Cloning, Subcellular Localization and Expression Analysis of the Transcription Factor Gene FtbHLH3 in Fagopyrum tataricum

doi:10.13560/j.cnki.biotech.bull.1985.2023-0034

Abstract

Abstract:

bHLH（basic helix-loop-helix）transcription factors play crucial roles in the biosynthesis of flavonoids in plants. In order to explore the function and molecular regulation mechanism of bHLH transcription factor in flavonoid biosynthesis in tartary buckwheat, RT-PCR was used to clone the FtbHLH3, one bHLH transcription factor. Then the bioinformatics subcellular localization, transcriptional activation activity, gene expression, gene co-expression, and the correlation between gene expression level and total flavonoid content of it were conducted. As results, the CDS length of FtbHLH3 was total 783 bp and it encoded total 260 amino acids. Conserved domain and phylogenetic analysis showed that FtbHLH3 was a member of non-IIIf subfamily of bHLH transcription factor family. Subcellular localization and transcriptional activation assay suggested that FtbHLH3 was located in the nucleus and had no transcriptional activation activity. Gene co-expression analysis revealed that FtbHLH3 was co-expressed with the Arabidopsis TT8 homologous gene FtTT8 and 12 structural genes of flavonoid biosynthesis in the tartary buckwheat. The correlation analysis between gene expression level and total flavonoids content in different tissues indicated that there was a highly positive correlation between the expressions of FtbHLH3 and the contents of total flavonoids. All these results suggest that FtbHLH3 is a non-IIIf subfamily bHLH transcription factor that was located in the nucleus and has no transcriptional activation activity. It may regulate the flavonoids biosynthesis in tartary buckwheat through interacting with other transcription factors.

Key words: tartary buckwheat, FtbHLH3, bHLH transcription factor, gene cloning, subcellular localization, transcriptional activation, flavonoid

LYU Qiu-yu, SUN Pei-yuan, RAN Bin, WANG Jia-rui, CHEN Qing-fu, LI Hong-you. Cloning, Subcellular Localization and Expression Analysis of the Transcription Factor Gene FtbHLH3 in Fagopyrum tataricum[J]. Biotechnology Bulletin, 2023, 39(8): 194-203.

Figures/Tables 8

References 37

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引物名称 Primer name	引物序列 Primer sequence（5'-3'）
FtbHLH3F1	ATGGATAACATCGGTGACGAGTAC
FtbHLH3R1	TCAATTGATCGTAGCGCTATGAGG
FtbHLH3F2	CAAGCTTGCATGCCTGCAGGTCGACATGGATAAC- ATCGGTGACGAG
FtbHLH3R2	CCTCGCCCTTGCTCACCATGGATCCATTGATCGT- AGCGCTATGAG
FtbHLH3F3	CCGGAATTCATGGATAACATCGGTGACGAGTAC
FtbHLH3R3	CGCGGATCCTCAATTGATCGTAGCGCTATGAGG
FtbHLH3qF	GGTGTTTGAATCACTGGAGC
FtbHLH3qR	TCGTAGCGCTATGAGGTTGA
FtActin7F	ATGTTCACTACCACCGCTGA
FtActin7R	TGAACCTCTCAGCACCAATC

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
FtbHLH3F1	ATGGATAACATCGGTGACGAGTAC
FtbHLH3R1	TCAATTGATCGTAGCGCTATGAGG
FtbHLH3F2	CAAGCTTGCATGCCTGCAGGTCGACATGGATAAC- ATCGGTGACGAG
FtbHLH3R2	CCTCGCCCTTGCTCACCATGGATCCATTGATCGT- AGCGCTATGAG
FtbHLH3F3	CCGGAATTCATGGATAACATCGGTGACGAGTAC
FtbHLH3R3	CGCGGATCCTCAATTGATCGTAGCGCTATGAGG
FtbHLH3qF	GGTGTTTGAATCACTGGAGC
FtbHLH3qR	TCGTAGCGCTATGAGGTTGA
FtActin7F	ATGTTCACTACCACCGCTGA
FtActin7R	TGAACCTCTCAGCACCAATC

基因名称 Gene name	FtbHLH3		FtbHLH14		FtbHLH29		FtbHLH33/FtTT8
基因名称 Gene name	r	P	r	P	r	P	r	P
FtCHS-1	0.948 4	0.032 7	0.640 4	0.028 5	0.722 8	0.046 4	0.898 6	0.045 8
FtCHS-2	0.961 4	0.002 5	0.746 7	0.002 1	0.746 1	0.004 2	0.930 4	0.004 1
FtCHS-3	0.760 0	0.000 5	0.615 4	0.000 4	0.911 9	0.001 0	0.947 5	0.001 0
FtCHI	0.890 8	0.001 7	0.752 6	0.000 9	0.833 7	0.011 1	0.975 7	0.008 4
FtF3H-1	0.849 8	0.000 2	0.825 2	0.000 2	0.676 1	0.000 3	0.880 3	0.000 3
FtF3H-2	0.522 4	3.15E-05	0.589 3	2.1E-05	0.701 6	0.000 1	0.798 4	8.19E-05
FtF3'H-1	0.810 2	0.001 0	0.595 8	0.000 7	0.929 6	0.002 6	0.976 7	0.002 3
FtF3'H-2	0.859 4	0.000 2	0.825 1	0.000 2	0.678 3	0.000 3	0.884 2	0.000 3
FtF3'5'H	0.631 9	0.048 0	0.257 7	0.019 5	0.528 5	0.301 8	0.719 8	0.262 8
FtFLS	-0.239 6	6.36E-05	0.179 1	5.78E-05	0.144 7	8.18E-05	0.042 0	8.05E-05
FtDFR	0.946 4	0.002 4	0.786 5	0.002 0	0.694 9	0.004 0	0.937 0	0.003 9
FtANS	0.700 0	0.000 1	0.747 0	0.000 1	0.760 8	0.000 2	0.852 2	0.000 2
FtANR	0.955 6	0.006 9	0.921 1	0.006 0	0.476 9	0.010 3	0.773 4	0.010 1
FtLAR	0.923 6	0.014 7	0.782 2	0.011 4	0.754 4	0.028 1	0.947 8	0.026 9

基因名称 Gene name	FtbHLH3		FtbHLH14		FtbHLH29		FtbHLH33/FtTT8
基因名称 Gene name	r	P	r	P	r	P	r	P
FtCHS-1	0.948 4	0.032 7	0.640 4	0.028 5	0.722 8	0.046 4	0.898 6	0.045 8
FtCHS-2	0.961 4	0.002 5	0.746 7	0.002 1	0.746 1	0.004 2	0.930 4	0.004 1
FtCHS-3	0.760 0	0.000 5	0.615 4	0.000 4	0.911 9	0.001 0	0.947 5	0.001 0
FtCHI	0.890 8	0.001 7	0.752 6	0.000 9	0.833 7	0.011 1	0.975 7	0.008 4
FtF3H-1	0.849 8	0.000 2	0.825 2	0.000 2	0.676 1	0.000 3	0.880 3	0.000 3
FtF3H-2	0.522 4	3.15E-05	0.589 3	2.1E-05	0.701 6	0.000 1	0.798 4	8.19E-05
FtF3'H-1	0.810 2	0.001 0	0.595 8	0.000 7	0.929 6	0.002 6	0.976 7	0.002 3
FtF3'H-2	0.859 4	0.000 2	0.825 1	0.000 2	0.678 3	0.000 3	0.884 2	0.000 3
FtF3'5'H	0.631 9	0.048 0	0.257 7	0.019 5	0.528 5	0.301 8	0.719 8	0.262 8
FtFLS	-0.239 6	6.36E-05	0.179 1	5.78E-05	0.144 7	8.18E-05	0.042 0	8.05E-05
FtDFR	0.946 4	0.002 4	0.786 5	0.002 0	0.694 9	0.004 0	0.937 0	0.003 9
FtANS	0.700 0	0.000 1	0.747 0	0.000 1	0.760 8	0.000 2	0.852 2	0.000 2
FtANR	0.955 6	0.006 9	0.921 1	0.006 0	0.476 9	0.010 3	0.773 4	0.010 1
FtLAR	0.923 6	0.014 7	0.782 2	0.011 4	0.754 4	0.028 1	0.947 8	0.026 9