梅花PmABCG9在苯甲醇挥发中的功能分析

doi:10.3864/j.issn.0578-1752.2023.13.011

摘要/Abstract

摘要：

【目的】挖掘与梅花重要芳香成分跨膜运输相关的基因，进一步完善梅花芳香成分转运机制。【方法】以梅花‘彩枝舞粉’为试材，在分析梅花不同开花时期苯甲醇的挥发量与内源含量基础上，通过加权基因共表达网络（WGCNA）、系统进化树、表达量分析，筛选与苯甲醇跨膜运输相关的ABC转运蛋白（ATP-binding cassette transporter）。利用亚细胞定位、转基因技术和底物孵育试验，验证PmABCG9参与转运苯甲醇的功能。【结果】将梅花品种‘彩枝舞粉’的开花进程分为7个时期，苯甲醛、苯甲醇和乙酸苯甲酯在各个时期的挥发物中都能检测到，进入初花期后，这3种成分的总相对含量超过80%，是‘彩枝舞粉’的主要芳香成分。定量分析各主要芳香成分的挥发量和内源含量，苯甲醛内源含量最高，但挥发量最高仅有13.17 ng·g^-1·h^-1，苯甲醇内源含量和挥发量最高时期为末花期，挥发量达到82.28 ng·g^-1·h^-1，乙酸苯甲酯挥发量最高时期为盛花期，挥发量为280.21 ng·g^-1·h^-1。利用WGCNA方法联合分析不同开花时期花朵转录组与芳香成分挥发量数据，获得与苯甲醇挥发量相关性高的turquoise模块，在该模块中鉴定到11个ABCG亚家族基因。通过不同部位、不同时期的表达模式分析，推测PmABCG9与梅花中苯甲醇跨膜转运相关。克隆PmABCG9，分析结果表明该基因具有典型的NBD-TMD结构域，亚细胞定位结果证实其能在细胞膜上表达。构建PmABCG9过表达载体并转化本氏烟草，利用苯甲醇溶液孵育转基因及野生型烟草叶片，顶空分析各株系苯甲醇挥发量，发现转基因烟草挥发量显著高于野生型，证明PmABCG9具有转运苯甲醇的功能。【结论】苯甲醛、苯甲醇、乙酸苯甲酯是梅花‘彩枝舞粉’花期主要的芳香成分。梅花PmABCG9属于ABCG亚家族的半分子转运蛋白类型，PmABCG9的表达与苯甲醇挥发量相关，其能有效地将苯甲醇通过细胞膜转运到空气中。

关键词: 梅花, 苯甲醇, PmABCG9, 转运, 花香

Abstract:

【Objective】The aim of this study was to improve the volatilization mechanism of floral scent by exploring the gene related to the transmembrane transport of important aromatic components in P. mume. 【Method】The P. mume Caizhiwufen was applied as material plant. Based on the analysis of volatile and endogenous contents of benzyl alcohol at different blooming stages, the ABC transporters (ATP-binding cassette transporter) involved in the transmembrane transport of benzyl alcohol were identified by weighted gene co-expression network analysis (WGCNA), phylogenetic analysis, and gene expression profiles. Finally, the functions of PmABCG9 were validated by subcellular localization, transgenic technology and incubation assay with substrates. 【Result】The flowering process of P. mume was divided into seven blooming stages, and the total relative content of benzaldehyde, benzyl alcohol and benzyl acetate reached more than 80% at the initial blooming stage, indicating that they were the key aromatic components of Caizhiwufen. The quantitative analysis showed that the endogenous content of benzaldehyde was the highest among the above three, while its volatile content was only 13.17 ng·g^-1·h^-1 at the highest level. The endogenous and volatile content of benzyl alcohol reached the highest at wither stage, up to 82.28 ng·g^-1·h^-1 of the volatile content. The highest volatile content of benzyl acetate was at full blooming stage and up to 280.21 ng·g^-1·h^-1. WGCNA between the transcriptome data of different blooming stages and the volatile content of aromatic components showed that the turquoise module highly correlated with the volatile content of benzyl alcohol, importantly. 11 ABCG subfamily genes were identified. Moreover, based on the expression patterns of PmABCG9 in different flower organs and blooming stages of P. mume, it was inferred that PmABCG9 was related to the transmembrane transport of benzyl alcohol in P. mume. Therefore, PmABCG9 gene was cloned, and the typical NBD-TMD domain of PmABCG9 gene was confirmed. Subcellular localization showed that PmABCG9 was localized on the cell membrane. The leaves of transgenic PmABCG9 Nicotiana benthamiana and wild-type plants were incubated with benzyl alcohol solution, and the volatile content of benzyl alcohol from each line were analyzed by GC-MS. It was noted that the volatile content of benzyl alcohol from transgenic tobaccos was significantly higher than that of wild-type ones, demonstrating that PmABCG9 had the function of transporting benzyl alcohol. 【Conclusion】 Benzaldehyde, benzyl alcohol and benzyl acetate were the key aromatic components at florescence of P. mume Caizhiwufen. PmABCG9 belonged to the semi-molecular transporters type from ABCG subfamily, PmABCG9 was closely related to volatile content benzyl alcohol, furthermore, it could effectively transport benzyl alcohol into the air through cell membrane.

Key words: Prunus mume, benzyl alcohol, PmABCG9, transport, floral scent

郝瑞杰, 邱晨, 耿晓云, 贾浩田, 张雅静, 常珺, 冯新新. 梅花PmABCG9在苯甲醇挥发中的功能分析[J]. 中国农业科学, 2023, 56(13): 2574-2585.

HAO RuiJie, QIU Chen, GENG XiaoYun, JIA HaoTian, ZHANG YaJing, CHANG Jun, FENG XinXin. The Function of PmABCG9 Transporter Related to the Volatilization of Benzyl Alcohol in Prunus mume[J]. Scientia Agricultura Sinica, 2023, 56(13): 2574-2585.

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引物名称 Primer name	引物序列 Prime sequence (5′-3′)	用途 Purpose
PmABCG9-F	TTGGAGAGGACACGC*ATGGAATTGCCAGTGACAATGC	PmABCG9克隆、超表达 Cloning and overexpression of PmABCG9
PmABCG9-R	GCAGGACTCTAGGGACTAGTCTCTGTTTCTGCAGCAT	PmABCG9克隆、超表达 Cloning and overexpression of PmABCG9
PmABCG9-GFP-F	AACACGGGGGACTTTGCAACATGGAATTGCCAGTGACAATGCCGG	PmABCG9亚细胞定位 Subcellular localization of PmABCG9
PmABCG9-GFP-R	CCTGAAGCGGCCGCTGTACAGTCTCTGTTTCTGCAGCATCTGTACC	PmABCG9亚细胞定位 Subcellular localization of PmABCG9
PmABCG9-qPCR-F	GGTTGGTTGGATTAAGGAGGAA	实时荧光定量 qRT-PCR
PmABCG9-qPCR-R	CACTGAAACAAGCCACAACAG	实时荧光定量 qRT-PCR
UBC-F	GCAGGTGGAGTGTTCCTTGTGA	内参引物 Internal reference primer
UBC-R	GGCAGGGCTCCACTGTTCTTT	内参引物 Internal reference primer