‘海黄’牡丹芳樟醇合酶基因PsTPS14的克隆及功能验证

doi:10.16420/j.issn.0513-353x.2023-0383

摘要/Abstract

摘要：

以芳香型牡丹品种‘海黄’（Paeonia × lemoinei‘High Noon’）为材料，根据转录组测序所得片段，克隆得到PsTPS14基因，该结构基因的完整CDS序列长903 bp，共编码300个氨基酸。氨基酸序列分析发现该蛋白具有较高的保守性，有1个保守结构域Terpene_cyclase_plant_C1。PsTPS14在‘海黄’牡丹半开期花瓣中表达量最高，与其花香挥发物释放规律一致。亚细胞定位分析表明PsTPS14主要定位于叶绿体中。利用瞬时表达方法将PsTPS14过表达载体注射到烟草叶片和淡香型牡丹‘凤丹’切花花瓣中，通过GC-MS方法可检测到烟草叶片和‘凤丹’花瓣中芳樟醇的释放，同时PsTPS14高表达，且芳樟醇合酶含量及其活性明显升高。研究结果表明PsTPS14在植物体内调控芳樟醇的合成。

关键词: 牡丹, 芳樟醇, 芳樟醇合酶基因, 基因克隆, 功能验证

Abstract:

The linalool synthase gene PsTPS14 was cloned from the aromatic tree peony‘High Noon’based on the transcriptome sequencing. The complete CDS sequence of this gene was 903 bp in length encoding 300 amino acids. The analysis of the amino acids sequence showed that the protein has one conservative domain（Terpene_cyclase_plant_C1）. The expression level of PsTPS14 was the highest in half opening stage of petals in tree peony‘High Noon’，consistent with the release pattern of floral volatiles. Subcellular localization analysis showed that PsTPS14 was mainly localized in the chloroplast. The overexpression vector of PsTPS14 was injected into tobacco leaves and the petals of cut flowers of ‘Fengdan’，a light scent tree peony cultivar，by using the transient expression method. The release of linalool in tobacco leaves and‘Fengdan’petals could be detected by GC-MS method. At the same time，the expression level of PsTPS14 was higher in tobacco leaves and‘Fengdan’petals，and the content and activity of linalool synthase were significantly increased. This study showed that PsTPS14 regulated the synthesis of linalool in plants.

Key words: tree peony, linalool, linalool synthase gene, gene cloning, functional verification

王佩云, 李子昂, 白杨, 杨萍, 尹承芃, 李传荣, 张馨文, 宋秀华. ‘海黄’牡丹芳樟醇合酶基因PsTPS14的克隆及功能验证[J]. 园艺学报, 2024, 51(6): 1273-1283.

WANG Peiyun, LI Ziang, BAI Yang, YANG Ping, YIN Chengpeng, LI Chuanrong, ZHANG Xinwen, SONG Xiuhua. Cloning and Functional Verification of Linalool Synthase Gene PsTPS14 in Tree Peony‘High Noon’[J]. Acta Horticulturae Sinica, 2024, 51(6): 1273-1283.

图/表 10

表1 本研究中所用引物序列

Table 1 Sequences of primers used in this study

基因名 Gene name	作用 Function	引物序列（5′-3′） Primer sequence
TPS14	基因克隆 Gene clone	F：ATGGCCTATTCCAGTGGCTCC；R：AGTGTTTAGAATTAAACCACTTTGCTT
TPS14	qRT-PCR	F：GCTCCTCTACTTTCTGGCGTTGTC；R：GCTGTGATCTTGGGCAAGGTTCC
ubiquintin	内参基因 Internal reference gene	F：GACCTATACCAAGCCGAAG；R：CGTTCCAGCACCACAATC

图1 牡丹PsTPS14的PCR扩增产物

Fig. 1 PCR amplification products of PsTPS14 gene from tree peony

图2 PsTPS14蛋白和同源蛋白的系统进化树分析 Perilla frutescens：紫苏；Salvia dorisiana：红鼠尾草；Lavandula × intermedia：薰衣草；Solanum lycopersicum：番茄；Ficus carica：无花果；Freesia hybrid cultivar：香雪兰；Rosa chinensis：中国月季；Citrus unshiu：蜜柑；Arabidopsis thaliana：拟南芥；Osmanthus fragrans：桂花；Paeonia suffruticosa：牡丹‘海黄’；Paeonia delavayi：滇牡丹；Camellia saluenensis：山茶；Citrus sinensis：甜橙；Malus × domestica：苹果.

Fig. 2 Phylogenetic tree analysis of PsTPS14 proteins and homologous proteins

图3 ‘海黄’牡丹不同开花期PsTPS14的表达量与芳樟醇的相对含量 S1：现蕾期；S2：风铃期；S3：圆桃期；S4：初开期；S5：半开期；S6：盛开期；S7：衰败期。不同小写字母表示差异显著（P < 0.05）。下同。

Fig. 3 The expression level of PsTPS14 and relative content of linalool in different flowering stages of tree peony‘High Noon’ S1：Squaring stage；S2：Wind bell stage；S3：Ballon stage；S4：Initial opening stage；S5：Half opening stage；S6：Full opening stage；S7：Declining stage. Different lowercase letters indicate significant differences at 0.05 level. The same below.

图4 ‘海黄’牡丹盛开期不同器官PsTPS14的表达量与芳樟醇的相对含量

Fig. 4 The expression level of PsTPS14 and relative content of linalool in different organs of tree peony ‘High Noon’during its full opening stage

图5 牡丹PsTPS14的亚细胞定位

Fig. 5 Subcellular localization of PsTPS14 in tree peony

图6 异源瞬时转化PsTPS14的烟草叶片中挥发物GC-MS分析

Fig. 6 GC-MS analysis of volatile compounds in tobacco leaves undergoing heterologous transient transformation of PsTPS14

图7 异源瞬时转化PsTPS14的烟草叶片中PsTPS14的相对表达量、芳樟醇合酶含量及其活性

Fig.7 Relative expression level，linalool synthase content and activity of PsTPS14 in tobacco leaves undergoing heterologous transient transformation of PsTPS14

图8 同源瞬时转化PsTPS14的‘凤丹’牡丹中挥发物GC-MS分析

Fig. 8 GC-MS analysis of volatile compounds in Paeonia ostia‘Fengdan’undergoing homologous transient transformation of PsTPS14

图9 同源瞬时转化PsTPS14的‘凤丹’牡丹中PsTPS14和PSY的相对表达量、芳樟醇合酶含量及其活性

Fig. 9 Relative expression levels of PsTPS14 and PSY，content and activity of linalool synthase in Paeonia ostia ‘Fengdan’undergoing homologous transient transformation of PsTPS14

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