Study On Using Various Strategies To Improve The Contents Of Tepenoid Indole Alkaloids In Catharanthus Roseus

The Apocynaceae Vinca plant Catharanthus roseus (L.) G. Don is amedicinal and ornamental plant. More than130kinds of terpenoid indolealkaloids with anti-tumor activity have been isolated and identified in it,such as catharanthine, vindoline, ajmalicine, vinblasitne and vincristine.However, the contents of most alkaloids are low and the most valuabledimeric indole alkaloids in C. roseus, vinblastine and vincristine areimpossible to be yielded by hairy root culture and suspend cell culturesystems, because of lacking some enzymes related to vindoline biosynthsisin them. At present, the chemical semi-synthetic method is normallyemployed to produce more vinblastine and vincristine based on theextraction of relatively higher abundance of vindoline and catharanthine innatural C. roseus. Commercial production of important TIAs is very difficult such as vinblastine and vincristine by chemical synthesis due to thecomplexity of their chemical structures, high-cost and low-output forsynthesis. Therefore, it is necessary and urgent to find more efficient waysto improve the contents of important TIAs in C. roseus.In this study, many ways on how to improve the contents of TIAs in C.roseus had been studied. They included over-expressing the key enzymegenes which involved in the TIAs biosynthetic pathway, CrPRX1andCrSTR in C. roseus plants by Agrobacterium tumefaciens-mediatedtransformation system, inducing tetraploid plants which have high contentsof TIAs by colchicine, and the plant growth regulator combinations applyingon the leaves of C. roseus during flower period had also been studied. Theresults are as follows:1) Monovalent expression vectors (pCAMBIA2300-STR, pCAMBIA2300--PRX1) containing the rate-limiting enzyme genes in the TIAs biosyntheticpathway in C. roseus, STR and PRX1, were constructed. Through sonicationassisted Agrobacterium tumefaciens-mediated genetic transformationsystem,11independent transgenic C. roseus plants containing PRX1geneand23independent transgenic C. roseus plants containing STR gene wereobtained. The genes which were integrated in the genomes of transgenic C.roseus plants were verified by southern blot. The tranformants were further analyzed by real-time PCR, and the results indicated that the genes wereover-expressed in transgenic C. roseus plants. The contents of three TIAs asvindoline, catharanthine and vinblastine in transgenic C. roseus plants weredetermined by HPLC, and high TIAs-yielding plants were screened out fromthese transgenic plants. Compared to the control (non-transgenic plants), thecontents of the TIAs such as vindoline, catharanthine and vinblastine intransgenic C. roseus plants were significantly enhanced, with the highestover-expressing STR gene containing more1.94-fold of vindoline,1.56-foldof catharanthine and the highest over-expressing PRX1gene containing2.88-fold of vinblastine as the control.2) The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained bycolchicine induction from seeds explants, and the ploidy of the plants wasidentified by flow cytometry. The optimal treatment is0.2%colchicinesolution treated for24hours and the induction rate reaches up to30%.Compared with morphological characteristics and growth habits betweentetraploids and the control, we found that tetraploids of C. roseus had largerstoma and more branches and leaves. Real-time PCR results showed that theexpression of enzymes involved in terpenoid indole alkaloids biosynthesispathway had increased in the tetraploid plants. HPLC analysis showedtetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Compared to the control (diploid plants which also treated withcolchicine), the contents of the TIAs such as vindoline, catharanthine andvinblastine in tetraploid C. roseus plants were significantly enhanced. Theywere increased1.31-fold,1.89-fold and1.23-fold respectively.3) The effects of plant growth regulator combinations on the contents ofvindoline, catharanthine and vinblastine were investigated by short-termspray on an ornamental and medicinal plant Catharanthus roseus during theblooming period for commercial use. The results showed the combinationgroups such as ethylene (0.1mM)+chlormequat chloride (0.1mM),salicylic acid (0.1mM)+chlormequat chloride (0.1mM) and salicylic acid(0.1mM)+ethylene (0.1mM)+chlormequat chloride (0.1mM) resulted ina significant increase of the three alkaloids contents. The combinationgroups salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequat chloride(0.01mM) and salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequatchloride (1.0mM), affected vindoline and catharanthine contents but had noeffect on vinblastine. Among the combination treatments, salicylic acid (0.1mM)+ethylene (0.1mM), ethylene (0.1mM)+chlormequat chloride (0.1mM) and salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequatchloride (0.1mM) could significantly increase the vinblastine content by209%at48h,246%at48h and213%at24h respectively. Thus compared to the single PGRs treatments, the combination treatments increase alkaloidaccumulations more effectively.