bHLH96的克隆及其在薄荷萜烯生物合成调控中的功能

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

摘要/Abstract

摘要：

【目的】薄荷精油是日化、医药和食品工业中的重要原料，bHLH转录因子在调控植物挥发性次生代谢产物合成中具有重要作用。探究bHLH蛋白在薄荷挥发性物质合成调控中的作用，为通过代谢或基因工程改良薄荷种质提供重要基因资源，拓展有关植物挥发性次生代谢产物合成途径与调控机制的认识。【方法】利用转录组测序和RT-qPCR技术分析bHLH96在薄荷根、茎、叶中的表达，并通过RT-PCR法克隆其全长序列，再利用DNA重组技术构建植物表达载体pBI121-bHLH96，利用农杆菌介导法在薄荷叶片中过表达，检测过表达bHLH96对薄荷叶片萜烯化合物含量和合成相关基因表达的影响。【结果】薄荷bHLH96的编码区全长861 bp，编码286个氨基酸残基。薄荷bHLH96是一个细胞核定位蛋白，植物bHLH96蛋白间的序列相似性为45.45%-73.68%，薄荷bHLH96与薰衣草LaMYC4和丹参SmbHLH94等唇形科植物bHLH蛋白的亲缘关系较近。在薄荷叶片中瞬时过表达bHLH96，显著影响15种萜烯化合物的相对含量。过表达bHLH96显著上调萜烯合成相关基因OC2、SM1、KS1、ND和EAO1的表达，显著下调NLS1、LS1和iPR的表达。【结论】薄荷bHLH96可能通过调控萜烯合成相关基因的表达，影响相关萜合成酶的活性，从而调控薄荷萜烯物质的合成。

关键词: 薄荷, 精油, 挥发性化合物, 合成调控, bHLH转录因子

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

王斌, 袁晓, 蒋园园, 王玉昆, 肖艳辉, 何金明. bHLH96的克隆及其在薄荷萜烯生物合成调控中的功能[J]. 生物技术通报, 2024, 40(1): 281-293.

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.

图/表 10

表1 引物信息

Table 1 Primer information

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

图1 薄荷bHLH家族基因的表达模式及bHLH96的表达 A：薄荷不同组织中差异表达的bHLH家族基因的表达热图；B：转录组测序得到的bHLH96表达量；C：RT-qPCR法测定的bHLH96表达量；L：叶片，S：茎，R：根；1-3表示3个生物学重复；黑色方框中显示3个在叶片中高表达的bHLH基因；不同小写字母表示不同组织间差异显著（P≤0.05）。下同

Fig. 1 Expression patterns of bHLH family genes and bHLH96 expression A: Expression heatmap of bHLH family genes differentially expressed in different tissues of peppermint. B: Expression values of bHLH96 from RNA-Seq. C: Expression level of bHLH96 measured by RT-qPCR method. L: Leaf. S: Shoot. R: Root. 1-3 represent three biological replicates; three bHLH genes that are highly expressed in leaves are shown in the black box. Different lowercase letters indicate significant differences between different tissues（P≤0.05）. The same below

图2 薄荷bHLH96的PCR扩增产物电泳图 A：以薄荷叶片cDNA为模板扩增的DNA片段；B：以重组质粒为模板扩增的DNA片段；M：不同分子量的DNA标准物；1-4：bHLH96全长序列的扩增产物（1：以cDNA为模板扩增的DNA片段；2-4：以重组质粒为模板扩增的DNA片段）

Fig. 2 Electrophoresis maps of PCR amplification products of peppermint bHLH96 gene A: DNA fragments amplified using peppermint leaf cDNA as a template. B: DNA fragments amplified using recombinant plasmids as templates. M: DNA marker. 1-4: DNA amplified products of the full-length sequence of bHLH96 gene（1: DNA fragment amplified using cDNA as a template, 2-4: DNA fragments amplified using recombinant plasmids as templates）

图3 薄荷bHLH96的核苷酸序列（浅绿色背景）和推导的氨基酸序列（浅蓝色背景） *代表终止密码子

Fig. 3 Nucleotide sequence（light green background）and the deduced amino acid sequence（light blue background）of peppermint bHLH96 gene * indicates stop codon

图4 薄荷bHLH96转录因子的亚细胞定位

Fig. 4 Subcellular localization of peppermint bHLH96 transcription factor

图5 薄荷bHLH96与模式植物bHLH96氨基酸序列比对（A）和保守结构域保守性分析（B）下划线显示bHLH蛋白的保守结构域序列；ObbHLH96：野生水稻，XP_006649528.1；AtbHLH96：拟南芥，NP_001320586.1；NtbHLH96：烟草，XP_016477918.1；SlbHLH96：番茄，XP_004250124.1。下同

Fig. 5 Amino acid sequence alignment（A）and conservation analysis of conserved domain（B）of peppermint bHLH96 and bHLH96 from model plants The underlined line indicates conserved domain sequences of bHLH proteins. ObbHLH96: Oryza brachyantha, XP_006649528.1. AtbHLH96: Arabidopsis thaliana, NP_001320586.1; NtbHLH96: Nicotiana tabacum, XP_016477918.1; SlbHLH96: Solanum lycopersicum, XP_004250124.1. The same below

表2 不同植物bHLH96氨基酸序列一致性比较

Table 2 Comparison in the amino acid sequences of bHLH96 proteins in different plant species %

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

图6 植物bHLH蛋白系统发育分析 ▲显示薄荷bHLH96；薰衣草LaMYC4的氨基酸序列见文献[20]

Fig. 6 Phylogenetic analysis of plant bHLH proteins ▲ indicates peppermint bHLH96. The amino acid sequence of lavender LaMYC4 can be found in the reference[20]

表3 CK和OE薄荷叶片中的挥发性化合物种类和相对含量

Table 3 Types and relative contents of volatile compounds in the leaves of CK and OE peppermint seedlings

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

图7 薄荷叶片中瞬时过表达bHLH96对相关TPS基因表达的影响 CK：过表达pBI121空载；OE：过表达bHLH96

Fig. 7 Effects of transient overexpression of bHLH96 in the peppermint leaves on the expressions of TPS genes CK: Overexpression of pBI121 plasmid. OE: Overexpression of bHLH96

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