中国水仙雄蕊发育相关转录因子NtTGAL11的基因克隆与功能研究

doi:10.7668/hbnxb.20196617

摘要/Abstract

摘要：

旨在探究中国水仙雄蕊发育的分子调控机制。基于马艳军水仙课题组前期中国水仙金盏银台雄蕊发育相关转录组数据,发现1个TGAL11同源序列在雄蕊中特异性高表达,克隆该同源基因并命名为NtTGAL11。结果表明,NtTGAL11序列全长1 020 bp,编码339个氨基酸。NtTGAL11蛋白分子质量为38.072 31 ku,是碱性亲水蛋白,该蛋白主要由α-螺旋和无规则卷曲构成,包括bZIP和DOG1 2种结构域,AtTGA9蛋白和AtTGA10蛋白与其亲缘关系最近。通过亚细胞定位分析,证实NtTGAL11定位于细胞核。RT-qPCR数据分析表明,NtTGAL11在金盏银台雄蕊中高表达,在其他花器官中,包括玉玲珑瓣化雄蕊中低表达,与转录组数据一致。双分子荧光互补试验证明,NtTGAL11能够与AG类基因NtMADS6互作。推测NtTGAL11可能通过调控AG基因的时空表达促进雄蕊发育,NtTGAL11的表达模式分析进一步表明NtTGAL11在中国水仙雄蕊发育过程中起关键调控作用。

关键词: 中国水仙, 雄蕊发育, TGAL, 生物信息学, 双分子荧光互补

Abstract:

It aimed to elucidate the molecular regulatory mechanism underlying stamen development in Chinese narcissus.Based on our group's previous transcriptome data from stamens of the Chinese narcissus cultivar Jinzhanyintai from the Narcissus Research Group led by Ma Yanjun,a TGAL11 homologous sequence that is specifically and highly expressed in stamens was identified.This gene was cloned and designated NtTGAL11.Sequence analysis showed that the full-length coding sequence of NtTGAL11 was 1 020 bp,encoding a protein of 339 amino acids with a molecular mass of 38.072 31 ku.NtTGAL11 was a basic,hydrophilic protein predicted to be predominantly composed of αhelices and random coils,and it contained both a bZIP domain and a DOG1 domain.Phylogenetic analysis revealed that NtTGAL11 was most closely related to AtTGA9 and AtTGA10.Subcellular localization assays confirmed that NtTGAL11 was targeted to the nucleus.RT-qPCR analysis demonstrated that NtTGAL11 was highly expressed in the stamens of Jinzhanyintai but was expressed at low levels in other floral organs,as well as in the petaloid stamens of the double-flowered cultivar Yulinglong,consistent with the transcriptomic data.Bimolecular fluorescence complementation(BiFC)assays further showed that NtTGAL11 physically interacted with the AGAMOUS-class MADS-box protein NtMADS6.Taken together,these findings suggest that NtTGAL11 might promote stamen development by modulating the spatiotemporal expression of AG-like genes,and its expression pattern underscores a key regulatory role in stamen morphogenesis in Chinese narcissus.

Key words: Narcissus tazetta var.chinensis, Stamen development, TGAL, Bioinformatics, BiFC

中图分类号:

S682.2+
1

赵鹏达, 胡陶, 张娜, 杨语婷, 常艳婷, 江泽慧, 马艳军. 中国水仙雄蕊发育相关转录因子NtTGAL11的基因克隆与功能研究[J]. 华北农学报, 2026, 41(5): 74-82. doi: 10.7668/hbnxb.20196617.

ZHAO Pengda, HU Tao, ZHANG Na, YANG Yuting, CHANG Yanting, JIANG Zehui, MA Yanjun. Cloning and Preliminary Functional Analysis of NtTGAL11,a Transcription Factor Gene Involved in Stamen Development of Narcissus tazetta var.chinensis[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(5): 74-82. doi: 10.7668/hbnxb.20196617.

http://www.hbnxb.net/CN/Y2026/V41/I5/74

图/表 10

表1 引物序列

Tab.1 Primer sequence

引物 Primers	序列(5'—3') Sequences(5'—3')
NtTGAL11	F:ATGGCCACACAAAATCATGAGCTC
	R:GAAGGCTGAAAATTGGCCGTG
PHG-NtTGAL11-EGFP	F:TCTCTCTCTCAAGCTATGGCCACACAAAATCATGAGCT
	R:CGGGTCATGAGCTCCGAAGGCTGAAAATTGGCCGTG
YNE-NtTGAL11	F:CTAGATCGGGAATTCATGGCCACACAAAATCATGAGCT
	R:ATGCAAGCTAAGCTTGAAGGCTGAAAATTGGCCGTG
YCE-NtMADS6	F:CTAGATCGGGAATTCATGGGTAGGGGGAAGATAGAGATCAAA
	R:ATGCAAGCTAAGCTTTCCCAGTTGAAGGGTAGTCTGC

图1 NtTGAL11基因扩增产物的琼脂糖凝胶电泳 M.2 000 bp DNA 标记;1,2.NtTGAL11 PCR产物。

Fig.1 Agarose gel electrophoresis of NtTGAL11 gene amplification products M.2 000 bp DNA Marker;1,2. NtTGAL11 PCR products.

图2 NtTGAL11 蛋白亲水性预测

Fig.2 Prediction result of hydrophilicity of NtTGAL11 protein

图3 NtTGAL11蛋白的二级结构(A)和三级结构(B)预测

Fig.3 Prediction of secondary structure(A)and tertiary structure(B)of NtTGAL11 protein

图4 NtTGAL11蛋白磷酸化位点预测

Fig.4 Prediction result of phosphorylation sites in NtTGAL11 protein

图5 NtTGAL11蛋白保守结构域分析

Fig.5 Conserved domain analysis of the NtTGAL11 protein

图6 NtTGAL11和拟南芥10个TGA蛋白的系统发育树(A)及多序列比对(B)

Fig.6 Phylogenetic tree(A)and multiple sequence alignment(B)of NtTGAL11 and 10 TGA proteins from A.thaliana

图7 NtTGAL11蛋白在烟草细胞中亚细胞定位

Fig.7 Subcellular localization of NtTGAL11 protein in tobacco cells

图8 双分子荧光互补检测NtTGAL11与NtMADS6的相互作用

Fig.8 BiFC assay for detecting the interaction of NtTGAL11 with NtMADS6

图9 NtTGAL11在不同组织部位的相对表达量误差线代表3个生物学重复的标准差(s)。不同小写字母表示差异显著(P<0.05)。

Fig.9 The relative expression levels of NtTGAL11 in different tissues Data are presented as means±s(n=3 biological replicates).Different lowercase letters indicate significant differences(P<0.05).

参考文献 32

[1]	Coen E S, Meyerowitz E M. The war of the whorls:genetic interactions controlling flower development[J]. Nature, 1991, 353(6339):31-37.doi:10.1038/353031a0.
[2]	Yanofsky M F, Ma H, Bowman J L, Drews G N, Feldmann K A, Meyerowitz E M. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors[J]. Nature, 1990, 346(6279):35-39.doi:10.1038/346035a0.
[3]	张璐. 中国水仙AGL6和TFL1基因功能分析[D]. 福州: 福建农林大学, 2020.doi:10.27018/d.cnki.gfjnu.2020.000392.
	Zhang L. Functional analysis of AGL6 and TFL1 genes in Narcissus tazetta[D]. Fuzhou: Fujian Agriculture and Forestry University, 2020.
[4]	武丹. 基于转录组测序的中国水仙成花基因的研究[D]. 福州: 福建农林大学, 2018.doi:10.27018/d.cnki.gfjnu.2018.000275.
	Wu D. Research on flowering genes in Narcissus tazetta based on transcriptome sequencing[D]. Fuzhou: Fujian Agriculture and Forestry University, 2018.
[5]	李洪有, 王婵, 李丽林, 赵瑞, 王永勤. 单子叶植物花器官发育的分子机制及修正的ABC模型[J]. 中国细胞生物学学报, 2013, 35(4):526-535.doi:10.11844/cjcb.2013.04.0012.
	Li H Y, Wang C, Li L L, Zhao R, Wang Y Q. The conservative property of flower development model and modified ABC model in monocotyledons[J]. Chinese Journal of Cell Biology, 2013, 35(4):526-535.
[6]	赵素珍. MADS-box基因在单子叶植物花发育中的功能研究[D]. 北京: 中国科学院植物研究所, 2005.
	Zhao S Z. Functional study of MADS-box genes in flower development of monocot plants[D]. Beijing: Institute of Botany,Chinese Academy of Sciences, 2005.
[7]	Li X F, Xu J, Yang R, Jia L Y, Deng X J, Xiong L J, Zhang X P, Fang Q, Zhang W, Sun Y, Xu L. Analysis of B-Class genes NAP3L3 and VNAP3L4 in Narcissus tazetta var.chinensis[J]. Plant Molecular Biology Reporter, 2013, 31(2):255-263.doi:10.1007/s11105-012-0493-8. URL
[8]	高志民, 陈段芬, 李雪平, 蔡春菊, 彭镇华. 一个中国水仙MADS-box基因的克隆与分析[J]. 园艺学报, 2008, 35(2):295-300.doi:10.3321/j.issn:0513-353X.2008.02.023.
	Gao Z M, Chen D F, Li X P, Cai C J, Peng Z H. Cloning and sequence analysis of a flowering-related MADS-box gene in Narcissus tazetta var.chinensis Roem[J]. Acta Horticulturae Sinica, 2008, 35(2):295-300.
[9]	Jakoby M, Weisshaar B, Dröge-Laser W, Vicente-Carbajosa J, Tiedemann J, Kroj T, Parcy F. bZIP transcription factors in Arabidopsis[J]. Trends in Plant Science, 2002, 7(3):106-111.doi:10.1016/S1360-1385(01)02223-3. pmid: 11906833
[10]	Fonseca A, Urzúa T, Jelenska J, Sbarbaro C, Seguel A, Duarte Y, Greenberg J T, Holuigue L, Blanco-Herrera F, Herrera-Vásquez A. The TGA transcription factors from clade Ⅱ negatively regulate the salicylic acid accumulation in Arabidopsis[J]. International Journal of Molecular Sciences, 2022, 23(19):11631.doi:10.3390/ijms231911631. URL
[11]	Murmu J, Bush M J, DeLong C, Li S T, Xu M L, Khan M, Malcolmson C, Fobert P R, Zachgo S, Hepworth S R. Arabidopsis basic leucine-zipper transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development[J]. Plant Physiology, 2010, 154(3):1492-1504.doi:10.1104/pp.110.159111. URL
[12]	Tomaž Š, Gruden K, Coll A. TGA transcription factors-structural characteristics as basis for functional variability[J]. Frontiers in Plant Science, 2022, 13:935819.doi:10.3389/fpls.2022.935819. URL
[13]	Chen J, Wang Y, Li X, Zhang H, Liu Y, Zhang L. Genome-wide identification and expression analysis of the TGA gene family in rose(Rosa chinensis)reveals their role in flower development[J]. Journal of Plant Growth Regulation, 2021, 40(3):1235-1249.doi:10.1007/s00344-020-10181-6.
[14]	Liu Y, Zhang H, Wang C, Li J, Chen X. Functional characterization of NtTGA1,a TGA transcription factor from Narcissus tazetta,in regulating floral organ development[J]. Plant Cell Reports, 2024, 43(2):56.doi:10.1007/s00299-023-03136-2.
[15]	Lu C F, Liu X Y, Tang Y Q, Fu Y Q, Zhang J M, Yang L T, Li P H, Zhu Z L, Dong P. A comprehensive review of TGA transcription factors in plant growth,stress responses,and beyond[J]. International Journal of Biological Macromolecules, 2024, 258:128880.doi:10.1016/j.ijbiomac.2023.128880. URL
[16]	Fan K K, Ma Y J, Chang Y T, Hu X M, Zhang W B, Deng Y Y, Xia M S, Jiang Z H, Hu T. Cloning and functional analysis of NtMYB9 in Jinzhanyintai of Narcissus tazetta var.chinensis[J]. Horticulturae, 2022, 8(6):528.doi:10.3390/horticulturae8060528. URL
[17]	胡雨莹, 孙茂, 汪骞, 黎志彬, 鲍锐, 桂敏, 钟秋月, 杜光辉, 吴丽艳. 野生蒜芥茄SsRPM1基因的生信分析、亚细胞定位及表达分析[J]. 华北农学报, 2025, 40(3):27-33.doi:10.7668/hbnxb.20193300.
	Hu Y Y, Sun M, Wang Q, Li Z B, Bao R, Gui M, Zhong Q Y, Du G H, Wu L Y. Bioinformatics,subcellular localization and expression analysis of SsRPM1 gene in wild Solanum sisymbriifolium Lam[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3):27-33.
[18]	Hu X M, Zhang N, Fan K K, Chang Y T, Zhang W B, Deng Y Y, Wang S H, Hu T, Ma Y J. Cloning and functional analysis of NtHDR in Jinzhanyintai of Narcissus tazetta var.chinensis M.Roem[J]. Horticulturae, 2023, 9(8):904.doi:10.3390/horticulturae9080904. URL
[19]	Zhang N, Zhao P D, Chu T K, Fan K K, Deng Y Y, Chang Y T, Zhang W B, Zhang X, Bai Y W, Ye Y Q, Jiang Z H, Hu T, Ma Y J. Functional analysis of NtMYB15 from Narcissus tazetta var.chinensis in transgenic aromatic plants(Petunia hybrida and Nicotiana tabacum)regulating the colour and aroma of the flowers[J]. Industrial Crops and Products, 2025, 237:122245.doi:10.1016/j.indcrop.2025.122245. URL
[20]	田义, 张彩霞, 康国栋, 李武兴, 张利益, 丛佩华. 植物TGA转录因子研究进展[J]. 中国农业科学, 2016, 49(4):632-642.doi:10.3864/j.issn.0578-1752.2016.04.003.
	Tian Y, Zhang C X, Kang G D, Li W X, Zhang L Y, Cong P H. Progress on TGA transcription factors in plant[J]. Scientia Agricultura Sinica, 2016, 49(4):632-642. doi: 10.3864/j.issn.0578-1752.2016.04.003
[21]	郭梦雪. OsTGAL4和OsTGAL11调控水稻花器官发育的功能分析[D]. 杭州: 浙江大学, 2023.doi:10.27461/d.cnki.gzjdx.2023.002817.
	Guo M X. Functional analysis of OsTGAL4 and OsTGAL11 in regulating rice floral organ development[D]. Hangzhou: Zhejiang University, 2023.
[22]	高倩, 秦迎迎, 杨致荣. 谷子TGA转录因子家族全基因组鉴定与分析[J]. 山西农业科学, 2023, 51(5):469-477.doi:10.3969/j.issn.1002-2481.2023.05.01.
	Gao Q, Qin Y Y, Yang Z R. Genome-wide identification and analysis of the TGA family of transcription factors in Setaria italica[J]. Journal of Shanxi Agricultural Sciences, 2023, 51(5):469-477.
[23]	孟新超, 殷恒梅, 张兴政, 汪林林, 曹昆, 朴京培, 刘剑锋. 玉米TGA转录因子家族全基因组鉴定及盐胁迫相关表达分析[J]. 江西农业大学学报, 2023, 45(3):631-642.doi:10.13836/j.jjau.2023059.
	Meng X C, Yin H M, Zhang X Z, Wang L L, Cao K, Piao J P, Liu J F. Genome-wide identification and salt stress-related expression analysis of maize TGA transcription factor family[J]. Acta Agriculturae Universitatis Jiangxiensis(Natural Sciences Edition), 2023, 45(3):631-642.
[24]	简子林, 王自力, 秦畅, 何方, 任明见. 小麦TGA转录因子家族全基因组鉴定及其响应非生物胁迫的表达分析[J]. 江苏农业学报, 2025, 41(10):1873-1885.doi:10.3969/j.issn.1000-4440.2025.10.001.
	Jian Z L, Wang Z L, Qin C, He F, Ren M J. Genome-wide identification of the TGA transcription factor family and expression analysis under abiotic stress in Triticum aestivum L.[J]. Jiangsu Journal of Agricultural Sciences, 2025, 41(10):1873-1885.
[25]	赵振南. 黑穗病菌胁迫下甘蔗lncRNA组学分析及共表达基因ScTGA1的功能鉴定[D]. 福州: 福建农林大学, 2023.doi:10.27018/d.cnki.gfjnu.2023.000496.
	Zhao Z N. LncRNA omics analysis and functional characterization of co-expressed gene ScTGA1 in sugarcane under smut pathogen stress[D]. Fuzhou: Fujian Agriculture and Forestry University, 2023.
[26]	袁学顺, 周佳玉, 刘淼, 周莹, 姚丹, 崔喜艳, 陈展宇. 大豆GmTGA15基因的克隆、转录激活活性分析及亚细胞定位研究[J]. 吉林农业大学学报, 2024, 46(3):358-365.doi:10.13327/j.jjlau.2024.5729.
	Yuan X S, Zhou J Y, Liu M, Zhou Y, Yao D, Cui X Y, Chen Z Y. Molecular cloning,transcriptional activation activity and subcellular localization of GmTGA15 gene from Glycine max[J]. Journal of Jilin Agricultural University, 2024, 46(3):358-365.
[27]	Lin P, Dong T, Chen W L, Zou N X, Chen Y L, Li Y Q, Chen K L, Wang M Y, Liu J F. Expression analysis of MaTGA8 transcription factor in banana and its defence functional analysis by overexpression in Arabidopsis[J]. International Journal of Molecular Sciences, 2021, 22(17):9344.doi:10.3390/ijms22179344. URL
[28]	郭双元, 任惠文, 张艳琴, 冯传欣, 冯浩, 王晓杰, 康振生, 张新梅. 小麦抗病相关基因TaTGA2.2在小麦-条锈菌互作中的表达分析及功能研究[J]. 麦类作物学报, 2020, 40(6):645-655.doi:10.7606/j.issn.1009-1041.2020.06.01.
	Guo S Y, Ren H W, Zhang Y Q, Feng C X, Feng H, Wang X J, Kang Z S, Zhang X M. Characterization and functional analyses of wheat disease resistance-related gene TaTGA2.2 in the interaction between wheat and stripe rust[J]. Journal of Triticeae Crops, 2020, 40(6):645-655.
[29]	Wang Z N, Chen D D, Lin X L, Wang J, Arif M, Li J L, Gong Y L, Lei Y, Wu X, Wu C X, Zhu S S, Li L H. OsROXY2 regulates stamen number through interaction with OsbZIP47 in rice[J]. Rice, 2025, 18(1):76.doi:10.1186/s12284-025-00833-0. pmid: 40801995
[30]	Ji S Y, Yang Y, Yang S M, Jiang S, Cheng H F, Liu T, Han Y, Yu C, Luo L, Cheng T R, Wang J, Zhang Q X, Pan H T. The RcAP2L-RcAS1 complex modulates petal number in roses by targeting the RcAGL80 promoter[J]. The Plant Journal, 2025, 124(3):e70549.doi:10.1111/tpj.70549. URL
[31]	Yang Y, Sun M, Yuan C Q, Zhang Q X. ECE-CYC1 transcription factor CmCYC1a may interact with CmCYC2 in regulating flower symmetry and stamen development in Chrysanthemum morifolium[J]. Genes, 2025, 16(2):152.doi:10.3390/genes16020152. URL
[32]	Ma Y J, Hu X M, Fan K K, Zhang N, Shang L L, Deng Y Y, Hu T, Zhang W B, Wang Y, Jiang Z H. Emergence of corona is independent of the four whorls of floral organs in Narcissus tazetta[J]. Plants, 2023, 12(7):1458.doi:10.3390/plants12071458. URL

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