烟草单萜合酶基因NtTPS2的克隆及功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-1492

摘要/Abstract

摘要：

萜类化合物是植物次生代谢产物中结构和功能最丰富多样的类群,在植物的生长发育、胁迫响应以及与环境中的生物因子和非生物因子的互作中具有重要作用。研究烟草（Nicotiana tabacum）单萜合酶基因的功能,为研究烟草萜类化合物的生物合成提供依据。从烟草中克隆得到一个单萜合酶基因,命名为NtTPS2。该基因的完整ORF为1 854 bp,编码617个氨基酸的蛋白质,具有N端信号肽以及保守的DDxxD和NSE/DTE功能结构域。进化分析表明NtTPS2属于TPS-II分支,与矮牵牛、柠檬及柑橘等物种中的单萜合酶具有较高同源性。NtTPS2定位于叶绿体中,在烟草的根和雌蕊中表达量较高,且能够响应低温、高盐、干旱和ABA处理。将NtTPS2转入含甲羟戊酸途径以及牻牛儿基焦磷酸合成酶的大肠杆菌中,发现该工程菌可以合成单萜类化合物香叶醇及其异构体橙花醇。NtTPS2在烟草单萜类化合物的生物合成代谢中具有重要作用。

关键词: 烟草, NtTPS2, 单萜合酶, 香叶醇, 橙花醇

Abstract:

Terpenoids constitute the most abundant and diverse family in structure and function of the secondary metabolites of plants. They play important roles in the regulation of plant growth and development,stress responses and interactions with environmental biotic and abiotic factors. To study the biosynthesis of terpenoids in tobacco（Nicotiana tabacum）,a monoterpene synthase was cloned from N. tabacum,named as NtTPS2. NtTPS2 contained an open reading frame（ORF）of 1 854 bp,encoding a protein of 617 amino acids with a N-terminal signal peptide and conserved DDxxD and NSE/DTE domains. Phylogenetic analysis revealed that NtTPS2 was in the TPS-II clade sharing high amino acid sequence identities with monoterpene synthase genes from petunia（Petunia hybrida）,lemon（Citrus limon）and citrus（Citrus reticulata Blanco）. NtTPS2 was located in the chloroplast in plant cells. Real-time PCR analysis showed that NtTPS2 expressed abundantly in the roots and pistils,and responded to the treatments of low temperature,high salt,drought and ABA. When NtTPS2 was co-expressed in Escherichia coli with mevalonate pathway and geranyl diphosphate synthase,the formation of monoterpene geraniol and its isomer nerol was detected. These results suggest that NtTPS2 plays a key role in the biosynthesis and metabolism of monoterpene in tobacco.

Key words: Nicotiana tabacum, NtTPS2, monoterpene synthase, geraniol, nerol

刘少华, 赵希胜, 杨晴, 杨长青, 潘旭浩, 张建会, 杨爱国, 李依婷. 烟草单萜合酶基因NtTPS2的克隆及功能鉴定[J]. 生物技术通报, 2021, 37(9): 132-141.

LIU Shao-hua, ZHAO Xi-sheng, YANG Qing, YANG Chang-qing, PAN Xu-hao, ZHANG Jian-hui, YANG Ai-guo, LI Yi-ting. Cloning and Functional Identification of Monoterpene Synthase Gene NtTPS2 in Tobacco[J]. Biotechnology Bulletin, 2021, 37(9): 132-141.

图/表 9

表1 NtTPS2基因克隆、载体构建及qPCR引物

Table 1 Primers for NtTPS2 gene cloning,vector construction and qPCR

引物名称 Primer	引物序列Sequence（5'-3'）
NtTPS2-F	ATGGCCACCTCCATAAGACCTGCAA
NtTPS2-R	TTATAGGGATGGATTGGGAGTCAAT
NtTPS2∷gfp-F	CAGTGGTCTCACAACATGGCCACCTCCATAAGACC
NtTPS2∷gfp-R	CAGTGGTCTCATACATAGGGATGGATTGGGAGTCA
NtTPS2-pYJM26-F	GAAGATCTATGGCCACCTCCATAAGACCTGCAA
NtTPS2-pYJM26-R	CCCTCGAGTTATAGGGATGGATTGGGAGTCAAT
Actin-F	TTCCGATGCCCTGAAGTCCT
Actin-R	TCTGCCTTTGCAATCCACAT
NtTPS2-qF	AAATGCCAAACGCTAATCCT
NtTPS2-qR	TTCCACCACCTTGATACATCTC

图1 NtTPS2的PCR扩增 M：RB 2 000 DNA分子标记;1：PCR扩增产物

Fig.1 PCR amplification of NtTPS2 M：RB 2 000 DNA molecular marker;1：PCR amplification product

图2 NtTPS2蛋白的保守结构域预测 A：萜类合酶,N末端区域;B：类异戊二烯合酶区域,萜类合酶金属结合结构域

Fig.2 Prediction of the conserved domain of NtTPS2 protein A：Terpene synthase,N-terminal domain;B：isoprenoid synthase domain,terpene synthase,metal-binding domain

图3 NtTPS2与其他植物萜烯合酶基因的多序列比对红色方框标注为萜烯合酶的保守结构域“DDxxD”和“（N,D）D（L,I,V）X（S,T）XXXE”。AcNES：猕猴桃橙花醇合成酶（AER36088.1）;MsLIS：薄荷柠檬烯合成酶（AAC37366.1）;ObZIS：罗勒α-松烯合成酶（AAV63788.1）;AaLIS：青蒿芳樟醇合成酶（AAF13356.1）;VvGES：葡萄香叶醇合成酶（NP_001267895.1）

Fig.3 Multiple sequence alignment of NtTPS2 and other plant terpene synthase genes The red boxes are marked as conserved domains of terpene synthase “DDxxD” and “（N,D）D（L,I,V）X（S,T）XXXE”. AcNES：Actinidia chinensis nerol synthase（AER36088.1）;MsLIS：Mentha spicata limonene synthase（AAC37366.1）;ObZIS：Ocimum basilicum α-pinene synthase（AAV63788.1）;AaLIS：Artemisia annua linalool synthase（AAF13356.1）;VvGES：Vitis vinifera geraniol synthase（NP_001267895.1）

图4 NtTPS2与其他物种萜烯合酶系统发育进化树分析

Fig.4 Phylogenetic tree analysis of NtTPS2 and terpene synthase of other species

图5 NtTPS2的亚细胞定位

Fig.5 Subcellular localization of NtTPS2

图6 NtTPS2的组织表达模式分析不同字母表示在P<0.05水平差异显著。下同

Fig.6 Tissue expression pattern analysis of NtTPS2 Different letters indicate significant differences at the P<0.05 level. The same below

图7 不同逆境胁迫下NtTPS2的表达模式分析

Fig.7 Expression analysis of NtTPS2 under different stresses

图8 NtTPS2的代谢产物分析 A：香叶醇和橙花醇混合标准品GC-MS图;B：NtTPS2工程菌合成出的香叶醇和橙花醇GC-MS图;C：对照工程菌合成产物的GC-MS图;D：橙花醇标准品GC-MS分子碎片质谱图;E：香叶醇标准品GC-MS分子碎片质谱图;F：NtTPS2工程菌合成出的橙花醇GC-MS分子碎片质谱图;G：NtTPS2工程菌合成出的香叶醇GC-MS分子碎片质谱图

Fig.8 Analysis of metabolites of NtTPS2 A：GC-MS analyses of mixed standard products of geraniol and nerol;B：GC-MS analyses of geraniol and nerol synthesized by NtTPS2 engineering bacteria;C：GC-MS analyses of products synthesized by control engineering bacteria;D：mass spectrum of nerol standard;E：mass spectrum of geraniol standard;F：mass spectrum of nerol synthesized by NtTPS2 engineering bacteria;G.：mass spectrum of geraniol synthesized by NtTPS2 engineering bacteria

参考文献 30

[1]	Benford HL, Frith JC, Auriola S, et al. Farnesol and geranylgeraniol prevent activation of caspases by aminobisphosphonates:biochemical evidence for two distinct pharmacological classes of bisphosphonate drugs[J]. Molecular Pharmacology, 1999, 56(1):131-140. pmid: 10385693
[2]	Kotti T, Ramirez D, Pfeiffer B, et al. Brain cholesterol turnover required for geranylgeraniol production and learning in mice[J]. Proceedings of the National Academy of ences of the United States of America, 2006, 103(10):3869-3874.
[3]	Ito M, Honda G. Geraniol synthases from perilla and their taxonomical significance[J]. Phytochemistry, 2007, 68(4):446-453. doi: 10.1016/j.phytochem.2006.11.006 URL
[4]	王凌健, 方欣, 杨长青, 等. 植物萜类次生代谢及其调控[J]. 中国科学:生命科学, 2013, 43(12):1030-1046.
	Wang LJ, Fang X, Yang CQ, et al. Biosynjournal and regulation of secondary terpenoid metabolism in plants[J]. Science China Life Sciences, 2013, 43(12):1030-1046.
[5]	梁宗锁, 方誉民, 杨东风. 植物萜类化合物生物合成与调控及其代谢工程研究进展[J]. 浙江理工大学学报:自然科学版, 2017, 37(2):255-264.
	Liang ZS, Fang YM, Yang DF. Biosynjournal, regulation and metabolic engineering of terpenoids in plants[J]. Journal of Zhejiang Sci-Tech University:Natural Sciences Edition, 2017, 37(2):255-264.
[6]	Mohammed MJ, Tadros MG, Michel HE. Geraniol protects against cyclophosphamide-induced hepatotoxicity in rats:Possible role of MAPK and PPAR-γ signaling pathways[J]. Food and Chemical Toxicology, 2020, 139:111251. doi: S0278-6915(20)30139-3 pmid: 32171873
[7]	吴凤礼, 彭彦峰, 徐毅诚, 等. 代谢工程改造微生物生产芳香族化合物的研究进展[J]. 生物加工过程, 2017, 15(5):9-23.
	Wu FL, Peng YF, Xu YC, et al. Advances in microbial metabolic engineering for producing aromatic chemicals[J]. Chinese Journal of Bioprocess Engineering, 2017, 15(5):9-23.
[8]	Wendt KUl, Schulz GE. Isoprenoid biosynjournal:manifold chemistry catalyzed by similar enzymes[J]. Structure, 1998, 6(2):127-133. pmid: 9519404
[9]	Peters RJ, Carter OA, Zhang Y, et al. Bifunctional abietadiene synthase:mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations[J]. Biochemistry, 2003, 42(9):2700-2707. doi: 10.1021/bi020492n URL
[10]	KoKsal M, Hu H, Coates RM, et al. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase[J]. Nature Chemical Biology, 2011, 7(7):431-433. doi: 10.1038/nchembio.578 URL
[11]	Zhou K, Peters RJ. Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants[J]. Phytochemistry, 2009, 70(3):366-369. doi: 10.1016/j.phytochem.2008.12.022 pmid: 19201430
[12]	Chen F, Tholl D, D’Auria JC, et al. Biosynjournal and emission of terpenoid volatiles from Arabidopsis flowers[J]. The Plant Cell, 2003, 15:481-494. doi: 10.1105/tpc.007989 URL
[13]	Tholl D, Chen F, Petri J, et al. Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers[J]. The Plant Journal, 2005, 42:757-771. doi: 10.1111/tpj.2005.42.issue-5 URL
[14]	Huang MS, Abel C, Sohrabi R, et al. Variation of herbivore-induced volatile terpenes among Arabidopsis ecotypes depends on allelic differences and subcellular targeting of two terpene synthases, TPS02 and TPS03[J]. Plant Physiology, 2010, 153:1293-1310. doi: 10.1104/pp.110.154864 URL
[15]	Raguso RA. Floral scent in a whole- plant context:moving beyond pollinator attraction[J]. Functional Ecology, 2009, 23:837-840 doi: 10.1111/fec.2009.23.issue-5 URL
[16]	Martin DM, Bohlmann J. Identification of Vitis vinifera(-)-α-terpineol synthase by in silico screening of full- length cDNA ESTs and functional characterization of recombinant terpene synthase[J]. Phytochemistry, 2004, 65(9):1223-1229. pmid: 15184006
[17]	庞强强, 蔡兴来, 孙晓东, 等. 大白菜TPS基因家族鉴定及其在高温胁迫下的表达分析[J]. 分子植物育种, 2020, 18(8):2452-2459.
	Pang QQ, Cai XL, Sun XD, et al. Identification of TPS gene family in Chinese cabbage(Brassica campestris L. )and its expression under heat stress[J]. Molecular Plant Breeding, 2020, 18(8):2452-2459.
[18]	Kayo Y, Shiduku T, Keiichiro T, et al. Rice terpene synthase 24(TPS24)encodes a jasmonate-responsive monoterpene synthase that produces an antibacterial γ-terpinene against rice pathogen[J]. Plant Physiology, 2016, 191:120-126.
[19]	Masaki K, Miho H, Kayo Y, et al. Rice terpene synthase 18(TPS18)encodes a sesquiterpene synthase that produces an antibacterial(E)-nerolidol against a bacterial pathogen of rice[J]. Gen Plant Pathol, 2018, 84:221-229.
[20]	吕婧, 陈浣, 孙亭亭, 等. 普通烟草TPS家族全基因组序列鉴定与表达分析[J]. 基因组学与应用生物学, 2017, 36(6):2518-2530.
	Lv J, Chen H, Sun TT, et al. Genome-wide sequence identification and expression analysis of the TPS gene family in nicotiana tobacum[J]. Genomics and Applied Biology, 2017, 36(6):2518-2530.
[21]	Yang JM, Nie QJ, Ren M, et al. Metabolic engineering of Esche-richia coli for the biosynjournal of alpha-pinene[J]. Biotechnology for Biofuels, 2013, 6(1):60. doi: 10.1186/1754-6834-6-60 URL
[22]	Yang JM, Zhao G, Sun YZ, et al. Bio-isoprene production using exogenous MVA pathway and isoprene synthase in Escherichia coli[J]. Bioresource Technology, 2011, 104:642-647. doi: 10.1016/j.biortech.2011.10.042 URL
[23]	唐亮, 马香, 周志钦. 植物萜类合成酶的进化研究[J]. 南大学学报:自然科学版, 2014, 36(4):89-96.
	Tang L, Ma X, Zhou Z Q. An evolutionary study of plant terpene synthases[J]. Journal of Southwest University:Natural Science Edition, 2014, 36(4):89-96.
[24]	谢翎, 汪章勋, 黄勃. 大豆TPS基因家族全基因组鉴定、分类与表达分析[J]. 中国油料作物学报, 2014, 36(2):160-167.
	Xie L, Wang Z X, Huang B. Genome-wide identification classification and expression of TPS family genes in soybean[J]. Chinese Journal of Oil Crop Sciences, 2014, 36(2):160-167.
[25]	Li HW, Zang BS, Deng XW, et al. Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice[J]. Planta, 2011, 234(5):1007-1018. doi: 10.1007/s00425-011-1458-0 URL
[26]	Xie DW, Wang XN, Fu LS, et al. Identification of the trehalose-6-phosphate synthase gene family in winter wheat and expression analysis under conditions of freezing stress[J]. Journal of Genetics, 2015, 94(1):55-65. pmid: 25846877
[27]	Han JL, Li ZQ, Liu BY, et al. Metabolic engineering of terpenoids in plants[J]. Chinese Journal of Biotechnology, 2007, 23(4):561-569.
[28]	王诗语, 王鹤蓉, 黄子豪, 等. 利用基因工程改造的大肠杆菌合成蒎烯[J]. 生物技术通讯, 2019, 30(4):455-463.
	Wang SY, Wang HR, Huang ZH, et al. Efficient synjournal of pinene by using genetically engineered Escherichia coli[J]. Letters in Biotechnology, 2019, 30(4):455-463.
[29]	袁雪峰. 大肠杆菌工程菌高密度发酵生产虾青素[D]. 保定:河北大学, 2020.
	Yuan XF. Production of astaxanthin by high-density fermentation of engineered E. coli[J]. Baoding:Hebei Universitiy, 2020.
[30]	李瑜, 李一萌, 杨丽, 等. 5L发酵罐高密度培养番茄红素工程菌及其发酵条件优化[J]. 现代食品科技, 2020, 36(6):137-146.
	Li Y, Li YM, Yang L, et al. High-density cultivation of lycopene-producing engineered bacteria in 5L fermenter and optimization of fermentation conditions[J]. Modern Food Science and Technology, 2020, 36(6):137-146.