烟草单萜合酶基因NtTPS2的克隆及功能鉴定

Cloning and Functional Identification of Monoterpene Synthase Gene NtTPS2 in Tobacco

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1854 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。

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 1854 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.