Function Study of NtMYC2 Gene on Tobacco Trichome Development

doi:10.13560/j.cnki.biotech.bull.1985.2024-0744

Abstract

Abstract:

Objective The regulatory mechanism of NtMYC2 transcription factor in JA-mediated on tobacco trichomes density improvement was studied. Method NtMYC2 gene was cloned from Nicotiana tabacum L. by homologous cloning method. Bioinformatics analysis and subcellular localization were performed to analyze NtMYC2 genetic characterization. Fluorescence quantitative PCR technology was used to detect its expressions files in different tissues and responses to different concentrations of MeJA treatment. NtMYC2 knockout materials were created to verify the function of regulating glandular trichomes. The yeast two-hybrid system was adopted to clarify the protein interaction between NtMYC2 and NtJAZ11. Result NtMYC2 had two homologous sequences, NtMYC2a and NtMYC2b, with open reading frame lengths of 2 040 and 2 046 bp, and coding protein lengths of 679 and 681 amino acids. The homology evolution analysis found that NtMYC2a was highly homologous to Nicotiana tomentosiformis and NtMYC2b was highly homologous to Nicotiana sylvestris, respectively. Both of them had the close relationship with homologous genes from Solanum lycopersicum, Solanum tuberosum and Capsicum annuum, but the distant relationship from Artemisia annua and Arabidopsis thaliana. The protein sequence alignment found that NtMYC2 had JID, TAD and bHLH domains and belonged to the bHLH gene family. Expression analysis clarified that both NtMYC2a and NtMYC2b had the highest expressions in leaves and the lowest expression levels in roots. Both of them were strongly induced by MeJA. NtMYC2 fusion protein was localized in the nucleus. Moreover, long and short glandular trichomes density ofthe leaf surface of NtMYC2 gene knockout lines decreased significantly, non-secretory trichomes disappeared. Yeast two-hybrid results verified that both NtMYC2a and NtMYC2b interacted with NtJAZ11. Conclusion NtMYC2 is involved in the regulation of JA-mediated tobacco trichomes development with protein interacting with NtJAZ11.

Key words: tobacco, NtMYC2, jasmonic acid, trichomes density, NtJAZ11

JI Ying-tong, GAO Jia-ning, QIAN Meng-ying, PAN Ning, ZHANG Jun, CUI Hong, YAN Xiao-xiao. Function Study of NtMYC2 Gene on Tobacco Trichome Development[J]. Biotechnology Bulletin, 2025, 41(4): 278-288.

Figures/Tables 9

References 43

1	崔迪, 孟丽君, 葛秀丽, 等. 铁皮石斛DobHLH51基因克隆及表达特性分析 [J]. 山东农业科学, 2024, 56(5): 9-18.
	Cui D, Meng LJ, Ge XL, et al. Cloning and expression analysis of DobHLH51 from Dendrobium officinale Kimura et Migo [J]. Shandong Agric Sci, 2024, 56(5): 9-18.
2	寇呈熹, 佟金泉, 马瑞, 等. 藜芦bHLH转录因子分析鉴定 [J]. 中草药, 2022, 53(14): 4476-4485.
	Kou CX, Tong JQ, Ma R, et al. Analysis of Veratrum nigrum bHLH transcription factor based on transcriptome [J]. Chin Tradit Herb Drugs, 2022, 53(14): 4476-4485.
3	Zhao MZ, Morohashi K, Hatlestad G, et al. The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci [J]. Development, 2008, 135(11): 1991-1999.
4	韩瑶. 基因差异表达与不同倍性小麦根毛长度变异的关系研究 [D]. 北京: 中国农业大学, 2015.
	Han Y. Study on the relationship between gene differential expression and root hair length variation of wheat with different ploidy [D]. Beijing: China Agricultural University, 2015.
5	王斌, 袁晓, 蒋园园, 等. bHLH96的克隆及其在薄荷萜烯生物合成调控中的功能 [J]. 生物技术通报, 2024, 40(1): 281-293.
	Wang B, Yuan X, Jiang YY, et al. Cloning of bHLH96 gene and its roles in regulating the biosynthesis of peppermint terpenes [J]. Biotechnol Bull, 2024, 40(1): 281-293.
6	Outchkourov NS, Carollo CA, Gomez-Roldan V, et al. Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves [J]. Front Plant Sci, 2014, 5: 519.
7	Liang YF, Ma F, Li BY, et al. A bHLH transcription factor, SlbHLH96, promotes drought tolerance in tomato [J]. Hortic Res, 2022, 9: uhac198.
8	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.
9	Wang YG, Wang S, Tian Y, et al. Functional characterization of a sugar beet BvbHLH93 transcription factor in salt stress tolerance [J]. Int J Mol Sci, 2021, 22(7): 3669.
10	Geng JJ, Liu JH. The transcription factor CsbHLH18 of sweet orange functions in modulation of cold tolerance and homeostasis of reactive oxygen species by regulating the antioxidant gene [J]. J Exp Bot, 2018, 69(10): 2677-2692.
11	Zhao Q, Xiang XH, Liu D, et al. Tobacco transcription factor NtbHLH123 confers tolerance to cold stress by regulating the NtCBF pathway and reactive oxygen species homeostasis [J]. Front Plant Sci, 2018, 9: 381.
12	高丽华, 刘博欣, 李金博, 等. 陆地棉bHLH转录因子GhMYC4基因的克隆及功能分析 [J]. 中国农业科技导报, 2016, 18(5): 33-41.
	Gao LH, Liu BX, Li JB, et al. Cloning and function analysis of b HLH transcription factor gene GhMYC4 from Gossypium hirsutism L [J]. J Agric Sci Technol, 2016, 18(5): 33-41.
13	Kazan K, Manners JM. MYC2: the master in action [J]. Mol Plant, 2013, 6(3): 686-703.
14	Gao CH, Qi SH, Liu KG, et al. MYC2, MYC3, and MYC4 function redundantly in seed storage protein accumulation in Arabidopsis [J]. Plant Physiol Biochem, 2016, 108: 63-70.
15	Niu YJ, Figueroa P, Browse J. Characterization of JAZ-interacting bHLH transcription factors that regulate jasmonate responses in Arabidopsis [J]. J Exp Bot, 2011, 62(6): 2143-2154.
16	Çevik V, Kidd BN, Zhang PJ, et al. MEDIATOR25 acts as an integrative hub for the regulation of jasmonate-responsive gene expression in Arabidopsis [J]. Plant Physiol, 2012, 160(1): 541-555.
17	Abe H, Urao T, Ito T, et al. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling [J]. Plant Cell, 2003, 15(1): 63-78.
18	Wei M, Liu QG, Wang ZC, et al. PuHox52-mediated hierarchical multilayered gene regulatory network promotes adventitious root formation in Populus ussuriensis [J]. New Phytol, 2020, 228(4): 1369-1385.
19	Zhang HT, Hedhili S, Montiel G, et al. The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus [J]. Plant J, 2011, 67(1): 61-71.
20	Zhang HB, Bokowiec MT, Rushton PJ, et al. Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis [J]. Mol Plant, 2012, 5(1): 73-84.
21	悦曼芳, 张春, 郑登俞, 等. 玉米转录因子ZmbHLH91对非生物逆境胁迫的应答 [J]. 作物学报, 2022, 48(12): 3004-3017.
	Yue MF, Zhang C, Zheng DY, et al. Response of maize transcriptional factor ZmbHLH91 to abiotic stress [J]. Acta Agron Sin, 2022, 48(12): 3004-3017.
22	Fu JY, Wang LP, Pei WZ, et al. ZmEREB92 interacts with ZmMYC2 to activate maize terpenoid phytoalexin biosynthesis upon Fusarium graminearum infection through jasmonic acid/ethylene signaling [J]. New Phytol, 2023, 237(4): 1302-1319.
23	袁铭远. AabHLH113整合JA和ABA信号调控青蒿素生物合成的功能研究 [D]. 重庆: 西南大学, 2023.
	Yuan MY. Study on the function of AabHLH113 integrating JA and ABA signals to regulate artemisinin biosynthesis [D]. Chongqing: Southwest University, 2023.
24	Hua B, Chang J, Wu ML, et al. Mediation of JA signalling in glandular trichomes by the woolly/SlMYC1 regulatory module improves pest resistance in tomato [J]. Plant Biotechnol J, 2021, 19(2): 375-393.
25	Xu JS, van Herwijnen ZO, Dräger DB, et al. SlMYC1 regulates type VI glandular trichome formation and terpene biosynthesis in tomato glandular cells [J]. Plant Cell, 2018, 30(12): 2988-3005.
26	余兰, 王浩然, 张莹, 等. 转录因子MYCs调控番茄表皮毛萜类化合物的分子机制研究进展 [J]. 中国农学通报, 2022, 38(6): 87-93.
	Yu L, Wang HR, Zhang Y, et al. Transcription factor MYCs regulating terpenoids in tomato trichomes: research progress on molecular mechanism [J]. Chin Agric Sci Bull, 2022, 38(6): 87-93.
27	Feng ZX, Sun L, Dong MM, et al. Identification and functional characterization of CsMYCs in cucumber glandular trichome development [J]. Int J Mol Sci, 2023, 24(7): 6435.
28	Yuan MY, Sheng YG, Bao JJ, et al. AaMYC3 bridges the regulation of glandular trichome density and artemisinin biosynthesis in Artemisia annua [J]. Plant Biotechnol J, 2024.
29	Guan YQ, Jiang L, Wang Y, et al. CmMYC2-CmMYBML1 module orchestrates the resistance to herbivory by synchronously regulating the trichome development and constitutive terpene biosynthesis in Chrysanthemum [J]. New Phytol, 2024, 244(3): 914-933.
30	孟盈, 闫筱筱, 王召军, 等. NtCycB2基因表达对烟草腺毛发生的影响 [J]. 中国烟草学报, 2019, 25(2): 85-92, 98.
	Meng Y, Yan XX, Wang ZJ, et al. Effects of NtCycB2 expression on tobacco trichomes [J]. Acta Tabacaria Sin, 2019, 25(2): 85-92, 98.
31	潘影, 张振远, 滕环瑜, 等. 烟草腺毛生物反应器材料的创制与性状分析 [J]. 烟草科技, 2023, 56(7): 1-7.
	Pan Y, Zhang ZY, Teng HY, et al. Creation and characterization of tobacco glandular trichome bioreactor material [J]. Tob Sci Technol, 2023, 56(7): 1-7.
32	Qi TC, Song SS, Ren QC, et al. The Jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate Jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana [J]. Plant Cell, 2011, 23(5): 1795-1814.
33	Hu HY, He X, Tu LL, et al. GhJAZ2 negatively regulates cotton fiber initiation by interacting with the R2R3-MYB transcription factor GhMYB25-like [J]. Plant J, 2016, 88(6): 921-935.
34	Hua B, Chang J, Xu ZJ, et al. HOMEODOMAIN PROTEIN8 mediates jasmonate-triggered trichome elongation in tomato [J]. New Phytol, 2021, 230(3): 1063-1077.
35	Yan XX, Cui LP, Liu XY, et al. NbJAZ3 is required for jasmonate-meditated glandular trichome development in Nicotiana benthamiana [J]. Physiol Plant, 2022, 174(2): e13666.
36	徐寒池, 徐梦晓, 滕环瑜, 等. 烟草NtCycB2基因通过茉莉酮酸途径调控腺毛发生 [J]. 中国烟草学报, 2023, 29(4): 96-104.
	Xu HC, Xu MX, Teng HY, et al. Tobacco NtCycB2 regulates glandular trichomes generation via jasmonic acid pathway [J]. Acta Tabacaria Sin, 2023, 29(4): 96-104.
37	张娇. 两个bHLH转录因子(AtLPl和AtLP2)在拟南芥细胞伸长生长中的功能研究 [D]. 武汉: 华中师范大学, 2019.
	Zhang J. Function of two bHLH transcription factors (AtLPl and AtLP2) in elongation and growth of Arabidopsis thaliana cells [D]. Wuhan: Central China Normal University, 2019.
38	Liu ZH, Chen Y, Wang NN, et al. A basic helix-loop-helix protein (GhFP1) promotes fibre elongation of cotton (Gossypium hirsutum) by modulating brassinosteroid biosynthesis and signalling [J]. New Phytol, 2020, 225(6): 2439-2452.
39	Vaughan Symonds V, Hatlestad G, Lloyd AM. Natural allelic variation defines a role for ATMYC1: trichome cell fate determination [J]. PLoS Genet, 2011, 7(6): e1002069.
40	Cheng ZW, Sun L, Qi TC, et al. The bHLH transcription factor MYC3 interacts with the Jasmonate ZIM-domain proteins to mediate jasmonate response in Arabidopsis [J]. Mol Plant, 2011, 4(2): 279-288.
41	Fernández-Calvo P, Chini A, Fernández-Barbero G, et al. The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses [J]. Plant Cell, 2011, 23(2): 701-715.
42	Wang S, Wang YB, Yang R, et al. Genome-wide identification and analysis uncovers the potential role of JAZ and MYC families in potato under abiotic stress [J]. Int J Mol Sci, 2023, 24(7): 6706.
43	Zhang HY, Li WJ, Niu DX, et al. Tobacco transcription repressors NtJAZ: Potential involvement in abiotic stress response and glandular trichome induction [J]. Plant Physiol Biochem, 2019, 141: 388-397.

引物名称 Primer name	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	用途 Usage
CLNtMYC2a	TCCACTCTCATTCCTCTCACTCA	TTAGCGTGTTTCAGCAACTCTG	NtMYC2克隆 NtMYC2 cloning
CLNtMYC2b	TGTCCACTCTCATTCCTCTCACTC	TTAGCGTGTTTCAGCAACTCTG	NtMYC2克隆 NtMYC2 cloning
CLNtMYC2	ATGACTGATTACAGCTTACCCACC	TTAGCGTGTTTCAGCAACTCTG	NtMYC2a和NtMYC2b 开放阅读框克隆 NtMYC2a and NtMYC2b Open reading frame cloning
L25	CCCCTCACCACAGAGTCTGC	AAGGGTGTTGTTGTCCTCAATCTT	RT-qPCR
qRT-NtMYC2a	ATGACTGATTACAGCTTACCCACC	GACAGTAGAAGCACAAGAAGACGG
qRT-NtMYC2b	CTAGTGGTACTACCGATGACAACG	ACAGTAGAGGCACAAGAAGACGG
pC2300-NtMYC2a/2b	GGGGTACCATGACTGATTACAGCTTACCCACC	CGGGATCCGCGTGTTTCAGCAACTCTGG	NtMYC2亚细胞定位载体构建 Vector construction of NtMYC2 subcellular localization
MT-NtMYC2a	AGCTTCTGGTGCGATGAAGTC	ATCTTGCTTCCAGTATGGCTAGAC	NtMYC2-KO株系检测 Detection of NtMYC2 knockout lines
MT-NtMYC2b	CAGGGGAGGAGAATAAGAACAAG	TCCAGTATGGCTAGACATCTTGTGA	NtMYC2-KO株系检测 Detection of NtMYC2 knockout lines
BD-NtJAZ11	CGGAATTCATGGAGAGAGATTTTATGGG	CGGGATCCTCACCTTGTGTCTACATTATTTTG	NtJAZ11-BD载体构建 Vector construction of NtJAZ11-BD
AD-NtMYC2a	TCCCCCGGGATGACTGATTACAGCTTACCCACC	CGGGATCCTTAGCGTGTTTCAGCAACTCTG	NtMYC2a-AD载体构建 Vector construction of NtMYC2a-AD
AD-NtMYC2b	GGAATTCCATATGATGACTGATTACAGCTTACCCACC	CGGGATCCTTAGCGTGTTTCAGCAACTCTG	NtMYC2b-AD载体构建 Vector construction of NtMYC2b-AD

引物名称 Primer name	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	用途 Usage
CLNtMYC2a	TCCACTCTCATTCCTCTCACTCA	TTAGCGTGTTTCAGCAACTCTG	NtMYC2克隆 NtMYC2 cloning
CLNtMYC2b	TGTCCACTCTCATTCCTCTCACTC	TTAGCGTGTTTCAGCAACTCTG	NtMYC2克隆 NtMYC2 cloning
CLNtMYC2	ATGACTGATTACAGCTTACCCACC	TTAGCGTGTTTCAGCAACTCTG	NtMYC2a和NtMYC2b 开放阅读框克隆 NtMYC2a and NtMYC2b Open reading frame cloning
L25	CCCCTCACCACAGAGTCTGC	AAGGGTGTTGTTGTCCTCAATCTT	RT-qPCR
qRT-NtMYC2a	ATGACTGATTACAGCTTACCCACC	GACAGTAGAAGCACAAGAAGACGG
qRT-NtMYC2b	CTAGTGGTACTACCGATGACAACG	ACAGTAGAGGCACAAGAAGACGG
pC2300-NtMYC2a/2b	GGGGTACCATGACTGATTACAGCTTACCCACC	CGGGATCCGCGTGTTTCAGCAACTCTGG	NtMYC2亚细胞定位载体构建 Vector construction of NtMYC2 subcellular localization
MT-NtMYC2a	AGCTTCTGGTGCGATGAAGTC	ATCTTGCTTCCAGTATGGCTAGAC	NtMYC2-KO株系检测 Detection of NtMYC2 knockout lines
MT-NtMYC2b	CAGGGGAGGAGAATAAGAACAAG	TCCAGTATGGCTAGACATCTTGTGA	NtMYC2-KO株系检测 Detection of NtMYC2 knockout lines
BD-NtJAZ11	CGGAATTCATGGAGAGAGATTTTATGGG	CGGGATCCTCACCTTGTGTCTACATTATTTTG	NtJAZ11-BD载体构建 Vector construction of NtJAZ11-BD
AD-NtMYC2a	TCCCCCGGGATGACTGATTACAGCTTACCCACC	CGGGATCCTTAGCGTGTTTCAGCAACTCTG	NtMYC2a-AD载体构建 Vector construction of NtMYC2a-AD
AD-NtMYC2b	GGAATTCCATATGATGACTGATTACAGCTTACCCACC	CGGGATCCTTAGCGTGTTTCAGCAACTCTG	NtMYC2b-AD载体构建 Vector construction of NtMYC2b-AD

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