烟草AP2/ERF转录因子NtESR2的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0894

摘要/Abstract

摘要：

目的 AP2/ERF（APETALA2/ethylene-responsive factor）转录因子家族中的ESR2基因对植物叶芽发育调控具有重要的作用。克隆烟草NtESR2基因并分析其功能，为烟草叶芽的发育调控提供靶标基因。方法采用同源克隆从栽培烟草（Nicotiana tabacum L.）中克隆NtESR2基因，并进行生物信息学、基因表达、激素响应和亚细胞定位分析，同时利用CRISPR/Cas9编辑技术创制NtESR2a和NtESR2b双突变体ntesr2，观察ntesr2突变体的表型。结果 NtESR2在栽培烟草中具有2个拷贝（NtESR2a和NtESR2b），其CDS长度分别为1 188 bp和1 200 bp，分别编码395和399个氨基酸；NtESR2a蛋白定位于细胞核，NtESR2b蛋白定位于细胞核和细胞膜上，2个蛋白都具有1个AP2结构域，属于ERF亚族，其中NtESR2a来自林烟草，NtESR2b来自绒毛状烟草。NtESR2基因在烟草的根、茎、叶、顶芽、腋芽和花中均有表达，其中在顶芽和腋芽表达较高。通过顺式作用元件分析和外源激素处理，表明该基因受到多种植物激素的调控。ntesr2突变体表现出子叶融合和分生组织缺失的性状，NtCUC2、NtLAS、NtREV、NtYUCCA和NtPIN1基因表达显著下调。结论从烟草中克隆得到ERF转录因子NtESR2的2个成员，在烟草顶芽和腋芽中表达较高，受到生长素等多种植物激素的调控，NtESR2通过调控生长素的运输来影响烟草叶芽的形态建立过程，研究结果为调控烟草叶芽发育提供理论依据。

关键词: 烟草, NtESR2, CRISPR/Cas9, 子叶融合, 分生组织, 生物信息学分析, 亚细胞定位

Abstract:

Objective ESR2 gene in the AP2/ERF (APETALA2/ethylene-responsive factor) transcription factor family plays an important role in the regulation of plant leaf bud development. To clone the tobacco NtESR2 gene and analyze its function, we aimed to provide a target gene for the regulation of tobacco leaf bud development. Method NtESR2 gene was cloned from cultivated tobacco (Nicotiana tabacum L.) by homologous cloning, and its bioinformatics, gene expression, hormone response, and subcellular localization analysis were performed. Meanwhile, CRISPR/Cas9 editing technology was used to create the NtESR2a and NtESR2b double mutant ntesr2, and the phenotype of the ntesr2 mutant was observed. Result NtESR2 has two copies in cultivated tobacco, NtESR2a and NtESR2b, with CDS lengths of 1 188 bp and 1 200 bp, encoding 395 and 399 amino acids, respectively. NtESR2a protein is located in the nucleus, while NtESR2b protein is located in the nucleus and cell membrane. Both proteins have only one AP2 domain and belong to the ERF subfamily, of which NtESR2a is from Nicotiana sylvestris and NtESR2b is from Nicotiana tomentosiformis. NtESR2 gene is expressed in the roots, stems, leaves, terminal buds, axillary buds and flowers of tobacco, among which the expression in terminal buds and axillary buds is higher, indicating that NtESR2 gene may have an important regulatory role in these tissues. Cis-acting element analysis and exogenous hormone treatment showed that the gene is regulated by a variety of plant hormones. The ntesr2 mutant showed the characteristics of cotyledon fusion and meristem loss, and the expression of NtCUC2, NtLAS, NtREV, NtYUCCA and NtPIN1 genes was significantly downregulated. Conclusion Two members of the ERF transcription factor NtESR2 are cloned from tobacco. They are highly expressed in apical buds and axillary buds and are regulated by a variety of plant hormones such as auxin. NtESR2 regulates the transport of auxin to affect the morphological establishment process of tobacco leaf buds. The research results provide a theoretical basis for regulating the development of tobacco leaf buds.

Key words: tobacco, NtESR2, CRISPR/Cas9, cotyledon fusion, meristem, bioinformatics analysis, subcellular localization

卢勇杰, 夏海乾, 李永铃, 张文建, 余婧, 赵会纳, 王兵, 许本波, 雷波. 烟草AP2/ERF转录因子NtESR2的克隆及功能分析[J]. 生物技术通报, 2025, 41(4): 266-277.

LU Yong-jie, XIA Hai-qian, LI Yong-ling, ZHANG Wen-jian, YU Jing, ZHAO Hui-na, WANG Bing, XU Ben-bo, LEI Bo. Cloning and Expression Analysis of AP2/ERF Transcription Factor NtESR2 in Nicotiana tabacum[J]. Biotechnology Bulletin, 2025, 41(4): 266-277.

图/表 12

表1 引物序列

Table 1 Primer Sequence

引物Primer	序列Sequence （5′‒3′）	用途Usage
NtESR2a-F	ATGGAAGATGCCATGAG	NtESR2a基因克隆 NtESR2a amplification
NtESR2a-R	TTAAGAATTCTGCAGCTTAG	NtESR2a基因克隆 NtESR2a amplification
NtESR2b-F	ATGCGCGGTGTTAAAGC	NtESR2b基因克隆 NtESR2b amplification
NtESR2b-R	TCAAGAATTCTGCAGCTTAG	NtESR2b基因克隆 NtESR2b amplification
Actin-Q-F	CGGAATCCACGAGACTACATA	RT-qPCR内标基因检测 RT-qPCR internal standard gene detection
Actin-Q-R	GGGAAGCCAAGATAGAGC	RT-qPCR内标基因检测 RT-qPCR internal standard gene detection
ESR2a-Q-F	CCAACTTCCACTAACGATGGGT	RT-qPCR检测NtESR2a基因表达量 RT-qPCR detection of NtESR2a gene expression
ESR2a-Q-R	GTGTCTGAAGAAAGGGAACTGGT
ESR2b-Q-F	GTGAACTTCTTAGTTCGAACCCCT	RT-qPCR检测NtESR2b基因表达量 RT-qPCR detection of NtESR2b gene expression
ESR2b-Q-R	GACTTGATGGGCTTGGGCT
NtESR2a-CRISPR test-F	CATCTGTTGGGGCGGTGAGGTAT	检测NtESR2a基因突变
NtESR2a-CRISPR test-R	CTGGTAGTATTGGAGGAATTG	Detection of NtESR2a gene mutation
NtESR2b-CRISPR test-F	CATCTGTCGGGGCGGTGAGGTAC	检测NtESR2b基因突变
NtESR2b-CRISPR test-R	CAAAGAATCATTGCTTTTCTGA	Detection of NtESR2b gene mutation
CUC2-Q-F	TCCTCTGTCGGTGCCAATTC	RT-qPCR检测NtCUC2基因表达量
CUC2-Q-R	TTGGGTCGAAACTGTGCGTA	RT-qPCR detection of NtCUC2 gene expression
LAS-Q-F	ACAGCTGCAGTAAAGGTGCC	RT-qPCR检测NtLAS基因表达量
LAS-Q-R	GCGAGGATACTCTCCCAGCA	RT-qPCR detection of NtLAS gene expression
REV-Q-F	CCACTGGGCCACACAATAGAA	RT-qPCR检测NtREV基因表达量
REV-Q-R	ACAGATCGTGCACTGTAGCC	RT-qPCR detection of NtREV gene expression
YUCCA-Q-F	TGCTAAGTACGACGAAGCTTGT	RT-qPCR检测NtYUCCA基因表达量
YUCCA-Q-R	ATGCCAGAATTTCCACAGCCTA	RT-qPCR detection of NtYUCCA gene expression
PIN1-Q-F	TCGAACCAAGTGAGGGCATC	RT-qPCR检测NtPIN1基因表达量
PIN1-Q-R	AGCTGCAATCAGAGAAGCGA	RT-qPCR detection of NtPIN1 gene expression

图1 NtESR2基因CDS扩增

Fig. 1 Amplification of NtESR2 CDS

表2 NtESR2蛋白的理化性质分析

Table 2 Analysis of the physicochemical properties of NtESR2 proteins

蛋白

Protein

分子式

Formula

分子量

Molecular weight/kD

理论等电点

Theoretical pI

不稳定系数

Instability index

总平均亲水性系数

GRAVY

NtESR2a

C₁₈₇₁H₂₈₉₉N₅₃₃O₆₁₁S₁₈

C₁₈₉₅H₂₉₄₀N₅₃₄O₆₂₀S₁₈

图2 NtESR2蛋白的二级及三级结构预测

Fig. 2 Prediction of secondary and tertiary structures of NtESR2 protein

图3 ESR2蛋白序列比对（A）、系统进化树构建及motif分析（B）NsESR2：林烟草，XP_009765569.1；NtomESR2：绒毛状烟草，XP_009598275.1；SlLFS：番茄，XP_004239483.2；StESR2：马铃薯，XP_006357626.1；ApESR2：穿心莲，XP_051122507.1；HbESR2：橡胶树，XP_021657412.1；AtESR2：拟南芥，NP_173864.1；BnESR2：欧洲油菜，XP_013726908.1；ZmESR2：玉米，XP_008678756.1；ObESR2：短花稻，XP_040377266.1

Fig. 3 ESR2 protein sequence alignment (A), phylogenetic tree construction and motif analysis (B)NsESR2: Nicotiana sylvestris, XP_009765569.1; NtomESR2: Nicotiana tomentosiformis, XP_009598275.1; SlLFS: Solanum lycopersicum, XP_004239483. 2; StESR2: Solanum tuberosum, XP_006357626. 1; ApESR2: Andrographis paniculata, XP_051122507.1; HbESR2: Hevea brasiliensis, XP_021657412.1; AtESR2: Arabidopsis thaliana, NP_173864.1; BnESR2: Brassica napus, XP_013726908.1; ZmESR2: Zea mays, XP_008678756.1; ObESR2: Oryza brachyantha, XP_040377266.1

图4 NtESR2及AtESR2、SlLFS启动子的顺式作用元件分析

Fig. 4 Analysis of cis-acting elements in the promoters of NtESR2, AtESR2 and SlLFS

图5 NtESR2基因在不同组织中的表达分析不同小写字母表示差异达到显著水平（P<0.05）。下同

Fig. 5 Expression analysis of NtESR2 gene in different tissuesDifferent lower letters indicate significant difference (P<0.05). The same below

图6 NtESR2基因在IAA、SA、ABA、MeJA和GA处理后的相对表达量

Fig. 6 Relative expressions of NtESR2 gene after treatment with IAA, SA, ABA, MeJA, and GA

图7 NtESR2基因的亚细胞定位

Fig. 7 Subcellular localization of NtESR2 gene

图8 ntesr2突变体的鉴定

Fig. 8 Identification of ntesr2 mutants

图9 突变体材料和野生型材料表型观察A，C：野生型WT；B，D：ntesr2突变体

Fig. 9 Phenotypic observation of mutant materials and wild-type materialsA, C: Wild type WT; B, D: ntesr2 double copy mutant

图10 ntesr2突变体与野生型WT的基因表达分析*表示显著性P<0.05，**表示显著性P<0.01

Fig. 10 Analysis of gene expression in ntesr2 mutant and wild‑type* indicates significance P<0.05, and ** indicates significance P<0.01

参考文献 31

1	Feng K, Hou XL, Xing GM, et al. Advances in AP2/ERF super-family transcription factors in plant [J]. Crit Rev Biotechnol, 2020, 40(6): 750-776.
2	Xie ZL, Nolan T, Jiang H, et al. The AP2/ERF transcription factor TINY modulates brassinosteroid-regulated plant growth and drought responses in Arabidopsis [J]. Plant Cell, 2019, 31(8): 1788-1806.
3	Owji H, Hajiebrahimi A, Seradj H, et al. Identification and functional prediction of stress responsive AP2/ERF transcription factors in Brassica napus by genome-wide analysis [J]. Comput Biol Chem, 2017, 71: 32-56.
4	Ghorbani R, Zakipour Z, Alemzadeh A, et al. Genome-wide analysis of AP2/ERF transcription factors family in Brassica napus [J]. Physiol Mol Biol Plants, 2020, 26(7): 1463-1476.
5	Chen K, Tang WS, Zhou YB, et al. AP2/ERF transcription factor GmDREB1 confers drought tolerance in transgenic soybean by interacting with GmERFs [J]. Plant Physiol Biochem, 2022, 170: 287-295.
6	Kirch T, Simon R, Grünewald M, et al. The DORNROSCHEN/ENHANCER OF SHOOT REGENERATION1 gene of Arabidopsis acts in the control of meristem cell fate and lateral organ development [J]. Plant Cell, 2003, 15(3): 694-705.
7	Marsch-Martinez N, Greco R, Becker JD, et al. BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathways [J]. Plant Mol Biol, 2006, 62(6): 825-843.
8	Nag A, Yang YZ, Jack T. DORNROSCHEN-LIKE an AP2 gene, is necessary for stamen emergence in Arabidopsis [J]. Plant Mol Biol, 2007, 65(3): 219-232.
9	Ikeda Y, Králová M, Zalabák D, et al. Post-embryonic lateral organ development and adaxial-abaxial polarity are regulated by the combined effect of ENHANCER OF SHOOT REGENERATION 1 and WUSCHEL in Arabidopsis shoots [J]. Int J Mol Sci, 2021, 22(19): 10621.
10	Capua Y, Eshed Y. Coordination of auxin-triggered leaf initiation by tomato LEAFLESS [J]. Proc Natl Acad Sci U S A, 2017, 114(12): 3246-3251.
11	Lu R, Hu SQ, Feng J, et al. The AP2 transcription factor BARE RECEPTACLE regulates floral organogenesis via auxin pathways in woodland strawberry [J]. Plant Cell, 2024, 36(12): 4970-4987.
12	Long JA, Moan EI, Medford JI, et al. A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis [J]. Nature, 1996, 379(6560): 66-69.
13	王兵, 赵会纳, 余婧, 等. 利用CRISPR/Cas9系统研究REVOLUTA参与烟草叶芽发育的调控 [J]. 生物技术通报, 2023, 39(10): 197-208.
	Wang B, Zhao HN, Yu J, et al. Regulation of leaf bud by REVOLUTA in tobacco based on CRISPR/Cas9 system [J]. Biotechnol Bull, 2023, 39(10): 197-208.
14	Zhang C, Wang J, Wenkel S, et al. Spatiotemporal control of axillary meristem formation by interacting transcriptional regulators [J]. Development, 2018, 145(24): dev158352.
15	Greb T, Clarenz O, Schafer E, et al. Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation [J]. Genes Dev, 2003, 17(9): 1175-1187.
16	Hibara KI, Karim MR, Takada S, et al. Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation [J]. Plant Cell, 2006, 18(11): 2946-2957.
17	Tian CH, Zhang XN, He J, et al. An organ boundary-enriched gene regulatory network uncovers regulatory hierarchies underlying axillary meristem initiation [J]. Mol Syst Biol, 2014, 10(10): 755.
18	Yamada M, Tanaka S, Miyazaki T, et al. Expression of the auxin biosynthetic genes YUCCA1 and YUCCA4 is dependent on the boundary regulators CUP-SHAPED COTYLEDON genes in the Arabidopsis thaliana embryo [J]. Plant Biotechnol, 2022, 39(1): 37-42.
19	Lee BH, Jeon JO, Lee MM, et al. Genetic interaction between GROWTH-REGULATING FACTOR and CUP-SHAPED COTYLEDON in organ separation [J]. Plant Signal Behav, 2015, 10(2): e988071.
20	Scofield S, Murison A, Jones A, et al. Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network [J]. Development, 2018, 145(9): dev157081.
21	Spinelli SV, Martin AP, Viola IL, et al. A mechanistic link between STM and CUC1 during Arabidopsis development [J]. Plant Physiol, 2011, 156(4): 1894-1904.
22	Keller T, Abbott J, Moritz T, et al. Arabidopsis REGULATOR OF AXILLARY MERISTEMS1 controls a leaf axil stem cell niche and modulates vegetative development [J]. Plant Cell, 2006, 18(3): 598-611.
23	Yang F, Wang Q, Schmitz G, et al. The bHLH protein ROX acts in concert with RAX1 and LAS to modulate axillary meristem formation in Arabidopsis [J]. Plant J, 2012, 71(1): 61-70.
24	Leitch IJ, Hanson L, Lim KY, et al. The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae) [J]. Ann Bot, 2008, 101(6): 805-814.
25	Matsuo N, Makino M, Banno H. Arabidopsis ENHANCER OF SHOOT REGENERATION (ESR)1 and ESR2 regulate in vitro shoot regeneration and their expressions are differentially regulated [J]. Plant Sci, 2011, 181(1): 39-46.
26	Möller B, Weijers D. Auxin control of embryo patterning [J]. Cold Spring Harb Perspect Biol, 2009, 1(5): a001545.
27	Brandt R, Salla-Martret M, Bou-Torrent J, et al. Genome-wide binding-site analysis of REVOLUTA reveals a link between leaf patterning and light-mediated growth responses [J]. Plant J, 2012, 72(1): 31-42.
28	O'Connor DL, Runions A, Sluis A, et al. A division in PIN-mediated auxin patterning during organ initiation in grasses [J]. PLoS Comput Biol, 2014, 10(1): e1003447.
29	Gälweiler L, Guan C, Müller A, et al. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue [J]. Science, 1998, 282(5397): 2226-2230.
30	Cheng YF, Dai XH, Zhao YD. Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis [J]. Plant Cell, 2007, 19(8): 2430-2439.
31	Cao X, Yang HL, Shang CQ, et al. The roles of auxin biosynthesis YUCCA gene family in plants [J]. Int J Mol Sci, 2019, 20(24): 6343.

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