The Establish System Of Hairyroots And Molecular Cloning And Expression Analysis Of A New Gene Encoding 3-hydroxy-3-methylglutaryl-CoA Synthase From Camptotheca Acuminata

C. acuminata, a Nyssaceae species with its needles containing high content of camptothecin and comparatively rapid growth speed, is one of the major source species currently for commercial production of camptothecin. In order to further understand the biosynthetic pathway and the molecular basis for higher camptothecin content in C. acuminata , gene cloning, expression regulation and content comparison were performed and reported as follows:1. Camptotheca acuminata is a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyses the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the CPT biosynthetic pathway. A full-length cDNA encoding HMGS (designated as CaHMGS, GenBank Accession No. EU677841) was firstly isolated from young leaves of Camptotheca acuminata by rapid amplification of cDNA ends (RACE). The full-length cDNA of CaHMGS was 1801 bp containing a 1413-bp open reading frame (ORF) encoding a polypeptide of 472 amino acids. Comparative and bioinformatic analyses revealed that CaHMGS showed extensive homology with HMGSs from other plant species. Molecular modeling showed that CaHMGS was a new HMGS with a spatial structure similar to other plant HMGSs. Phylogenetic tree analysis indicated that CaHMGS belonged to the plant HMGS super-family and had the closest relationship with HMGS from Hevea brasiliensis. Tissue expression pattern analysis revealed that CaHMGS expressed in hypocotyls and cotyledons with strong signal, but no expression could be detected in roots. The expression of CaHMGS could be regulated by exogenous elicitor including salicylic acid (SA) and methyl jasmonate (MJ), suggesting that CaHMGS was elicitor-responsive. This work will be helpful to understand more about the role of HMGS involved in the CPT biosynthesis at the molecular level.2. In order to induce hairy roots from Camptotheca acuminata, different explants(including leaves, steams, cotyledons and hypocotyls) , different ages of explants and different strains of Agrobacterium rhizogenes (including A4, 15834, R1601 and C58C1) were tested. Transgenic roots were confirmed by PCR using rolB specific primers. The results showed that the appropriate explant was hypocotyls, the most suitable age of hypocotyls was 5-10d and 15834 was the best one among the four A.rhizogenes strains.3. Obtained the transformed hairy roots with the key genes(G10H, ORCA3, STR+ G10H) which in involved in CPT biosynthetic pathway. CPT in different hairy roots was detected by HPLC. It was revealed that the production of CPT in root lines transformed with STR+G10H was about 5 times higher compared to non-transformed roots, and 10 times higher than one mouth young plants. This work effectively develops a promising approach to produce camptothecin and also provide good basement to metabolize regulation of in hairy roots by genetic engineering.