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| 黄瓜转录因子CsWIP1互作蛋白的筛选与鉴定 |
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罗小明1, 李杰林1, 付朝婷1, 王晓敏1,2,3,4, 李建设1,2,3,4, 李征5, 郭亚路1,2,3,4
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1.宁夏大学 葡萄酒与园艺学院，宁夏 银川 750021;2.农业农村部园艺作物节水抗逆高效生产创新重点实验室（省部共建），宁夏 银川 750021;3.宁夏现代设施园艺工程技术研究中心，宁夏 银川 750021;4.宁夏优势特色作物现代分子育种重点实验室，宁夏 银川 750021;5.西北农林科技大学 园艺学院，陕西 杨凌 712100
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| 摘要: |
| 【目的】筛选黄瓜隐性全雌控制基因g（CsWIP1）编码蛋白的互作蛋白，为进一步阐明其影响黄瓜性别 决定的分子机制奠定基础。【方法】基于本实验室已构建的黄瓜顶芽酵母双杂交cDNA文库，使用同源重组法构建CsWIP1-pGBKT7诱饵表达载体，检测其自激活活性后，将CsWIP1-pGBKT7与黄瓜cDNA文库进行酵母双杂交筛 选；对获得的阳性克隆进行检测及BLAST分析，筛选CsWIP1蛋白的互作蛋白；对筛选到的互作蛋白CsIDD-3的氨基酸序列进行分析，并构建系统进化树。对CsWIP1与CsIDD-3的互作关系进行酵母双杂交（Y2H）点对点回转验证、双 分子荧光互补（BiFC）验证和荧光素酶互补成像（LCI）验证。【结果】诱饵表达载体CsWIP1-pGBKT7构建成功，该载体无自激活活性；筛选到的互作蛋白包括热激蛋白类、组蛋白类、蛋白激酶类和转录因子类等，选取CsIDD-3进行后续试 验；CsIDD-3基因的CDS序列编码363个氨基酸，具有2个C2H2型锌指蛋白结构域，IDD-3基因在其他物种中的氨基酸 序列相对保守，黄瓜IDD-3基因与甜瓜的亲缘关系最近。互作验证试验表明，CsWIP1与CsIDD-3存在互作关系。【结论】推测CsWIP1与CsIDD-3蛋白互作参与黄瓜的性型分化通路，CsIDD-3基因可能具有调控雌雄花比例的功能。 |
| 关键词: 黄瓜 性型分化 CsWIP1 转录因子 互作蛋白 |
| DOI：10. 13207/j. jnwafu. 2027. 01. 001 |
| 分类号: |
| 基金项目:宁夏自然科学基金项目（2024AAC03122）；中央引导地方专项（2024FRD05010）；宁夏回族自治区科技创新团队项目（2024CX-TD010） |
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| Screening and identification of proteins interacting with the CsWIP1 transcription factor in cucumber |
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Luo Xiaoming1, Li Jielin1, Fu Chaoting1, Wang Xiaomin1,2,3,4,5, Li Jianshe1,2,3,4,5, Li Zheng5, Guo Yalu1,2,3,4,5
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1.School of Enology and Horticulture，Ningxia University，Yinchuan，Ningxia 750021，China;2.Key Laboratory of Water-Saving and Stress-Resistant High-Efficiency Production Innovation for Horticultural Crops，Ministry of Agriculture and Rural Affairs（Provincial-Ministerial Co-Construction），Yinchuan，Ningxia 750021，China;3.Ningxia Modern Facility Horticulture Engineering Technology Research Center，Yinchuan，Ningxia 750021，China;4.Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia，Yinchuan，Ningxia 750021，China;5.College of Horticulture，Northwest A&F University，Yangling，Shaanxi 712100，China
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| Abstract: |
| 【Objective】This study aims to screen proteins interacting with the product of recessive all-female control gene g（CsWIP1） in cucumber，thereby laying a foundation for further elucidating the molecular mechanism underlying cucumber sex determination.【Method】Based on the cucumber terminal bud yeast twohybrid（Y2H） cDNA library constructed in this laboratory，the CsWIP1-pGBKT7 bait vector was constructed by homologous recombination. After detecting autoactivation，CsWIP1-pGBKT7 and cucumber cDNA library were screened using Y2H approach. Positive clones were detected and analyzed by BLAST to identify proteins interacting with CsWIP1. The amino acid sequence of the interacting protein CsIDD-3 was characterized，fo-llowed by phylogenetic tree construction. The interaction between CsWIP1 and CsIDD-3 was verified by Y2H point-to-point assay，bimolecular fluorescence complementation（BiFC） and luciferase complementation ima-ging（LCI）.【Result】The bait vector CsWIP1-pGBKT7 was successfully constructed and showed no autoacti-vation. The identified interacting proteins included heat shock proteins，histones，protein kinases and transcrip?tion factors. Among them，CsIDD-3，which was also a transcription factor，was selected for subsequent experiments. The CDS sequence of CsIDD-3 encodes 363 amino acids and contains two C2H2-type zinc finger do?mains.The IDD-3 amino acid sequence is relatively conserved across other species，with the cucumber IDD-3 showing the closest phylogenetic relationship to that of melon. Interaction assays confirmed that CsWIP1 inter?acts with CsIDD-3.【Conclusion】These findings suggest that the protein interaction between CsWIP1 and CsIDD-3 participates in cucumber sexual differentiation，and the CsIDD-3 gene may function in regulating the fe?male-to-male flower ratio. |
| Key words: cucumber sexual differentiation CsWIP1 transcription factor interacting protein |
