Cloning and expression of the gene and promoter of <i>LsUFGT</i>1 from <i>Lycoris sprengeri</i>

YIN Suya; ZHOU Yangli; ZHAO Mengjing; GAO Yanhui

doi:10.11833/j.issn.2095-0756.20240508

Volume 42 Issue 4

Aug. 2025

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YIN Suya, ZHOU Yangli, ZHAO Mengjing, et al. Cloning and expression of the gene and promoter of LsUFGT1 from Lycoris sprengeri[J]. Journal of Zhejiang A&F University, 2025, 42(4): 714−724 doi: 10.11833/j.issn.2095-0756.20240508

Citation:

YIN Suya, ZHOU Yangli, ZHAO Mengjing, et al. Cloning and expression of the gene and promoter of LsUFGT1 from Lycoris sprengeri[J]. Journal of Zhejiang A&F University, 2025, 42(4): 714−724 doi: 10.11833/j.issn.2095-0756.20240508

Cloning and expression of the gene and promoter of LsUFGT1 from Lycoris sprengeri

DOI: 10.11833/j.issn.2095-0756.20240508

College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

Received Date: 2024-08-27
Accepted Date: 2025-03-17
Rev Recd Date: 2024-11-13

Available Online: 2025-08-01

Publish Date: 2025-08-01

Abstract

Objective Anthocyanins are important components that determine flower color in plants. Flavonoid-3-O-glycosyltransferase (UFGT) plays a role in the downstream of anthocyanins biosynthesis, which converts unstable pigments into stable anthocyanins. This study analyzed the function of LsUFGT1 gene in Lycoris sprengeri, which would provide a theoretical basis for further studying the flower coloration mechanism in L. sprengeri. Method Several molecular biology techniques such as gene cloning, reverse transcription PCR (RT-PCR), genome walking, histological staining, and dual luciferase reporter assay, were used to study the functions of LsUFGT1 and its promoter. Result (1) The cDNA sequence of LsUFGT1 was obtained with a length of 1 632 bp, with an open reading frame of 1 398 bp, encoding 465 amino acids; the homology between LsUFGT1 and NtUFGT1 in Narcissus ntazettatorium was up to 60.57%. (2) The expression of LsUFGT1 was consistent with the trend of total anthocyanin contents, and was the most highest at blooming period, whereas the difference in expression between the pink and blue petal parts was not significant. (3) The LsUFGT1 promoter with 1 184 bp length was cloned, which contained cis-acting elements such as MYB binding site, response to light, hormone and adverse stress. β-glucosidase (GUS) histological staining in Arabidopsis thaliana showed that the LsUFGT1 promoter had promoter activity. (4) Dual luciferase reporter assays revealed that LsMYB4 and LsMYB5 were able to significantly inhibit the activity of the LsUFGT1 promoter (P＜0.05). Conclusion LsMYB4 and LsMYB5 modulated anthocyanin accumulation by directly binding to the LsUFGT1 promoter in L. sprengeri. [Ch, 9 fig. 2 tab. 38 ref.]
- Lycoris sprengeri,
- flavonoid-3-O-glucosyltransferase (UFGT),
- anthocyanin,
- functional analysis

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References

[1]	TANIKAWA N, HONMA K, TATSUZAWA F. Flavonoids of the rose-pink, blue, and white flowers of Nigella damascena L. (Ranunculaceae)[J/OL]. Scientia Horticulturae, 2019, 257: 108609[2024-08-15]. DOI: 10.1016/j.scienta.2019.108609.
[2]	ZHI Yajing, WANG Wenhe, LENG Pingsheng, et al. Components and content of flower pigments in the petals of different color Lilium species [J]. Northern Horticulture, 2017(9): 62−69.
[3]	HUI Bodi. Carotenoid Chemistry and Biochemistry[M]. Beijing: China Light Industry Press, 2005.
[4]	SU Zushang. Anthocyanins and flavonoids of Vaccinium L. [J]. Pharmaceutical Crops, 2012, 3: 7−37.
[5]	PETRONI K, TONELLI C. Recent advances on the regulation of anthocyanin synthesis in reproductive organs [J]. Plant Science, 2011, 181(3): 219−229.
[6]	LI Chonghui, YIN Junmei, Genetic engineeringpProgress and breeding tactics on blue flowers[J]. Biotechnology Bulletin, 2019, 35(11): 160−168.
[7]	SUN Ye, BAO Jianzhong, LIU Chungui, et al. Progresses on genetic engineering in orchid floral color [J]. Journal of Nuclear Agricultural Sciences, 2015, 29(9): 1701−1710.
[8]	ZHANG Hengbin, HUANG Ling, HU Xianmei, et al. Cloning and functional identification of the dihydroflavonol 4-reductase genes TgDFR1 and TgDFR2 in tulips [J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2025, 51(1): 118−127.
[9]	HALL D, YUAN Xiaoxin, MURATA J, et al. Molecular cloning and biochemical characterization of the UDP-glucose: flavonoid 3-O-glucosyltransferase from concord grape (Vitis labrusca) [J]. Phytochemistry, 2012, 74: 90−99.
[10]	MONTEFIORI M, ESPLEY R V, STEVENSON D, et al. Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis) [J]. The Plant Journal, 2011, 65(1): 106−118.
[11]	GAO Ziqi, WANG Jia, TANG Yuchen, et al. Cloning and functional analysis of the gene NtUFGT in Nitraria tangutorum [J]. Acta Prataculturae Sinica, 2020, 29(5): 159−170.
[12]	YIN Jianmei, WANG Li, ZHANG Peitong, et al. Analysis of genes involved in key enzymes of anthocyanin biosynthesis in Chinese yam (Dioscorea alata) [J]. Acta Agriculturae Jiangxi, 2018, 30(7): 1−6.
[13]	TANG Mingjia, YANG Yan, WANG Yu, et al. Identification and characterization of glucosyltransferase UGT71AN1 from Myrica rubra [J]. Chinese Medicinal Biotechnology, 2019, 14(5): 385−393.
[14]	CHEN Min, LIU Jing, LIU Zhirou, et al. Cloning and functional verification of BsUFGT gene in Begonia semperflorens [J]. Journal of Agricultural Biotechnology, 2023, 31(10): 2087−2097.
[15]	LI Chunxia, QIAO Zhongquan, DENG Fuyuan, et al. Cloning and bioinformatics analysis of LiUFGT gene of Lagerstroemia indica [J]. Hunan Forestry Science & Technology, 2023, 50(1): 24−30.
[16]	NIU Tiequan, DONG Yanmei, LIU Haixia, et al. The regulations of the MYBA1 in UFGT and DFR from the grape berries [J]. Scientia Agricultura Sinica, 2018, 51(12): 2368−2377.
[17]	LAI Biao, WANG Qi, LUO Gangjun, et al. Identification of interaction region between LcMYB1 and promoters of anthocyanin biosynthetic genes LcDFR and LcUFGT1 in Litchi chinensis [J]. Chinese Journal of Tropical Crops, 2021, 42(8): 2151−2157.
[18]	KOU Mingrui, DOU Yaqi, WANG Ying, et al. Cloning, sequence analysis and interaction analysis of MiUFGT43 promoter and MiMYB1 genes in Mango fruit [J]. Molecular Plant Breeding, 2024, 22(15): 4873−4883.
[19]	KAUR R, KAPOOR N, ASLAM L, et al. Molecular characterization of PgUFGT gene and R2R3-PgMYB transcription factor involved in flavonoid biosynthesis in four tissues of wild pomegranate (Punica granatum L. )[J/OL]. Journal of Genetics, 2019, 98(4): 94[2024-08-15]. DOI: 10.1007/s12041-019-1141-y.
[20]	XU Zhenyuan, GAO Yanhui, ZHOU Fenjing, et al. Cloning and expression analysis of LsMYB4 gene in Lycoris sprengeri [J]. Acta Horticulturae Sinica, 2014, 41(11): 2281−2290.
[21]	XU Zhenyuan. Cloning and Expression Analysis of F3′H and MYB4 Homologous Genes in Lycoris sprengeri[D]. Hangzhou: Zhejiang A&F University, 2014.
[22]	HOU Shuo, GAO Yanhui, TONG Zaikang. Cloning and expression analysis of LsMYB5 gene in Lycoris sprengeri [J]. Journal of Agricultural Biotechnology, 2019, 27(12): 2164−2174.
[23]	ZHOU Yangli, HOU Shuo, ZHENG Zhengquan, et al. Study on LsMYBs gene function in Lycoris sprengeri based on VIGS gene silencing system [J]. Journal of Agricultural Biotechnology, 2020, 28(6): 974−983.
[24]	XUE Huimin, ZHOU Yangli, GAO Yanhui. Cloning and promoter function analysis of the anthocyanins synthase gene (LsANS) in Lycoris sprengeri [J]. Journal of Agricultural Biotechnology, 2022, 30(8): 1468−1479.
[25]	FAN Qingling, ZHOU Ribao, LIU Xiangdan, et al. Comparative study on RNA extraction methods in different organs of Lycoris aurea herb [J]. Journal of Anhui Agricultural Sciences, 2020, 48(18): 169−172.
[26]	JIANG Tingting, GAO Yanhui, TONG Zaikang. Selection of reference genes for quantitative real-time PCR in Lycoris [J]. Acta Horticulturae Sinica, 2015, 42(6): 1129−1138.
[27]	LABRA M, VANNINI C, GRASSI F, et al. Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip [J]. Theoretical and Applied Genetics, 2004, 109(7): 1512−1518.
[28]	GAO Jianqiang, LIANG Hua, ZHAO Jun. Progress on the floral-dip method of agribacterium-mediated plant transformation [J]. Chinese Agricultural Science Bulletin, 2010, 26(16): 22−25.
[29]	JAAKOLA L. New insights into the regulation of anthocyanin biosynthesis in fruits [J]. Trends in Plant Science, 2013, 18(9): 477−483.
[30]	YING Zhen, LI Jiyuan, YIN Hengfu, et al. Analysis of gene expressing and metabolites for synthesising anthocyanins influencing the red color of leaves of Camellia cultivar [J]. Forest Research, 2017, 30(6): 1034−1040.
[31]	NIE Zhuxin, LÜ Jikun, MA Ziqiao, et al. Cloning and expression analysis of 3-O-flavonoid glucosyltransferase LrUFGT1 from Lycium ruthenicum Murr. [J]. Molecular Plant Breeding, 2023-12-27[2024-08-15]. https://link.cnki.net/urlid/46.1068.S.20231227.1005.006.
[32]	CHEN Xinna, YU Xin, ZHANG Liying, et al. Cloning and expression analysis of chrysanthemum CmUFGT gene and its promoter [J/OL]. Molecular Plant Breeding, 2022-09-28 [2024-08-15]. https://kns.cnki.net/kcms/detail/46.1068.S.20220927.1231.004.html.
[33]	CHEN W H, HSU C Y, CHENG Haoyun, et al. Downregulation of putative UDP-glucose: flavonoid 3-O-glucosyltransferase gene alters flower coloring in Phalaenopsis [J]. Plant Cell Reports, 2011, 30(6): 1007−1017.
[34]	WANG Yiping, LIU Yongqiang, ZHANG Lianxi, et al. A novel R2R3-MYB transcription factor FaMYB10-like promotes light-induced anthocyanin accumulation in cultivated strawberry[J/OL]. International Journal of Molecular Sciences, 2023, 24(23): 16561[2024-08-15]. DOI: 10.3390/ijms242316561.
[35]	YANG Xiaona, ZHAO Changling, LI Yun, et al. Research advances in the methods of cloning and function-analyzing of promoters [J]. Journal of Yunnan Agricultural University (Natural Science), 2010, 25(2): 283−290.
[36]	LI Qinqin, DONG Shanrong, LUO Jianrang, et al. Cloning and functional analysis of PqDFR and PqANS genes and its promoters from Paeonia qiui [J]. Acta Horticulturae Sinica, 2024, 51(6): 1256−1272.
[37]	JIANG Yu, WANG Xueting, WANG Chunnan, et al. Cloning and expression analysis of VvAGL12 gene promoter in Vitis vinifera [J]. Journal of Shaanxi Normal University (Natural Science Edition), 2024, 52(5): 31−41.
[38]	ZHOU Lin, YUAN Meng, QI Yu, et al. Promoter functional analysis of the key gene PsDFR involved in Paeonia suffruticosa anthocyanin biosynthesis [J]. Forest Research, 2024, 37(2): 16−26.

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植物丰富的花色由植物组织和器官的特定细胞色素所呈现，随类黄酮、类胡萝卜素和甜菜色素等次生代谢产物含量变化而变化^[1−2]。类黄酮包括原花青素、花色素、异黄酮、黄酮醇、黄烷酮五大类^[3−4]，其中花色素生物合成途径^[5]已被研究得较为清晰。花色苷生物合成是类黄酮途径的一个分支，以丙二酰辅酶A (malonyl-CoA)和苯丙氨酸(phenylalanine)为底物，经查尔酮合成酶(chalcone synthase, CHS)、查尔酮异构酶(chalcone isomerase, CHI)、黄烷酮3-羟化酶(flavanone 3-hydroxylase, F3H)、黄烷酮3′-羟化酶(flavanone 3′-hydroxylase, F3′H)、类黄酮-3′, 5′-羟化酶(flavonoid-3′, 5′-hydroxylase, F3′5′H)、二氢黄酮醇还原酶(dihydroflavonol 4-reductase, DFR)等合成酶及不同底物分别形成无色天竺葵色素、矢车菊色素和飞燕草色素；经花青素合成酶(anthocyanidin synthase, ANS)催化生成有色花色素，再经花色素酰基转移酶、糖基转移酶和甲基转移酶的转化形成不同颜色的矢车菊色素、天竺葵色素以及飞燕草色素^[6−8]，最后经类黄酮糖基转移酶(flavonoid-3-O-glucosyltransferase，UFGT)将不稳定的色素转变为稳定的花色苷，由谷胱甘肽S-转移酶(glutathione S-transferase, GST)转运至液泡中储存。

UFGT是类黄酮生物合成下游途径最后一个关键酶，可催化UDP-葡萄糖上的葡萄糖基转移至花色素C环C₃羟基上，主要参与花色素支路和黄酮醇支路的糖基化过程^[9]。近年来，有关植物UFGT的表达与花色苷积累的报道较多，UFGT可催化花色苷的形成，正调控花色苷积累。猕猴桃Actinidia chinensis糖基转移酶基因F3G77和F3GG77参与花色苷的合成^[10]；唐古特白刺Nitraria tangutorum NtUFGT可将不稳定的花色素转变为稳定的花色苷，积累花色素抵抗逆境胁迫^[11]；UFGT是调控紫山药Dioscorea alata颜色的关键酶基因^[12]；杨梅Morella rubra UGT71AN1可利用酶促或全细胞生物催化进行糖基化结构修饰^[13]；四季秋海棠Begonia semperflorens BsUFGT能够促进花色苷的合成和积累^[14]；紫薇Lagerstroemia indica LiUFGT对紫薇花色基因具有直接调控的作用，在红色花瓣中表达量最高，在白色中最低^[15]。大量研究表明UFGT可能与其他基因共同调控花色苷的积累。葡萄Vitis vinifera VvMYBA1通过激活VvUFGT启动子活性，调控花色苷合成积累^[16]；荔枝Litchi chinensis LcMYB1直接结合LcDFR和LcUFGT1启动子激活基因表达，进而调控花色苷的生物合成^[17]；芒果Mangifera indica MiMYB1可直接结合MiUFGT43启动子，激活MiUFGT43表达，从而影响采后芒果花青苷的合成^[18]；石榴Punica granatum 转录因子R2R3-PgMYB通过调控PgUFGT表达来调节花色苷合成^[19]。

换锦花Lycoris sprengeri为石蒜科Amaryllidaceae石蒜属Lycoris多年生球根花卉，是良好的花园地面覆盖植物，其花色为特殊的红蓝复合色，是研究多色花形成的较好材料。近年来，学者们从花色苷形成相关的关键酶基因LsF3′H和LsANS及R2R3-MYB类调控基因的克隆和表达深入探究换锦花花色的形成机制^[20−24]，基于病毒介导的基因沉默(virus-induced gene silencing，VIGS)的LsMYB4和LsMYB5基因沉默后可上调LsUFGT表达^[23]，这为换锦花花色育种和新品种选育奠定了一定的理论基础。为进一步研究和解析LsUFGT1对换锦花花瓣呈色的作用，本研究采用逆转录PCR (reverse transcription-PCR, RT-PCR)、RACE (rapid amplification of cDNA ends)和染色体步移 (genome walking)技术分离换锦花LsUFGT1基因及启动子序列，并利用双荧光素酶报告实验分析LsMYB4和LsMYB5转录因子对LsUFGT1基因启动子的激活功能，以期为进一步解析换锦花花瓣呈色的分子机制提供基础，为石蒜属植物基因工程花色改良提供技术指导。

1. 材料与方法

1.1. 材料

于2021年8—9月，于浙江农林大学石蒜属植物种质资源圃，采集盛花期换锦花新鲜花瓣(粉色和蓝色部分)和花发育不同时期(小花苞、大花苞、盛花期)花瓣，液氮速冻，于−80 ℃冰箱保存备用；拟南芥Arabidopsis thaliana (抽薹7 d)用于β-葡萄糖苷酶(GUS)染色。

1.2. 方法

1.2.1. 总RNA的提取和LsUFGT1基因的克隆

使用改良十六烷基三甲基溴化铵(CTAB)+Trizol法^[25]提取换锦花花瓣总RNA，用质量浓度为1.0%的琼脂糖凝胶电泳检测其质量及完整性。参照SMART^TM RACE cDNA Amplification Kit (Takara)说明书合成5′-RACE -Ready cDNA第1链。根据换锦花花瓣转录组的LsUFGT1 (GenBank: MT664242.1)序列信息设计LsUFGT1-5′ GSP1和LsUFGT1-5′ GSP2引物(表1)，PCR扩增LsUFGT1序列5′端的未知序列，用质量分数为1.0%的琼脂糖凝胶电泳检测PCR扩增产物，回收目的片段并连接到pMD-19T载体(Takara)上，转化大肠埃希菌Escherichia coli DH5α感受态细胞，挑取阳性克隆送杭州擎科生物技术有限公司测序。

引物名称	引物序列(5′→3′)	引物用途
LsUFGT1-5′GSP1	AAGACCATAGAAAGAGGACCCCGC	LsUFGT1基因5′端的未知序列扩增
LsUFGT1-5′GSP2	CGCTCGCTTCCAATCCCTCTGCC	LsUFGT1基因5′端的未知序列扩增
LsUFGT1全长-F	TAGCCTGTGCCACTTCCCTT	LsUFGT1基因扩增
LsUFGT1全长-R	GCTGGAGAGTTCGCAATACA	LsUFGT1基因扩增
Q-LsUFGT1-F	GGTGGTGAAGGATGAGGAAGGTAGG	实时荧光定量PCR(RT-qPCR)
Q-LsUFGT1-R	GTTGAACCGCTCGAACCGCAATC
Q-LsACTIN-F	CAGACTTTCAATGTGCCCG
Q-LsACTIN-R	CACCATCACCAGAATCCAGC
LsUFGT1-R-SP1	CGCTGATTAGACACGTGATCCTCAA	启动子克隆
LsUFGT1-R-SP2	CTCGAAGATCCTGAGGTTGGGT
LsUFGT1-R-SP3	TGGCGGTGGTGGCAATCTAATC
LsUFGT1-2-R-SP1	GGGAGGGGAAATATCCAAAATGGAG
LsUFGT1-2-R-SP2	TTCTGATTGGGCCACGTCGTCGTTC
LsUFGT1-2-R-SP3	GGTGAAGCTGCTGTTCGGTGACGTGT
LsUFGT1-pro-F	AAATAAAGGAAGGAGGGAGTGG	LsUFGT1启动子扩增
LsUFGT1-pro-R	GGCAATCTAATCCCATGGT	LsUFGT1启动子扩增
pBI121-LsUFGT1-pro::GUS-F	TGATTACGCCAAGCTTAAATAAAGGAAGGAGGGAGTGG	pBI121载体构建
pBI121-LsUFGT1-pro::GUS-R	CCGGGGATCCTCTAGATGGCAATCTAATCCCATGGT	pBI121载体构建
pGreenⅡ 62-SK-LsMYB4-F	TGGCGGCCGCTCTAGATGGGTAGGTCTCCATGCTG	双荧光素酶报告实验
pGreenⅡ 62-SK-LsMYB4-R	CAGCGTACCGAATTGTCAAACACTACTTCTAAAGTCCAAT
pGreenⅡ 62-SK-LsMYB5-F	TGGCGGCCGCTCTAGATGGGTAGATCTCCATGTTG
pGreenⅡ 62-SK-LsMYB5-R	CAGCGTACCGAATTGCTATCTAATTACATGTGGATTGAT
pGreenⅡ 0800-LsUFGT1-pro-LUC-F	CGGTATCGATAAGCTGTCGAGTGATATGAAAAGTACTCC
pGreenⅡ 0800-LsUFGT1-pro-LUC-R	TAGAACTAGTGGATCGGTGGATGCGCAGGTTAG

Table 1. Sequences of the primers

利用Primer Premier 5.0设计拼接的LsUFGT1未知序列扩增引物(表1)，以cDNA第1链为模板PCR扩增验证LsUFGT1序列是否正确。通过在线软件ORF Finder (https://www.ncbi.nlm.nih.gov/gorf/gorf.htmL)查找LsUFGT1 cDNA序列开放阅读框(open reading frame, ORF)；利用美国国家生物技术信息中心(NCBI)数据库(https://blast.ncbi.nlm.nih.gov/)检索近缘物种的UFGT序列信息；利用ProtParam (https://web.expasy.org/protparam/)分析蛋白质理化性质；利用NPSA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.htmL)预测蛋白质二级结构；通过DNAMAN软件进行氨基酸序列比对；使用MEGA7.0构建系统进化树。

1.2.2. 换锦花LsUFGT1表达分析

采用Prime Script^RT reagent Kit Perfect Real Time (TaKaRa)试剂盒合成cDNA第1链，利用Primer 5.0设计实时荧光定量PCR (RT-qPCR)引物(表1)，使用RT-qPCR技术检测LsUFGT1在换锦花盛花期花瓣和不同花发育时期花瓣表达模式，以LsActin为内参基因^[26]，用2^−ΔΔCt计算各基因的相对表达量。

1.2.3. 花色苷提取和测定

将采集的换锦花花瓣避光冻干7 d后研磨，精密称量换锦花花瓣干粉0.040 0 g，加入2 mL 体积分数为1%的CH₄-HCl溶液，充分摇匀，密塞，20 ℃超声波提取30 min，12 000 r·m⁻¹ 离心15 min，收集上清液，0.45 μm微孔滤膜过滤，滤液于−20 ℃冰箱保存待用，每个样本重复3次。总花色苷测定方法采用UltimateR LP-C18 column (4.6 mm×250.0 mm，5 µm)^[24]，以矢车菊素-3-O-葡萄糖苷为标准品，重复3次，SPSS 20.0处理数据和分析差异显著性，并利用GraphPad Prism 8.0绘图。

1.2.4. LsUFGT1启动子克隆和序列分析

采用染色体步移法分离LsUFGT1启动子序列。采用CTAB法提取换锦花基因组DNA，以质量浓度为1.0%的琼脂糖凝胶电泳检测DNA的浓度和质量。利用Primer Premier 5.0设计LsUFGT1基因启动子克隆和扩增引物(表1)，以换锦花基因组DNA为模板，采用Genome Walking Kit (Takara)扩增LsUFGT1启动子序列，以质量浓度为1%的琼脂糖凝胶电泳检测并回收目的片段，连接到pMD-19T载体，转化大肠埃希菌DH5α感受态细胞，挑取阳性克隆检测并测序。通过伯克利果蝇基因组项目(BDGP) (https://www.fruit-fly.org/seq_tools/promoter.html)预测启动子转录起始位点，Plant CARE (https://bioinfor-matics.psb.ugent.be/webtools/plantcare/htm L/)分析启动子顺式作用元件。

1.2.5. pBI121-LsUFGT1-pro::GUS载体构建及农杆菌侵染拟南芥

采用同源重组法构建pBI121-LsUFGT1-pro::GUS表达载体，将重组质粒转入农杆菌 GV3101感受态细胞，采用整染法^[27−28]瞬时侵染拟南芥植株48 h后，洗净拟南芥植株表面菌液，用GUS染色液37 ℃染色过夜，将植株于脱色液(无水乙醇和冰乙酸体积比为3∶1)中脱色2~4 h后，置于体积分数为70%的乙醇保存，观察LsUFGT1启动子在拟南芥植株的表达并拍照。

1.2.6. 双荧光素酶报告实验

采用无缝克隆技术构建pGreenⅡ 0800-LsUFGT1-pro-LUC 和pGreenⅡ 62-SK-LsMYB4、pGreenⅡ 62-SK-LsMYB5表达载体，使用冻融法将pGreen Ⅱ 62-SK、pGreenⅡ 0800-LUC 空载及重组质粒转入农杆菌Agrobacterium tumefaciens GV3101感受态细胞，注射烟草叶片暗培养 24 h后，正常光照培养 48 h 后用6 mm 打孔器取样；液氮研磨，根据Dual-Luciferase® Reporter Assay System试剂盒的说明书用GloMax 20/20 2020化学发光检测仪(Promega)检测萤火虫荧光素酶(Luc)和海肾荧光素酶(Ren)活性，并计算Luc/Ren比值。

3. 讨论

花青素是一种重要的植物色素，作为类黄酮途径的末端产物，由内质网膜系列酶催化合成，其中，UFGT在花青素生物合成途径中催化植物细胞中不稳定的花青素转化成稳定的花色苷^[29]。已有研究发现：红叶山茶‘金华美女’ Camellia japonica‘Jinhua Meinü’叶片生长过程中，CjUFGT持续催化花青素合成，花青素苷大量积累，导致叶片颜色变深^[30]；四季秋海棠BsUFGT在烟草Nicotiana tabacum ‘NC89’中过表达能够提高花瓣的颜色和花色苷矢车菊素-3-O-芸香糖苷(cyanidin-3-O- rutinoside, Cy-3R)含量^[14]；黑果枸杞Lycium ruthenicum LrUFGT1的表达具有组织特异性，且主要在花和紫果中表达，表达量随花朵的发育先增加后减少，表明 LrUFGT1 可能参与了黑果枸杞花朵的呈色，影响黑果枸杞果实和花瓣的着色^[31]；菊花Chrysanthemum morifolium CmUFGT表达与彩斑突变体CQ17-mu植株花瓣中紫色条斑呈色有关^[32]；特异性敲除蝴蝶兰Phalaenopsis equestris PeUFGT3，其花朵中花色苷含量显著降低，同时花朵颜色变浅^[33]。本研究发现LsUFGT1与换锦花花瓣花色苷含量呈正相关，换锦花不同发育期LsUFGT1表达差异显著，呈递减趋势。

花色苷生物合成还受转录因子MYB、bHLH和WD40单独或形成MBW复合体协同调控，转录因子往往直接结合到花色苷形成基因启动子调控花色苷积累^[34]。启动子是基因转录调控的核心组件，位于基因上游区域，专门负责激活RNA聚合酶，从而精准调控基因的表达^[35]。常通过瞬时表达法将启动子表达载体导入植物受体，短时间内通过检测报告基因在受体植物中的表达位置和活性，从而研究启动子片段功能。李琴琴等^[36]利用GUS组织化学染色发现瞬时转化烟草中卵叶牡丹Paeonia qiui proPqDFR和proPqANS均具有启动子活性；姜宁等^[37]研究发现proVvAGL12启动子片段在拟南芥中具有启动活性，能够激活GUS在转基因拟南芥植株叶片、茎段、花器官、根和果荚部位的表达；周琳等^[38]经GUS组织化学染色和酶活性检测，发现牡丹PsDFR启动子缺失片段都具有启动子活性。本研究克隆的LsUFGT1pro启动子序列能够启动GUS在拟南芥植株的表达，说明LsUFGT1基因起始密码子上游1 184 bp序列具有启动下游基因的活性。此外，前期研究发现换锦花LsMYB4和LsMYB5基因沉默后会导致换锦花花瓣LsUFGT1的表达上调^[23]，本研究双荧光素酶报告实验发现LsMYB4和LsMYB5能够显著抑制LsUFGT1启动子活性，这与荔枝LcUFGT1的启动子受到LcMYB1的调控而影响花色苷的积累相一致^[17]。

4. 结论

本研究克隆到长1 632 bp的LsUFGT1基因cDNA和1 184 bp启动子序列，LsUFGT1基因在换锦花花发育和不同花色花瓣的基因表达与总花色苷含量的变化相一致，LsMYB4和LsMYB5转录因子对LsUFGT1基因的表达起着负调控的作用。本研究结果为进一步研究换锦花花色形成的机制提供了理论依据，对深入研究换锦花花色相关基因调控模式具有重要的基础意义。

Reference (38)

[1]	TANIKAWA N, HONMA K, TATSUZAWA F. Flavonoids of the rose-pink, blue, and white flowers of Nigella damascena L. (Ranunculaceae)[J/OL]. Scientia Horticulturae, 2019, 257: 108609[2024-08-15]. DOI: 10.1016/j.scienta.2019.108609.
[2]	智雅静, 王文和, 冷平生, 等. 不同花色百合色素种类和含量分析[J]. 北方园艺, 2017(9): 62−69.	ZHI Yajing, WANG Wenhe, LENG Pingsheng, et al. Components and content of flower pigments in the petals of different color Lilium species [J]. Northern Horticulture, 2017(9): 62−69.
[3]	惠伯棣. 类胡萝卜素化学及生物化学[M]. 北京: 中国轻工业出版社, 2005.	HUI Bodi. Carotenoid Chemistry and Biochemistry[M]. Beijing: China Light Industry Press, 2005.
[4]	SU Zushang. Anthocyanins and flavonoids of Vaccinium L. [J]. Pharmaceutical Crops, 2012, 3: 7−37.
[5]	PETRONI K, TONELLI C. Recent advances on the regulation of anthocyanin synthesis in reproductive organs [J]. Plant Science, 2011, 181(3): 219−229.
[6]	李崇晖, 尹俊梅. 蓝色花形成的基因工程进展与育种策略[J]. 生物技术通报, 2019, 35(11): 160−168.	LI Chonghui, YIN Junmei, Genetic engineeringpProgress and breeding tactics on blue flowers[J]. Biotechnology Bulletin, 2019, 35(11): 160−168.
[7]	孙叶, 包建忠, 刘春贵, 等. 兰花花色基因工程研究进展[J]. 核农学报, 2015, 29(9): 1701−1710.	SUN Ye, BAO Jianzhong, LIU Chungui, et al. Progresses on genetic engineering in orchid floral color [J]. Journal of Nuclear Agricultural Sciences, 2015, 29(9): 1701−1710.
[8]	张恒滨, 黄玲, 胡先梅, 等. 郁金香二氢黄酮醇4-还原酶基因TgDFR1和TgDFR2的克隆与功能鉴定[J]. 浙江大学学报(农业与生命科学版), 2025, 51(1): 118−127.	ZHANG Hengbin, HUANG Ling, HU Xianmei, et al. Cloning and functional identification of the dihydroflavonol 4-reductase genes TgDFR1 and TgDFR2 in tulips [J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2025, 51(1): 118−127.
[9]	HALL D, YUAN Xiaoxin, MURATA J, et al. Molecular cloning and biochemical characterization of the UDP-glucose: flavonoid 3-O-glucosyltransferase from concord grape (Vitis labrusca) [J]. Phytochemistry, 2012, 74: 90−99.
[10]	MONTEFIORI M, ESPLEY R V, STEVENSON D, et al. Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis) [J]. The Plant Journal, 2011, 65(1): 106−118.
[11]	高子奇, 王佳, 汤宇晨, 等. 唐古特白刺类黄酮-3-O-葡萄糖基转移酶基因(NtUFGT)的克隆与功能分析[J]. 草业学报, 2020, 29(5): 159−170.	GAO Ziqi, WANG Jia, TANG Yuchen, et al. Cloning and functional analysis of the gene NtUFGT in Nitraria tangutorum [J]. Acta Prataculturae Sinica, 2020, 29(5): 159−170.
[12]	殷剑美, 王立, 张培通, 等. 山药花青素合成关键酶基因的解析[J]. 江西农业学报, 2018, 30(7): 1−6.	YIN Jianmei, WANG Li, ZHANG Peitong, et al. Analysis of genes involved in key enzymes of anthocyanin biosynthesis in Chinese yam (Dioscorea alata) [J]. Acta Agriculturae Jiangxi, 2018, 30(7): 1−6.
[13]	唐铭袈, 杨燕, 王宇, 等. 杨梅糖基转移酶UGT71AN1的克隆与功能鉴定[J]. 中国医药生物技术, 2019, 14(5): 385−393.	TANG Mingjia, YANG Yan, WANG Yu, et al. Identification and characterization of glucosyltransferase UGT71AN1 from Myrica rubra [J]. Chinese Medicinal Biotechnology, 2019, 14(5): 385−393.
[14]	陈敏, 刘静, 刘至柔, 等. 四季秋海棠BsUFGT基因克隆及功能验证[J]. 农业生物技术学报, 2023, 31(10): 2087−2097.	CHEN Min, LIU Jing, LIU Zhirou, et al. Cloning and functional verification of BsUFGT gene in Begonia semperflorens [J]. Journal of Agricultural Biotechnology, 2023, 31(10): 2087−2097.
[15]	李春霞, 乔中全, 邓涪元, 等. 紫薇LiUFGT基因的克隆与生物信息学分析[J]. 湖南林业科技, 2023, 50(1): 24−30.	LI Chunxia, QIAO Zhongquan, DENG Fuyuan, et al. Cloning and bioinformatics analysis of LiUFGT gene of Lagerstroemia indica [J]. Hunan Forestry Science & Technology, 2023, 50(1): 24−30.
[16]	牛铁泉, 董燕梅, 刘海霞, 等. 葡萄果实MYBA1与UFGT、DFR的作用机制[J]. 中国农业科学, 2018, 51(12): 2368−2377.	NIU Tiequan, DONG Yanmei, LIU Haixia, et al. The regulations of the MYBA1 in UFGT and DFR from the grape berries [J]. Scientia Agricultura Sinica, 2018, 51(12): 2368−2377.
[17]	赖彪, 王琪, 罗刚军, 等. LcMYB1激活荔枝花色苷生物合成关键基因LcDFR和LcUFGT1的启动子区域分析[J]. 热带作物学报, 2021, 42(8): 2151−2157.	LAI Biao, WANG Qi, LUO Gangjun, et al. Identification of interaction region between LcMYB1 and promoters of anthocyanin biosynthetic genes LcDFR and LcUFGT1 in Litchi chinensis [J]. Chinese Journal of Tropical Crops, 2021, 42(8): 2151−2157.
[18]	寇明睿, 窦雅琪, 王莹, 等. 芒果MiUFGT43启动子和MiMYB1基因的克隆、序列及互作分析[J]. 分子植物育种, 2024, 22(15): 4873−4883.	KOU Mingrui, DOU Yaqi, WANG Ying, et al. Cloning, sequence analysis and interaction analysis of MiUFGT43 promoter and MiMYB1 genes in Mango fruit [J]. Molecular Plant Breeding, 2024, 22(15): 4873−4883.
[19]	KAUR R, KAPOOR N, ASLAM L, et al. Molecular characterization of PgUFGT gene and R2R3-PgMYB transcription factor involved in flavonoid biosynthesis in four tissues of wild pomegranate (Punica granatum L. )[J/OL]. Journal of Genetics, 2019, 98(4): 94[2024-08-15]. DOI: 10.1007/s12041-019-1141-y.
[20]	许振渊, 高燕会, 周芬静, 等. 换锦花LsMYB4基因的克隆与表达分析[J]. 园艺学报, 2014, 41(11): 2281−2290.	XU Zhenyuan, GAO Yanhui, ZHOU Fenjing, et al. Cloning and expression analysis of LsMYB4 gene in Lycoris sprengeri [J]. Acta Horticulturae Sinica, 2014, 41(11): 2281−2290.
[21]	许振渊. 换锦花F3′H和MYB4同源基因的克隆与表达分析[D]. 杭州: 浙江农林大学, 2014.	XU Zhenyuan. Cloning and Expression Analysis of F3′H and MYB4 Homologous Genes in Lycoris sprengeri[D]. Hangzhou: Zhejiang A&F University, 2014.
[22]	侯朔, 高燕会, 童再康. 换锦花LsMYB5基因的克隆与表达分析[J]. 农业生物技术学报, 2019, 27(12): 2164−2174.	HOU Shuo, GAO Yanhui, TONG Zaikang. Cloning and expression analysis of LsMYB5 gene in Lycoris sprengeri [J]. Journal of Agricultural Biotechnology, 2019, 27(12): 2164−2174.
[23]	周洋丽, 侯朔, 郑正权, 等. 基于VIGS基因沉默体系的换锦花LsMYBs基因功能研究[J]. 农业生物技术学报, 2020, 28(6): 974−983.	ZHOU Yangli, HOU Shuo, ZHENG Zhengquan, et al. Study on LsMYBs gene function in Lycoris sprengeri based on VIGS gene silencing system [J]. Journal of Agricultural Biotechnology, 2020, 28(6): 974−983.
[24]	薛惠敏, 周洋丽, 高燕会. 换锦花花青素合成酶基因(LsANS)的克隆及启动子功能分析[J]. 农业生物技术学报, 2022, 30(8): 1468−1479.	XUE Huimin, ZHOU Yangli, GAO Yanhui. Cloning and promoter function analysis of the anthocyanins synthase gene (LsANS) in Lycoris sprengeri [J]. Journal of Agricultural Biotechnology, 2022, 30(8): 1468−1479.
[25]	樊青玲, 周日宝, 刘湘丹, 等. 忽地笑不同器官RNA提取方法比较研究[J]. 安徽农业科学, 2020, 48(18): 169−172.	FAN Qingling, ZHOU Ribao, LIU Xiangdan, et al. Comparative study on RNA extraction methods in different organs of Lycoris aurea herb [J]. Journal of Anhui Agricultural Sciences, 2020, 48(18): 169−172.
[26]	蒋婷婷, 高燕会, 童再康. 石蒜属植物实时荧光定量PCR内参基因的选择[J]. 园艺学报, 2015, 42(6): 1129−1138.	JIANG Tingting, GAO Yanhui, TONG Zaikang. Selection of reference genes for quantitative real-time PCR in Lycoris [J]. Acta Horticulturae Sinica, 2015, 42(6): 1129−1138.
[27]	LABRA M, VANNINI C, GRASSI F, et al. Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip [J]. Theoretical and Applied Genetics, 2004, 109(7): 1512−1518.
[28]	高建强, 梁华, 赵军. 植物遗传转化农杆菌浸花法研究进展[J]. 中国农学通报, 2010, 26(16): 22−25.	GAO Jianqiang, LIANG Hua, ZHAO Jun. Progress on the floral-dip method of agribacterium-mediated plant transformation [J]. Chinese Agricultural Science Bulletin, 2010, 26(16): 22−25.
[29]	JAAKOLA L. New insights into the regulation of anthocyanin biosynthesis in fruits [J]. Trends in Plant Science, 2013, 18(9): 477−483.
[30]	应震, 李纪元, 殷恒福, 等. 红叶山茶品种花青素苷相关基因表达水平及代谢产物分析[J]. 林业科学研究, 2017, 30(6): 1034−1040.	YING Zhen, LI Jiyuan, YIN Hengfu, et al. Analysis of gene expressing and metabolites for synthesising anthocyanins influencing the red color of leaves of Camellia cultivar [J]. Forest Research, 2017, 30(6): 1034−1040.
[31]	聂祝欣, 吕冀堃, 马子乔, 等. 黑果枸杞3-O-类黄酮糖基转移酶LrUFGT1的克隆与表达分析[J/OL]. 分子植物育种, 2023-12-27[2024-08-15]. https://link.cnki.net/urlid/46.1068.S.20231227.1005.006.	NIE Zhuxin, LÜ Jikun, MA Ziqiao, et al. Cloning and expression analysis of 3-O-flavonoid glucosyltransferase LrUFGT1 from Lycium ruthenicum Murr. [J]. Molecular Plant Breeding, 2023-12-27[2024-08-15]. https://link.cnki.net/urlid/46.1068.S.20231227.1005.006.
[32]	陈新娜, 喻锌, 张利英, 等. 菊花CmUFGT基因及其启动子的克隆与表达分析[J/OL]. 分子植物育种, 2022-09-28[2024-08-15]. https://kns.cnki.net/kcms/detail/46.1068.S.20220927.1231.004.html.	CHEN Xinna, YU Xin, ZHANG Liying, et al. Cloning and expression analysis of chrysanthemum CmUFGT gene and its promoter [J/OL]. Molecular Plant Breeding, 2022-09-28 [2024-08-15]. https://kns.cnki.net/kcms/detail/46.1068.S.20220927.1231.004.html.
[33]	CHEN W H, HSU C Y, CHENG Haoyun, et al. Downregulation of putative UDP-glucose: flavonoid 3-O-glucosyltransferase gene alters flower coloring in Phalaenopsis [J]. Plant Cell Reports, 2011, 30(6): 1007−1017.
[34]	WANG Yiping, LIU Yongqiang, ZHANG Lianxi, et al. A novel R2R3-MYB transcription factor FaMYB10-like promotes light-induced anthocyanin accumulation in cultivated strawberry[J/OL]. International Journal of Molecular Sciences, 2023, 24(23): 16561[2024-08-15]. DOI: 10.3390/ijms242316561.
[35]	杨晓娜, 赵昶灵, 李云, 等. 启动子序列克隆和功能分析方法的研究进展[J]. 云南农业大学学报(自然科学版), 2010, 25(2): 283−290.	YANG Xiaona, ZHAO Changling, LI Yun, et al. Research advances in the methods of cloning and function-analyzing of promoters [J]. Journal of Yunnan Agricultural University (Natural Science), 2010, 25(2): 283−290.
[36]	李琴琴, 董山榕, 罗建让, 等. 卵叶牡丹PqDFR和PqANS及启动子克隆与功能分析[J]. 园艺学报, 2024, 51(6): 1256−1272.	LI Qinqin, DONG Shanrong, LUO Jianrang, et al. Cloning and functional analysis of PqDFR and PqANS genes and its promoters from Paeonia qiui [J]. Acta Horticulturae Sinica, 2024, 51(6): 1256−1272.
[37]	姜宁, 王雪婷, 王春楠, 等. 葡萄VvAGL12基因启动子克隆及表达活性分析[J]. 陕西师范大学学报(自然科学版), 2024, 52(5): 31−41.	JIANG Yu, WANG Xueting, WANG Chunnan, et al. Cloning and expression analysis of VvAGL12 gene promoter in Vitis vinifera [J]. Journal of Shaanxi Normal University (Natural Science Edition), 2024, 52(5): 31−41.
[38]	周琳, 袁梦, 齐宇, 等. 牡丹花青素苷合成关键基因PsDFR启动子活性分析[J]. 林业科学研究, 2024, 37(2): 16−26.	ZHOU Lin, YUAN Meng, QI Yu, et al. Promoter functional analysis of the key gene PsDFR involved in Paeonia suffruticosa anthocyanin biosynthesis [J]. Forest Research, 2024, 37(2): 16−26.

元件	植物	基序	数量	功能
ABRE	拟南芥	ACGTG	1	脱落酸响应元件
ARE	玉米	AAACCA	1	厌氧诱导所必需顺式作用元件
Box-4	皱叶欧芹	ATTAAT	2	参与光响应的保守DNA部分
CAAT-box	豌豆、拟南芥	CAAAT、CCAAT	3	启动子和增强子顺式作用元件
CAT-box	拟南芥	GCCACT	1	分生组织表达相关的顺式作用调控元件
CCAAT-box	大麦	CAACGG	1	大麦MYB结合位点
CGTCA-motif	大麦	CGTCA	1	茉莉酸甲酯响应顺式作用元件
G-box	玉米	CACGTC	1	光响应顺式作用元件
GA-motif	拟南芥	ATAGATAA	1	部分光响应元件
GC-motif	玉米	CCCCCG	2	缺氧特异诱导的增强子样元件
GT1-motif	燕麦	GGTTAAT	1	光响应元件
LTR	大麦	CCGAAA	2	低温反应的顺式作用元件
MBS	拟南芥、烟草	CAACAG	1	参与干旱调控MYB结合位点
TATA-box	拟南芥、水稻、欧洲油菜	TACAAAA、ATTATA、TATA、ATATAT	5	转录起始位点上游-30的启动子核心元件
TCA-element	烟草	CCATCTTTTT	1	水杨酸反应的顺式作用元件
TCT-motif	拟南芥	TCTTAC	1	部分光响应元件
TGA-element	甘蓝	AACGAC	1	生长素响应元件
说明：拟南芥. Arabidopsis thaliana，玉米. Zea mays，‌豌豆. Pisum sativum，水稻. Oryza sativa，大麦. Hordeum vulgare，燕麦. Avena sativa‌，欧洲油菜. Brassica napus，‌烟草. Nicotiana tabacum，甘蓝. Brassica oleracea‌。