Cloning And Sequence Analysis Of Full-length CDNA Of 1-deoxy-D-xylulose-5-phosphate Reductoisomerase(DXR) Gene From Tobacco(Nicotiana Tabacum)

Terpenoids, also called isoprenoids, are a group of natural products which all formally built from isoprenic unit. Those kinds of compound are biosynthesized by archaebacterium, bacterium, fungi, and plant. Many of these compounds are of important economic value. There are three key fragrant terpenoids which are synthesized through DOXP/MEP pathway in tobacco, including Cembranoids, Carotenoids, Labdaniods. Their biosynthesis pathway and regulation of concerned emzymes should be elucidated. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the first key enzyme in the process of the DOXP/MEP pathway for terpenoid biosynthesis. It catalyzes conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2C-methyl-D-erythritol-4-phosphate (MEP), that represents the committed step in the production of isopentenylallyl diphosphate (IPP). It will determine the outputs of downstream products. DXR gene has been isolated from different species, including bacteria, protozoa and higher plants. The amino acid identity among DXR from different species is high.In this work, reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques had been applied to clone the DXR gene (GenBank accession No.: DQ839130) using tobacco (Nicotiana tabacum) leaves as the target material for the first time. The full-length cDNA was 1,804bp and the biggest open reading frame (ORF) was 1,422bp and encoded 473 amino acids which had a theoretical molecular weight of 51.2 kDa, an isoelectric point of 6.04 and a plastid transit peptide which was rich in Pro residue at its N-terminal. Secondary structure predicting reveals thatα-helix and random coil were the main structural conformations in DXR protein. Through sequence analysis by Blast P online, the deduced amino acid sequence showed highly homologous to the DXR which was from other plant species. The identification that compared with Nicotiana benthamiana (AM236596), Lycopersicon esculentum(AF331705), Catharanthus roseus(AF250235), Antirrhinum majus(AY770406) and Mentha×piperita(AF116825) were 97%, 92%, 84%, 82% and 80% respectively.The phylogenetic tree showed that tobacco DXR was closely related to tomato DXR, and basically reflect the phylogenetic relationships of these DXR in ten plants.The cloning and analysis of tobacco DXR gene provided a good base for research on functional expression and potential application in improving the fragrant quality of tobacco leaves, and for construction of high yield key fragrant terpenoids genetic engineering tobacco.