1 119 bp and encoded 372 amino acids. Multiple sequence alignment and phylogenetic tree analyses showed that FtMYBF belonged to R2R3-MYB SG7 subfamily. Subcellular localization results suggested that the FtMYBF protein was located in the nucleus. Gene coexpression analysis demonstrated that FtMYBF was coexpressed with several structural genes of flavonol biosynthesis in F. tataricum. The correlation analysis between gene expression and total flavonoids content indicated that there was a highly positive correlation between the FtMYBF expression level and the total flavonoid content in different tissues. The results of this study suggest that FtMYBF is a member of R2R3-MYB SG7 subfamily and is a nuclear localization transcription factor,which might positively regulate the flavonols biosynthesis by regulating the expression of the flavonol biosynthesis structural genes in F. tataricum.

Key words: Fagopyrum tataricum, FtMYBF, R2R3-MYB transcription factor, gene cloning, subcellular localization, flavonoid

孙小倩, 王佳蕊, 陈庆富, 李洪有. 苦荞转录因子FtMYBF的克隆、亚细胞定位及表达分析[J]. 生物技术通报, 2021, 37(3): 10-17.

SUN Xiao-qian, WANG Jia-rui, CHEN Qing-fu, LI Hong-you. Gene Cloning,Subcellular Localization and Expression Analyses of FtMYBF Transcription Factor in Fagopyrum tataricum[J]. Biotechnology Bulletin, 2021, 37(3): 10-17.

图/表 7

表1 本研究所用引物序列

引物名称	引物序列（5'-3'）
FtMYBF-f1	ATGGGAAGAGCACCTTGCTG
FtMYBF-r1	CTCAAGAAAGAAGCCAGGCAAC
FtMYBF-f2	CAAGCTTGCATGCCTGCAGGTCGACATGGGAA- GAGCACCTTGCTG
FtMYBF-r2	CCTCGCCCTTGCTCACCATGGATCCAGAAAGA- AGCCAGGCAACCA
FtMYBF-qf	GGGAGAGAGTAGCTGCAATG
FtMYBF-qr	CCCACATTAGCCACGATAGG
FtActin7-f	ATGTTCACTACCACCGCTGA
FtActin7-r	TGAACCTCTCAGCACCAATC

图1 FtMYBF全长CDS克隆 M：DNA maker 2000;1和2：扩增片段

图2 FtMYBF蛋白序列多序列比对

图3 FtMYBF系统进化树分析

图4 FtMYBF亚细胞定位

图5 FtMYBF与苦荞黄酮类化合物生物合成结构基因表达聚类热图种1：灌浆前种子;种2：灌浆期种子;种3：乳熟期种子

图6 FtMYBF在苦荞不同组织部位中的表达量与总黄酮含量间的相关性 A：FtMYBF在不同组织部位中的表达;B：黑苦013不同组织部位的总黄酮含量;C：FtMYBF在不同组织部位表达量与总黄酮含量间的相关性。叶1：成年植株顶端1叶;叶2：成年植株顶端3叶;种1：灌浆前种子;种2：灌浆期种子;种3：乳熟期种子

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