牡丹PoLPAT2基因的克隆及表达分析

doi:10.3969/j.issn.1004-1524.20240504

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (2): 321-328.DOI: 10.3969/j.issn.1004-1524.20240504

牡丹PoLPAT2基因的克隆及表达分析

张美莹¹^,²(), 莫倩¹^,³, 齐秀双³, 佟宁宁², 孔凡², 刘政安², 吕长平⁴^,⁵^,^*(), 彭丽平²^,³^,^*()

1.湖南农业大学园艺学院,湖南长沙 410128
2.中国科学院植物研究所北方资源植物重点实验室,国家植物园,北京 100093
3.天香源(辽宁)生物科技有限责任公司,辽宁葫芦岛 125300
4.湖南农业大学风景园林与艺术设计学院,湖南长沙 410128
5.湖南农业大学湖南省中亚热带优质花木繁育与利用工程技术研究中心,湖南长沙 410128

收稿日期:2024-06-09 出版日期:2025-02-25 发布日期:2025-03-20
作者简介:彭丽平,E-mail:pengliping@ibcas.ac.cn
*吕长平,E-mail:changpinglv@sina.com;
张美莹(1999—),女,山西临汾人,硕士研究生,研究方向为高油牡丹选育。E-mail: zmy317506431@163.com
通讯作者: 吕长平,彭丽平

Cloning and expression analysis of peony PoLPAT2 gene

ZHANG Meiying¹^,²(), MO Qian¹^,³, QI Xiushuang³, TONG Ningning², KONG Fan², LIU Zheng’an², LYU Changping⁴^,⁵^,^*(), PENG Liping²^,³^,^*()

1. College of Horticulture, Hunan Agricultural University, Changsha 410128, China
2. Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, China National Botanical Garden, Beijing 100093, China
3. Tianxiangyuan (Liaoning) Biotechnology Co., Ltd., Huludao 125300, Liaoning, China
4. College of Landscape Architecture and Horticultural Design, Hunan Agricultural University, Changsha 410128, China
5. Hunan Mid-Subtropical Quality Plant Breeding and Utilization Engineering Technology Research Center, Hunan Agricultural University, Changsha 410128, China

Received:2024-06-09 Online:2025-02-25 Published:2025-03-20
Contact: LYU Changping,PENG Liping

摘要/Abstract

摘要： 溶血磷脂酸酰基转移酶(lysophosphatidic acid acyltransferase,LPAT)催化溶血磷脂酸(lysophosphatidic acid,LPA)sn-2位酰基化形成磷脂酸(phosphatidic acid,PA),是油脂合成过程中的关键限速酶之一。牡丹(Paeonia suffruticosa)是新型木本油料,研究其LPAT基因家庭的进化和表达模式,对进一步探索牡丹种子中油脂合成的规律具有重要意义。本研究从不同时期的牡丹中克隆了PoLPAT2基因,并利用生物信息学方法对其理化性质、系统进化、基因结构和表达模式等进行了分析。结果表明,PoLPAT2的CDS长为1 194 bp,编码397个氨基酸,由20种氨基酸组成,定位于质膜上。蛋白质的相对分子质量为44.82 ku,分子式为C₂₀₆₅H₃₂₃₅N₅₄₁O₅₄₄S₁₅,共有6 400个原子,理论等电点为9.67,磷酸化位点为37个,拥有较高的脂肪族性质,属于亲水蛋白,跨膜区域为3个,没有信号肽,不平衡指数为43.68,属于不稳定蛋白。蛋白质结构预测表明,PoLPAT2蛋白由4种二级结构组成,分别是无规卷曲、α螺旋、β转角和延伸链。系统发育分析结果显示,牡丹PoLPAT2与圆叶葡萄、核桃的亲缘关系最近。qRT-PCR结果表明,该基因在牡丹种子发育后期105 d时表达量最高,整体趋势呈先降低后升高趋势,表明PoLPAT2可能与发育时期有关。研究结果可为牡丹PoLPAT2基因的功能验证、提高牡丹种子含油量提供参考。

关键词: 牡丹, 溶血磷脂酸酰基转移酶(LPAT), 基因克隆, 生物信息学分析

Abstract:

Lysophosphatidic acid acyltransferase (LPAT) catalyzes the acylation of lysophosphatidic acid (LPA) at the sn-2 position to form phosphatidic acid (PA), which is one of the key rate-limiting enzymes in oil synthesis. Peony (Paeonia suffruticosa) is an emerging woody oil plant. Studying the evolution and expression patterns of the peony LPAT gene family is of great significance for further exploring the mechanisms of oil synthesis in peony seeds. In this study, we cloned the PoLPAT2 gene from peony seeds and analyzed its physicochemical properties, phylogenetic relationships, gene structure, and expression patterns using bioinformatics methods. The results showed that the coding sequence length of PoLPAT2 was 1 194 bp, encoding 397 amino acids, consisting of 20 different amino acids, and located on the plasma membrane. The relative molecular weight of the protein is 44.82 ku, with a molecular formula of C₂₀₆₅H₃₂₃₅N₅₄₁O₅₄₄S₁₅, a total of 6 400 atoms, a theoretical isoelectric point of 9.67. It contains 37 phosphorylation sites and exhibits high aliphatic properties. Classified as a hydrophilic protein, it possesses 3 transmembrane regions, lacks signal peptides, and has an instability index of 43.68, indicating it is an unstable protein. Protein structure prediction indicates that PoLPAT2 protein consists of four secondary structures, namely irregular coil, alpha helix, beta helix, and elongation chain. Phylogenetic analysis show that PoLPAT2 in peony is most closely related to homologs in grapes and walnuts. qRT-PCR results showed that the expression level of this gene was peaked at 105 d post-late seed development, and the overall trend showed a decrease followed by an increase, indicating that PoLPAT2 may be related to the stage of seed development. This findings provide reference for the functional verification of the PoLPAT2 gene and the improvement of oil content in peony seeds.

Key words: peony, lysophosphatidyl acyltransferase (LPAT), gene cloning, bioinformatics analysis

中图分类号:

S685.11

张美莹, 莫倩, 齐秀双, 佟宁宁, 孔凡, 刘政安, 吕长平, 彭丽平. 牡丹PoLPAT2基因的克隆及表达分析[J]. 浙江农业学报, 2025, 37(2): 321-328.

ZHANG Meiying, MO Qian, QI Xiushuang, TONG Ningning, KONG Fan, LIU Zheng’an, LYU Changping, PENG Liping. Cloning and expression analysis of peony PoLPAT2 gene[J]. Acta Agriculturae Zhejiangensis, 2025, 37(2): 321-328.

图/表 9

图1 牡丹PoLPAT2琼脂糖凝胶电泳检测

Fig.1 Agarose gel electrophoresis of PoLPAT2

图2 PoLPAT2与其他LPAT2的氨基酸序列比对

Fig.2 Sequence analysis of amino acids PoLPAT2 and other LPAT2s

图3 PoLPAT2和其他植物同源蛋白系统发育分析

Fig.3 Phylogenetic analysis of PoLPAT2 with its homologues from other species

图4 PoLPAT2蛋白的亲/疏水性分布曲线

Fig.4 Hydrophilic/hydrophobic distribution curve of PoLPAT2 protein

图5 PoLPAT2蛋白的信号肽预测

Fig.5 Signal peptide prediction of PoLPAT2 protein

图6 PoLPAT2蛋白的跨膜域预测

Fig.6 Transmembrane domain prediction of PoLPAT2 protein

图7 PoLPAT2蛋白二级结构预测蓝色,α螺旋;绿色,β转角;红色,延伸链;紫色,无规卷曲。

Fig.7 Secondary structure prediction of PoLPAT2 protein Blue, α-helix;Green, β-turn:Red, Extended strand;Purple, Random coli.

图8 PoLPAT2 蛋白三级结构预测

Fig.8 Prediction of tertiary structure of PoLPAT2 protein

图9 PoLPAT2在各个发育时期种子中的相对表达量

Fig.9 Relative expression levels of PoLPAT2 in seeds at different developmental stages

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牡丹PoLPAT2基因的克隆及表达分析

Cloning and expression analysis of peony PoLPAT2 gene

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