Plant Regeneration System Optimization And Squalene Synthase Function Verification In Tobacco Hair Roots Of Panax Japonicus C.A.Meyer

Panax japonicus C. A. Meyer is one of the most precious medicines in Chinese traditional herb system. The rhizomes have been used as a substitute for Ginseng roots. Because of its important pharmacological action, most of wild resource was destroyed severely in recent years. The extant nature resource is impossible to satisfy the increasing demand. Thus, establishing and optimizing the regeneration system for protecting the wild resource which about to extinct is imminent.Saponins have been clarified to be the most important active component in rhizomes of P. japonicus. Squalene synthase is a key enzyme in triterpenoid saponin metabolism pathway catalyzed farnesyl pyrophosphoric acid to form triterpenoid saponin and phytosterol. With the developing of gene engineering in recent years, through regulating the key enzyme expression level to alter metabolism pathway increase target metabolites content show a bright prospect. Base on hair roots engineering was consummate and the superduper characteristics of hair roots, through hair roots culture to obtain some valuable metabolites and to study biological metabolism in hair roots show an extensive application prospect.To confirm the function of squalene synthase in saponin metabolic pathway in hair roots is very important and significance to obtain SS gene modified hair root lines of P. japonicus.that have a high synthesis efficient in saponin.To study culture in vitro and plant regeneration of P. japonicus. Embryos, stems and leaves of P. japonicus were used for explants, effects of different hormones for callus induction and plant regeneration were studied and optimized.The optimal way to obtain sterile explant for Seeds were sterilized in 75%ethyl alcohol for 60 s then 0.1% HgCL2 for 12 min; stems and leaves were sterilized in 75%ethyl alcohol for 15 s then 5%NaClO for 5 min.Use MS as basic medium,the optimal hormones combination for callus induction of embryos were 1.5 mg/LNAA+1.5 mg/L 2,4-D+0.1 mg/LKT;stems were 1.5 mg/L NAA+1.0 mg/L 2,4-D+0.1 mg/LKT; leaves were 1.5 mg/L NAA+1.0 mg/L 2,4-D+0.2 mg/L KT under the illumination.But under the darkness, the optimal callus induction hormones combination for embryos were 1.0 mg/L NAA+1.5 mg/L 2,4-D+0.2 mg/L KT;stems were 1.5 mg/L NAA+1.5 mg/L 2,4-D+0.1 mg/L KT;leaves were 1.5 mg/LNAA+1.0 mg/L 2,4-D+0.1 mg/L.The optimal medium for germination was MS+3.0 mg/L 6-BA+1.0 mg/L GA3. The optimal medium for roots generation was MS+1.0 mg/L 6-BA+3.0 mg/L IB A. The optimized regeneration system offered an efficient way to obtain sufficient seedling for enlarging the resourse of P. japonicus C. A. MeyerIn order to have a further study on the function of squalene synthase in hair roots. Total RNA was isolated from leaves of 30 days-old seedling oi P. japonicus cultivated in vitro. The completely open reading flame of Squalene synthase gene of P. japonicus. was amplified, The PCR product was purified and the resulting 1291bp PCR product was ligated into the binary vector pCXSN which was digested by Xcm I, the restriction sites was between a cauli flower mosaic virus 35S promoter and followed by NOS terminator. The norientation recombinant vector was confirmed by PCR detection and verified by automated DNA sequencing and then transformed into Agrobacterium tumefaciens strain C58C1.Tobacco leaves was infected by A. rhizogenes strain C58C1 harboring the recombinant binary vector pCXSN-SS to form hair roots, Genomic DNA was isolated from hair roots of non-transgenic type and transgenic lines of P. japonicus. use the method of CTAB. Genomic DNA were used as template for PCR analysis of rolb hyg,virD and SS to confirm SS were integrated into the tobacco hair roots genome.Total RNA was isolated from leaves of wild-type tobacco,wild-type hair root of tobacco induction by C58C1 and transgenic hair roots lines, and then reverse transcription synthesize cDNA,18S rRNA gene as an internal, adjust the concentration of the same cDNA template.SS transcript levels were detemined by reverse transcription polymerase chain reaction (RT-PCR), SS transcript level were increased in the transgenic hair roots lines contrasting to wild-type tobacco plant and non-transgenic hair root line.Finally,13 positive transformed hair roots lines were obtained, transformation efficiency reached to 40.63%.Rhizomes of P. japonicus was used as the material, the sonication extraction method for ginsenoside Re was optimized. The best extraction method was methanol:H2O(75:35); material:dissolvent(100mg:3ml); 45min;2 times. The total saponin isalated from wild type tobacco, wild type hair roots and 10 transgenic hair roots lines were analysised by spectrophotometric method. The total saponin isalated from 10 transgenic hair roots lines were 1.67-2.8 fold higher than wild type hair roots and 2.76-4.62 fold higher than wild type tobacco respectively. The result shows that SS gene from P. japonicus over-expression in tobacco hair roots can enhance saponin accumulation in hair roots.