卵叶牡丹花色苷合成相关基因bHLH的克隆与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0149

摘要/Abstract

摘要：

【目的】以卵叶牡丹（Paeonia qiui）为试验材料，研究PqbHLH1基因在叶片发育过程中对花色苷合成的调控机制。【方法】基于前期获得的卵叶牡丹叶片转录组数据设计引物，利用PCR技术从卵叶牡丹叶片中克隆得到bHLH1基因，采用生物信息学方法分析PqbHLH1基因的理化性质、亲疏水性、理论等电点、蛋白二级结构以及物种的进化关系。通过荧光定量PCR技术检测其在不同组织、不同时期的表达量。利用农杆菌介导法使其在拟南芥（Arabidopsis thaliana）中过表达，检测过表达PqbHLH1对卵叶牡丹叶片花色苷的影响。【结果】PqbHLH1基因的编码区全长为1 920 bp，编码639个氨基酸。PqbHLH1蛋白属于亲水性蛋白，同时不含有信号肽和跨膜结构，且定位在细胞核上。系统进化分析表明卵叶牡丹PqbHLH1蛋白与葡萄VvMYCA1的亲缘关系最近。荧光定量PCR分析表明PqbHLH1基因在卵叶牡丹叶片中的表达量呈先降低后增高的趋势，在叶片S6时期表达量显著，且花色苷合成结构基因PqCHS、PqF3'H、PqDFR和PqANS随着叶片发育其表达水平呈现出先上升后下降的趋势。PqbHLH1基因过表达的拟南芥株系中，与花色苷合成相关结构基因的表达量显著高于野生型拟南芥，且花色苷含量与结构基因AtCHI、AtF3'H和AtUFGT的表达趋势一致，黄酮醇含量与结构基因AtFLS的表达趋势一致。【结论】PqbHLH1基因通过调控花色苷合成相关基因的表达，从而正向调控卵叶牡丹叶片中花色苷和黄酮醇的合成。

关键词: 卵叶牡丹, bHLH转录因子, 花色苷, 表达分析

Abstract:

【Objective】Using Paeonia qiui as the experimental material, the regulatory mechanism of PqbHLH1 gene on anthocyanin synthesis during leaf development was studied.【Method】Based on the transcriptome data obtained in the early stage, primers were designed and the bHLH1 gene was cloned from the leaves of Paeonia qiui using PCR technology. Bioinformatics methods were used to analyze the physicochemical properties, hydrophobicity, theoretical isoelectric points, protein secondary structure, and evolutionary relationships of the PqbHLH1 gene. Fluorescence quantitative PCR technology was used to detect its expressions in different tissues and stages. Agrobacterium mediated method was employed to overexpress it in Arabidopsis thaliana and detect the effect of overexpressing PqbHLH1 on the anthocyanins of peony leaves.【Result】The total length of the coding region of PqbHLH1 gene was 1 920 bp, encoding 639 amino acids. The PqbHLH1 protein was hydrophilic and does not contain signal peptides or transmembrane structures, and was localized in the nucleus. Phylogenetic analysis showed that the PqbHLH1 protein in peony ovale had the closest genetic relationship with VvMYCA1 in grapes. Fluorescence quantitative PCR analysis showed that the expression of PqbHLH1 gene in peony leaves had a trend of first decreasing and then increasing, and the expression was significant in the S6 stage of leaves. The expressions of anthocyanin synthesis structural genes PqCHS, PqF3'H, PqDFR, and PqANS showed a trend of first increasing and then decreasing with leaf development. In A. thaliana strains overexpressing the PqbHLH1 gene, the expressions of structural genes related to anthocyanin synthesis was significantly higher than that of wild-type Arabidopsis, and the expression trend of anthocyanin content was consistent with that of structural genes AtCHI, AtF3'H, and AtUFGT, while the expression trend of flavonol content was consistent with that of structural gene AtFLS.【Conclusion】The PqbHLH1 gene positively regulates the synthesis of anthocyanins and flavonols in peony leaves by regulating the expressions of genes related to anthocyanin synthesis.

Key words: Paeonia qiui, bHLH transcription factor, anthocyanin, expression analysis

李雨晴, 吴楠, 罗建让. 卵叶牡丹花色苷合成相关基因bHLH的克隆与功能分析[J]. 生物技术通报, 2024, 40(8): 174-185.

LI Yu-qing, WU Nan, LUO Jian-rang. Cloning and Functional Analysis of bHLH Gene Related to Anthocyanin Synthesis in Paeonia qiui[J]. Biotechnology Bulletin, 2024, 40(8): 174-185.

图/表 12

图1 卵叶牡丹花朵（A）及叶片（B）表型 S1：萌芽5 d；S2：萌芽后15 d；S3：萌芽后35 d；S4：萌芽后45 d；S5：萌芽后60 d；S6：萌芽后80 d。下同

Fig. 1 Phenotypes of flowers (A) and leaves（B）of P. qiui S1: Sprout for 5 d; S2: 15 d after sprouting; S3: 35 d after sprouting; S4: 45 d after sprouting; S5: 60 d after sprouting; S6: 80 d after sprouting. The same below

图2 PqbHLH1基因PCR扩增结果以卵叶牡丹cDNA为模板扩增的DNA片段；M：不同分子量的DNA标准物；1：bHLH1全长序列的扩增产物

Fig. 2 PCR amplification of gene PqbHLH1 DNA fragments amplified using cDNA from peony as a template; M: DNA standards with different molecular weights; 1: amplification products of bHLH1 full-length sequence

图3 PqbHLH1蛋白的生物信息学分析 A：蛋白亲/疏水性预测；B：信号肽位点预测；C：氨基酸跨膜预测；D：蛋白三级结构预测；E: 蛋白二级结构预测；F：结构域分析

Fig. 3 Bioinformatics analysis of PqbHLH1 A: Hydrophilic and hydrophobic prediction; B: signal peptide prediction; C: amino acid transmembrane prediction; D: tertiary structure prediction of protein; E: secondary structure prediction of protein; F: structure domain analysis

图4 PqbHLH1推导的氨基酸序列比对分析

Fig. 4 Alignment of PqbHLH1-deduced amino acid sequences

图5 PqbHLH1蛋白的系统进化树分析

Fig. 5 Phylogenetic tree analysis of PqbHLH1 protein

图6 PqbHLH1的亚细胞定位分析

Fig. 6 Subcellular localization analysis of PqbHLH1

图7 PqbHLH1在叶片不同发育时期（A）和不同组织中（B）的表达量所有图表根据Ducan多重检验，每列上方的不同字母表示差异显著，P<0.05。下同

Fig. 7 Expressions of PqbHLH1 during leaf development（A）and in various tissues（B） According to the Ducan multiple test, the different letters above each column indicate significant differences, P<0.05. The same below

图8 卵叶牡丹在叶片不同发育时期的花色苷含量

Fig. 8 Anthocyanin contents in peony leaves at different developmental stages

图9 卵叶牡丹叶片6个发育时期花色苷合成结构基因的表达量

Fig. 9 Expressions of anthocyanin synthesis structural genes during the development of six stages in P. qiui leaves

图10 PqbHLH1转化拟南芥的PCR检测及转基因拟南芥表型 M：DL5000 DNA marker；1-2：阴性对照；3-5：转基因拟南芥PCR产物

Fig. 10 PCR detection of PqbHLH1 transformed Arabi-dopsis thaliana and transgenic Arabidopsis pheno-type M：DL5000 DNA marker; 1-2: negative control; 3-5: the product PCR of transformed Arabidopsis

图11 过表达PqbHLH1基因促进拟南芥花色苷积累及相关基因表达水平 A：PqbHLH1基因在转基因拟南芥中的表达水平；B：转基因拟南芥的PCR鉴定（M：DNA marker；WT：野生型拟南芥PCR产物；OE-1-3：转基因拟南芥PCR产物）；C：花色苷合成相关基因在转基因拟南芥株系中的表达分析

Fig. 11 Overexpression of PqbHLH1 gene promotes anthocyanin accumulation and related gene expressions in Arabidopsis A: Expression of PqbHLH1 gene in transgenic Arabidopsis; B: PCR identification of transgenic Arabidopsis（M: DNA marker; WT: wild type Arabidopsis PCR product; OE-1-3: PCR product of transgenic Arabidopsis）; C: expression analysis of genes related to anthocyanin synthesis in transgenic Arabidopsis lines

图12 转基因拟南芥花色苷含量和黄酮醇含量分析

Fig. 12 Analysis of anthocyanin content and flavonol content in transgenic Arabidopsis

参考文献 47

[1]	段晶晶. 卵叶牡丹叶片花青素合成相关MYB筛选、克隆与功能验证[D]. 杨凌: 西北农林科技大学, 2019.
	Duan JJ. Screening, cloning and functional verification of MYB related to leaf anthocyanin synthesis in Paeonia qiui[D]. Yangling: Northwest A & F University, 2019.
[2]	贾赵东, 马佩勇, 边小峰, 等. 植物花青素合成代谢途径及其分子调控[J]. 西北植物学报, 2014, 34(7): 1496-1506.
	Jia ZD, Ma PY, Bian XF, et al. Biosynthesis metabolic pathway and molecular regulation of plants anthocyanin[J]. Acta Bot Boreali Occidentalia Sin, 2014, 34(7): 1496-1506.
[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]	高文, 闫庚洋, 左俊凯, 等. 蜀葵ArbHLH148基因克隆与表达分析[J]. 河南农业科学, 2023, 52(9): 133-140.
	Gao W, Yan GY, Zuo JK, et al. Cloning and expression analysis of ArbHLH148 gene in Alcea rosea L[J]. J Henan Agric Sci, 2023, 52(9): 133-140.
[6]	Pires N, Dolan L. Origin and diversification of basic-helix-loop-helix proteins in plants[J]. Mol Biol Evol, 2010, 27(4): 862-874. doi: 10.1093/molbev/msp288 pmid: 19942615
[7]	杜杨梅. 紫花椰菜MYB-TT8-TTG1转录因子调控烟草花青素积累的研究[D]. 重庆: 西南大学, 2017.
	Du YM. Study anthocyanin accumulation in tobacco by constitutively expression MYB2, TT8, TTG1 transcription factors from purple cauliflower[D]. Chongqing: Southwest University, 2017.
[8]	王勇江, 陈克平, 姚勤. bHLH转录因子家族研究进展[J]. 遗传, 2008, 30(7): 821-830.
	Wang YJ, Chen KP, Yao Q. Progress of studies on bHLH transcription factor families[J]. Hereditas, 2008, 30(7): 821-830.
[9]	Wu YY, Wu SH, Wang XQ, et al. Genome-wide identification and characterization of the bHLH gene family in an ornamental woody plant Prunus mume[J]. Hortic Plant J, 2022, 8(4): 531-544.
[10]	韩婷, 杜方. 植物萜烯类合成的转录调控研究进展[J]. 山西农业科学, 2020, 48(10): 1686-1692.
	Han T, Du F. Research progress on regulation of transcription factors related to plant terpene sythesis[J]. J Shanxi Agric Sci, 2020, 48(10): 1686-1692.
[11]	张鹏飞, 丁楠, 李萍. 花色素苷合成酶的生物信息学分析[J]. 生物化工, 2017, 3(5): 10-15.
	Zhang PF, Ding N, Li P. Bioinformatics analysis of anthocyanin synthesis in plants[J]. Biol Chem Eng, 2017, 3(5): 10-15.
[12]	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
[13]	Quattrocchio F, Wing JF, van der Woude K, et al. Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes[J]. Plant J, 1998, 13(4): 475-488. doi: 10.1046/j.1365-313x.1998.00046.x pmid: 9680994
[14]	Spelt C, Quattrocchio F, Mol JN, et al. anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes[J]. Plant Cell, 2000, 12(9): 1619-1632. doi: 10.1105/tpc.12.9.1619 pmid: 11006336
[15]	Feyissa DN, Løvdal T, Olsen KM, et al. The endogenous GL3, but not EGL3, gene is necessary for anthocyanin accumulation as induced by nitrogen depletion in Arabidopsis rosette stage leaves[J]. Planta, 2009, 230(4): 747-754.
[16]	Espley RV, Hellens RP, Putterill J, et al. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10[J]. Plant J, 2007, 49(3): 414-427. doi: 10.1111/j.1365-313X.2006.02964.x pmid: 17181777
[17]	Hichri I, Heppel SC, Pillet J, et al. The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine[J]. Mol Plant, 2010, 3(3): 509-523. doi: 10.1093/mp/ssp118 pmid: 20118183
[18]	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
[19]	Wang LH, Tang W, Hu YW, et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang[J]. Plant J, 2019, 99(2): 359-378.
[20]	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
[21]	Wang MJ, Ou Y, Li Z, et al. Genome-wide identification and analysis of bHLH transcription factors related to anthocyanin biosynthesis in Cymbidium ensifolium[J]. Int J Mol Sci, 2023, 24(4): 3825.
[22]	Deng J, Li JJ, Su MY, et al. A bHLH gene NnTT8 of Nelumbo nucifera regulates anthocyanin biosynthesis[J]. Plant Physiol Biochem, 2021, 158: 518-523.
[23]	Zhao R, Song XX, Yang N, et al. Expression of the subgroup IIIf bHLH transcription factor CpbHLH1 from Chimonanthus praecox(L.)in transgenic model plants inhibits anthocyanin accumulation[J]. Plant Cell Rep, 2020, 39(7): 891-907.
[24]	宁改星. 苹果MdbHLH51和MdbHLH155的基因克隆及其在花青素合成中的作用[D]. 兰州: 甘肃农业大学, 2020.
	Ning GX. Cloning and elucidation of the functional role of apple MdbHLH51 and MdbHLH155 in anthocyanin biosynthesis[D]. Lanzhou: Gansu Agricultural University, 2020.
[25]	Park KI, Ishikawa N, Morita Y, et al. A bHLH regulatory gene in the common morning glory, Ipomoea purpurea, controls anthocyanin biosynthesis in flowers, proanthocyanidin and phytomelanin pigmentation in seeds, and seed trichome formation[J]. Plant J, 2007, 49(4): 641-654.
[26]	Qi Y, Zhou L, Han LL, et al. PsbHLH1, a novel transcription factor involved in regulating anthocyanin biosynthesis in tree peony(Paeonia suffruticosa)[J]. Plant Physiol Biochem, 2020, 154: 396-408.
[27]	Luan YT, Tang YH, Wang X, et al. Tree peony R2R3-MYB transcription factor PsMYB30 promotes petal blotch formation by activating the transcription of the anthocyanin synthase gene[J]. Plant Cell Physiol, 2022, 63(8): 1101-1116. doi: 10.1093/pcp/pcac085 pmid: 35713501
[28]	Zhang C, Wang WN, Wang YJ, et al. Anthocyanin biosynthesis and accumulation in developing flowers of tree peony(Paeonia suffruticosa)‘Luoyang Hong’[J]. Postharvest Biol Technol, 2014, 97: 11-22.
[29]	吴楠. 卵叶牡丹叶片花色苷合成相关bHLH筛选、克隆与功能分析[D]. 杨凌: 西北农林科技大学, 2021.
	Wu N. Screening, cloning and functional analysis of bHLH related to anthocyanin synthesis of leaf in Paeonia qiui[D]. Yangling: Northwest A & F University, 2021.
[30]	朱璐璐, 周波. bHLH蛋白在植物发育及非生物胁迫中的调控[J]. 分子植物育种, 2022, 20(20): 6750-6760.
	Zhu LL, Zhou B. Regulation of bHLH protein in plant development and abiotic stress[J]. Mol Plant Breed, 2022, 20(20): 6750-6760.
[31]	刘晓月, 王文生, 傅彬英. 植物bHLH转录因子家族的功能研究进展[J]. 生物技术进展, 2011, 1(6): 391-397. doi: 10.3969/j.issn.2095-2341.2011.06.02
	Liu XY, Wang WS, Fu BY. Research progress of plant bHLH transcription factor family[J]. Curr Biotechnol, 2011, 1(6): 391-397.
[32]	Hong GJ, Xue XY, Mao YB, et al. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression[J]. Plant Cell, 2012, 24(6): 2635-2648.
[33]	Liang YF, Ma F, Li BY, et al. A bHLH transcription factor, SlbHLH96, promotes drought tolerance in tomato[J]. Hortic Res, 2022, 9: uhac198.
[34]	Wang S, Zhang Z, Li LX, et al. Apple MdMYB306-like inhibits anthocyanin synthesis by directly interacting with MdMYB17 and MdbHLH33[J]. Plant J, 2022, 110(4): 1021-1034.
[35]	Matus JT, Poupin MJ, Cañón P, et al. Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine(Vitis vinifera L.)[J]. Plant Mol Biol, 2010, 72(6): 607-620. doi: 10.1007/s11103-010-9597-4 pmid: 20112051
[36]	Lai B, Du LN, Liu R, et al. Two LcbHLH transcription factors interacting with LcMYB1 in regulating late structural genes of anthocyanin biosynthesis in Nicotiana and Litchi chinensis during anthocyanin accumulation[J]. Front Plant Sci, 2016, 7: 166.
[37]	安红立, 何霞红, 芮蕊. 林下三七PnbHLH23基因的克隆及表达模式分析[J/OL]. 分子植物育种, 2023. http://kns.cnki.net/kcms/detail/46.1068.S.20231221.1821.010.html.
	An HL, He XH, Rui R. Cloning and expression pattern analysis of PnbHLH23 gene in Panax notoginseng under forest[J/OL]. Mol Plant Breed, 2023. http://kns.cnki.net/kcms/detail/46.1068.S.20231221.1821.010.html.
[38]	Seo JS, Joo J, Kim MJ, et al. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice[J]. Plant J, 2011, 65(6): 907-921.
[39]	Liu ZJ, Wu XQ, Wang EH, et al. PHR1 positively regulates phosphate starvation-induced anthocyanin accumulation through direct upregulation of genes F3'H and LDOX in Arabidopsis[J]. Planta, 2022, 256(2): 42.
[40]	Zhu BJ, Wang Q, Wang JH, et al. DFR and PAL gene transcription and their correlation with anthocyanin accumulation in Rhodomyrtus tomentosa(Aiton.)Hassk[J]. Turk J Biochem, 2019, 44(3): 289-298.
[41]	Li P, Chen X, Sun F, et al. Tobacco TTG2 and ARF8 function concomitantly to control flower colouring by regulating anthocyanin synthesis genes[J]. Plant Biol, 2017, 19(4): 525-532.
[42]	Sun W, Shen H, Xu H, et al. Chalcone isomerase a key enzyme for anthocyanin biosynthesis in Ophiorrhiza japonica[J]. Front Plant Sci, 2019, 10: 865.
[43]	Li G, Michaelis DF, Huang JJ, et al. New insights into the genetic manipulation of the R2R3-MYB and CHI gene families on anthocyanin pigmentation in Petunia hybrida[J]. Plant Physiol Biochem, 2023, 203: 108000.
[44]	Mo RL, Han GM, Zhu ZX, et al. The ethylene response factor ERF5 regulates anthocyanin biosynthesis in ‘zijin’ mulberry fruits by interacting with MYBA and F3H genes[J]. Int J Mol Sci, 2022, 23(14): 7615.
[45]	Dai MJ, Kang XR, Wang YQ, et al. Functional characterization of flavanone 3-hydroxylase(F3H)and its role in anthocyanin and flavonoid biosynthesis in mulberry[J]. Molecules, 2022, 27(10): 3341.
[46]	白蓓蓓, 郑燕萍, 陈黎明, 等. 不同着色火龙果成熟过程中花青素代谢途径相关酶活分析[J/OL]. 分子植物育种, 2024. http://kns.cnki.net/kcms/detail/46.1068.S.20240222.1143.004.html.
	Bai BB, Zheng YP, Chen LM, et al. Analysis of enzyme activity related to anthocyanin metabolism pathways during the ripening process of different colored dragon fruits[J/OL]. Mol Plant Breed, 2024. http://kns.cnki.net/kcms/detail/46.1068.S.20240222.1143.004.html.
[47]	Liu HL, Su BB, Zhang H, et al. Identification and functional analysis of a flavonol synthase gene from grape hyacinth[J]. Molecules, 2019, 24(8): 1579.