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

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

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

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

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 Online:2026-05-25 Published:2026-05-26
Contact: CHANG Wei, DING Yong

Abstract

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

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.

Figures/Tables 6

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

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

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

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

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

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

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

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