Functional Study Of NtDXS And NtSPS In Tobacco Solanol Biosynthesis

Solanesol is an important secondary metabolite in plants,it plays an important regulation role in the growth and development of plants.It is a trans-long-chain isoprene containing 45 carbon atoms,which belongs to the category of polyterpenes according to the classification of terpenoids.In addition,because of its important economic value and medicinal value,its research is also extensive.In this study,the pBI121-SPS and pBI121-DXS-SPS overexpression vectors were constructed and the key enzyme gene SPS during the synthesis of solanesol was cloned.through the genetic transformation of tobacco,the content of solanesol in gene overexpressing plants was analyzed to explore the regulation mechanism of solanesol biosynthesis.This is the result of the study:（1）This is cloned SPS gene sequence from tobacco K326 and it is 1206 bp,the homologous phylogenetic analysis showed that tobacco K326 SPS belongs to SPS3,encoding a total of 401 amino acids,no signal peptide nor transmembrane domain.It contains 15 phosphorylation sites,subcellular localization in chloroplasts,and two binding to Mg²⁺conserved domain,one of the members of Isoprenoid_Biosyn_C1superfamily.The SPS protein is highly consistent with fluffy tobacco,forest tobacco and common tobacco with 100%homology,and the consistency with other SPS proteins of pepper,potato and tomato are More than 95%.It has the domains of DDxxD,which is typical of isopentenyl pyrophosphate synthase,have the function of isopentenyl pyrophosphate synthase.（2）The pBI121-SPS expression vector and the pBI121-DXS-SPS double gene co-expression vector were successfully constructed,and the transgenic plants overexpressing the SPS gene and the transgenic plants overexpressing the DXS-SPS double gene were obtained.The content of gene and solanesol was analyzed by real-time PCR and high performance liquid chromatography.The expression of SPS gene in plants overexpressing SPS gene was up to 3.25 times,and the expression of DXS gene was up by 9.3 times,solanesol content increased by up to 3 times compared with the control.For plants overexpressing DXS and SPS genes,the highest expression of DXS gene and SPS gene in seedling stage were 10.2 times and 5.3 times of that of the control,respectively,and the content of solanesol was 2-2.5 times of the control compared with the control.This will provide an effective method for improving tobacco biosynthesis of solanesol.The comprehensive development and utilization of tobacco natural active ingredients has important theoretical significance and high economic value.（3）The high-efficiency strains of tobacco K326 were studied by Agrobacterium rhizogenes C58C1,ATCC15834 and A4.The C58C1 strain is the best rooting effect of tobacco,and the C58C1 strain had the highest SPS gene transformation efficiency.The transformed C58C1 strain with SPS genewas successfully constructed,and the rooting rate of the explanted tobacco explants of C58C1 strain transformed with SPS gene reached 53.2%.The hairy roots in MS solid medium containing 0.01mg/L NAA+50mg/L SM+50mg/L Rif+50mg/L Kana grow well,which sets up a good experimental foundation for the biosynthesis of solanesol in tobacco root culture system.