Molecular Cloning,Expression Analysis And Transformation Of The Gene Of Saponins Synthesis Pathway In Medicago Sativa L.

Alfalfa(Medicago sativa L.)is known as the "king of the grass" which widely distributed throughout the world, recognized as high yield and good quality forage. In recent years the second metabolites-alfalfa saponins is considered to have several medicinal functions: lower cholesterol or blood fat,antiinflammatory, antitumor, antibacterial, insecticidal and other medicinal and biological activity. At present, the utilization of alfalfa main focused on hay and livestock feed, alfalfa saponins and other secondary metabolites, however, rarely being developed into a specialized product or additives used in the production. Through cloning,expression analysis and transformation of the gene of saponins synthesis pathway in Medicago sativa L. in order to explore candidate gene improvement and breeding new varieties. The main progress is as follows:1. Using homology cloning,a full-length c DNA sequence of SS gene was isolated from Medicago sativa L..The gene contain a 1242 bp open reading frame and encoding a 47 k Da ploypeptide of 413 amino acids.The c DNA sequence of SS is 97.91% identical to that of squalene synthases from Medicago truncatula L.,the encoding protein is also highly homology of 93.53%,indicated that the sequence from alfalfa is the squalene synthase gene,named Ms SS.Through bioinformatics analysis, the predict protein of Ms SS has two possible transmembrane helical area, the hydrophobic regions in the Cterminal may be the membrane boundarea; There are 6 conservative domains in Ms SS,including three highly conservative.2. Real-time quantitative PCR was performed to analysis the transcript level of MsSS in different tiusse and unser different abiotic stress.The results indicated tha the Ms SS has expressed highest in the root, followed by leaves, the least in stems.Furthermore, the Ms SS expression induced under ABA, GA3,UV damage and continuous darkness.3. The MeJA can induced increasing the transcript level of MsSS in different tiusse,while also increasing the total saponins and squalene synthase activity in alfalfa in time-dependent m anner, respectively.Preliminary experiment proved that Me JA could indued the transcript lev el of Ms SS,which can affects the squalene synthase activity and saponin synthesis.4. Full-length or truncating 30 amino acids of hydrophobic region at the carboxy terminus Ms SS c DNA was subcloned into prokaryotic expression vector p EASY-E2 and the constructed plasmid was introduced to Transetta(DE3) for induced overexpress-ion by IPTG.The resu ltsshow that the spheroidin with expected molecular mass was observed in E.coli containi ngthe full-length squalene synthase c DNA; while the soluble protein was successful express in E.coli containing the C-terminal truncated Ms SS c DNA. Through the analysis of the su bce-llular localization preliminary judgment Ms SS was localized in the cytoplasm.5. The full-Ms SS coding domain sequence was inserted into over-expression vector p BI121,the recombinant vector transfered into Medicago sativa L. by agrobacterium-mediated method.The results shows that overexpression of Ms SS gene increased the transcript level of Ms SS in transgenic plants,the toatal saponins content of transgenic plants was higher than the wil-d plants.6. To establish a method for rapid determination of alfalfa saponins using solid phase extracti on(SPE) followed by UV-spectrophotometry.The results showed that the optimal extractio n conditions were as follows:70% of ethanol concentration,ultrasonic extracting twice, each time 30 min, centrifugate 30 min at 10000 g.The supernatant was passed through a C18 S PE extraction cartridge,then washed with 5 m L each of 100%water and 30%Me OH.The sap onins were then eluted with 4 m L of 100%Me OH and detected by UV-spectrophotometry.