Establishment Of Exogenous Gene Expression In Hairy Roots Of Rauvolfia Verticillata (Lour.) Baill.

Plant alkaloids, which constitute one of the largest group of natural products, provide many pharmacologically active compounds. With some key enzymes being cloned in the TIAs biosynthetic pathway, it is possible to produce the alkaloids in the plant cell by the metabolic engineering. Rauvolfia verticillata (Lour.)Baill., belonging to the family Apocynaceae, is a rare important medicinal woody plant because there is reserpine in its root which is one of the medication for antihypertension widely used in clinic.The strictosidine synthase (STR) is important enzymes involved in catalyzing key steps in the biosynthesis of terpenoid indole alkaloids(TIAs). In this research, plant expression cassettes of gene from catharanthus roseus driven by the CaMV 35S promoter were covalently constructed within the T-borders of a binary Ti plasmid vector and synchronously introduced into the genome mediated by the disarmed Agrobacterium tumefaciens strain C58C1 which also carries Agrobacterium rhizogenes Ri plasmid pRiA4. We used the former strain as the carrier, and introduced str gene into the nucleus genome of Rauvolfia verticillata (Lour. )Baill. in order to obtain the hairy root lines of high yield of reserpine.Single transgenic hairy root lines and transformed roots induced by A. rhizogenes strain A4 (pRiA4) as the control were also obtained at the same time.The conditions of transformation via Agrobacterium rhizogenes were optimized.The srain had strong ability to induce hairy roots when its' OD was about 0.6. Among different partsexplants, such as leaves, cotyledons, stems and leaves sterilized, leaves had the highest induced rate as 85.71%.Explants pre-cultured for 2 days and co-cultured with Agrobacterium rhizogenes for 3 days had the highest inducing rate. Added 100/miol/L AS in the medium also promoted the transferring rate(83.33%).Cultured conditions of hairy root in vitro were optimized. It showed that hairy root grew best in the 1/2 MS medium supplemented with 0.2 mg/mL IBA and 0.1 mg/mL NAA. Subsequently hairy root was selected in the above medium supplemented with 20 mg/mL hygromycin. Then hairy root with str gene was obtained primarily.The genomic DNA PCR analysis was confirmed that rolB and rolC integrated into Rauvolfiaverticillata (Lour.) Baill. genome, resulting in the generation of hairy roots. At the same time, PCR analysis showed that str gene were integrated into the host's genome, and further confirmed that the hairy roots were transformed with exogenous str gene.The contents of reserpine in the samples were determined by HPLC. It showed that the maximum amount of reserpine( 1.367% DW) in the hairy roots with str gene had 14.24 times than that the hairy roots without str gene( 0.096% DW) and had 52.58 times than that in the nature plant roots(0.026% DW). So the result effectively showed that str gene is one of the key enzymes in the TIAs biosynthetic pathway and one of the most important targets in the TIAs metabolic engineering. There are important study and business values.