Overexpression Of1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase(NtDXR)in Tobacco(Nicotiana Tabacum)

Terpenoids are important aroma substances and precursors for the flue-cured tobacco.To regulate the biosynthesis of tobacco terpenoids,In the study,Nt DXR isolated from tobacco(Nicotiana tabacum L.),were constitutively overexpressed or trichome-specific overexpressed in tobacco.The positive transgenetic tobacco plants were obtained via DNA-PCR、RT-PCR、Southern Blot、GUS stain、q PCR and were carried out morphological and physiological and gene expressions analysis.The results were as follows: 1.The ORFs of DXR was fused to green fluorescent protein(GFP)and transferred into tobacco by Agrobacterium-mediated transformation.Leaf epidermal cells were examined for green fluorescence.The results showed that Nt DXR-GFP was present in chloroplasts,which verified that DXR was located in chloroplasts.2.The constitutive expression vector(p CAMBIA-35S-Nt DXR-GUS)and trichome-specific expression vector(p CAMBIA1391-CYP-Nt DXR-GUS)were separately transformed into A.tumefaciens strain EHA105 and then transferred into tobacco variety K326 by Agrobacterium-mediated transformation.Positive transgenic plants were first screened on kanamycin plates and then identified by DNA-PCR and RT-PCR.Positive transgenic plants leaves were confirmed by GUS histochemical staining assays.It showed that all tissues of 35S-Nt DXR plants leaves and the only glandular trichome of CYP-Nt DXR plants leaves were stained blue.3.Southern blot analyses were performed to detect the integration of exogenous genes.It showed that Nt DXR gene was inserted into tobacco genome successfully.4.Morphological assays indicated that the plant heights of 35S:Nt DXR transformed plants were significantly longer than those of controls.Compared with controls,CYP:Nt DXR plants had shorter stalk glandular trichomes and larger glandular head cells.5.Overexpressing Nt DXR under control of the 35 S promoter resulted in enhancement of various photosynthetic pigments and hormones in leaves.In contrast,there were no significant changes in cembrane-related diterpenoids synthesised in glandular trichomes.To further elucidate the function of Nt DXR in the biosynthesis of diterpenoids,overexpression vectors for Nt DXR under the control of a trichome-specific CYP promoter was transferred to tobacco plants.CYP-Nt DXR tobaccos exhibited larger glandular cells and increased cembrane-related diterpenoids in leaf glandular trichomes.6.Transcripts of eight MEP pathway genes were significantly upregulated in Nt DXR-overexpressing tobacco plants,indicating that overexpression of Nt DXR could boost the expression of downstream genes in the MEP pathway.Our results suggested that overexpression of Nt DXR could increase the levels of photosynthetic pigments,leaf surface exudates,and hormones though the MEP pathway.The research is useful for the molecular regulation of terpenoid biosynthesis and quality improvement in tobacco.