Functional Identification of the Transcription Factor Gene FtbHLH3 in Regulating Flavonoid Biosynthesis in Fagopyrum tataricum

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

Abstract

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

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.

Figures/Tables 6

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

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

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

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)

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

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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