西瓜卵形家族蛋白编码基因ClOFP16的克隆及功能研究

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

摘要/Abstract

摘要：

卵形家族蛋白（ovate family proteins，OFP）是一类植物中特有的转录因子，参与植物细胞内的信号转导和生物合成。以西瓜‘TN07011’为材料，克隆ClOFP16的编码基因（ClOFP16，Cla97C10G206030.1）并对其进行功能分析。结果表明：ClOFP16为1 020 bp，共编码339个氨基酸，其蛋白N端含有DNA结合结构域，C端含有Ovate保守结构域。亚细胞定位结果显示ClOFP16定位于细胞核中。利用RT-qPCR分析发现，ClOFP16在盐和4 ℃低温胁迫下上调表达，在干旱胁迫中下调表达。在拟南芥中过表达ClOFP16，T₃代拟南芥种子纵径减小；T₃代植株在盐和低温胁迫下ClOFP16表达量显著上调，且其SOD、POD和CAT酶活性显著上升，叶绿素和可溶性蛋白含量显著提高，而MDA的累积降低。综上，ClOFP16在植物生长发育和非生物胁迫响应中具有重要功能。

关键词: 西瓜, 转录因子, ClOFP16, 非生物胁迫, 功能验证

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

蔡泽宇, 李爱, 朱佳伟, 邓满, 靳羽璇, 张卫华, 吴颖. 西瓜卵形家族蛋白编码基因ClOFP16的克隆及功能研究[J]. 园艺学报, 2026, 53(5): 1329-1342.

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.

图/表 12

表1 ClOFP16基因PCR及RT-qPCR所用引物

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

图1 OFP的系统发育进化树（A）、保守基序（B）和保守基序的序列特征（C）

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

图2 ClOFP与其他物种OFP的多序列比对

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

图3 ClOFP16在烟草表皮中的亚细胞定位

Fig. 3 Subcellular localization of ClOFP16 in tobacco epidermis

图4 ClOFP16在胁迫（A、B、C）和不同组织（D）中的表达模式 A：盐胁迫；B：干旱胁迫；C：低温胁迫；D：不同组织。*和**分别表示在P < 0.05、P < 0.01 下存在显著差异；ns表示无显著差异。下同

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

图5 拟南芥过表达ClOFP16株系（OE#）的鉴定

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

图6 拟南芥种子平均纵径长度比较

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

图7 非生物胁迫下14 d的T3代拟南芥OE#1、OE#2和OE#3的表型

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

图8 非生物胁迫下过表达ClOFP16拟南芥中基因表达量

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

图9 拟南芥在盐胁迫（A）、干旱胁迫（B）和低温胁迫（C）下的NBT和DAB染色观察

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

图10 非生物胁迫下OE#ClOFP16株系中SOD、POD和CAT酶活性

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

图11 非生物胁迫下OE#ClOFP16株系的丙二醛、可溶性蛋白和叶绿素含量

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

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