苦荞转录因子基因FtbHLH3调控类黄酮生物合成的功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2024-1017

摘要/Abstract

摘要：

目的苦荞是一种重要的药食两用作物，而类黄酮是苦荞中一种重要的生物活性成分。bHLH转录因子在类黄酮生物合成过程中发挥着重要的调控作用，前期研究发现FtbHLH3与类黄酮合成大部分结构基因的转录水平显著正相关，探究该转录因子在苦荞类黄酮生物合成中的作用机制，可以丰富苦荞类黄酮生物合成调控机理的研究，并为苦荞高类黄酮品种的育种提供优异的基因资源。方法构建35S：FtbHLH3过表达载体，转化到拟南芥attt8突变体中分析其功能。结果 attt8突变体的幼苗无色素积累，种皮呈淡黄色，FtbHLH3过表达株系幼苗有色素积累，种子表皮颜色恢复至野生型的表型。花色苷含量测定表明FtbHLH3过表达株系幼苗和种子中的花色苷与野生型的都显著高于attt8突变体的。原花青素染色和含量分析表明，在幼苗中三者的含量均较低，且无显著差异，但在种子中，FtbHLH3过表达株系中原花青素的含量最高，显著高于attt8突变体。此外三者黄酮的含量在幼苗和种子中均较低，且无显著差异。RT-qPCR表明在幼苗和种子中，FtbHLH3过表达株系中的花色苷合成基因的表达水平比attt8突变体和野生型均显著上升，关键基因AtANS和AtUFGT极显著上调。而三者幼苗中AtANR和AtFLS的表达水平无显著差异。FtbHLH3过表达株系种子中的原花青素合成分支上的关键基因AtANR的表达水平显著高于野生型，但黄酮合成关键基因AtFLS也没有显著差异，这与表型和含量测定结果一致。结论 FtbHLH3是花色苷和原花青素合成过程中的正调控因子，但不影响黄酮的合成。

关键词: 苦荞, bHLH转录因子, 花色苷, 原花青素, 黄酮, 遗传转化

Abstract:

Objective Tartary buckwheat is an important medicinal and edible crop, and flavonoids are an important bioactive component in Tartary buckwheat. The bHLH transcription factor plays an important regulatory role in the flavonoid biosynthesis. Previous studies have found that the transcription level of FtbHLH3 is significantly positively correlated with the transcription levels of most structural genes in flavonoid synthesis. Exploring the mechanism of this transcription factor in flavonoid biosynthesis in Tartary buckwheat may enrich the studies on the regulation mechanism of flavonoid biosynthesis in Tartary buckwheat, and provide excellent gene resource for breeding Tartary buckwheat cultivars with high flavoniod. Method The 35S:FtbHLH3 overexpression vector was constructed and transformed into Arabidopsis attt8 mutant to analyze its function. Result There were no pigment accumulation in the seedling of att8 mutans, and their seed coat was light yellow; while the seedlings of FtbHLH3-overexpressed line had pigment accumulation, and the seed coat color returned to the wild type's phenotype. Measurement of total anthocyanin content showed that the anthocyanins in the seedlings and seeds of the FtbHLH3-overexpressed line and the wild type were significantly higher than that of the attt8 mutant. Proanthocyanidin staining and content analysis showed that the contents of three types of Arabidopsis seedlings were all very low with no significant difference. However, the content of proanthocyadins in the seeds of FtbHLH3-overexpressed line was the highest, which was significantly higher than that of the attt8 mutant. In addition, the total flavones contents of these three types of Arabidopsis seedlings and seeds had no significant difference. RT-qPCR showed that the expressions of anthocyanin synthesis genes significantly increased in the seedlings of FtbHLH3-overexpressed lines, and the key genes AtANS and AtUFGT were significantly upregulated while compared with that of the mutant and wild-type Arabidopsis. There was no significant difference in the expressions of AtANR and AtFLS among the three type Arabidopsis seedlings. The expression of AtANR, a key gene on the branch of proanthocyanidin biosynthesis, in the seeds of the FtbHLH3-overexpressed line was significantly higher than that of wild type, but there was no significant difference in the key gene AtFLS for flavones synthesis, which was consistent with the phenotype and content. Conclusion FtbHLH3 is a positive regulator in the biosynthesis of anthocyanin and proanthocyanidin, but does not affect the synthesis of flavones.

Key words: Tartary buckwheat, bHLH transcription factor, anthocyanins, proanthocyanidins, flavonoids, genetic transformation

杨朝结, 张兰, 陈红, 黄娟, 石桃雄, 朱丽伟, 陈庆富, 李洪有, 邓娇. 苦荞转录因子基因FtbHLH3调控类黄酮生物合成的功能鉴定[J]. 生物技术通报, 2025, 41(4): 134-144.

YANG Chao-jie, ZHANG Lan, CHEN Hong, HUANG Juan, SHI Tao-xiong, ZHU Li-wei, CHEN Qing-fu, LI Hong-you, DENG Jiao. Functional Identification of the Transcription Factor Gene FtbHLH3 in Regulating Flavonoid Biosynthesis in Fagopyrum tataricum[J]. Biotechnology Bulletin, 2025, 41(4): 134-144.

图/表 6

表1 本研究所用引物序列

Table 1 Primer sequences used in the study

引物名称 Primer name	引物序列 Primer sequences（5′-3′）	用途 Purpose
FtbHLH3-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGATAACATCGGTGACGAGTAC	遗传转化
FtbHLH3-R	GGGGACCACTTTGTACAAGAAAGCTGGGTGTCAATTGATCGTAGCGCTATGAGG	遗传转化
AtCHS	F：AATGGTGATGGCTGGTGCTT；R：TGGTGATGCGGAAGTAGTAGTC	实时荧光定量
AtCHI	F：GCCTCCTCCAATCCATTATTCC；R：CCTTCCACTTGACAGATAGAGAA
AtF3H	F：CATCGTCTCTAGTCACCTCCAG；R：CTCACTATACTCCTCCGTCACTT
AtDFR	F：CGCCAAGACGCTACTCACT；R：CGGCTTTATCACTTCGTTCTCA
AtANS	F：GTGATTACATAGAAGCAACGAGTG；R：CTAAACCTAGACCGACAGAGAGA
AtANR	F：CGGCGATACAAGGAGTGA；R：AAGGCTTCTCCTCTGTGA
AtUFGT	F：CAACTGGTTTTCCGTTTCTGGTT；R：GCTTCCTCGACGGTTGATACAC
AtFLS	F：TCACAACATTCCGAGGTCCAA；R：CTTCGTCGGGATCGCTTAGA
AtActin2	F：TGCTGGATTCTGGTGATGGT；R：AAGGTCAAGACGGAGGATGG

图1 FtbHLH3与其他物种中调控类黄酮生物合成的bHLH转录因子的系统进化树及保守基序分析桃PpTT8（Prunus persica，XP_007200710.2）和PpbHLH3（AIE57508.1）；樱桃李PcbHLH3（Prunus cerasifera， AKV89647.1）；李子PsbHLH3（Prunus salicina， AXU25495.1）；甜樱桃PabHLH3（Prunus avium， AJB28481.1）；草莓FabHLH3（Fragaria × ananassa， USN18571.1）；枫香LfTT8（Liquidambar formosana， QVX18577.1）；苦荞FtbHLH1（Fagopyrum tataricum， KT737454）和FtTT8 （FtPinG0008212200.01.T02）；拟南芥AtTT8（Arabidopsis thaliana， NP_192720.2），AtbHLH35（AT5G57150.4）和AtbHLH27 （AT4G29930.3）；枣ZjbHLH35（Ziziphus jujuba， XP_048323445.2）

Fig. 1 Phylogenetic tree and conserved motif analysis of FtbHLH3 and bHLH transcription factor regulating flavonoid biosynthesis in other species

图2 苦荞FtbHLH3与其他调控类黄酮生物合成的bHLH蛋白的序列分析

Fig. 2 Sequence analysis of Tartary buckwheat FtbHLH3 and other bHLH proteins regulating flavonoid biosynthesis

图3 过表达载体pK7WG2D-FtbHLH3（A）及转基因拟南芥阳性苗鉴定（B）A：过表达载体pK7WG2D-FtbHLH3；B：FtbHLH3回补拟南芥tt8突变体T1代苗。M：DL 2 000 marker；1-6：各转基因植株的gDNA作为模板；C-：阴性对照（以水为模板）；WT：阴性对照（以野生型gDNA为模板）

Fig. 3 Overexpression vector pK7WG2D-FtbHLH3 (A) and identification of positive seedlings of transgenic Arabidopsis (B)A: Overexpression vector pK7WG2D-FtbHLH3; B: FtbHLH3 complements the T1 generation seedlings of Arabidopsistt8 mutant. M: DL 2 000 marker; 1-6: the gDNA of each transgenic plant used as a template; C-: negative control (using water as template); WT: negative control (using wild-type gDNA as template)

图4 FtbHLH3转拟南芥attt8突变体植株的幼苗分析A：WT、attt8突变体、35S：FtbHLH3系的幼苗表型观察；B：幼苗DMACA染色和显微镜观察（40×）；C：花色苷含量测定；D：PAs含量测定；E：总黄酮含量测定；F：RT-qPCR分析。WT：野生型；tt8：拟南芥attt8突变体；35S：FtbHLH3：FtbHLH3回补拟南芥attt8突变体；*代表P<0.05；**代表P<0.01；***代表P<0.001。下同

Fig. 4 Seedling analysis of FtbHLH3 transgenic Arabidopsis thalianaattt8 mutantA: Phenotypic observation of seedlings of WT, attt8 mutant, and 35S:FtbHLH3 line. B: DMACA staining of seedlings and microscope observation (40×). C: Determination of anthocyanin content. D: Determination of PAs content. E: Determination of total flavonoid content. F: RT-qPCR analysis. WT: Wild type; tt8: Arabidopsis attt8 mutant; 35S: FtbHLH3: FtbHLH3 complements the Arabidopsisattt8 mutant; *P<0.05; **P<0.01; ***P<0.001. The same below

图5 FtbHLH3转拟南芥attt8突变体植株的种子分析A：WT、attt8突变体、35S：FtbHLH3系的种子表型观察；B：DMACA染色和显微镜观察（40×）；C：花色苷含量测定；D：PAs含量测定；E：总黄酮含量测定；F：RT-qPCR分析

Fig. 5 Seed analysis of FtbHLH3 transgenic Arabidopsis thalianaattt8 mutantA: Observation of seed phenotypes of WT, attt8 mutant, and 35S:FtbHLH3 line. B: DMACA staining and microscope observation (40×). C: Determination of anthocyanin content. D: Determination of PAs content. E: Determination of total flavonoid content. F: RT-qPCR analysis

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