Cloning of bHLH Transcription Factor UNE10 and Its Regulatory Roles in the Biosynthesis of Volatile Compounds in Clove Basil

doi:10.13560/j.cnki.biotech.bull.1985.2025-0218

Abstract

Abstract:

Objective bHLH transcription factors are key regulators in the biosynthesis of secondary metabolites in aromatic plants. The investigation into the regulatory roles of the bHLH transcription factor UNE10 in the biosynthesis of volatile compounds in clove basil (Ocimum gratissimum) provides critical regulatory factors for fine-tuning the essential oil components of this plant. Method The bHLH family gene OgUNE10, which has significantly high expression in the leaves of clove basil, was identified. The coding sequence of OgUNE10 was amplified using PCR, followed by its overexpression in the leaves of clove basil through Agrobacterium-mediated transformation to assess its impacts on the biosynthesis of volatile compounds. Result OgUNE10 expression in clove basil demonstrated notable tissue specificity, with particularly high levels in the leaves. The OgUNE10 gene was successfully cloned from the leaves and encoded a protein consisting of 161 amino acid residues, featuring a conserved bHLH domain. This protein was localized in the nucleus and presented low homology with bHLH amino acid sequences from model plants. The overexpression of OgUNE10 in the leaves significantly affected the contents of major components in the essential oil of clove basil, resulting in an increase in eugenol content while decreasing the contents of estragole and methyl cinnamate. Furthermore, OgUNE10 overexpression led to significant upregulations on three PAL (phenylalanine ammonia lyase) and two MES (methylesterase) genes, but downregulations on one MTA (methyltransferases) gene, while also affecting the expression of six terpene synthase-related genes. Conclusion OgUNE10 regulates the biosynthesis of volatile compounds by integrating the phenylpropanoid pathway, MEP and MVA pathways in clove basil.

Key words: clove basil, volatile compounds, biosynthesis regulation, bHLH transcription factor, UNE subfamily

WANG Bin, LIN Chong, YUAN Xiao, JIANG Yuan-yuan, WANG Yu-kun, XIAO Yan-hui. Cloning of bHLH Transcription Factor UNE10 and Its Regulatory Roles in the Biosynthesis of Volatile Compounds in Clove Basil[J]. Biotechnology Bulletin, 2025, 41(9): 207-218.

Figures/Tables 11

References 40

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基因名称 Gene name	正向序列 Forward sequence（5′-3′）	反向序列 Reverse sequence（5′-3′）	实验用途 Experimental purpose
OgUNE10	ggactctagaggatccccgggATGAAGACATTGCAGAAGTTGGTC	ataagggactgaccacccgggGTTCTTG GAGGCAGAAGCCG	TA克隆、亚细胞定位和过表达 TA cloning， subcellular localization， and overexpression
OgACTIN7	GGAGCTCGTCTTTGCTGTCT	GAGCGGGAAATTGTGAGGGA	RT-qPCR
OgUNE10	TGTCTATGATGGGCATGGGC	GCCTGACAAGCAAAGAAGGC
OgPAL1	TTTTTCGCCCGTCAAGAAGC	CTGGAGAGTGGGAATCCAGC
OgPAL2	GGATCCCCTGCAGAAACCAA	ATCAATCAGCGGGTTGTCGT
OgPAL4	ATGCCGGAGAAACCTTGGAG	ATTCTCCCTCGCTCAGCAAC
OgMES1	GATAGGCCCTCGTGGAGTTG	TAACTGCATCAACGCCCAGT
OgMES2	CTCCATGGTCCGGGCTATTC	TGTCCCCTCAAATCCAAGGC
OgMAT1	TCTACACACGCACACTCGAC	GTACCAGCACCATGCTCCAT
OgEFS1	CCACACTTGTTGCTCCTTGC	GCTGGTAGTTTCCAGAGCGT
OgMD1	GGTCTTCAACAGCCCGATCA	ACGTCGGAACTTTTGACGGA
OgCyt P450	AATCCGATAATGGGTCCGCC	TTGCTGCAGGAGAAGTAGCC
OgG8H1	TGGGTCCGTCAAATCCTTGG	ACTGCCGGAAAAGTAGCCTC
OgiPR1	GCAAAGTGAAGAGGCCAACG	TTCTGCCGAATGAATGGGCT
OgGD2	ATCTTGGCGTGGACGAGTTT	TCAGCAACGGAACAATGGGA

基因名称 Gene name	正向序列 Forward sequence（5′-3′）	反向序列 Reverse sequence（5′-3′）	实验用途 Experimental purpose
OgUNE10	ggactctagaggatccccgggATGAAGACATTGCAGAAGTTGGTC	ataagggactgaccacccgggGTTCTTG GAGGCAGAAGCCG	TA克隆、亚细胞定位和过表达 TA cloning， subcellular localization， and overexpression
OgACTIN7	GGAGCTCGTCTTTGCTGTCT	GAGCGGGAAATTGTGAGGGA	RT-qPCR
OgUNE10	TGTCTATGATGGGCATGGGC	GCCTGACAAGCAAAGAAGGC
OgPAL1	TTTTTCGCCCGTCAAGAAGC	CTGGAGAGTGGGAATCCAGC
OgPAL2	GGATCCCCTGCAGAAACCAA	ATCAATCAGCGGGTTGTCGT
OgPAL4	ATGCCGGAGAAACCTTGGAG	ATTCTCCCTCGCTCAGCAAC
OgMES1	GATAGGCCCTCGTGGAGTTG	TAACTGCATCAACGCCCAGT
OgMES2	CTCCATGGTCCGGGCTATTC	TGTCCCCTCAAATCCAAGGC
OgMAT1	TCTACACACGCACACTCGAC	GTACCAGCACCATGCTCCAT
OgEFS1	CCACACTTGTTGCTCCTTGC	GCTGGTAGTTTCCAGAGCGT
OgMD1	GGTCTTCAACAGCCCGATCA	ACGTCGGAACTTTTGACGGA
OgCyt P450	AATCCGATAATGGGTCCGCC	TTGCTGCAGGAGAAGTAGCC
OgG8H1	TGGGTCCGTCAAATCCTTGG	ACTGCCGGAAAAGTAGCCTC
OgiPR1	GCAAAGTGAAGAGGCCAACG	TTCTGCCGAATGAATGGGCT
OgGD2	ATCTTGGCGTGGACGAGTTT	TCAGCAACGGAACAATGGGA

植物类型Plant species	丁香罗勒Ocimum gratissimum	水稻Oryza sativa	拟南芥Arabidopsis thaliana	烟草Nicotiana tabacum	番茄Solanum lycopersicum
丁香罗勒 Ocimum gratissimum	100
水稻 Oryza sativa	23.55	100
拟南芥 Arabidopsis thaliana	29.51	52.57	100
烟草 Nicotiana tabacum	24.31	47.90	52.89	100
番茄 Solanum lycopersicum	22.87	47.98	53.05	79.45	100

植物类型Plant species	丁香罗勒Ocimum gratissimum	水稻Oryza sativa	拟南芥Arabidopsis thaliana	烟草Nicotiana tabacum	番茄Solanum lycopersicum
丁香罗勒 Ocimum gratissimum	100
水稻 Oryza sativa	23.55	100
拟南芥 Arabidopsis thaliana	29.51	52.57	100
烟草 Nicotiana tabacum	24.31	47.90	52.89	100
番茄 Solanum lycopersicum	22.87	47.98	53.05	79.45	100

序号 No.	化合物 Compound	保留时间 Retention time （min）	相对含量 Relative content （%）
序号 No.	化合物 Compound	保留时间 Retention time （min）	35S：：GFP	35S：：OgUNE10-GFP
1	3-己烯醛 3-Hexenal	4.05	0.27±0.01	0.38±0.08*
2	α-蒎烯 α-Pinene	6.19	0.23±0.04	0.25±0.04
3	莰烯 Camphene	6.45	-	0.10±0.01
4	香桧烯 Sabinene	6.84	0.13±0.03	-
5	β-蒎烯 β-Pinene	6.90	0.24±0.04	0.27±0.02
6	β-月桂烯 β-Myrcene	7.08	0.14±0.01	0.19±0.03
7	D-枞油烯 D-sylvestrene	7.73	0.10±0.01	0.15±0.01*
8	桉叶油醇 Eucalyptol	7.78	0.64±0.08	0.71±0.06
9	反式-β-罗勒烯 Trans-β-Ocimene	8.00	0.77±0.03	0.74±0.03
10	茴香酮 Fenchone	8.71	0.23±0.04	1.02±0.05*
11	芳樟醇 Linalool	8.81	1.53±0.05	4.65±0.19*
12	（+）-2-莰酮（+）-2-Bornanone	9.60	0.28±0.03	0.71±0.12*
13	L-4-松油醇 L-terpinen-4-ol	10.06	0.21±0.03*	0.14±0.03
14	L-α-松油醇 L-α-Terpineol	10.25	0.09±0.01	0.10±0.02
15	草蒿脑 Estragole	10.35	17.43±1.33*	1.08±0.03
16	顺式肉桂酸甲酯 Methyl cis-cinnamate	11.87	0.17±0.05	-
17	丁香酚 Eugenol	12.59	20.10±5.75	46.58±1.11*
18	肉桂酸甲酯 Methyl cinnamate	12.94	51.39±5.28*	34.43±0.28
19	β-榄香烯 β-Elemene	13.09	0.13±0.01	0.28±0.01*
20	甲基丁香酚 Methyl eugenol	13.16	1.48±0.23	3.73±0.04*
21	反式-α-佛手柑油烯 Trans-α-Bergamotene	13.64	0.84±0.09	3.73±0.04*
22	α-愈创木烯 α-Guaiene	13.71	-	0.09±0.01
23	蛇麻烯 Humulene	13.95	0.21±0.05	0.35±0.03*
24	顺式-4（15），5-衣兰油二烯 Cis-Muurola-4（15），5-diene	14.06	0.19±0.02	0.19±0.04
25	大根香叶烯D Germacrene D	14.29	0.95±0.11	0.97±0.06
26	β-环大根香叶烷 β-Cyclogermacrane	14.48	0.22±0.02	0.33±0.02*
27	δ-愈创木烯 δ-Guaiene	14.58	-	0.14±0.02
28	γ-愈创木烯 γ-Cadinene	14.68	0.62±0.03	0.83±0.04*
29	τ-杜松醇 τ-Cadinol	16.18	0.83±0.08	0.77±0.06
总鉴定率 Total identification rate （%）			99.42	99.49