Analysis Of Key Enzyme Gene Expression Patterns In Tobacco Solanesol Synthesis And Function Research Of NtSPS Gene

Tobacco is not only an important economic crop,but also has important medical value.There are many beneficial secondary metabolites,such as polyphenols,nicotine,solanesol and so on.Solanesol is a medically important noncyclic terpene alcohol that is synthesized by the condensation of nine isoprene units.it is a precursor of ubiquinone,also known as coenzyme Q10,vitamin K2,and anticancer drugs,including N-solanesyl-N,N?-bis(3,4-dimethoxybenzyl)ethylenediamine(SDB).At present solanesol used in medicine maily comes from tobacco leaves.So it is important to study the solanesol synthesis in tobacco.In this study,we used ultra high performance liquid chromatography and real-time quantitative polymerase chain reaction(RT-PCR)to determine the content of solanesol in roots,stems and leaves and the expression of key enzymes of terpenoids metabolism in 6 developmental stages of Honghuadajinyuan and Zhongyan90.The correlation between the accumulation of solanesol and the expression of key enzymes was analyzed.In addition,the genes of Solanesyl Pyrophosphate(SPS)Synthase were obtained by cloning,and the gene sequences were analyzed by bioinformatics.Then,the function of the gene was analyzed by constructing the overexpression vector.The main findings are as follows:(1)The trends of solanesol accumulation were consistent at different development stage in different organs of these cultivars i.e.,the solanesol content of leaf was highest while that of root was the lowest.The expression patterns of different terpenoid synthetic enzymes were not completely identical in these two cultivars.The expression level of FPS,DXR,IPI,SPS,GGPPS increased gradually from seeding stage to flowering stage.The expression level was highest at the budding period in root,at the flowering period in stem and leaf,and then declined rapidly.The correlation analysis showed that the expression levels of DXS and GGPPS was negative correlations with solanesol content,but FPS,DXR,SPS and IPI exhibited extremely significant positive correlation with solanesol content.The higher solanesol content may be caused by the higher expression level of the genes in HD.In addition,the expression level of IPI showed extremely significant positive correlation with that of DXS,FPS and SPS in tobacco root.The expression level of SPS has significantly positive correlation with FPS and IPI,but has significantly negative correlation with DXS in leaf.The expression level of IPI has extremely significant positive correlation with HMGR in leaf.(2)The two SPS genes were obtained by cloning in the N.tabacum,named Nt SPS1 and Nt SPS2 respectively.Nt SPS1 contained an open reading fram of 1209 bp encoding 402 amino acids and Nt SPS2 contained an ORF of 1248 bp encoding 416 amino acids.The bioinformatics analysis indicated that all the proteins encoded by the two genes belonged to the hydrophilic protein and had no transmembrane structure.Nt SPS1 was located in the chloroplast and Nt SPS2 was located in the mitochondria.Furthermore Nt SPS1 Encoded protein instability index is high,is unstable protein,Nt SPS2 encoded protein instability index is low,are stable protein.(3)The overexpression vectors of Nt SPS1 and Nt SPS2 were constructed and the 16,8 transgenic plants of these two genes were obtained respectively by Agrobacterium tumefaciens-mediated tobacco genetic transformation.The results of q RT-PCR showed that the expression level was 6-15 fold higher than that of CK,and the expression level of SPS1 transgenic plants was 3-5 fold higher than that of Nt SPS2 transgenic plants.The solanesol contant of transgenic plants was higher 20-30% than the solanesol contant of CK.