3种兜兰FAD2基因克隆及响应零上低温表达的差异分析

doi:10.16420/j.issn.0513-353x.2025-0442

园艺学报 ›› 2026, Vol. 53 ›› Issue (5): 1371-1381.doi: 10.16420/j.issn.0513-353x.2025-0442

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

3种兜兰FAD2基因克隆及响应零上低温表达的差异分析

黄久妍¹, 段晓芊¹, 付月¹, 常玮²^,^*(), 丁勇¹^,^*()

¹ 西南林业大学生物与食品工程学院，云南省高校大健康类森林资源开发利用工程研究中心，昆明 650224
² 中国科学院昆明植物研究所资源植物与生物技术重点实验室，昆明 650204

收稿日期:2025-06-20 修回日期:2025-12-09 出版日期:2026-05-25 发布日期:2026-05-26
通讯作者:
^* E-mail：dingyongSWFU@163.com，
changwei@mail.kib.ac.cn
基金资助:
云南省中青年学术与技术带头人后备人才项目(202405AC350032); 云南省教育厅科学研究基金项目(2023Y0786); 中国科学院技术支撑人才项目(E4233811); 生物学省级学科建设经费资助项目(32004217)

Cloning of FAD2 Genes in Three Paphiopedilum Species and Comparative Analysis of Their Expression in Responses to Chilling Stress

HUANG Jiuyan¹, DUAN Xiaoqian¹, FU Yue¹, CHANG Wei²^,^*(), DING Yong¹^,^*()

¹ College of Biological Science and Food Engineering/Forest Resources Exploitation and Utilization Engineering Research Center for Grand Health of Yunnan Provincial Universities， Southwest Forestry University， Kunming 650224， China
² Key Laboratory of Economic Plants and Biotechnology， Kunming Institute of Botany，Chinese Academy of Sciences， Kunming 650201， China

Received:2025-06-20 Revised:2025-12-09 Published:2026-05-25 Online:2026-05-26
Contact:
^* E-mail：dingyongSWFU@163.com，
changwei@mail.kib.ac.cn

摘要/Abstract

摘要：

低温是制约兜兰属（Paphiopedilum）植物生存繁衍的重要环境因子，而脂肪酸去饱和酶2（fatty acid desaturase 2，FAD2）通过动态调节细胞膜脂不饱和度在植物响应零上低温胁迫中发挥重要作用。本研究中选取具有典型生态适应差异的紫纹兜兰（P. purpuratum）、长瓣兜兰（P. dianthum）和杏黄兜兰（P. armeniacum）为研究对象，结合基因克隆、生物信息分析及qRT-PCR技术，揭示了3种兜兰FAD2基因的序列特征及其在低温下的表达调控规律。研究显示，3种兜兰FAD2蛋白的理化参数、空间构象及跨膜特性高度保守，但其亚细胞定位呈现明显物种特异性，杏黄兜兰PaFAD2主要定位于质体，紫纹兜兰PpFAD2和长瓣兜兰PdFAD2分别富集于质膜/内质网和多种内膜系统。在4 ℃低温胁迫下，PaFAD2基因在胁迫初期（24 h）快速被激活并维持高水平表达，而PpFAD2和PdFAD2基因分别表现出应答滞后和弱响应特征。结合物种地理分布特征推测，杏黄兜兰PaFAD2基因表达的快速响应机制可能与其对高海拔生境长期适应密切相关，而紫纹兜兰和长瓣兜兰则可能通过FAD2的差异表达策略平衡抗寒与能量消耗。

关键词: 兜兰, FAD2, 低温胁迫, 基因克隆, 表达特征

Abstract:

Low temperature is a critical environmental factor limiting the survival and reproduction of Paphiopedilum species. Fatty acid desaturase 2（FAD2）plays a pivotal role in plant chilling tolerance by dynamically regulating the unsaturation of membrane lipids. Here，the sequence characteristics of FAD2genes and their expression regulation under low-temperature conditions were systematically elucidated in

three Paphiopedilum species with distinct ecological adaptations（P. purpuratum，P. dianthum，and P. armeniacum） through gene cloning，bioinformatics analysis，and qRT-PCR methods. The results revealed that the physicochemical parameters，spatial conformations，and transmembrane characteristics of the three FAD2 proteins were highly conserved. However，their subcellular localization predicted by WoLF PSORT exhibited marked species specificity：PaFAD2 from P. armeniacum showed predominant plastid localization，while PpFAD2 and PdFAD2 exhibited preferential accumulation in the plasma membrane/ endoplasmic reticulum and diverse endomembrane systems，respectively. Under 4 ℃ chilling stress，the PaFAD2 gene was rapidly activated at the early stress stage（24 h treatment group）and maintained high expression levels；whereas，PpFAD2 or PdFAD2 genes displayed delayed response or weak induction characteristics，respectively. Integrating biogeographical distribution patterns，it was hypothesized that the rapid transcriptional response of PaFAD2 in P. armeniacum might be evolutionarily associated with long-term adaptation to high-altitude habitats，whereas P. purpuratum and P. dianthum appeared to utilize differential FAD2 expression strategies to achieve an adaptive trade-off between cold resistance and metabolic efficiency.

Key words: Paphiopedilum, fatty acid desaturase 2, low-temperature stress, gene cloning, expression characteristics

黄久妍, 段晓芊, 付月, 常玮, 丁勇. 3种兜兰FAD2基因克隆及响应零上低温表达的差异分析[J]. 园艺学报, 2026, 53(5): 1371-1381.

HUANG Jiuyan, DUAN Xiaoqian, FU Yue, CHANG Wei, DING Yong. Cloning of FAD2 Genes in Three Paphiopedilum Species and Comparative Analysis of Their Expression in Responses to Chilling Stress[J]. Acta Horticulturae Sinica, 2026, 53(5): 1371-1381.

图/表 6

表1 本研究中所用引物信息

Table 1 Primer information in this study

目的 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因克隆 Gene cloning	PpFAD2-F	AGTGAATTCGAGCTCGGTACATGGGCGCCGGCGGACAC
	PpFAD2-R	GCCCGGGATCCGATATCTAGTCAGAGCTCGTTGCGGTACCAG
	PdFAD2-F	AGTGAATTCGAGCTCGGTACATGGGTGCTGGCGGACACATG
	PdFAD2-R	GCCCGGGATCCGATATCTAGTCAGAACTCGTTGCGGTACCAGAAC
	PaFAD2-F	AGTGAATTCGAGCTCGGTACCATGGGTGCCGGCGGACAC
	PaFAD2-R	GCCCGGGATCCGATATCTAGATCAGAGCTCGTTGCGGTACCAG
荧光定量 PCR Quantitative Real-time PCR	FAD2-F	CTCCCACTTTGACCCCTACG
	FAD2-R	CAGTCCCACTCCGATGAGTC
内参基因荧光定量 PCR Internal reference genes Quantitative Real-time PCR	PpGAPDH-F	TCTCACTTGAAGGGTGGTGC
	PpGAPDH-R	ATGTTCTGACTTGCTCCCCG
	PdGAPDH-F	CTGTTGCGGTCTTTGGCATC
	PdGAPDH-R	TGTCGTGAATGACCTTGGCA
	PaGAPDH-F	AACAGTTGGTGGTGCAGCTA
	PaGAPDH-R	CAGGGTGGTGCTAAGAAGGT

图1 3种兜兰FAD2基因PCR扩增结果 Pa：杏黄兜兰；Pp：紫纹兜兰；Pd：长瓣兜兰。下同

Fig. 1 PCR amplification results of FAD2 genes in 3 Paphiopedilum species Pa：Paphiopedilum armeniacum；Pp：Paphiopedilum purpuratum；Pd：Paphiopedilum dianthum. The same below

图2 3种兜兰与不同植物FAD2蛋白质保守基序与系统进化分析 As：深圳拟兰；Pa：杏黄兜兰；Pp：紫纹兜兰；Pd：长瓣兜兰；Dc：黄石斛；Pe：小兰屿蝴蝶兰；Ip：香根鸢尾；Ao：芦笋；Zm：玉蜀黍；Ci：美人蕉；Mu：三明野生蕉；Ma：大米七香蕉

Fig. 2 Conserved motifs and phylogenetic analysis of FAD2 proteins in 3 Paphiopedilum species and other plants As：Apostasia shenzhenica；Pa：Paphiopedilum armeniacum；Pp：Paphiopedilum purpuratum；Pd：Paphiopedilum dianthum；Dc：Dendrobium catenatum；Pe：Phalaenopsis equestris；Ip：Iris pallida；Ao：Asparagus officinalis；Zm：Zea mays；Ci：Canna indica；Mu：Musa AB Group；Ma：Musa acuminata AAA Group

图3 3种兜兰FAD2基因编码蛋白二级（A）与三级（B）结构预测 N和C分别表示蛋白质多肽链的氨基端与羧基端

Fig. 3 Prediction of secondary（A）and tertiary（B）structures of FAD2-encoded proteins in 3 Paphiopedilum species N and C denote the N-terminus and C-terminus of the protein polypeptide chain respectively

图 4 3种兜兰FAD2基因编码蛋白跨膜区域（A）与信号肽（B）预测

Fig. 4 Prediction of transmembrane domains（A）and signal peptides（B）in FAD2-encoded proteins from 3 Paphiopedilum species

图5 3种兜兰FAD2基因在低温胁迫下的表达变化

Fig. 5 Expression patterns of FAD2 genes in 3 Paphiopedilum species under different durations of low-temperature stress

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3种兜兰FAD2基因克隆及响应零上低温表达的差异分析

Cloning of FAD2 Genes in Three Paphiopedilum Species and Comparative Analysis of Their Expression in Responses to Chilling Stress

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3种兜兰FAD2基因克隆及响应零上低温表达的差异分析

Cloning of FAD2 Genes in Three Paphiopedilum Species and Comparative Analysis of Their Expression in Responses to Chilling Stress

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