Cloning Of MCT, HDS And SGD Genes From Rauvolfia Verticillata And Establishment Of Genetic Transformation System

Rauvolfia verticillata is a medicinal shrub belonging to the family Apocynaceae, from which more than 90 kinds of Terpenoid Indole Alkaloids (TIAs) have been extracted including ajmalicine, reserpine and yohimbin etc. The low content of TIAs in natural plants has greatly handicapped its commercial production, thus the demand of TIAs in international market cannot be met. Even though the chemical synthesis of reserpine is possible, it is not feasible because of the high costs. With the progress in cloning genes involved in biosynthetic pathway, metabolic engineering is deemed as a promising strategy to enhance the production of ajmalicine. To unveil the biosynthetic pathway of TIAs as well as to provide new target genes, MCT, HDS and SGD were cloned from R. verticillata for the first time. Meanwhile, the highly efficient regeneration system and hairy root system of this species were also investigated.2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MCT, EC:2.7.7.60), the third enzyme of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, catalyzes MEP and CDP to yield 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. The full-length cDNA of MCT was isolated from R. verticillata using Rapid Amplification of cDNA Ends (RACE) technique and characterized. The new cDNA was designated as RvMCT and submitted to GenBank(?) to be assigned with an accession number:JF966732. The full-length RvMCT was 1524 bp containing a 945 bp coding region encoding a 314 amino acids peptide with a calculated molecular mass of 34.45 kDa and an isoelectric point of 7.34. Seconday structure prediction revealed that RvMCT consists of 52.87% random coil,15.61% extened strand and 31.53% alpha helix. RvMCT was predicted to possess a plastid transit peptide in its N terminal. Comparative and bioinformatic analyses revealed that RvMCT had extensive identity with MCTs from other plants.Phylogenetic analysis suggested that MCTs can be divided into three groups including bacterial, gymnosperm and angiosperm. RvMCT belongs to angiosperm MCTs family. Quantitative RT-PCR analysis showed that RvMCT expressed highest in tender leaves, followed by old leaves, bark, roots and tender stems. 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS, EC:1.17.4.3) catalyzes the sixth enzymatic reaction of MEP pathway. The full-length cDNA of HDS was cloned and characterized from R.verticillata.The new cDNA was designated as RvHDS and submitted to GenBank(?) to be assigned with an accession number:HQ659759. The full-length cDNA of RvHDS was 2645 bp, containing a 2223 bp ORF encoding 740 amino acids with a calculated molecular mass of 82.0 kDa and an isoelectric point of 6.28. Bioinformatic analysis revealed that RvHDS had extensive homology with HDSs from other plant species and contained a conserved transit peptide for plastids. The phylogenetic analysis indicated that RvHDS belonged to plant HDSs family. Quantitative RT-PCR showed that expression level of RvHDS was highest in old leaves, followed by barks, tender leaves, roots and tender leaves.Scrictosidine-β-D-glucosidase (SGD, EC 3.2.1.105) catalyzes the deglucosylation reaction of scrictosidine, contributing to the diversity of TIAs in different species. The full-length cDNA of SGD was cloned and characterized from R. verticillata. The new cDNA was designated as RvSGD and submitted to GenBank(?) to be assigned with an accession number:JF966733. The full-length cDNA of RvSGD was 1859 bp, containing a 1608 bp ORF encoding 536 amino acids with a calculated molecular mass of 61.0 kDa and an isoelectric point of 6.16. Bioinformatic analysis revealed that RvSGD showed high similarity with SGDs from C. roseus and Rauvolfia serpentina at the amio acid level, and three conserved catalytic sites His-161, Glu-207 and Glu-419 were also present in RvSGD. Quantitative RT-PCR showed that expression level of RvSGD was highest in barks, followed by old leaves, roots, tender leaves and tender stems. The present study will be helpful to understand more about the functions of the three genes at the level of molecular genetics, as well as providing new targets for molecular regulation of TIAs biosynthesis.Due to the short supply of R. verticillata, metabolic engineering is one of the most promising strategies to solve this problem with the help of further elucidation of biosynthetic pathway. Transgenic plants and hairy roots obtained through genetic transformation are alternative sources of TIAs. Establishment of transformation system is indispensable for carrying on transgenic research; however, R. verticillata is a kind of woody plant which is difficult for establishing highly efficient regeneration system. In order to facilitate the transgenic research in the future, the present research investigated the establishment of regeneration system and hairy root transformation system. The result showed that regeneration system can be developed through cultivating stems on half strength MS medium containing 0.8 mg/L IBA firstly, followed by cultivating on MS medium supplemented with 2.0 mg/L 6-BA and 0.05 mg/L NAA. Through infecting tender leaves with Agrobacterium rhizogenes C58C1, hairy root system of R, verticillata was established. HPLC detection showed that ajmalicine production in hairy root was 170.99μg/g (DW). The present study will facilitate the research on metabolic engineering of TIAs in R. rauvolfia in the future.