Cloning and Functional Analysis of Citrullus lanatus Ovate Family Protein Gene ClOFP16

doi:10.16420/j.issn.0513-353x.2025-0571

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (5): 1329-1342.doi: 10.16420/j.issn.0513-353x.2025-0571

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

Cloning and Functional Analysis of Citrullus lanatus Ovate Family Protein Gene ClOFP16

CAI Zeyu, LI Ai, ZHU Jiawei, DENG Man, JIN Yuxuan, ZHANG Weihua^*(), WU Ying^*()

College of Horticulture and Landscape， Tianjin Agricultural University， Tianjin 300384， China

Received:2025-09-28 Revised:2026-01-21 Online:2026-05-25 Published:2026-05-26
Contact: ZHANG Weihua, WU Ying

Abstract

Abstract:

Ovate family proteins（OFP）constitute a class of plant-specific transcription factors implicated in intracellular signal transduction and biosynthetic processes in plants. Utilizing the watermelon cultivar‘TN07011’，the coding sequence（CDS）of ClOFP16 were isolated and performed functional characterization. Results demonstrate that ClOFP16 comprises 1 020 base pairs，encoding a protein of 339 amino acids. The encoded protein harbors an N-terminal DNA-binding domain and a C-terminal conserved Ovate domain. Subcellular localization assays confirmed the nuclear localization of ClOFP16. RT-qPCR analysis demonstrated that ClOFP16 transcript abundance was significantly upregulated under salt and 4 ℃ cold stress，but downregulated under drought stress. Arabidopsis thaliana overexpressing ClOFP16 exhibited reduced seed longitudinal diameter in T₃ generation. Under salt and low-temperature stress conditions，T₃ generation plants exhibited a significant upregulation of ClOFP16 expression. Concurrently，the activities of the antioxidant enzymes SOD，POD，and CAT were markedly increased. Furthermore，total chlorophyll content and soluble protein content were significantly elevated，while the accumulation of malondialdehyde（MDA）was reduced. Collectively，these findings implicate ClOFP16 in pivotal roles governing plant growth and development，as well as in mediating adaptive responses to abiotic stress.

Key words: watermelon, transcription factor, ClOFP16, abiotic stress, functional verification

CAI Zeyu, LI Ai, ZHU Jiawei, DENG Man, JIN Yuxuan, ZHANG Weihua, WU Ying. Cloning and Functional Analysis of Citrullus lanatus Ovate Family Protein Gene ClOFP16[J]. Acta Horticulturae Sinica, 2026, 53(5): 1329-1342.

Figures/Tables 12

Table 1 Primers for PCR and RT-qPCR of ClOFP16 gene

引物名称 Primer name	引物序列（5'-3'） Primer sequences	引物用途 Primer purpose
ClOFP16-F	ATGAAGAAGCAAAGGCAAAAAAG	目标基因克隆
ClOFP16-R	CTAATAATGAACTCCAATATCC	Target gene cloning
ClOFP16-MQ-F	GGGGTACCATGAAGAAGCAAAGGCAAAAAAG	植物过表达载体构建
ClOFP16-MQ-R	GCGTCGACCTAATAATGAACTCCAATATCC	Construction of plant overexpression vector
ClOFP16-Y-R	GCGTCGACATAATGAACTCCAATATCCGTCATG	亚细胞定位载体构建 Construction of subcellular localization vector
ClOFP16-Q-F	AAAAGAACCGTGGTGGGC
ClOFP16-Q-R	TGTGATCTGGGCGTGAAA
Atactin-F	GCCATCCAAGCTGTTCTCTC	内参引物
Atactin-R	GCTCGTAGTCAACAGCAACAA	Reference gene
ClACTIN-F	TTGTGTTGGACTCTGGTGATGG
ClACTIN-R	CCGTTCAGCAGTGGTGGTG

Fig. 1 Phylogenetic tree of OFP（A），conserved motifs（B），and sequence characteristics of conserved motifs（C）

Fig. 2 Multi sequence alignment of ClOFP with OFP from other species

Fig. 3 Subcellular localization of ClOFP16 in tobacco epidermis

Fig. 4 Expression patterns of ClOFP16 under stress treatments（A，B，C）and in different tissues（D） A：Salt；B：Drought；C：Low temperature；D：Different tissues. * and ** indicate significant differences at P < 0.05，P < 0.01 respectively；ns indicates no significant difference. The same below

Fig. 5 Identification of Arabidopsis thaliana overexpressing ClOFP16 strain（OE#）

Fig. 6 Comparison of average longitudinal diameter length of Arabidopsis seeds

Fig. 7 Phenotypes of OE#1，OE#2，and OE#3 in T3 Arabidopsis thaliana under abiotic stresses for 14 d

Fig. 8 Overexpression of ClOFP16 gene expression in Arabidopsis under abiotic stresses

Fig. 9 Observation of NBT and DAB staining in Arabidopsis thaliana under salt stress（A），drought stress（B），and low temperature stress（C）

Fig. 10 SOD，POD，and CAT enzyme activities in OE#ClOFP16 strain under abiotic stress

Fig. 11 Analysis of MDA，SP，and Chl content in the OE#ClOFP16 strain under abiotic stress

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Cloning and Functional Analysis of Citrullus lanatus Ovate Family Protein Gene ClOFP16

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