蜡梅CpBEAT的克隆、表达分析及功能验证

doi:10.16420/j.issn.0513-353x.2024-0008

园艺学报 ›› 2025, Vol. 52 ›› Issue (1): 160-170.doi: 10.16420/j.issn.0513-353x.2024-0008

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

蜡梅CpBEAT的克隆、表达分析及功能验证

王飞龙¹^,^*, 梁前艳²^,³^,^*, 尚均忠¹, 陈龙清⁴, 向林¹^,^**()

¹ 华中农业大学果蔬园艺作物种质创新与利用全国重点实验室，武汉 430070
² 中国长江三峡集团有限公司长江生物多样性研究中心，武汉 430014
³ 长江经济带生态环境国家工程研究中心，北京100038
⁴ 西南林业大学园林园艺学院，昆明 650224

收稿日期:2024-05-17 修回日期:2024-09-23 出版日期:2025-01-25 发布日期:2025-01-19
通讯作者:
** E-mail：xianglin@mail.hzau.edu.cn
作者简介:
* 共同第一作者
基金资助:
国家自然科学基金项目(32372755)

Cloning，Expression and Functional Identification of CpBEAT Gene in Chimonanthus praecox

WANG Feilong¹, LIANG Qianyan²^,³, SHANG Junzhong¹, CHEN Longqing⁴, XIANG Lin¹^,^**()

¹ National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops，Huazhong Agricultural University，Wuhan 430070，China
² Yangtze River Biodiversity Research Center of China Three Gorges Corporation Limited，Wuhan 430014，China
³ National Engineering Research Center of Eco-environment Protection for Yangtze River Economic Belt，Beijing 100038，China
⁴ College of Landscape Architecture and Horticulture Sciences，Southwest Forestry University，Kunming 650224，China

Received:2024-05-17 Revised:2024-09-23 Published:2025-01-25 Online:2025-01-19
Contact:
** E-mail：xianglin@mail.hzau.edu.cn

摘要/Abstract

摘要：

以蜡梅（Chimonanthus praecox）为材料，采用顶空固相微萃取—气相色谱质谱技术对其5个花期的花器官挥发物含量进行了测定。结果表明，随着花朵的发育，苯甲醇和乙酸苄酯的含量逐渐升高，在盛开期最高。从花中克隆出结构基因CpBEAT，GenBank登录号为OQ503489，开放阅读框为1 392 bp，编码463个氨基酸。CpBEAT的表达水平随着花发育逐渐升高，表达趋势与乙酸苄酯的挥发趋势基本一致。将CpBEAT导入烟草异源表达，在外施底物苯甲醇的条件下能够合成乙酸苄酯。进一步克隆CpBEAT的启动子，通过双荧光素酶试验发现CpMYC2正向调控CpBEAT启动子转录活性。

关键词: 蜡梅, 花香, 苯甲醇乙酰基转移酶（BEAT）, 乙酸苄酯, MYC2

Abstract:

In this study，the contents of benzyl alcohol and benzyl acetate in the tepals were determined from flower of Chimonanthus praecox at five developmental stages by headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry（HS-SPME-GC-MS）. These results showed that the content of benzyl alcohol and benzyl acetate continued to increase along with the development of flowers，and the highest content was in full flowering stage flower. The sequence of CpBEAT was cloned from C. praecox and the GenBank accession number was OQ503489. CpBEAT contained an open reading frame of 1 392 bp encoding a putative protein of 463 amino acids. The expression level of CpBEAT showed a trend of rising during the flower development. The expression pattern was consistent with the volatilization of benzyl acetate in the five stages of flower development. CpBEAT could synthesize benzyl acetate under the condition of external substrate benzyl alcohol in transgenic plants. The promoter sequence of CpBEAT was also cloned. A dual luciferase assay showed that CpMYC2 positively regulated transcription activity on the CpBEAT promoter.

Key words: Chimonanthus praecox, floral fragrance, benzyl alcohol acetyltransferase（BEAT）, benzyl acetate, MYC2

王飞龙, 梁前艳, 尚均忠, 陈龙清, 向林. 蜡梅CpBEAT的克隆、表达分析及功能验证[J]. 园艺学报, 2025, 52(1): 160-170.

WANG Feilong, LIANG Qianyan, SHANG Junzhong, CHEN Longqing, XIANG Lin. Cloning，Expression and Functional Identification of CpBEAT Gene in Chimonanthus praecox[J]. Acta Horticulturae Sinica, 2025, 52(1): 160-170.

图/表 9

图1 蜡梅花朵发育不同阶段

Fig. 1 Different stages of development of Chimonanthus praecox

图2 蜡梅不同发育时段苯甲醇和乙酸苄酯挥发量采用t检测进行差异显著性分析，* P < 0.05，** P < 0.01。下同

Fig. 2 Volatilization of benzyl alcohol and benzyl acetatein Chimonanthus praecox different periods flower development Significant difference were evaluated using a t test，* P < 0.05，** P < 0.01. The same below

图3 蜡梅CpBEAT与其他植物BEAT氨基酸同源序列比对 Cp：蜡梅；Bh：冬瓜；Pd：海枣；Jr：胡桃；Cm：甜瓜。红色框表示HXXXD和DFGWG保守结构域

Fig. 3 Alignment of the deduced amino acid sequences of CpBEATfrom Chimonanthus praecox and BEAT from other plant species Cp：Chimonanthus praecox；Bh：Benincasa hispida；Pd：Phoenix dactylifera；Jr：Juglans regia；Cm：Cucumis melo. The red rectangular show the conserved domains HXXXD and DFGWG

图4 蜡梅CpBEAT与其他BAHD类基因系统进化树 Lp：大苞野芝麻；Vh：美女樱；Pf：紫苏；Gs：龙胆；Ss：一串红；Dp：大丽花；At：拟南芥；Cr：长春花；Cb：仙女扇；Rs：蛇根木；Cm：甜瓜；Fa：草莓；Rh：蔷薇；Hv：大麦；Tc：东北红豆杉；Tc：加拿大红豆杉；Mp：大蕉；Os：水稻；Mp：苹果；Zm：玉米；Pd：山杨；Cp：蜡梅。各节点处数字表示Bootstrap值（重复1 000次）

Fig. 4 Phylogenetic analysis of CpBEAT with other BAHD genes Lp：Lamium purpureum；Vh：Verbena hybrida；Pf：Perilla frutescens；Gs：Gentiana scabra；Ss：Salvia splendens；Dp：Dahlia pinnata；At：Arabidopsis thaliana；Cr：Catharanthus roseus；Cb：Clarkia breweri；Rs：Rauvolfia serpentine ；Cm：Cucumis melo；Fa：Fragaria × ananassa；Rh：Rosa hybrid；Hv：Hordeum vulgare；Tc：Taxus cuspidate；Tc：Taxus canadensis；Mp：Musa × paradisiaca；Os：Oryza sativa；Mp：Malus pumila；Zm：Zea mays；Pd：Populus davidiana；Cp：Chimonanthus praecox. The numbers at node represent the Bootstrap values（1000 replicates）

图 5 CpBEAT在蜡梅5个时期的表达

Fig. 5 The gene expression of the CpBEAT in flowers at five developmental stages of Chimonanthus praecox

图6 转CpBEAT 早花烟草阳性植株的半定量检测 M：DS2000分子量标记；A ~ K：转入CpBEAT基因植株；W：空载对照

Fig. 6 Semi-quantitative detection of CpBEAT transgenic in Nicotiana tabacum M：DS2000 marker；A-K：Plant transferred with CpBEAT gene；W：Empty vector

图7 苯甲醇处理后早花烟草野生型、转空载pCAMBIA1300、转CpBEAT基因植株挥发物的GC-MS测定及其乙酸苄酯挥发量

Fig. 7 GC-MS analysis of the volatiles from wild type，transgenic empty vector pCAMBIA1300 and transgenic CpBEAT Nicotiana tabacum after treatment with benzyl alcohol and the volatilization of benzyl acetate

图8 CpBEAT基因启动子元件

Fig. 8 Analysis of CpBEAT gene promoter elements

图9 相对双荧光素酶活性以空载体作为对照，活性定位1进行相对活性计算

Fig. 9 Statistical graph of dual-luciferase assay results Using the empty vector as a blank control，the relative activity of the experimental group was calculated with active targeting

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蜡梅CpBEAT的克隆、表达分析及功能验证

Cloning，Expression and Functional Identification of CpBEAT Gene in Chimonanthus praecox

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蜡梅CpBEAT的克隆、表达分析及功能验证

Cloning，Expression and Functional Identification of CpBEAT Gene in Chimonanthus praecox

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