NtMYC2调控烟草表皮毛发育的功能研究

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

摘要/Abstract

摘要：

目的研究转录因子NtMYC2在JA介导的烟草表皮毛密度改良过程中的调控机制。方法在普通烟草中对NtMYC2进行同源克隆、生物信息学分析和亚细胞定位分析，利用实时荧光定量PCR分析其在不同组织及不同浓度MeJA处理下的表达模式，通过创制敲除材料对NtMYC2功能进行研究，利用酵母双杂交技术验证其与NtJAZ11的蛋白互作关系。结果 NtMYC2有2条同源序列NtMYC2a和NtMYC2b，编码区长度分别为2 040和2 046 bp，分别编码679和681个氨基酸。进化树结果显示，NtMYC2a与祖先种绒毛烟草高度同源，NtMYC2b与祖先种林烟草高度同源，二者与番茄、马铃薯和辣椒中的同源基因亲缘关系较近，与青蒿和拟南芥中的同源基因亲缘关系较远。经蛋白序列比对发现，NtMYC2具有JID、TAD和bHLH保守结构域，属于bHLH基因家族。表达分析发现NtMYC2a与NtMYC2b均在叶中表达量最高，在根中表达量最低，二者均受MeJA的强烈诱导。亚细胞定位结果表明，NtMYC2融合蛋白定位在细胞核。对转基因株系进行观察发现，NtMYC2敲除株系叶面长柄和短柄腺毛密度显著降低，非分泌型表皮毛消失。酵母双杂交结果表明，NtMYC2a和NtMYC2b与JA信号关键抑制子NtJAZ11均存在蛋白互作关系。结论 NtMYC2通过与NtJAZ11蛋白结合共同参与调控JA介导的烟草表皮毛发育。

关键词: 烟草, NtMYC2, 茉莉酸, 表皮毛密度, NtJAZ11

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

季盈彤, 高嘉宁, 钱孟英, 潘宁, 张筠, 崔红, 闫筱筱. NtMYC2调控烟草表皮毛发育的功能研究[J]. 生物技术通报, 2025, 41(4): 278-288.

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.

图/表 9

表1 引物序列

Table 1 Primer sequences

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

图1 NtMYC2的克隆及生物信息学分析A：NtMYC2克隆PCR电泳图；B：NtMYC2及其同源蛋白系统进化树；C：NtMYC2与近缘物种同源蛋白的多序列比对；Nt：普通烟草；Nsyl：林烟草；Ntom：绒毛状烟草；Nb：本氏烟；St：马铃薯；Sl：番茄；Ca：辣椒；Aa：青蒿；At：拟南芥。黑色方框分别为JID domain、TAD motif和bHLH domain

Fig. 1 NtMYC2 gene clone and bioinformatics analysisA: PCR electrophoresis of NtMYC2 gene. B: The phylogenetic tree of NtMYC2 and homologous proteins. C: Multiple sequence alignment of NtMYC2 and homologous proteins of related species; Nt: Nicotiana tabacum; Nsyl: Nicotiana sylvestris; Ntom: Nicotianatomentosiformis; Nb: Nicotiana benthamiana; St: Solanum tuberosum; Sl: Solanum lycopersicum; Ca: Capsicum annuum; Aa: Artemisia annua; At: Arabidopsis thaliana. The black boxes refer to JID domain, TAD motif and bHLH domain

图2 NtMYC2在不同组织中的表达模式分析小写字母表示在P<0.05水平差异显著。下同

Fig. 2 Analysis of the expression patterns in different tissues of NtMYC2Lower case letters indicate significant differences at P<0.05. The same below

图3 NtMYC2应答不同浓度MeJA处理后的表达分析

Fig. 3 Expression analysis of NtMYC2 after treatments with different concentrations of MeJA

图4 NtMYC2蛋白的亚细胞定位

Fig. 4 Subcellular localization of the NtMYC2 protein

图5 NtMYC2敲除纯合株系鉴定A：敲除纯合单株sgRNA区域测序分析；B：纯合敲除单株中NtMYC2蛋白翻译预测。红色方框代表翻译提前终止

Fig. 5 Identification of NtMYC2 knockout homozygous linesA: Sequencing analysis of sgRNA regions of the homozygous knockout plants. B: Protein translation prediction of NtMYC2 homozygous knockout plants. The red boxes indicate the early termination of translation

图6 敲除株系叶面表皮毛形态观察和密度统计

Fig. 6 Morphological observation and trichomes density statistics of NtMYC2-knockout plants

图7 pGKBT7-NtJAZ11诱饵蛋白自激活检测

Fig. 7 Identification of self-activation of pGKBT7-NtJAZ11

图8 NtJAZ11蛋白和NtMYC2a、NtMYC2b的Y2H验证

Fig. 8 Y2H results of protein interactions between protein NtJAZ11 and NtMYC2a, NtMYC2b

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