Construction And Genetic Transformation Of C4H And HCT Gene Overexpression Vectors In The Chlorogenic Acid Synthesis Pathway Of Lonicerae Japonica Thunb

Chlorogenic acid,one of the main functional components of the natural medicinal plant---Lonicera japonica Thunb,is possesses anti-oxidative,antibacterial disinfection,cardiovascular disease prevention and atherosclerosis prevention activities.It has been widely applied in food,health care products,medicines,daily cosmetics and other fields.Therefore,more and more attention has been paid to the key genes in the efficient biosynthetic pathway of chlorogenic acid and the genetic transformation application of key genes.The current paper was based on the overexpression of key genes in the chlorogenic acid synthesis pathway of Lonicerae japonicae Thunb explored by our previous research group.Firstly,the accumulation of secondary metabolites,changes in enzyme activity and antioxidant capacity under the regulation of key genes overexpression were further explored.Secondly,RT-PCR was used to clone the key genes in the chlorogenic acid synthesis pathway of Lonicera japonica Thunb cells and the overexpression vectors were constructed.Finally,Agrobacterium-mediated callus transformation method was used to obtain transgenic callus and the chlorogenic acid content in the transgenic callus was determined.The key experimental results are as follows:（1）Changes of secondary metabolites,enzyme activities and antioxidant capacity in suspension cells of Lonicerae japonica Thunb under the regulation of key genes overexpression:Under the regulation of key genes overexpression,the accumulation of chlorogenic acids,polyphenols and flavonoids,POD and PPO activities,DPPH and ABTS free radical scavenging ability and Fe³⁺reducing ability in suspension cells all showed a trend of first increasing,then decreasing and gradually became stable,with the maximum value occurring on the 6th or 7th day after elicited.The increased multiples for each index compared with control and field-grown buds are as follow:4.52-fold and3.13-fold of CGAs,3.04-fold and 5.32-fold of flavonoids,6.56-fold and 4.84-fold of flavonoids,3.05-fold and 6.40-fold of peroxidase（POD）,3.26-fold and 2.56-fold of polyphenol oxidase（PPO）,2.62-fold and 4.91-fold of EC₅₀.DPPH,3.50-fold and6.17-fold of EC₅₀.ABTS,2.55-fold and 7.12-fold of ferric reducing antioxidant power（FRAP）,respectively.（2）The clone of key genes in the chlorogenic acid synthesis pathway of Lonicera japonica Thunb cells and construction of overexpression vector:The cDNA of Lonicera japonica Thunb cells was used as template to clone C4H and HCT genes by RT-PCR method.PstI and ECoRI were selected as restriction enzyme sites to construct pCAMBIA1302-HCT and pCAMBIA1302-C4H overexpression vectors,and then introduced into Agrobacterium EHA105 competent cells to lay a foundation for the transfer of target gene into rice callus.（3）Agrobacterium-mediated transformation of target gene and determination of chlorogenic acid content in transgenic callus:The seeds of the japonica rice Nipponbare was used as a model in this experiment to establish rice callus induction and multiplication culture system.Hyg and Carb are used as bacteriostats and screening agents in the transgene screening process,and the optimal concentrations of Hyg and Carb are optimized to be 40 mg/L and 200 mg/L,respectively.The overexpression vectors pCAMBIA1302-HCT and pCAMBIA1302-C4H were introduced into rice callus by Agrobacterium-mediated callus transformation,and the content of chlorogenic acid was determined by HPLC.There was no significant difference between the chlorogenic acid content of transgenic rice callus and the control group.