Cloning of bHLH96 Gene and Its Roles in Regulating the Biosynthesis of Peppermint Terpenes

doi:10.13560/j.cnki.biotech.bull.1985.2023-0641

Abstract

Abstract:

【Objective】Peppermint essential oil is an important raw material in the cosmetics, pharmaceutical, and food industries. bHLH transcription factors（TFs）play a crucial role in regulating the biosynthesis of volatile secondary metabolites（VSMs）in plants. Exploring the role of bHLH TFs in the regulation of VSM biosynthesis in peppermint may provide important gene resources for improving peppermint germplasm through metabolic or genetic engineering, and expand our understanding of the pathways and regulatory mechanisms of plant VSM biosynthesis. 【Method】The expression pattern of bHLH96 in peppermint roots, stems, and leaves was analyzed using transcriptome sequencing and qRT-PCR techniques. The full-length of bHLH96 coding sequence（CDS）was cloned using the RT-PCR method. Subsequently, a plant expression vector, pBI121-bHLH96, was constructed using DNA recombination technology, and the overexpression of bHLH96 in the peppermint leaves was achieved through Agrobacterium-mediated transformation. The effects of bHLH96 overexpression on the content of terpenoid compounds and the expression of related genes involved in terpenoid biosynthesis in the peppermint leaves were examined. 【Result】The results showed that the full length of bHLH96 CDS was 861 bp encoding 286 amino acids. Peppermint bHLH96 protein localized at the nucleus, and the sequence similarity among plant bHLH96 proteins ranged from 45.45% to 73.68%. Peppermint bHLH96 had a close relationship with plant bHLH proteins from the Labiatae family such as Lavandula angustifolia LaMYC4 and Salvia miltiorrhiza SmbHLH94. Transient overexpression of the bHLH96 gene in peppermint leaves significantly influenced relative contents of 15 terpenoid compounds. bHLH96 overexpression significantly upregulated the expressions of 5 terpene synthesis-related genes, such as OC2, SM1, KS1, ND, and EAO1, while downregulated the expressions of NLS1, LS1 and iPR. 【Conclusion】Peppermint bHLH96 might regulate terpene synthesis by modulating the expressions of terpene synthesis-related genes and influencing the activities of terpene synthases.

Key words: peppermint, essential oil, volatile compound, synthetic regulation, bHLH transcription factor

WANG Bin, YUAN Xiao, JIANG Yuan-yuan, WANG Yu-kun, XIAO Yan-hui, HE Jin-ming. Cloning of bHLH96 Gene and Its Roles in Regulating the Biosynthesis of Peppermint Terpenes[J]. Biotechnology Bulletin, 2024, 40(1): 281-293.

Figures/Tables 10

References 38

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引物名称 Primer name	正向序列 Forward sequence（5'-3'）	反向序列 Reverse sequence（5'-3'）	用途 Usage
bHLH96	ATGTCACTAGAGGCTGCGATGG	TTAGCTACAAATTGATTCTTCTTGAATC	TA克隆TA cloning
bHLH96-GFP	acgggggactctagaggatccATGTCACTAGAGGCTGCGATGG	ggactgaccacccggggatccGCTACAAATTGATTCTTCTTGAATCAT	亚细胞定位 Subcellular localization
Actin	AGCAAAAACAAGCTCTGCCG	TGGAATAGGACCTCAGGGCA	实时荧光定量
bHLH96	AGCCCACGAAGAACAAGGAG	TAGGAAGGGGGCATGATGGA	Quantitative real-time PCR
OC2	TGTAAAATCCCCAACCCCCG	ACGGTGCTTACAAAGGAGCA
SM1	GGGGATGTCAACTAGGCACC	CATGTCCCACTTCGTCGGAA
KS1	TTGGAAGCAGGCGAGGAAAT	TACCGATTTGCCCCTCGAAC
ND	GACATCGTCGATCCTGCCTC	GTGCCTGCATTGTTCACCAG
EAO1	TTGAGCGCAGTGTACGTCTT	GCTCTCCACCAATGGGAACA
NLS1	CACCAGATTGGGCTCACCAT	AGTACTCTTTTGCCCCTGGC
LS	AACTTTGTGAACGACGCAGC	GCATCCACCAAATCCCTCCA
iPR	TCCTCCTCCTTCGGGAGTTT	CAGACTATCCTCGTCCCCCA

引物名称 Primer name	正向序列 Forward sequence（5'-3'）	反向序列 Reverse sequence（5'-3'）	用途 Usage
bHLH96	ATGTCACTAGAGGCTGCGATGG	TTAGCTACAAATTGATTCTTCTTGAATC	TA克隆TA cloning
bHLH96-GFP	acgggggactctagaggatccATGTCACTAGAGGCTGCGATGG	ggactgaccacccggggatccGCTACAAATTGATTCTTCTTGAATCAT	亚细胞定位 Subcellular localization
Actin	AGCAAAAACAAGCTCTGCCG	TGGAATAGGACCTCAGGGCA	实时荧光定量
bHLH96	AGCCCACGAAGAACAAGGAG	TAGGAAGGGGGCATGATGGA	Quantitative real-time PCR
OC2	TGTAAAATCCCCAACCCCCG	ACGGTGCTTACAAAGGAGCA
SM1	GGGGATGTCAACTAGGCACC	CATGTCCCACTTCGTCGGAA
KS1	TTGGAAGCAGGCGAGGAAAT	TACCGATTTGCCCCTCGAAC
ND	GACATCGTCGATCCTGCCTC	GTGCCTGCATTGTTCACCAG
EAO1	TTGAGCGCAGTGTACGTCTT	GCTCTCCACCAATGGGAACA
NLS1	CACCAGATTGGGCTCACCAT	AGTACTCTTTTGCCCCTGGC
LS	AACTTTGTGAACGACGCAGC	GCATCCACCAAATCCCTCCA
iPR	TCCTCCTCCTTCGGGAGTTT	CAGACTATCCTCGTCCCCCA

品种 Species	薄荷 Peppermint	短花药野生稻 Oryza brachyantha	拟南芥 Arabidopsis	烟草 Tobacco	番茄 Tomato
薄荷 Peppermint	100
短花药野生稻 Oryza brachyantha	46.71	100
拟南芥 Arabidopsis	46.44	45.45	100
烟草 Tobacco	55.74	60.52	47.04	100
番茄 Tomato	52.73	48.62	48.03	73.68	100

品种 Species	薄荷 Peppermint	短花药野生稻 Oryza brachyantha	拟南芥 Arabidopsis	烟草 Tobacco	番茄 Tomato
薄荷 Peppermint	100
短花药野生稻 Oryza brachyantha	46.71	100
拟南芥 Arabidopsis	46.44	45.45	100
烟草 Tobacco	55.74	60.52	47.04	100
番茄 Tomato	52.73	48.62	48.03	73.68	100

序号 No.	化合物 Compound	保留时间 Retention/min	相对含量Relative content/%
序号 No.	化合物 Compound	保留时间 Retention/min	CK	OE
1	己醛 Hexanal	3.80	0.36±0.03b	0.65±0.05a
2	（E）-2-己烯醛（E）-2-Hexenal	4.76	1.10±0.07b	2.66±0.06a
3	α-蒎烯 α-Pinene	6.61	2.48±0.02b	4.23±0.07a
4	香桧烯 Sabinene	7.63	0.89±0.02b	1.32±0.32a
5	L-β-蒎烯 L-β-Pinene	7.72	1.91±0.08b	3.40±0.04a
6	β-月桂烯 β-Myrcene	8.07	0.99±0.05b	1.78±0.04a
7	3-辛醇 3-Octanol	8.17	1.10±0.06	1.00±0.20
8	正辛醛 Octanal	8.40	0.07±0.00b	0.11±0.00a
9	D-柠檬烯 D-Limonene	9.12	0.46±0.08b	0.85±0.01a
10	桉叶油醇 Eucalyptol	9.21	0.31±0.02b	0.53±0.00a
11	顺式-β-罗勒烯 cis-β-Ocimene	9.36	0.14±0.00b	0.35±0.02a
12	异胡薄荷醇 Isopulegol	12.45	0.47±0.00a	0.34±0.02b
13	左旋薄荷酮 L-Menthone	12.71	7.51±0.44	7.96±1.38
14	顺式薄荷酮 cis-Menthone	13.00	3.45±0.27	3.54±0.24
15	薄荷醇 Menthol	13.40	63.1±4.38	64.16±4.25
16	异薄荷醇 Isomenthol	13.58	0.12±0.01	0.12±0.01
17	α-松油醇 α-Terpineol	13.77	0.16±0.01	0.18±0.01
18	顺式-3-己烯异戊酸cis-3-Hexenyl isovalerate	14.97	0.16±0.01a	0.11±0.01b
19	（+）-胡薄荷酮（±）-Pulegone	15.13	10.23±6.00a	3.45±0.17b
20	胡椒酮 Piperitone	15.52	0.51±0.08a	0.37±0.00b
21	石竹烯Caryophyllene	19.92	0.44±0.04	0.52±0.05
22	大根香叶烯 Germacrene D	21.46	1.27±0.01b	1.79±0.23a
23	双环大牛儿烯 Bicyclogermacrene	21.83	0.15±0.02	0.19±0.04