苦荞转录因子基因FtbHLH3的克隆、亚细胞定位及表达分析

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

摘要/Abstract

摘要：

bHLH（basic helix-loop-helix）转录因子在植物黄酮化合物生物合成中具有重要的调控作用。为探究苦荞[Fagopyrum tataricum（L.）Gaertn]中bHLH转录因子在黄酮化合物生物合成中的功能和分子调控机制，利用RT-PCR（reverse transcription-polymerase chain reaction）技术从苦荞中克隆了一个bHLH转录因子基因FtbHLH3，并对其进行生物信息学、亚细胞定位、转录激活活性、基因表达、基因共表达和基因表达量与总黄酮含量相关性分析。结果表明，FtbHLH3全长CDS序列为783 bp，编码260个氨基酸。保守结构域和系统进化分析表明，FtbHLH3是一个非IIIf亚家族bHLH转录因子成员。亚细胞定位和转录激活活性分析显示，FtbHLH3定位于细胞核，不具有转录激活活性。基因共表达分析表明，FtbHLH3与苦荞中拟南芥TT8同源基因FtTT8和12个黄酮化合物生物合成结构基因共表达。不同组织部位中基因表达量与总黄酮含量间相关性分析表明，FtbHLH3的表达量与总黄酮含量间具有较高的正相关性。本研究结果表明，FtbHLH3是一个定位于细胞核，不具有转录激活活性的非IIIf亚家族bHLH转录因子，可能通过与其他转录因子互作来调控苦荞黄酮化合物的生物合成。

关键词: 苦荞, FtbHLH3, bHLH转录因子, 基因克隆, 亚细胞定位, 转录激活, 黄酮

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

吕秋谕, 孙培媛, 冉彬, 王佳蕊, 陈庆富, 李洪有. 苦荞转录因子基因FtbHLH3的克隆、亚细胞定位及表达分析[J]. 生物技术通报, 2023, 39(8): 194-203.

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.

图/表 8

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 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

图1 FtbHLH3的克隆 M：DNA maker DL2000；1和2：FtbHLH3扩增片段

Fig. 1 Cloning of FtbHLH3 M: DNA maker DL2000. 1 and 2: Amplified fragments of FtbHLH3

图2 FtbHLH3与已知的调控类黄酮生物合成的bHLH蛋白的系统进化树

Fig. 2 Phylogenetic tree of FtbHLH3 and known bHLH protein regulating flavonoid biosynthesis

图3 FtbHLH3亚细胞定位

Fig. 3 Subcellular localization of FtbHLH3

图4 FtbHLH3在酵母中转录自激活活性鉴定

Fig. 4 Identification of transcriptional self-activation activity of FtbHLH3 in yeast

图5 FtbHLH3与苦荞种子差异表达bHLH转录因子基因和类黄酮生物合成结构基因的表达聚类热图种子1：授粉后7 d的种子；种子2：授粉后13 d的种子；种子3：授粉后16 d的种子；-1和-2表示生物学重复1和2。红色框表示的是与bHLH3共表达的苦荞黄酮生物合成结构基因和bHLH转录因子基因

Fig. 5 Expression cluster heat map of FtbHLH3, differentially expressed transcription factor bHLH genes and flavonoid biosynthesis structure genes in tartary buckwheat seeds Seed 1: Seeds 7 d after pollination. Seed 2: Seeds 13 d after pollination. Seed 3: Seeds 16 d after pollination. - 1 and - 2 represent biological repeat 1 and 2. The red box shows the flavonoid biosynthesis structure genes and bHLH transcription factor genes co-expressed with bHLH3 of tartary buckwheat

表2 FtbHLH3与类黄酮生物合成结构基因表达相关系数

Table 2 Correlation coefficient between FtbHLH3 and flavonoid biosynthesis structure gene expression

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

图6 FtbHLH3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

Fig. 6 Correlation between the expressions of FtbHLH3 in different tissue parts of tartary buckwheat and the contents of total flavonoids

参考文献 37

[1]	Zhang KX, He M, Fan Y, et al. Resequencing of global Tartary buckwheat accessions reveals multiple domestication events and key loci associated with agronomic traits[J]. Genome Biol, 2021, 22(1): 23. doi: 10.1186/s13059-020-02217-7 pmid: 33430931
[2]	Li HY, Lyu QY, Liu AK, et al. Comparative metabolomics study of Tartary(Fagopyrum tataricum(L.)Gaertn)and common(Fagop-yrum esculentum Moench)buckwheat seeds[J]. Food Chem, 2022, 371: 131125. doi: 10.1016/j.foodchem.2021.131125 URL
[3]	Xu WJ, Dubos C, Lepiniec L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes[J]. Trends Plant Sci, 2015, 20(3): 176-185. doi: 10.1016/j.tplants.2014.12.001 pmid: 25577424
[4]	高国应, 伍小方, 张大为, 等. MBW复合体在植物花青素合成途径中的研究进展[J]. 生物技术通报, 2020, 36(1): 126-134. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0738
	Gao GY, Wu XF, Zhang DW, et al. Research progress on the MBW complexes in plant anthocyanin biosynthesis pathway[J]. Biotechnol Bull, 2020, 36(1): 126-134.
[5]	Chandler VL, Radicella JP, Robbins TP, et al. Two regulatory genes of the maize anthocyanin pathway are homologous: isolation of B utilizing R genomic sequences[J]. Plant Cell, 1989, 1(12): 1175-1183. pmid: 2535537
[6]	Zhang BP, Schrader A. TRANSPARENT TESTA GLABRA 1-dependent regulation of flavonoid biosynthesis[J]. Plants(Basel), 2017, 6(4): 65.
[7]	Escaray FJ, Passeri V, Perea-García A, et al. The R2R3-MYB TT2b and the bHLH TT8 genes are the major regulators of proanthocyanidin biosynthesis in the leaves of Lotus species[J]. Planta, 2017, 246(2): 243-261. doi: 10.1007/s00425-017-2696-6 URL
[8]	Lim SH, Kim DH, Kim JK, et al. A radish basic helix-loop-helix transcription factor, RsTT8 acts a positive regulator for anthocyanin biosynthesis[J]. Front Plant Sci, 2017, 8: 1917. doi: 10.3389/fpls.2017.01917 URL
[9]	Li CH, Qiu J, Ding L, et al. Anthocyanin biosynthesis regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals[J]. Plant Physiol Biochem, 2017, 112: 335-345. doi: 10.1016/j.plaphy.2017.01.019 URL
[10]	Zhao Y, Zhang YY, Liu H, et al. Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis[J]. BMC Plant Biol, 2019, 19(1): 497. doi: 10.1186/s12870-019-2109-z pmid: 31726984
[11]	Heendeniya RG, Gruber MY, Lei Y, et al. Biodegradation profiles of proanthocyanidin-accumulating alfalfa plants coexpressing Lc-bHLH and C1-MYB transcriptive flavanoid regulatory genes[J]. J Agric Food Chem, 2019, 67(17): 4793-4799. doi: 10.1021/acs.jafc.9b00495 URL
[12]	Wang LH, Tang W, Hu YW, et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarpof red-centered kiwifruit Actinidia chinensis cv. Hongyang[J]. Plant J, 2019, 99(2): 359-378. doi: 10.1111/tpj.2019.99.issue-2 URL
[13]	Su WQ, Tao R, Liu WY, et al. Characterization of four polymorphic genes controlling red leaf colour in lettuce that have undergone disruptive selection since domestication[J]. Plant Biotechnol J, 2020, 18(2): 479-490. doi: 10.1111/pbi.13213 pmid: 31325407
[14]	Tao RY, Yu WJ, Gao YH, et al. Light-induced basic/helix-loop-helix64 enhances anthocyanin biosynthesis and undergoes CONSTITUTIVELY PHOTOMORPHOGENIC1-mediated degradation in pear[J]. Plant Physiol, 2020, 184(4): 1684-1701. doi: 10.1104/pp.20.01188 pmid: 33093233
[15]	Zhao PC, Li XX, Jia JT, et al. bHLH92 from sheepgrass acts as a negative regulator of anthocyanin/proanthocyandin accumulation and influences seed dormancy[J]. J Exp Bot, 2019, 70(1): 269-284. doi: 10.1093/jxb/ery335 pmid: 30239820
[16]	Burr FA, Burr B, Scheffler BE, et al. The maize repressor-like gene intensifier1 shares homology with the r1/b1 multigene family of transcription factors and exhibits missplicing[J]. Plant Cell, 1996, 8(8): 1249-1259. pmid: 8776895
[17]	Liu XF, Yin XR, Allan AC, et al. The role of MrbHLH1 and MrMYB1 in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry(Myrica rubra)during anthocyanin biosynthesis[J]. Plant Cell Tiss Organ Cult, 2013, 115(3): 285-298. doi: 10.1007/s11240-013-0361-8 URL
[18]	Hichri I, Barrieu F, Bogs J, et al. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway[J]. J Exp Bot, 2011, 62(8): 2465-2483. doi: 10.1093/jxb/erq442 pmid: 21278228
[19]	Bai YC, Li CL, Zhang JW, et al. Characterization of two Tartary buckwheat R2R3-MYB transcription factors and their regulation of proanthocyanidin biosynthesis[J]. Physiol Plant, 2014, 152(3): 431-440. doi: 10.1111/ppl.2014.152.issue-3 URL
[20]	Luo XP, Zhao HX, Yao PF, et al. An R2R3-MYB transcription factor FtMYB15 involved in the synthesis of anthocyanin and proanthocyanidins from Tartary buckwheat[J]. J Plant Growth Regul, 2018, 37(1): 76-84. doi: 10.1007/s00344-017-9709-3 URL
[21]	Huang YJ, Wu Q, Wang S, et al. FtMYB8 from Tartary buckwheat inhibits both anthocyanin/proanthocyanidin accumulation and marginal Trichome initiation[J]. BMC Plant Biol, 2019, 19(1): 263. doi: 10.1186/s12870-019-1876-x pmid: 31215400
[22]	Dong QX, Zhao HX, Huang YJ, et al. FtMYB18 acts as a negative regulator of anthocyanin/proanthocyanidin biosynthesis in Tartary buckwheat[J]. Plant Mol Biol, 2020, 104(3): 309-325. doi: 10.1007/s11103-020-01044-5 pmid: 32833148
[23]	Wang L, Deng RY, Bai YC, et al. Tartary buckwheat R2R3-MYB gene FtMYB3 negatively regulates anthocyanin and proanthocyanin biosynthesis[J]. Int J Mol Sci, 2022, 23(5): 2775. doi: 10.3390/ijms23052775 URL
[24]	Zhang D, Jiang CL, Huang CH, et al. The light-induced transcription factor FtMYB116 promotes accumulation of rutin in Fagopyrum tataricum[J]. Plant Cell Environ, 2019, 42(4): 1340-1351. doi: 10.1111/pce.13470
[25]	Yao PF, Huang YJ, Dong QX, et al. FtMYB6, a light-induced SG7 R2R3-MYB transcription factor, promotes flavonol biosynthesis in Tartary buckwheat(Fagopyrum tataricum)[J]. J Agric Food Chem, 2020, 68(47): 13685-13696. doi: 10.1021/acs.jafc.0c03037 URL
[26]	Sun ZX, Bin LH, Hou SY, et al. Tartary buckwheat FtMYB31 gene encoding an R2R3-MYB transcription factor enhances flavonoid accumulation in tobacco[J]. J Plant Growth Regul, 2020, 39(2): 564-574. doi: 10.1007/s00344-019-10000-7
[27]	Hou SY, Du W, Hao YR, et al. Elucidation of the regulatory network of flavonoid biosynthesis by profiling the metabolome and transcriptome in Tartary buckwheat[J]. J Agric Food Chem, 2021, 69(25): 7218-7229. doi: 10.1021/acs.jafc.1c00190 URL
[28]	孙小倩, 王佳蕊, 陈庆富, 等. 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. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0861
	Sun XQ, Wang JR, Chen QF, et al. Gene cloning, subcellular localization and expression analyses of FtMYBF transcription factor in Fagopyrum tataricum[J]. Biotechnol Bull, 2021, 37(3): 10-17.
[29]	Zhou ML, Sun ZM, Ding MQ, et al. FtSAD2 and FtJAZ1 regulate activity of the FtMYB11 transcription repressor of the phenylpropanoid pathway in Fagopyrum tataricum[J]. New Phytol, 2017, 216(3): 814-828. doi: 10.1111/nph.2017.216.issue-3 URL
[30]	Zhang KX, Logacheva MD, Meng Y, et al. Jasmonate-responsive MYB factors spatially repress rutin biosynthesis in Fagopyrum tataricum[J]. J Exp Bot, 2018, 69(8): 1955-1966. doi: 10.1093/jxb/ery032 URL
[31]	Li JB, Zhang KX, Meng Y, et al. FtMYB16 interacts with Ftimportin-α1 to regulate rutin biosynthesis in Tartary buckwheat[J]. Plant Biotechnol J, 2019, 17(8): 1479-1481. doi: 10.1111/pbi.13121 pmid: 30963665
[32]	Li HY, Lv QY, Ma C, et al. Metabolite profiling and transcriptome analyses provide insights into the flavonoid biosynthesis in the developing seed of Tartary buckwheat(Fagopyrum tataricum)[J]. J Agric Food Chem, 2019, 67(40): 11262-11276. doi: 10.1021/acs.jafc.9b03135 URL
[33]	Zhang LJ, Li XX, Ma B, et al. The Tartary buckwheat genome provides insightsinto rutin biosynthesis and abiotic StressTolerance[J]. Mol Plant, 2017, 10(9): 1224-1237. doi: 10.1016/j.molp.2017.08.013 URL
[34]	吕丹, 黎瑞源, 郑冉, 等. 苦荞“翅米荞×野苦荞”重组自交系群体籽粒黄酮含量及籽粒性状的分析[J]. 贵州师范大学学报: 自然科学版, 2019, 37(6): 40-46.
	Lv D, Li RY, Zheng R, et al. Analysis of flavonoids content in grains and grain traits on ‘Chimiqiao’ and ‘Yekuqiao’ recombinant inbred lines population of Tartary buckwheat[J]. J Guizhou Norm Univ Nat Sci, 2019, 37(6): 40-46.
[35]	Li CL, Bai YC, Li SJ, et al. Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in Tartary buckwheat[J]. J Agric Food Chem, 2012, 60(20): 5161-5168. doi: 10.1021/jf205192q URL
[36]	Xu HT, Jiang ZQ, Lin ZM, et al. FtUGT79A15 is responsible for rutinosylation in flavonoid diglycoside biosynthesis in Fagopyrum tataricum[J]. Plant Physiol Biochem, 2022, 181: 33-41. doi: 10.1016/j.plaphy.2022.04.004 URL
[37]	Yin QG, Han XY, Han ZX, et al. Genome-wide analyses reveals a glucosyltransferase involved in rutin and emodin glucoside biosynthesis in Tartary buckwheat[J]. Food Chem, 2020, 318: 126478. doi: 10.1016/j.foodchem.2020.126478 URL