茄子SmWRKY4的克隆及耐冷性功能验证

doi:10.16420/j.issn.0513-353x.2023-0880

摘要/Abstract

摘要：

前期研究发现茄子SmWRKY4的表达受到冷信号抑制。进一步以‘成都墨茄’为材料克隆出SmWRKY4全长序列，分析结果显示其开放阅读框为1 518 bp，编码505个氨基酸，含有2个WRKY结构域，属于第Ⅰ类WRKY转录因子。SmWRKY4启动子含有茉莉酸甲酯和水杨酸等激素响应元件及缺氧和低温等逆境胁迫响应元件。系统进化树分析表明，SmWRKY4与番茄和马铃薯的同源蛋白在进化关系上更近。表达模式分析显示SmWRKY4能够响应脱落酸、水杨酸和茉莉酸甲酯等外源植物生长调节剂和冷胁迫，并在茄子茎和花药中高表达。敲除SmWRKY4的突变体能够增强光合作用，降低细胞通透性，提高耐冷性。推测SmWRKY4负调控茄子耐冷性。

关键词: 茄子, SmWRKY4, 耐冷性, 克隆, 功能验证, 光合作用, 细胞通透性, 激素

Abstract:

Previous study showed that the expression level of SmWRKY4 was inhibited by cold stress. The full-length sequence of SmWRKY4 was cloned from‘Chengdu Moqie’in this study. Analysis results revealed that the open reading frame of the SmWRKY4 gene was 1 518 bp，encoding 505 amino acids which contained two WRKY domains. Consequently，SmWRKY4 protein belonged to group Ⅰ WRKY transcription factor. SmWRKY4 promoter contained hormone-response elements including MeJA and SA and stress-response elements including oxygen deficit and low temperature. Phylogenetic tree analysis suggested that SmWRKY4 protein was more closely related to its homologous proteins in tomato and potato. Expression pattern analysis showed that SmWRKY4 could respond to ABA，SA，MeJA and cold stress，and was highly expressed in eggplant stem and anther. SmWRKY4 mutation could enhance photosynthesis，reduce cell permeability and improve cold tolerance. These results suggested that SmWRKY4 negatively regulated cold tolerance in eggplant.

Key words: eggplant, SmWRKY4, cold tolerance, gene cloning, functional verification, photosynthesis, cell permeability, hormones

杨艳, 刘军, 周晓慧, 刘松瑜, 庄勇. 茄子SmWRKY4的克隆及耐冷性功能验证[J]. 园艺学报, 2025, 52(1): 101-110.

YANG Yan, LIU Jun, ZHOU Xiaohui, LIU Songyu, ZHUANG Yong. Cloning and Functional Verification of SmWRKY4 Gene in Cold Tolerance of Solanum melongena[J]. Acta Horticulturae Sinica, 2025, 52(1): 101-110.

图/表 9

表1 本研究中所用的引物

Table 1 Primers used in this study

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因克隆 Gene cloning	SmWRKY4-CDS F	TACAAATCTATCTCTCTCGAGATGGATGACGAAATCGACAGCT
基因克隆 Gene cloning	SmWRKY4-CDS R	GCCCTTGCTCACCATGGATCCGCAAGGCTTTATTTCAAATCCAA
启动子克隆 Promoter cloning	SmWRKY4-pro F	CTGGGTTCCCGACGAAGAAT
启动子克隆 Promoter cloning	SmWRKY4-pro R	GATTCCGACGAGCACACATTG
基因检测 Gene detection	35S-F	GATGTGACATCTCCACTGACGT
基因检测 Gene detection	SmWRKY4 5′ Reverse	TGATGTTCCGTTCGTGCTTG
实时荧光定量PCR qRT-PCR	SmWRKY4 qRT-PCR F	GTGATGGGTACAATTGGCGG
	SmWRKY4 qRT-PCR R	ACTTTTGCCATTGGTAGGCG
	SmActin qRT-PCR F	GCAGCTCCTCCATCGAAAAG
	SmActin qRT-PCR R	CCGATCAGCAATACCAGGG

图1 SmWRKY4的克隆

Fig. 1 The cloning of SmWRKY4

图2 WRKY4同源蛋白系统进化树

Fig. 2 Phylogenetic tree of WRKY4 homologous proteins

图3 茄子SmWRKY4在不同组织的表达模式用ANOVA进行显著性分析，不同小写英文字母表示组间具有显著差异（P < 0.05）。下同

Fig. 3 The expression pattern of SmWRKY4 in different tissues Significance analyses were performed using ANOVA. Different lowercase letters indicate a significant difference between different tissues（P < 0.05）. The same below

图4 SmWRKY4在不同外源生长调节剂处理下的表达模式

Fig. 4 The expression pattern of SmWRKY4 under treatments with different exogenous growth regulators

图5 SmWRKY4在4 ℃冷胁迫下的表达模式

Fig. 5 The expression pattern of SmWRKY4 under cold treatment

图6 常温和冷胁迫下茄子野生型（WT）和wrky4-3突变体的表型

Fig. 6 The phenotypes of wild type and mutant wrky4-3 under normal growth temperature and cold stress

图7 冷胁迫7 d对茄子野生型（WT）和突变体wrky4-3植株叶绿素相对含量（A）和Fv/Fm（B）的影响

Fig. 7 Effects of cold stress for 7 d on chlorophyll content（A）and Fv/Fm（B）of wild-type（WT）and SmWRKY4 gene mutation

图8 冷胁迫7 d对茄子野生型（WT）和突变体wrky4-3植株相对电导率的影响

Fig. 8 Effects of cold stress for 7 d on relative electrolytic leakage of wild-type（WT）and SmWRKY4 gene mutation

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