烟草ARF-GEF基因NtGNL2a启动子的克隆与表达分析

doi:10.3969/j.issn.1004-1524.20240850

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (10): 2032-2041.DOI: 10.3969/j.issn.1004-1524.20240850

烟草ARF-GEF基因NtGNL2a启动子的克隆与表达分析

金鑫¹^,²(), 林瑞², 刘岩², 许嘉盛²^,³, 陈琼琳², 袁璐², 薛大伟¹, 郑鹏²^,^*(), 徐盛春²^,⁴

1.杭州师范大学生命与环境科学学院,浙江杭州311121
2.湘湖实验室,浙江杭州311231
3.长江大学农学院,湖北荆州434025
4.浙江省农业科学院数字农业研究所,浙江杭州 310021

收稿日期:2024-10-08 出版日期:2025-10-25 发布日期:2025-11-13
作者简介:金鑫(2001—),女,河南驻马店人,硕士研究生,主要从事生物合成与植物代谢研究。E-mail:jxinaaa@163.com
通讯作者: *郑鹏,E-mail:zhengpeng@xhlab.ac.cn
基金资助:
国家自然科学基金(32401217);浙江省“三农九方”科技协作计划项目(2024SNJF037)

Promoter cloning and expression analysis of the ARF-GEF gene NtGNL2a from Nicotiana tabacum

JIN Xin¹^,²(), LIN Rui², LIU Yan², XU Jiasheng²^,³, CHEN Qionglin², YUAN Lu², XUE Dawei¹, ZHENG Peng²^,^*(), XU Shengchun²^,⁴

1. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
2. Xianghu Laboratory, Hangzhou 311231, China
3. College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China
4. Institute of Digital Agriculture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2024-10-08 Online:2025-10-25 Published:2025-11-13

摘要/Abstract

摘要：

ADP核糖基化因子鸟嘌呤核苷酸交换因子(ADP-ribosylation factor guanine nucleotide exchange factor, ARF-GEF)基因家族在植物细胞中调控囊泡运输和细胞骨架动态变化,对细胞极性生长至关重要。本研究以烟草品种中烟100(Nicotiana tabacum cv. Zhongyan 100)为材料,通过生物信息学分析发现,烟草基因组含有10个ARF-GEF亚家族成员,系统进化分析表明,NtGNL2a与拟南芥AtGNL2为同源基因。组织表达模式分析结果显示,NtGNL2a在烟草花中特异性表达,其在花粉中表达量最高,在其他组织中痕量表达。构建pCambia1301-NtGNL2apro::GUS植物表达载体并转化野生型中烟100,GUS组织化学染色结果表明,NtGNL2a启动子驱动GUS报告基因在转基因烟草花粉中特异性表达。本研究为后续开展NtGNL2a基因的功能研究提供了依据,同时为利用基因工程技术驱动目的基因在花粉中特异表达提供了新的调控元件。

关键词: 烟草, NtGNL2a, 花粉特异性表达, GUS组织化学染色

Abstract:

The ADP-ribosylation factor guanine nucleotide exchange factor (ARF-GEF) gene family plays a crucial role in regulating vesicle transport and cytoskeletal dynamics in plant cells, which is essential for polarized cell growth. In this study, using the tobacco variety Zhongyan 100 (Nicotiana tabacum cv. Zhongyan 100), we identified a total of 10 ARF-GBF subfamily members in the tobacco genome through bioinformatics methods. Phylogenetic analysis revealed that NtGNL2a is a homolog of the Arabidopsis AtGNL2. The tissue expression pattern analysis showed that NtGNL2a is specifically expressed in tobacco flower, with the highest expression in pollen and trace expression in other tissues. A recombinant plant expression vector, pCambia1301-NtGNL2apro::GUS, was constructed and transformed into the wild-type Zhongyan 100. GUS histochemical staining results indicated that the NtGNL2a promoter drives GUS gene expression specifically in the pollen of transgenic tobacco. This study provides a basis for subsequent functional studies of the NtGNL2a gene and offers a new regulatory element for driving the specific expression of target genes in pollen through genetic engineering techniques.

Key words: Nicotiana tabacum, NtGNL2a, pollen specific expression, GUS histochemical staining

中图分类号:

S572

金鑫, 林瑞, 刘岩, 许嘉盛, 陈琼琳, 袁璐, 薛大伟, 郑鹏, 徐盛春. 烟草ARF-GEF基因NtGNL2a启动子的克隆与表达分析[J]. 浙江农业学报, 2025, 37(10): 2032-2041.

JIN Xin, LIN Rui, LIU Yan, XU Jiasheng, CHEN Qionglin, YUAN Lu, XUE Dawei, ZHENG Peng, XU Shengchun. Promoter cloning and expression analysis of the ARF-GEF gene NtGNL2a from Nicotiana tabacum[J]. Acta Agriculturae Zhejiangensis, 2025, 37(10): 2032-2041.

图/表 8

参考文献 22

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引物名称 Primer name	序列(5'→3') Primer sequence (5'→3')	功能 Function
NtGNL2a-CDS-F	GGTTTTGATGTCTTGAGATGATTAATG	序列验证Sequence verification
NtGNL2a-CDS-R	CTGTTCATCACAACACTCTAATCA	序列验证Sequence verification
NtGNL2a-RTF	GTTTTGCTCTTGGTGAGGATT	表达量检测Expression level detection
NtGNL2a-RTR	CTTCTTCAACAGCATAGGGATAG	表达量检测Expression level detection
NtEF1a-RTF	CTGCAACAAGATGGATGCTAC	表达量检测Expression level detection
NtEF1a-RTR	ACCAACAGGGACAGTACCAAT	表达量检测Expression level detection
NtGNL2apro-EASY-F	CAATTTGTCTAATATTGTTCAAT	启动子克隆Promoter cloning
NtGNL2apro-EASY-R	TAATCATCTCAAGACATCAAA	启动子克隆Promoter cloning
NtGNL2apro-F	acgacggccagtgcc aagcttCAATTTGTCTAATATTGTTCAATGTCCT	植物表达载体构建 Construction of plant expression vector
NtGNL2apro-R	aaatttaccctcagatcta ccatggTAATCATCTCAAGACATCAAAACCATCC	植物表达载体构建 Construction of plant expression vector
GUS-test-F	AAGGGATCTTCACTCGCGAC	阳性鉴定Positive identification
M13R-48	AGCGGATAACAATTTCACACAGGA	阳性鉴定Positive identification

引物名称 Primer name	序列(5'→3') Primer sequence (5'→3')	功能 Function
NtGNL2a-CDS-F	GGTTTTGATGTCTTGAGATGATTAATG	序列验证Sequence verification
NtGNL2a-CDS-R	CTGTTCATCACAACACTCTAATCA	序列验证Sequence verification
NtGNL2a-RTF	GTTTTGCTCTTGGTGAGGATT	表达量检测Expression level detection
NtGNL2a-RTR	CTTCTTCAACAGCATAGGGATAG	表达量检测Expression level detection
NtEF1a-RTF	CTGCAACAAGATGGATGCTAC	表达量检测Expression level detection
NtEF1a-RTR	ACCAACAGGGACAGTACCAAT	表达量检测Expression level detection
NtGNL2apro-EASY-F	CAATTTGTCTAATATTGTTCAAT	启动子克隆Promoter cloning
NtGNL2apro-EASY-R	TAATCATCTCAAGACATCAAA	启动子克隆Promoter cloning
NtGNL2apro-F	acgacggccagtgcc aagcttCAATTTGTCTAATATTGTTCAATGTCCT	植物表达载体构建 Construction of plant expression vector
NtGNL2apro-R	aaatttaccctcagatcta ccatggTAATCATCTCAAGACATCAAAACCATCC	植物表达载体构建 Construction of plant expression vector
GUS-test-F	AAGGGATCTTCACTCGCGAC	阳性鉴定Positive identification
M13R-48	AGCGGATAACAATTTCACACAGGA	阳性鉴定Positive identification

分类 Classification	名称 Name	功能 Function	数量 Quantity
光响应 Light response	Box 4	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness	4
	G-box	参与光反应的顺式作用调控元件cis-acting regulatory element involved in light responsiveness	3
	GT1-motif	光响应元件Light responsive element	2
	TCT-motif	光响应元件的一部分Part of a light responsive element	1
激素响应 Hormone response	ABRE	参与脱落酸响应的顺式作用元件cis-acting element involved in the abscisic acid responsiveness	1
	CGTCA-motif	参与茉莉酸甲酯(MeJA)响应的顺式作用调控元件 cis-acting regulatory element involved in the MeJA-responsiveness	2
	TCA-element	参与水杨酸响应的顺式作用元件cis-acting element involved in salicylic acid responsiveness	1
胁迫响应 Stress response	LTR	参与低温响应的顺式作用元件cis-acting element involved in low-temperature responsiveness	1
	ARE	对厌氧诱导至关重要的顺式作用调控元件 cis-acting regulatory element essential for the anaerobic induction	1
	TC-rich repeats	参与防御和应激反应的顺式作用元件 cis-acting element involved in defense and stress responsiveness	1
生长发育 Growth and development	O2-site	参与玉米醇溶蛋白代谢调控的顺式作用调控元件 cis-acting regulatory element involved in zein metabolism regulation	1

分类 Classification	名称 Name	功能 Function	数量 Quantity
光响应 Light response	Box 4	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness	4
	G-box	参与光反应的顺式作用调控元件cis-acting regulatory element involved in light responsiveness	3
	GT1-motif	光响应元件Light responsive element	2
	TCT-motif	光响应元件的一部分Part of a light responsive element	1
激素响应 Hormone response	ABRE	参与脱落酸响应的顺式作用元件cis-acting element involved in the abscisic acid responsiveness	1
	CGTCA-motif	参与茉莉酸甲酯(MeJA)响应的顺式作用调控元件 cis-acting regulatory element involved in the MeJA-responsiveness	2
	TCA-element	参与水杨酸响应的顺式作用元件cis-acting element involved in salicylic acid responsiveness	1
胁迫响应 Stress response	LTR	参与低温响应的顺式作用元件cis-acting element involved in low-temperature responsiveness	1
	ARE	对厌氧诱导至关重要的顺式作用调控元件 cis-acting regulatory element essential for the anaerobic induction	1
	TC-rich repeats	参与防御和应激反应的顺式作用元件 cis-acting element involved in defense and stress responsiveness	1
生长发育 Growth and development	O2-site	参与玉米醇溶蛋白代谢调控的顺式作用调控元件 cis-acting regulatory element involved in zein metabolism regulation	1

烟草ARF-GEF基因NtGNL2a启动子的克隆与表达分析

Promoter cloning and expression analysis of the ARF-GEF gene NtGNL2a from Nicotiana tabacum

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