薰衣草LaGGPPS5基因在大肠杆菌中催化萜类物质合成

doi:10.13304/j.nykjdb.2023.0759

摘要/Abstract

摘要：

异戊烯基焦磷酸合酶（isoprenyl diphosphate synthase， IDS）在植物萜类多样化产物合成中发挥重要作用。克隆了薰衣草LaGGPPS5基因，其开放阅读框为1 038 bp，编码345个氨基酸，蛋白的相对分子量为37.20 kD，在薰衣草的茎和叶中表达量显著高于其他组织，在花蕾和花中表达量极低。利用包含MVA途径上游基因的载体共转化构建重组大肠杆菌菌株，经IPTG诱导后，重组蛋白LaGGPPS5表达条带37 kD左右，与预测蛋白分子量一致。GC-MS检测发酵产物显示，在MVA途径基础上，重组LaGGPPS5的BL21 （DE3）菌株合成了包括香叶醇乙酯、反，反-金合欢乙酯、香茅醇乙酯、橙花醇乙酯、2，3-二氢金合欢醇乙酯、反式-金合欢醇、香叶醇等25种单萜和倍半萜及其衍生物，萜类组分占挥发性物质总量的88.2%，其中香叶醇乙酯和反，反-金合欢乙酯的相对含量各占总含量的43.21%和20.36%，单萜类和倍半萜类挥发性物质分别占萜类总量的65.38%和34.62%，表明LaGGPPS5兼具香叶基焦磷酸合成酶（GPPS）和法尼基焦磷酸合成酶（FPPS）的功能。研究结果为LaGGPPS5基因在植物芳香组分分子改良以及大肠杆菌工程菌发酵生产萜烯类物质提供参考。

关键词: 薰衣草, 萜类, 异戊烯基焦磷酸合酶, 发酵

Abstract:

Isoprenyl diphosphate synthase （IDS） plays an important role in the the diversification of plant terpene synthesis.. LaGGPPS5 was cloned from lavender plant， and its open reading frame of LaGGPPS5 was 1 038 bp and encoded 345 amino acids with a relative molecular mass of 37.20 kD. LaGGPPS5 expressed significantly higher level in lavender stems and leaves compared to other plant tissues， while it nearly was absent in flower buds and flowers. A recombinant Escherichia coli strain was constructed through multiplasmid co-transformation， utilizing fundamental vectors contained upstream genes in MVA pathway. The recombination LaGGPPS5 displayed a 37 kD band following induction by IPTG， consistent with the anticapted molecular weight of the protein. The GC-MS analysis of fermentation products demonstrated that the BL21 （DE3） strain of recombinant LaGGPPS5 successfully synthesized a total of 25 monoterpenes， sesquiterpenes and their derivatives， including geranyl acetate， trans， trans-farnesyl acetate， citronellol acetate， nerol acetate， 2，3-dihydro farnesyl acetate， trans-farnesol， geraniol， etc. The terpenoid components constituted a significant proportion of 88.2% of the overall volatile matter. Notably， geranyl acetate and trans， trans-farnesyl acetate accounted for 43.21% and 20.36% of the total content， and the volatile compounds of monoterpenoids and sesquiterpenes accounted for 65.38% and 34.62% of the total terpenoids， respectively. These findings indicated that LaGGPPS5 had dual functionality as both geranyl diphosphate synthase （GPPS） and farnesyl diphosphate synthase （FPPS）. The results of this study offered valuable insights for enhancing the molecular composition of plant aromatic constituents and the fermentation of terpenes through the manipulation of E. coli engineering bacteria utilizing LaGGPPS5 gene.

Key words: lavender, terpenoids, isoprenyl diphosphate synthase, ferment

中图分类号:

岳俊齐, 约日耶提·萨力, 克拉热木·克里木江, 陈永坤. 薰衣草LaGGPPS5基因在大肠杆菌中催化萜类物质合成[J]. 中国农业科技导报, 2023, 25(12): 85-92.

Junqi YUE, Yueriyeti Sali, Kelaremu Kelimujiang, Yongkun CHEN. Lavender LaGGPPS5 Gene Catalyzing Terpenoids Synthesis in Escherichia coli[J]. Journal of Agricultural Science and Technology, 2023, 25(12): 85-92.

图/表 7

参考文献 28

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引物命名 Primer name	序列信息 Sequence information （5’-3’）	片段大小Fragment size/bp	引物用途 Primer purpose
GGPPS5F	TAAGGAGATATACCATGGGCATGAGGTCTATGAATCTGGGTGTTC	1 077	LaGGPPS5克隆 LaGGPPS5 clone
GGPPS5R	CGAGCTCGAATTCGGATCCTTAGTTCTGCCGGTGAGCAATGTAA	1 077	LaGGPPS5克隆 LaGGPPS5 clone
RSFdetcF	TGTAGAAATAATTTTGTTTAAC	1 166	pRSFDuet-1载体检测 pRSFDuet-1 vector check
RSFdetcR	TACGATTACTTTCTGTTCGA	1 166	pRSFDuet-1载体检测 pRSFDuet-1 vector check
qGGPPS5F	CCGATGTGGGATTGAAGACATTAG	123	LaGGPPS5荧光定量 LaGGPPS5 fluorescent quantitation
qGGPPS5R	TCAACTTCTCCACCTGTTCATCACT	123	LaGGPPS5荧光定量 LaGGPPS5 fluorescent quantitation
ActinF	GGTCGTACAACTGGTATTGTTC	139	内参基因LaActin Reference gene LaActin
ActinR	TCTTCATGAGGGAGTCGGTGAGA	139	内参基因LaActin Reference gene LaActin
ACYdetcF	CAGTGTGGAAGTCGGTATTG	712	pYJM16载体检测 pYJM16 vector check
ACYdetcR	AATCAATGACTCGGCGTCTG	712	pYJM16载体检测 pYJM16 vector check
TRCdetcF	ATGACTGCCGACAACAATAGTAT	1 942	pYJM14载体检测 pYJM14 vector check
TRCdetcR	TCCTTCGGTCCTCCGATCGTTGT	1 942	pYJM14载体检测 pYJM14 vector check

引物命名 Primer name	序列信息 Sequence information （5’-3’）	片段大小Fragment size/bp	引物用途 Primer purpose
GGPPS5F	TAAGGAGATATACCATGGGCATGAGGTCTATGAATCTGGGTGTTC	1 077	LaGGPPS5克隆 LaGGPPS5 clone
GGPPS5R	CGAGCTCGAATTCGGATCCTTAGTTCTGCCGGTGAGCAATGTAA	1 077	LaGGPPS5克隆 LaGGPPS5 clone
RSFdetcF	TGTAGAAATAATTTTGTTTAAC	1 166	pRSFDuet-1载体检测 pRSFDuet-1 vector check
RSFdetcR	TACGATTACTTTCTGTTCGA	1 166	pRSFDuet-1载体检测 pRSFDuet-1 vector check
qGGPPS5F	CCGATGTGGGATTGAAGACATTAG	123	LaGGPPS5荧光定量 LaGGPPS5 fluorescent quantitation
qGGPPS5R	TCAACTTCTCCACCTGTTCATCACT	123	LaGGPPS5荧光定量 LaGGPPS5 fluorescent quantitation
ActinF	GGTCGTACAACTGGTATTGTTC	139	内参基因LaActin Reference gene LaActin
ActinR	TCTTCATGAGGGAGTCGGTGAGA	139	内参基因LaActin Reference gene LaActin
ACYdetcF	CAGTGTGGAAGTCGGTATTG	712	pYJM16载体检测 pYJM16 vector check
ACYdetcR	AATCAATGACTCGGCGTCTG	712	pYJM16载体检测 pYJM16 vector check
TRCdetcF	ATGACTGCCGACAACAATAGTAT	1 942	pYJM14载体检测 pYJM14 vector check
TRCdetcR	TCCTTCGGTCCTCCGATCGTTGT	1 942	pYJM14载体检测 pYJM14 vector check

序号 Serial No.	化合物名称 Compound name	保留时间 Retention time/min	峰面积占比 Peak area ratio/%
1	香茅醇乙酯 Citronellol acetate	19.223	5.26
2	香叶醇乙酯 Geranyl acetate	20.124	43.21
3	反，反-金合欢乙酯 trans， trans-farnesyl acetate	28.882	20.36
4	香茅醇 Citronellol	15.789	1.91
5	香叶醇 Geraniol	16.533	2.22
6	橙花醇乙酯 Nerol acetate	19.517	2.69
7	（E）-β-金合欢烯（E）-β-farnesene	21.894	1.12
8	E-橙花叔醇 E-nerolidol	24.278	1.29
9	2，3-二氢金合欢醇 2，3-dihydrofarnesol	26.561	1.72
10	反式-金合欢醇 trans-farnesol	27.080	2.29
11	2，3-二氢金合欢醇乙酯2，3-dihydro farnesyl acetate	28.387	2.43

序号 Serial No.	化合物名称 Compound name	保留时间 Retention time/min	峰面积占比 Peak area ratio/%
1	香茅醇乙酯 Citronellol acetate	19.223	5.26
2	香叶醇乙酯 Geranyl acetate	20.124	43.21
3	反，反-金合欢乙酯 trans， trans-farnesyl acetate	28.882	20.36
4	香茅醇 Citronellol	15.789	1.91
5	香叶醇 Geraniol	16.533	2.22
6	橙花醇乙酯 Nerol acetate	19.517	2.69
7	（E）-β-金合欢烯（E）-β-farnesene	21.894	1.12
8	E-橙花叔醇 E-nerolidol	24.278	1.29
9	2，3-二氢金合欢醇 2，3-dihydrofarnesol	26.561	1.72
10	反式-金合欢醇 trans-farnesol	27.080	2.29
11	2，3-二氢金合欢醇乙酯2，3-dihydro farnesyl acetate	28.387	2.43

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