Study On The Dxps Gene Cloning And Expression Of Solanesol Biosynthesis Pathway In Tobacco

Solanesol is one of terpenoids in plant secondary metabolites,which has medicinal value itself, and it is the key intermediate of coenzyme Q10,so it has been paid an increasing attention.Generally,solanesol exists in many plants with little ratio in general.Among them,solanesol of mulberry leaves and potato leaves are higher than usual,but lower than tobacco leaves. This leads to an extreme short supply in the international market.Isopentenyl pyrophosphate,derived from deoxy-xylulose 5-phosphate pathway, where deoxy-xylulose-5-phosphate synthase (DXPS) is a key enzyme,which can synthesis Solanesol in tobacco.Experment shows you can reduce the level of metabolites by inhibiting the synthetic of deoxy-xylulose-5-phosphate synthase,while the introduction of outer dxps genes will increase the level of terpenoids.Hairy roots have the characteristics of growing fast without exogenous growth hormone;high content of secondary metabolites and genetic characteristics of stability,which make it to be used for the production of secondary metabolites in bioreactors.Thus,it has certain research significance to improve the content of solanesol through combining both genetic engineering and cell culture.The increasing content of solanesol is in need of two approaches,which are the introduction of foreign dxps genes and the cultivation of hairy roots.Specific methods are as follows:1.The acquisition of a target gene:extract total Arabidopsis RNA,design primers for RT-PCR,obtain the target gene dxps,then connect it with intermediate vector pMD 19,and undertake the transformation in E. coli,replication,sequencing.2. Expression vector:Re-design the primers with restriction sites,then connect with pMD19,undertake the transformation in E. coli and detection,connect it with pBI121 carrier,construct pBI121-pMD19dxps vector. Introduce it into rhizogenes A4 through the freeze-thawed nerve(FTN).3.Establishment of hairy root cultures of tobacco:confirm the optimum transformation materials and conditions by studying the effect factors of hairy root Inducing,and established an efficient system of hairy root induction and an efficient cultures system of hairy root by classifying growth medium. All the above provide new channels to the development and use of secondary metabolites of tobacco.Through the experiment,we successfully construct the plant expression vector and discuss the various factors that induce rooting,which makes the necessary preparations for the next step in the experiment. However,there are some unknown factors in the increaseing of material contents through plant metabolic processes by transgenic technology. Currently,the experiments about which both transgenic and plant cell culture method can be combined to increase the content of solanesol are few,but will be further discussed in one day.