牡丹PoLEC1基因的克隆及其功能

doi:10.11707/j.1001-7488.LYKX20250123

摘要/Abstract

摘要：

目的: LEAFY COTYLEDON 1（LEC1）基因是植物胚胎发育中特定表达的转录因子，本研究旨在探索该基因在调控牡丹高频体细胞胚发生的分子机制，为构建牡丹高效、稳定的优质再生体系和遗传转化体系提供理论依据。方法: 基于牡丹‘凤丹’基因组和实验室前期的转录组数据，从‘凤丹白’中克隆获得牡丹PoLEC1基因，并对其编码的蛋白进行结构和系统进化分析；通过实时定量 PCR 技术分析PoLEC1基因在牡丹不同组织和种胚不同发育时期中的表达特征；构建PoLEC1基因的过表达载体，采用花序浸染法进行拟南芥遗传转化，获得T3代转基因植株，观察转基因植株表型。结果: PoLEC1包含CBFD_NFYB_HMF结构域，系统进化分析表明，PoLEC1与葡萄和可可的亲缘关系较近，PoLEC1基因在胚胎发育早期和晚期的表达量较高，在牡丹的各组织中均有表达，在茎中的表达量最高，其次是叶；通过农杆菌介导法将PoLEC1基因在拟南芥中异源过表达，结果显示PoLEC1转拟南芥叶片大小无明显差异，叶片数量略多于野生型，开花晚于野生型。亚细胞定位确定PoLEC1定位于细胞核和细胞质中。结论: PoLEC1可能参与了体胚发育和开花过程，为牡丹分子育种的研究提供了重要的基因资源。

关键词: 牡丹, PoLEC1, 功能验证, 亚细胞定位, 体胚发生

Abstract:

Objective: The LEAFY COTYLEDON1 (LEC1) gene is a transcription factor specifically expressed in plant embryos. The aim of this study is to explore the molecular mechanism of this gene in regulating high frequency somatic embryogenesis of peony, providing a theoretical basis for constructing an efficient and stable high-quality regeneration system and genetic transformation system of tree peony. Method: Based on the genome of Paeonia ostii and the transcriptome data from our laboratory, PoLEC1 gene was cloned from P. ostii, and the structure and phylogenetic analysis of its encoded protein was performed. The expression characteristics of PoLEC1 gene in different tissues and embryo development were analyzed by real-time quantitative PCR. PoLEC1 gene overexpression vector was constructed, and Arabidopsis thaliana was genetically transformed by inflorescence impregnation method to obtain T3 transgenic plants, and the phenotype of transgenic plants was observed. Result: PoLEC1 contained the CBFD_NFYB_HMF domain. Phylogenetic analysis showed that PoLEC1 was closely related to grape (Vitis vinifera) and cocoa (Theobroma cacao), and PoLEC1 gene was highly expressed in early and late embryonic development. It was expressed in all tissues of tree peony, with the highest expression in stems, followed by leaves. PoLEC1 was heterogeneically overexpressed in A. thaliana by Agrobacteria-mediated method. The results showed that there was no significant difference in leaf size between PoLEC1 transgenic plants and wild type plants. The number of leaves of PoLEC1 transgenic plants was slightly more than that of wild type, and PoLEC1 transgenic plants bolted later than wild type. Subcellular localization determined that PoLEC1 was localized in the nucleus and cytoplasm. Conclusion: PoLEC1 may be involved in somatic embryo development and flowering, providing important genetic resources for molecular breeding of peony.

Key words: Paeonia ostii, PoLEC1, functional validation, subcellular localization, somatic embryogenesis

中图分类号:

常艳婷,胡陶,张雪,江泽慧,马艳军,邓雅云,张闻博. 牡丹PoLEC1基因的克隆及其功能[J]. 林业科学, 2025, 61(9): 138-145.

Yanting Chang,Tao Hu,Xue Zhang,Zehui Jiang,Yanjun Ma,Yayun Deng,Wenbo Zhang. Cloning and Function of PoLEC1 Gene in Paeonia ostii[J]. Scientia Silvae Sinicae, 2025, 61(9): 138-145.

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引物名称 Primer name	引物序列 Primers sequence	用途 Application
PoLEC1-F	ATGGCGGAGTCAGACGACG	CDS 扩增引物 CDS amplification primers
PoLEC1-R	CTACCTGGGCCTTCCAGTGG	CDS 扩增引物 CDS amplification primers
PHG-PoLEC1-F	TCTCTCTCTCAAGCTTATGGCGGAGTCAGACGACG	表达载体引物 Expression vector primers
PHG-PoLEC1-R	CGGGTCATGAGCTCCTGCAGCCTGGGCCTTCCAGTGG	表达载体引物 Expression vector primers
Q-PoLEC1-F	GCGAACGGCGAGTTGTCACA	实时荧光定量引物 qRT-PCR primers
Q-PoLEC1-R	ACGCACTCCTGTACGGTCTCTT	实时荧光定量引物 qRT-PCR primers

基本理化性质 Physical and chemical properties	PoLEC1基因 PoLEC1 gene
开放阅读框Open reading frame/bp	579
蛋白长度Protein Length/aa	193
相对分子质量Molecular weight MWs/Da	20 583.61
理论等电点Isoelectric point PI	5.98
不稳定系数II Instability coefficient	29.10
脂肪系数Aliphatic index	50.05
总平均亲水性GRAVY	–0.865
亚细胞定位预测Prediction of subcellular localization	细胞核Nucleus

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