Cloning and Functional Verification of SmWRKY4 Gene in Cold Tolerance of Solanum melongena

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

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (1): 101-110.doi: 10.16420/j.issn.0513-353x.2023-0880

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Cloning and Functional Verification of SmWRKY4 Gene in Cold Tolerance of Solanum melongena

YANG Yan¹^,², LIU Jun², ZHOU Xiaohui², LIU Songyu², ZHUANG Yong¹^,²^,^*()

¹ College of Horticulture，Nanjing Agricultural University，Nanjing 210095，China
² Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement，Institute of Vegetable Crops，Jiangsu Academy of Agricultural Sciences，Nanjing 210014，China

Received:2024-09-01 Revised:2024-10-21 Online:2025-01-25 Published:2025-01-19
Contact: ZHUANG Yong

Abstract

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

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.

Figures/Tables 9

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

Fig. 1 The cloning of SmWRKY4

Fig. 2 Phylogenetic tree of WRKY4 homologous proteins

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

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

Fig. 5 The expression pattern of SmWRKY4 under cold treatment

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

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

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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Cloning and Functional Verification of SmWRKY4 Gene in Cold Tolerance of Solanum melongena

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