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

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

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

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

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 Online:2025-01-25 Published:2025-01-19
Contact: XIANG Lin

Abstract

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

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.

Figures/Tables 9

Fig. 1 Different stages of development of Chimonanthus praecox

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

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

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）

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

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

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

Fig. 8 Analysis of CpBEAT gene promoter elements

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

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