青稞HvNAC53的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0002

摘要/Abstract

摘要：

目的 NAC（NAM/ATAF1/2/CUC2）转录因子作为植物中一类重要的调控蛋白，广泛参与植物的生长发育、生物胁迫和非生物胁迫响应。克隆青稞HvNAC53基因，分析其序列特性和表达模式，并进行功能验证，为青稞抗逆分子育种提供理论依据。方法以‘昆仑14号’为材料克隆HvNAC53基因，通过生物信息学及RT-qPCR方法，分析HvNAC53基因表达和蛋白特性，利用拟南芥异源过表达方法研究其在低温、干旱胁迫中的功能。结果 HvNAC53基因开放阅读框全长1 550 bp，编码305个氨基酸，无跨膜结构域和信号肽，属于亲水性不稳定蛋白。启动子区域存在多个响应光、激素和胁迫的顺式作用元件。系统进化分析表明，HvNAC53与长穗偃麦草TeNAC53亲缘关系最近；RT-qPCR结果显示，HvNAC53基因表达水平在根和叶中相对较高，且能被干旱和低温胁迫显著诱导上调表达。亚细胞定位结果显示，HvNAC53蛋白定位于细胞核。此外，低温、干旱胁迫下的功能验证结果显示，HvNAC53拟南芥异源过表达转基因株系耐冷性和耐旱性显著高于野生型。结论 HvNAC53在青稞低温、干旱胁迫响应中具有正调控作用。

关键词: 青稞, HvNAC53, 低温, 干旱, 异源过表达, 亚细胞定位

Abstract:

Objective NAC (NAM/ATAF1/2/CUC2) transcription factors are a crucial class of regulatory proteins in plants, extensively involved in growth, development, and responses to biotic and abiotic stresses. This study aimed to clone the HvNAC53 gene from hulless barley (Hordeum vulgare var. nudum), analyze its sequence characteristics and expression patterns, and functionally characterize it, thereby providing a theoretical basis for stress-tolerant molecular breeding in hulless barley. Methods The HvNAC53 gene was cloned from the hulless barley cultivar ‘Kunlun 14’. Bioinformatics analysis and RT-qPCR were used to analyze gene expression and protein characteristics. The role of HvNAC53 in response to low-temperature and drought stress was investigated through heterologous overexpression in Arabidopsis thaliana. Results The open reading frame of HvNAC53 is 1 550 bp in length, encoding a protein of 305 amino acids. The protein lacks transmembrane domains and signal peptides and is classified as a hydrophilic, unstable protein. Multiple cis-acting elements responsive to light, hormones, and stress were identified in the promoter region of HvNAC53. Phylogenetic analysis indicated that HvNAC53 is most closely related to TeNAC53 from Thinopyrum elongatum (tall wheatgrass). RT-qPCR results revealed high expression levels of HvNAC53 in roots and leaves compared to other tissues, and its expression was significantly induced by drought and cold stress. Subcellular localization demonstrated that HvNAC53 is localized in the nucleus. Furthermore, Arabidopsis thaliana lines heterologously overexpressing HvNAC53 exhibited significantly higher cold and drought tolerance than wild-type plants. Conclusion HvNAC53 positively regulates low-temperature and drought stress responses in hulless barley.

Key words: hulless barley, HvNAC53, low temperature, drought, heterologous overexpression, subcellular localization

何婧婧, 朋毛拉专, 姚晓华, 姚有华, 吴昆仑, 崔永梅. 青稞HvNAC53的克隆及功能分析[J]. 生物技术通报, 2026, 42(8): 142-152.

HE Jing-jing, PENG Mao-la-zhuan, YAO Xiao-hua, YAO You-hua, WU Kun-lun, CUI Yong-mei. Cloning and Functional Characterization of HvNAC53 from Hulless Barley[J]. Biotechnology Bulletin, 2026, 42(8): 142-152.

图/表 7

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元件 Site name	序列 Sequence	功能 Function	数量 Amount
Box 4	ATTAAT	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	3
LTR	CCGAAA	参与低温响应性的顺式作用元件 Cis-acting element involved in low-temperature responsiveness	1
TATA-box	ATTATA；TATAA；TATA；TATAAA	启动子核心元件 Core promoter element	12
TGACG-motif	TGACG	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	3
GC-motif	CCCCCG	参与缺氧特异性诱导的增强子样元件 Enhancer-like element involved in anoxic specific inducibility	1
CGTCA-motif	CGTCA	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	3
A-box	CCGTCC	顺式作用调节元件 Cis-acting regulatory element	4
MBS	CAACTG	MYB结合位点参与干旱诱导 MYB binding site involved in drought-inducibility	3
I-box	gGATAAGGTG	光响应元件 Part of a light responsive element	1
G-box	CACGTC；TACGTG；CACGAC； GCCACGTGGA；CACGTG； ACACGTGT；TCCACATGGCA；	参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness	18
GA-motif	ATAGATAA	光响应元件 Part of a light responsive element	1
LAMP-element	CCTTATCCA	光响应元件 Part of a light responsive element	1
TCA-element	CCATCTTTTT	参与水杨酸反应性的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness	1
ABRE	ACGTG；CGTACGTGCA； CACGTG； GACACGTGGC； CGCACGTGTC	参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness	15
CAAT-box	CCAAT；TGCCAAC；CAAAT	启动子和增强子区域中的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions	6
MRE	AACCTAA	MYB结合位点参与光响应 MYB binding site involved in light responsiveness	1
ACE	GACACGTATG	参与光响应的顺式作用调节元件 Cis-acting element involved in light responsiveness	1

元件 Site name	序列 Sequence	功能 Function	数量 Amount
Box 4	ATTAAT	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	3
LTR	CCGAAA	参与低温响应性的顺式作用元件 Cis-acting element involved in low-temperature responsiveness	1
TATA-box	ATTATA；TATAA；TATA；TATAAA	启动子核心元件 Core promoter element	12
TGACG-motif	TGACG	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	3
GC-motif	CCCCCG	参与缺氧特异性诱导的增强子样元件 Enhancer-like element involved in anoxic specific inducibility	1
CGTCA-motif	CGTCA	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	3
A-box	CCGTCC	顺式作用调节元件 Cis-acting regulatory element	4
MBS	CAACTG	MYB结合位点参与干旱诱导 MYB binding site involved in drought-inducibility	3
I-box	gGATAAGGTG	光响应元件 Part of a light responsive element	1
G-box	CACGTC；TACGTG；CACGAC； GCCACGTGGA；CACGTG； ACACGTGT；TCCACATGGCA；	参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness	18
GA-motif	ATAGATAA	光响应元件 Part of a light responsive element	1
LAMP-element	CCTTATCCA	光响应元件 Part of a light responsive element	1
TCA-element	CCATCTTTTT	参与水杨酸反应性的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness	1
ABRE	ACGTG；CGTACGTGCA； CACGTG； GACACGTGGC； CGCACGTGTC	参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness	15
CAAT-box	CCAAT；TGCCAAC；CAAAT	启动子和增强子区域中的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions	6
MRE	AACCTAA	MYB结合位点参与光响应 MYB binding site involved in light responsiveness	1
ACE	GACACGTATG	参与光响应的顺式作用调节元件 Cis-acting element involved in light responsiveness	1

蛋白名称 Protein name	注释 Annotation
M0XWR0_HORVV	含NAC结构域的蛋白
M0ZFS5_HORVV	WD_REPEATS_REGION含有结构域的蛋白质
A0A287LB51	EGF_CA含有结构域的蛋白质
A0A287IMF4	Ipi1_N含有结构域的蛋白质
F2DHF8_H0RVV	Pescadillo同源物；核糖体RNA成熟和大核糖体亚基形成所必需的
A0A287EA93	核糖体生物发生蛋白BOP1同源物；核糖体RNA成熟和大核糖体亚基形成所必需的；属于WD重复 BOP1/ERB1 家族
M0XXL5_HORVV	生长素反应因子
M0XAA8_HORVV	生长素反应因子
A0A287TNV8	生长素反应因子

蛋白名称 Protein name	注释 Annotation
M0XWR0_HORVV	含NAC结构域的蛋白
M0ZFS5_HORVV	WD_REPEATS_REGION含有结构域的蛋白质
A0A287LB51	EGF_CA含有结构域的蛋白质
A0A287IMF4	Ipi1_N含有结构域的蛋白质
F2DHF8_H0RVV	Pescadillo同源物；核糖体RNA成熟和大核糖体亚基形成所必需的
A0A287EA93	核糖体生物发生蛋白BOP1同源物；核糖体RNA成熟和大核糖体亚基形成所必需的；属于WD重复 BOP1/ERB1 家族
M0XXL5_HORVV	生长素反应因子
M0XAA8_HORVV	生长素反应因子
A0A287TNV8	生长素反应因子