Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (1): 281-293.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0641
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WANG Bin1(), YUAN Xiao1,2, JIANG Yuan-yuan1, WANG Yu-kun1, XIAO Yan-hui1, HE Jin-ming1()
Received:
2023-07-05
Online:
2024-01-26
Published:
2024-02-06
Contact:
HE Jin-ming
E-mail:b_wang@sgu.edu.cn;hjm@sgu.edu.cn
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.
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 |
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 |
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
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)
Fig. 3 Nucleotide sequence(light green background)and the deduced amino acid sequence(light blue background)of peppermint bHLH96 gene * indicates stop codon
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
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 |
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 |
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]
序号 No. | 化合物 Compound | 保留时间 Retention/min | 相对含量Relative content/% | |
---|---|---|---|---|
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 |
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/% | |
---|---|---|---|---|
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 |
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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