Vector Construction Of Panax Ginseng CAS Gene And Panax Notoginseng DS Gene By CRISPR/Cas9 And Establishment Of Transformation System

Gene editing is a genetic engineering technique which can be used to modify specific target genes in an organism's genome.Since its inception,it has been widely used in plant breeding,population improvement,mutant construction and other fields.Compared with field crops,gene editing technology is less used in medicinal plants.Panax ginseng C.A.Mey.and Panax notoginseng as important medicinal herbs of panax panicae,the establishment and application of its gene editing system are of far-reaching significance.This study was based on the metabolic pathways of the precursor substance 2,3-squalene oxide during the synthesis of P.ginseng and P.notoginseng saponins.The CRISPR/Cas9 expression vectors of P.ginseng CAS and P.notoginseng DS genes were constructed.The callus of P.ginseng was transformed by agrobacterium tumefaciens.After co-culture,the suitable bacteriostatic pressure and screening pressure were studied,and the genetic transformation system of ginseng callus was optimized and improved.The protoplasts of P.notoginseng with high yield and vigor were prepared as the receptor materials for PEG transduction.The study lay a foundation for the establishment of P.ginseng and P.notoginseng gene editing system.The main findings are as follows:1.The pRGEB32 plasmid was used as the basic framework,the CRISPR/Cas9 expression vectors of P.ginseng CAS gene and P.notoginseng DS gene were constructed by homologous recombination method with the designed target sequence fragment connected with the linearized carrier.The pCRISPR-CAS was introduced into EHA105 competent cells for callus transformation of P.ginseng.2.The effects of different concentrations of Cef and Kan on callus growth of P.ginseng were investigated to determine the optimal bacteriostatic pressure and screening pressure in agrobacterium-mediated genetic transformation.The callus was infected by agrobacterium,and the method of degerming was optimized after co-culture.The results showed that during bacteriostatic culture of ginseng callus the concentration of Cef in the medium was better between 100 mg/L and 300 mg/L,and the concentration of Kan in the medium should not be less than 200 mg/L for screening resistant callus.The callus after co-culture was cultured in medium with Cef concentration of 500 mg/L for 3 days first.Then the callus was transferred to medium with Cef concentration of 300 mg/L for 5 days.After that the callus was transferred to medium with Cef concentration of 100 mg/L.In this way,the material growed stably under the condition of low resistance concentration.3.The 200 mg/L Kan medium was used to screen the P.ginseng callus.The expression of marker genes and the mutation of target sites were detected.The results showed that the P.ginseng callus with the expression of KanR gene were obtained after the material was screened by the resistance medium.The target sites were amplified but no mutation was found after sequencing.4.The effects of different preparation conditions on the separation of protoplasts were statistically analyzed by single factor experiments on the subculture time of callus,ratio of enzyme solution,enzymatic hydrolysis time,osmotic pressure and centrifugal force.The protoplasts with high yield and vitality were prepared as transforming materials The results are as follows: The callus of P.notoginseng subcultured for 15 days,separated at the enzyme composition solution with 1.5% cellulase R-10 and 0.7% pectinase Y-23 in CPW-0.7mol/L mannitol for 7 h,then collected at 800r/min during centrifugation and purification could obtain high yield and vitality protoplast.