生物技术通报 ›› 2021, Vol. 37 ›› Issue (9): 132-141.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1492
刘少华1(), 赵希胜1,2, 杨晴1, 杨长青1, 潘旭浩1, 张建会3, 杨爱国1(), 李依婷1()
收稿日期:
2020-12-08
出版日期:
2021-09-26
发布日期:
2021-10-25
作者简介:
刘少华,男,硕士研究生,研究方向:植物次生代谢调控;E-mail: 基金资助:
LIU Shao-hua1(), ZHAO Xi-sheng1,2, YANG Qing1, YANG Chang-qing1, PAN Xu-hao1, ZHANG Jian-hui3, YANG Ai-guo1(), LI Yi-ting1()
Received:
2020-12-08
Published:
2021-09-26
Online:
2021-10-25
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。
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.
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 |
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
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