牡丹PoLEC1基因的克隆及其功能

doi:10.11707/j.1001-7488.LYKX20250123

Abstract

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

CLC Number:

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.

Figures/Tables 10

Table 1

Fig.1

Table 2

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

Fig.7

Fig.8

References 0

	郭海滨, 郝立冬. 玉米AINTEGUMENTA-LIKE 1 (AIL1)基因的克隆和表达模式分析. 分子植物育种, 2021, 22 (21): 6980- 6985.
	Guo H B, Hao L D. Cloning and expression pattern analysis of maize AINTEGUMENTA-LIKE 1(AIL1) gene. Molecular Plant Breeding, 2021, 22 (21): 6980- 6985.
	何桂梅, 成仿云, 李　萍. 两种牡丹胚珠与幼胚离体培养的初步研究. 园艺学报, 2006, 33 (1): 185. doi: 10.3321/j.issn:0513-353X.2006.01.047
	He G M, Cheng F Y, Li P. Preliminary studies on culturein vitro of ovule and immature embryo of two tree-peony cultivars. Acta Horticulturae Sinica, 2006, 33 (1): 185. doi: 10.3321/j.issn:0513-353X.2006.01.047
	刘　豪, 王艳丽, 孟晓丹, 等. 小麦TaLEC1基因的克隆及其表达特性分析. 西北植物学报, 2019, 39 (5): 904- 910. doi: 10.7606/j.issn.1000-4025.2019.05.0904
	Liu H, Wang Y L, Meng X D, et al. Cloning and expression analysis of TaLEC1 gene from wheat. Acta Botanica Boreali-Occidentalia Sinica, 2019, 39 (5): 904- 910. doi: 10.7606/j.issn.1000-4025.2019.05.0904
	文书生, 何绒绒, 郑佳康, 等. 牡丹组织培养技术研究进展. 林业科学, 2018, 54 (10): 143- 155. doi: 10.11707/j.1001-7488.20181017
	Wen S S, He R R, Zheng J K, et al. Research advances in tissue culture of tree peony. Scientia Silvae scincae, 2018, 54 (10): 143- 155. doi: 10.11707/j.1001-7488.20181017
	周秀梅. 2008. 牡丹体细胞胚胎发生研究. 北京: 北京林业大学.
	Zhou X M. 2008. Studies on somatic embryogenesis of tree peony. Beijing: Beijing Forestry University. ［in Chinese］
	Aregawi K, Shen J Q, Pierroz G, et al. Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing. Plant Biotechnology Journal, 2022, 20 (4): 748- 760. doi: 10.1111/pbi.13754
	Boulard C, Thévenin J, Tranquet O, et al. LEC1 (NF-YB9) directly interacts with LEC2 to control gene expression in seed. Biochimica et Biophysica Acta (BBA)–Gene Regulatory Mechanisms, 2018, 1861 (5): 443- 450. doi: 10.1016/j.bbagrm.2018.03.005
	Braybrook S A, Stone S L, Park S, et al. Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103 (9): 3468- 3473.
	Gao J, Xue J Q, Xue Y Q, et al. Transcriptome sequencing and identification of key callus browning-related genes from petiole callus of tree peony (Paeonia suffruticosa cv. Kao) cultured on media with three browning inhibitors. Plant Physiology and Biochemistry, 2020, 149, 36- 49. doi: 10.1016/j.plaphy.2020.01.029
	Hesami M, Pepe M, de Ronne M, et al. Transcriptomic profiling of embryogenic and non-embryogenic callus provides new insight into the nature of recalcitrance in Cannabis. International Journal of Molecular Sciences, 2023, 24 (19): 14625. doi: 10.3390/ijms241914625
	Hofmann F, Schon M A, Nodine M D. The embryonic transcriptome of Arabidopsis thaliana. Plant Reproduction, 2019, 32 (1): 77- 91. doi: 10.1007/s00497-018-00357-2
	Ikeuchi M, Favero D S, Sakamoto Y, et al. Molecular mechanisms of plant regeneration. Annual Review of Plant Biology, 2019, 70, 377- 406. doi: 10.1146/annurev-arplant-050718-100434
	Lotan T, Ohto M A, Yee K M, et al. Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell, 1998, 93 (7): 1195- 1205. doi: 10.1016/S0092-8674(00)81463-4
	Lowe K, La Rota M, Hoerster G, et al. Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis. In Vitro Cellular & Developmental Biology–Plant, 2018, 54 (3): 240- 252.
	Lv S Z, Cheng S, Wang Z Y, et al. Draft genome of the famous ornamental plant Paeonia suffruticosa. Ecology and Evolution, 2020, 10 (11): 4518- 4530. doi: 10.1002/ece3.5965
	Ochoa-Alejo N. 2016. The uses of somatic embryogenesis for genetic transformation//Loyola-Vargas V M, Ochoa-Alejo N. eds. Somatic embryogenesis: fundamental aspects and applications. Cham: Springer, 415-434.
	Quiroz-Figueroa F R, Rojas-Herrera R, Galaz-Avalos R M, et al. 2006. Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell, Tissue and Organ Culture, 86(3): 285-301.
	Ramakrishnan M, Zhou M B, Ceasar S A, et al. Epigenetic modifications and miRNAs determine the transition of somatic cells into somatic embryos. Plant Cell Reports, 2023, 42 (12): 1845- 1873. doi: 10.1007/s00299-023-03071-0
	Stone S L, Braybrook S A, Paula S L, et al. Arabidopsis LEAFY COTYLEDON2 induces maturation traits and auxin activity: implications for somatic embryogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105 (8): 3151- 3156.
	Xie L H, Niu L X, Zhang Y L, et al. Pollen sources influence the traits of seed and seed oil in Paeonia ostii ‘Feng Dan’. HortScience, 2017, 52 (5): 700- 705. doi: 10.21273/HORTSCI11803-17
	Yazawa K, Takahata K, Kamada H. Isolation of the gene encoding carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis. Plant Physiology and Biochemistry, 2004, 42 (3): 215- 223. doi: 10.1016/j.plaphy.2003.12.003
	Yuan H Y, Kagale S, Ferrie A M R. Multifaceted roles of transcription factors during plant embryogenesis. Frontiers in Plant Science, 2024, 14, 1322728. doi: 10.3389/fpls.2023.1322728
	Yuan J H, Jiang S J, Jian J B, et al. Genomic basis of the giga-chromosomes and giga-genome of tree peony Paeonia ostii. Nature Communications, 2022, 13 (1): 7328. doi: 10.1038/s41467-022-35063-1
	Zhang X X, Liu X, Zhou M H, et al. PacBio full-length sequencing integrated with RNA-seq reveals the molecular mechanism of waterlogging and its recovery in Paeonia ostii. Frontiers in Plant Science, 2022, 13, 1030584. doi: 10.3389/fpls.2022.1030584

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

[1]	Xingqiao Liu,Xiaolei Ma,Huili Ma,Nannan Zhang,Shuo Wei,Chengwei Song,Di Yang,Xiaogai Hou. Effects of Nitrogen Spraying Time on the Biomass Allocation and Nutrient Utilization of Paeonia ostii ‘Fengdan’ Twigs [J]. Scientia Silvae Sinicae, 2024, 60(12): 47-57.
[2]	Xiaoxue Wu,Aijing Zhang,Ying Gai,Xiangning Jiang. Effects of Exogenous Hormones on Different Stages of Somatic Embryogenesis of Larix kaempferi [J]. Scientia Silvae Sinicae, 2021, 57(1): 30-39.
[3]	Wu Lifang, Wei Xiaomei, Lu Weidong. Embryonic Callus Induction and Somatic Embryogenesis of Sophora davidii [J]. Scientia Silvae Sinicae, 2019, 55(7): 170-177.
[4]	Chen Tingting, Wang Pengkai, Zhang Jiaji, Shi Jisen, Cheng Tielong, Chen Jinhui. Effects of Combined ABA and ZT Treatment on Somatic Embryogenesis and Development of Liriodendron sino-americanum [J]. Scientia Silvae Sinicae, 2019, 55(3): 64-71.
[5]	Zhang Jiaqi, Hu Hengkang, Xu Chuanmei, Hu Yuanyuan, Huang Youjun, Xia Guohua, Huang Jianqin, Chang Yingying, Ye Lei, Lou Heqiang, Zhang Qixiang. Cloning, Subcellular Localization and Function Verification of Gibberellin 2-Oxidase Gene in Walnut (Juglans regia) [J]. Scientia Silvae Sinicae, 2019, 55(2): 50-60.
[6]	Chengzhong Wang,Xueyan Ni,Wei Zhu,Hanze Ma,Jianlin Qian,Ji Yang,Yonghong Hu,Zhiping Song. Seasonal Changes of Carbon, Nitrogen, Phosphorus and Soluble Sugar Concentrations in Plant of 'Fengdan' (Paeonia ostii) Chronosequence [J]. Scientia Silvae Sinicae, 2019, 55(12): 50-60.
[7]	Song Yue, Li Shujuan, Zhang Hanguo, Bai Xiaoming, Bi Xianyu, Dong Shiwei, Dong Hao. Establishment and Optimization of Embryogenic Callus Suspension Culture System of Larix [J]. Scientia Silvae Sinicae, 2018, 54(7): 146-154.
[8]	Gu Zhanying, Yang Ruonan, Chen Hao. The Establishment of Isolation and Transient Transformation Methods of Protoplasts of Vernicia fordii Mesophyll Cells [J]. Scientia Silvae Sinicae, 2018, 54(1): 46-53.
[9]	Xu Jianxiu, Wu Xiaoqin, Ye Jianren, Zhu Lihua, Wu Jing. Development, Maturation and Germination of Somatic Embryo of Nematode-Resistant Pinus densiflora [J]. Scientia Silvae Sinicae, 2017, 53(12): 41-49.
[10]	Guo Lili, Yin Weilun, Guo Dalong, Hou Xiaogai. Variations of Bacterial Biodiversity in Rhizosphere Soils of Oil Tree Peony Cropping Continuously for Different Years [J]. Scientia Silvae Sinicae, 2017, 53(11): 131-141.
[11]	Song Yue, Zhen Cheng, Zhang Hanguo, Li Shujuan. Embryogenic Callus Induction and Somatic Embryogenesis from Immature Zygotic Embryos of Larix olgensis [J]. Scientia Silvae Sinicae, 2016, 52(10): 45-54.
[12]	Zhang Jianwei, Wang Junhui, Li Qingfen, Ma Jianwei. Somatic Embryogenesis of Picea asperata Induced from Immature Embryos [J]. Scientia Silvae Sinicae, 2014, 50(4): 39-46.
[13]	Chen Jinhui;Zhang Yanjuan;Wu Yayun;Wang Pengkai;Wang Guangping;Shi Jisen. Effects of Phytosulfokine on the Somatic Embryogenesis of Liriodendron hybrids(L. chinense×L. tulipifera) [J]. , 2013, 49(2): 33-38.
[14]	Zeng Yanling, Tan Xiaofeng, Jiang Yao, Liu Min, Wang Jianyong, Zhou Junqin. Molecular Characterization and Expression Analysis of Fructose-1, 6-Diphosphate Aldolase Gene (CoFBA4) from Camellia oleifera [J]. Scientia Silvae Sinicae, 2013, 49(11): 164-170.
[15]	Liang Yan;Shen Hailong;Li Yuhua;Yang Ling;Yang Xiaojie. Role of Ethylene and Polyamines in Plant Somatic Embryogenesis [J]. Scientia Silvae Sinicae, 2012, 48(9): 145-153.

Cloning and Function of PoLEC1 Gene in Paeonia ostii

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 10

References 0

Related Articles 15

Recommended Articles

Metrics

Comments