Cloning And Expression Analysis Of Dxr Gene In Nicotina Tobacum

To investigate the expression characteristic of 1-Deoxy-D-xylulose-5- phosphate reductoisomerases(dxr)gene in tobacco, the cDNA of dxr was cloned from tobacco leaves(Nicotina tobacco L K326) through RT-PCR techonoledge. An 1422bp long open reading frame(ORF) which encode 473 amino acids was obtained. Sequence analysis by Clustal W declared that this fragment is highly homologous to dxr gene of other species. It had 84% amino acid homologous to Lycopersicon esculentum,85% to Catharanthus roseus, 94% to Antirrhinum majus, 88% to Mentha x piperita, 87% to Arabidopsis thaliana,85% to Zea mays and 63% to Nostoc sp. PCC 7120. Prokaryotic expression and Western Blot analysis confirmed DXR protein was about 51.36kD. Use Phylip version 3.6(alpha)to analysis the DXR protein Evolutionary relationship,including tobacco and other 18 species. The results show that ,the DXR protein sequence has apparente ethnic-specific,the tobacco DXR protein belong to the plant branch and is closed to Catharanthus . Expression pattern of dxr gene in tobacco was investigated also by RT-PCR. It highly expressed in flowers, leaves and stems, low expressed in trichomes and seeds, much lower expressed in roots. This preliminarily study will be the first step to efficiently regulate trepanned metabolism in tobacco by dxr gene manipulation.Tobacco dxr gene was cloned and sequenced right. Inserted encoded frame of the dxr gene into the expression vector pCAMBIA2300-35S-OCS to construct the plant constitutive expression vector pCAMBIA2300-35S-OCS-DXR which under CaMV35S promoter. The leave discs of tobacco (Nicotinan tabacium L. cv. K326) were transformed with dxr gene by Agrobacterium tumefaciens strain LBA4404 which transformed by the plasmid pCAMBIA2300-35S-OCS-DXR,than the leave discs were regenerated on Differentiation medium supplemented with 30 mg/L Kan and the shoots rooted on Rooting medium with 20 mg / L Kan. At the end we obtained 27 Kanamycin resistance plantlets. PCR analysis proved foreign dxr gene intergration in 7 Kanamycin resistance plantlets. The DXR Transgenic Plants play a very important role at the further study of the DXR enzyme in the regulation of terpenoid synthetic substances basised the molecular regulation to improve the quality of tobacco flavor.