茶树光敏色素互作因子CsPIF3a的克隆及其与光温逆境的响应

doi:10.13560/j.cnki.biotech.bull.1985.2024-0794

生物技术通报 ›› 2025, Vol. 41 ›› Issue (4): 256-265.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0794

• 研究报告 • 上一篇

茶树光敏色素互作因子CsPIF3a的克隆及其与光温逆境的响应

班秋艳¹(), 赵鑫月¹, 迟文静¹, 黎俊生¹, 王琼¹, 夏瑶¹, 梁丽云¹, 贺巍¹, 李叶云²(), 赵广山³()

^1.河南农业大学园艺学院，郑州 450046
^2.安徽农业大学茶树种质创新与资源利用全国重点实验室，合肥 230036
^3.河南农业大学食品科学技术学院，郑州 450002

收稿日期:2024-08-17 出版日期:2025-04-26 发布日期:2025-04-25
通讯作者: 李叶云，男，博士，教授，研究方向：茶树抗逆栽培及育种；E-mail: lyy@ahau.edu.cn
赵广山，男，博士，讲师，研究方向：茶叶加工、精深加工与健康；E-mail: gszhao@henau.edu.cn
作者简介:班秋艳，女，博士，讲师，研究方向：茶树响应逆境胁迫的分子机制；E-mail: qyban717@henau.edu.cn
基金资助:
茶树生物学与资源利用国家重点实验室开放基金(SKLTOF20230111);河南省科技攻关(242102110155);河南省豫南茶树资源综合开发重点实验室开放基金(HNKLTOF2020005)

Cloning of Phytochrome Interaction Factor CsPIF3a and Its Response to Light and Temperature Stress in Camellia sinensis

BAN Qiu-yan¹(), ZHAO Xin-yue¹, CHI Wen-jing¹, LI Jun-sheng¹, WANG Qiong¹, XIA Yao¹, LIANG Li-yun¹, HE Wei¹, LI Ye-yun²(), ZHAO Guang-shan³()

^1.College of Horticulture, Henan Agricultural University, Zhengzhou 450046
^2.National Key Laboratory for Tea Plant Germplasm Innovation and Resource Utilization, Anhui Agricultural University, Hefei 230036
^3.College of Food Science and Technology, Henan Agricultural University, Zhengzhou 450002

Received:2024-08-17 Published:2025-04-26 Online:2025-04-25

摘要/Abstract

摘要：

目的 PIFs（phytochrome interacting factors）是光敏色素介导光温信号中的核心成员。研究茶树（Camellia sinensis（L.） O.Kuntze）PIF3的功能，为利用光信号途径避免或缓解逆境对茶树造成的危害提供重要的理论指导。方法利用同源克隆方法从茶树中克隆CsPIF3a，并对其进行生物信息学分析，利用实时荧光定量PCR（real-time quantitative PCR, RT-qPCR）分析其在不同组织及不同光温处理的表达差异，同时，利用烟草瞬时表达和酵母双杂技术观察其蛋白定位和转录活性。结果 CsPIF3a开放阅读框长度为2 163 bp，编码721个氨基酸，蛋白分子相对质量为76.97 kD，理论等电点为5.70，具有亲水性；系统发育树分析表明，CsPIF3a与毛花猕猴桃（Actinidia eriantha Bentham.）关系最近；CsPIF3a蛋白含有1个bHLH结构域、1个APB结构域和1个APA结构域；二级结构中含α-螺旋18.17%、β-折叠结构6.80%、β-转角1.53%、无规则卷曲73.51%，此外，还含无序化区域46.87%；CsPIF3a蛋白被定位于细胞核中，并具有转录活性。RT-qPCR分析表明，CsPIF3a在老叶和茎中表达水平较高，响应温度胁迫表达，且表达量与光照强度相关。结论 CsPIF3a位于细胞核中，具有转录活性。CsPIF3a响应温度胁迫，表达量与光照强度有关。

关键词: CsPIF3a, 基因克隆, 生物信息学分析, 亚细胞定位, 转录活性, 表达分析

Abstract:

Objective PIFs (Phytochrome interacting factors) are the core components in the photopigment-mediated photothermal signaling pathway. Studying the function of PIF3 in tea plant (Camellia sinensis (L.) O. Kuntze) provides important theoretical guidance for using light signaling pathways to avoid or mitigate the harm caused by adverse conditions. Method The CsPIF3a gene was cloned from tea plants through homology cloning and subsequently analyzed using bioinformatics tools. Expression patterns of CsPIF3a across various tissues and under different light and temperature conditions were assessed using real-time quantitative PCR (RT-qPCR). Additionally, tobacco transient expression assays and yeast two-hybrid assays were conducted to determine the protein localization and transcriptional activity of CsPIF3a. Result The open reading frame (ORF) of CsPIF3a was 2 163 bp in length, encoding a protein of 721 amino acids. The predicted molecular mass of the encoded protein was 76.97 kD. The theoretical isoelectric point was 5.70, having hydrophilic properties. The phylogenetic tree analysis showed that CsPIF3a was closely related to Actinidia eriantha Bentham. and contained a basic helix-loop-helix (bHLH) domain, an active phytochrome binding (APB) domain, and an APA domain. The secondary structure analysis of CsPIF3a revealed that it comprised 18.17% α-helix, 6.80% β-sheet, 1.53% β-turn, and 73.51% random coil, with 46.87% of the regions being disordered. Subcellular localization assays demonstrated that CsPIF3a was localized in the nucleus and presented transcriptional activity. Reverse transcription quantitative PCR (RT-qPCR) analysis indicated that CsPIF3a was predominantly expressed in the old leaves and stems of tea plants. Additionally, its expression was found to be responsive to temperature stress and correlated with light intensity. Conclusion CsPIF3a is localized in the nucleus and exhibits transcriptional activity. In response to temperature stress, the expression level of CsPIF3a gene was related to light intensity.

Key words: CsPIF3a, gene cloning, bioinformatics analysis, subcellular localization, transcriptional activity, expression analysis

班秋艳, 赵鑫月, 迟文静, 黎俊生, 王琼, 夏瑶, 梁丽云, 贺巍, 李叶云, 赵广山. 茶树光敏色素互作因子CsPIF3a的克隆及其与光温逆境的响应[J]. 生物技术通报, 2025, 41(4): 256-265.

BAN Qiu-yan, ZHAO Xin-yue, CHI Wen-jing, LI Jun-sheng, WANG Qiong, XIA Yao, LIANG Li-yun, HE Wei, LI Ye-yun, ZHAO Guang-shan. Cloning of Phytochrome Interaction Factor CsPIF3a and Its Response to Light and Temperature Stress in Camellia sinensis[J]. Biotechnology Bulletin, 2025, 41(4): 256-265.

图/表 10

图1 茶树CsPIF3a核苷酸序列与推测的氨基酸序列

Fig. 1 The nucleotide and amino acid sequences of CsPIF3a from tea plant

图2 CsPIF3a蛋白与其他植物同源PIF3蛋白氨基酸序列比对

Fig. 2 Deduced amino acid sequence alignment between CsPIF3a and homologous PIF3 proteins of other plants

图3 茶树CsPIF3a蛋白的进化树分析

Fig. 3 Phylogenetic tree analysis of tea plant CsPIF3a protein

表1 不同植物中CsPIF3a氨基酸组成成分及理化性质分析

Table 1 Composition and chemical characterization analysis of amino acids of CsPIF3a among the different plants

植物 Plant	GenBank登录号 GenBank accession	氨基酸数 Number of amino acids	相对分子量 Relative molecular weight/kD	理论等电点 Theoretical isoelectric point	碱性氨基酸 Basic amino acids/%	酸性氨基酸 Acidic amino acids/%	芳香族氨基酸 Aromatic amino acid/%	脂肪族氨基酸 Aliphatic amino acids/%	总平均疏水性 Total average hydrophobicity
茶树C. sinensis	XP_028108728.1	721	76.96	5.70	11	11	3	16	-0.547
杨梅M. rubra	KAB1199165.1	739	78.80	6.46	13	11	4	16	-0.506
胡桃J. microcarpa	XP_041007950.1	738	78.93	5.98	12	11	4	15	-0.551
欧榛C. avellana	XP_059456869.1	744	79.75	6.05	12	11	3	16	-0.502
夏栎Q. robur	XP_050273354.1	749	80.23	6.05	11	10	4	15	-0.546
酸枣Z. jujuba	XP_015874808.1	729	77.78	5.64	11	11	4	15	-0.553
水蜜桃P. persica	XP_020422550.1	729	78.19	5.67	11	11	4	15	-0.579
欧洲甜樱桃P. avium	XP_021830447.1	728	78.16	5.77	11	11	4	15	-0.584
橡胶树H. brasiliensis	XP_058000598.1	745	79.20	5.73	11	10	4	15	-0.555
葡萄V. vinifera	XP_010659764.1	709	75.86	6.30	12	10	4	15	-0.598
君迁子D. lotus	XP_052204847.1	721	76.31	5.97	11	10	4	16	-0.511
狭叶油菜C. lanceoleosa	KAI8022635.1	627	68.01	5.64	11	11	4	15	-0.636
红马银花R. vialii	XP_058191728.1	700	75.50	5.94	12	11	5	16	-0.471
拟南芥Arabidopsis thaliana	NP_001318964.1	524	56.99	6.11	13	12	5	15	-0.728
毛花猕猴桃A. eriantha	XP_057494369.1	723	77.24	6.56	12	10	4	16	-0.584

图4 茶树CsPIF3a折叠状态的分析

Fig. 4 Analysis of the folding state of CsPIF3a in tea plant

图5 CsPIF3a二级结构（A）和三级结构（B）预测

Fig. 5 Prediction of secondary structure (A) and tertiary structure (B) of CsPIF3a

图6 CsPIF3a的亚细胞定位

Fig. 6 Subcellular localization of CsPIF3a

图7 酵母单杂交验证CsPIF3a的转录活性

Fig. 7 Transcriptional activity of CsPIF3a verified by yeast one hybrid

图8 CsPIF3a在茶树不同组织中的表达分析不同小写字母表示在0.05水平上差异显著

Fig. 8 Expression analysis of CsPIF3a gene in different tissues of tea plantDifferent lowercase letters indicate significant differences at the 0.05 level

图9 CsPIF3a在光温胁迫下的表达分析A：低温处理；B：高温处理。不同小写字母表示相同光照处理下不同天数的相对表达量之间的差异（P<0.05）。不同大写字母表示相同天数不同光照处理后的相对表达量之间的差异（P<0.05）

Fig. 9 Expression analysis of CsPIF3a gene under light and temperature stressA: Low temperature treatment. B: High temperature treatment. Different lowercase letters indicate the difference in relative expression between different days under the same light treatment (P<0.05). Different capital letters indicate the difference in the relative expression of the same number of days after different light treatments (P<0.05)

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茶树光敏色素互作因子CsPIF3a的克隆及其与光温逆境的响应

Cloning of Phytochrome Interaction Factor CsPIF3a and Its Response to Light and Temperature Stress in Camellia sinensis

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