Regulation Mechanism Of Catharanthine Biosynthesis Pathway To The Overexpression Of Gmmybz2 And Nitric Oxide/meja Elicitation In Catharanthus Roseus Hairy Root Culture

Catharanthus roseus belongs to Apocynaceae family, is known to biosynthesize more than 130 alkaloids, most of which appear to have pharmacological activity. It is especially true that the dimeric terpenoid indole alkaloids, such as 3',4'-anhydro-vinblastine, vincristine and vinblastine, have powerful effects as anticancer drugs. These dimeric terpenoid indole alkaloids are synthesized from vindoline and cathar-anthine. Most indole alkaloids are present in C. roseus leaves only in very small amounts and usually have some chiral centers. They are therefore difficult to synth-esize chemically, making them expensive to produce commercially. Many efforts have been made to improve the production of therapeutically important alkaloids of C. roseus in the last few years, however, these alkaloids still cannot be successfully industrialized mostly due to its sophisticated molecular mechanism. Unraveling the complexity of the genetic, catalytic and transport processes of TIAs biosynthesis is one of the most stimulating intellectual challenges in C. roseus.The soil-borne gram-negative bacterium Agrobacterium rhizogenes A4 contain a plasmid called Ri (Root inducing). Hairy roots cultures of C.roseus were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Nine independent root clones were evaluated for number of integration of Ri T-DNA genes and catharanthine contents. Based on the integration of the Ri TL-DNA (RolAB) and TR-DNA (Ags), there were two categories of independent hairy root RolAB+/Ags-(4 clones) and RolAB+/Ags+(5 clones). Catharanthine was extracted from hairy root cultures (RolAB+/Ags+) and analyzed by HPLC. The results show that catharanthine levels in these lines were highly variable, ranging from 0.38 to 0.56 mg/g FW. However, catharanthine levels in wild type lines were undetectable. A variety of gene transcripts of TIAs pathway were analyzed by relative quantitative real-time PCR. A1 line culture showed the decrease in the mRNA levels for inhibitors ZCT1 and ZCT2 compared to the wild type line, however, the mRNA levels of activator of ORCA2 and ORCA3 were increased. Except that the mRNA levels of PGGT-I were no change, other key enzymes (ASa, TDC, DXS, DXR, HDS, MECS, G10H, SLS, STR, SGD, CPR) were increased in their mRNA levels. This demonstrated that the T-DNA region gene of Ri plasmid could actively regulate TIAs biosynthesis pathway.We introduced plasmid pCAMBIA2301 contained GmMYBZ2 (R2R3 type MYB transcription factor cloned from zhongdou 27) into Agrobacterium rhizogenes A4 using the method of electroporation. Hairy roots cultures of C.roseus were established by infecting the leaves with Agrobacterium rhizogenes A4 strain contained GmMYBZ2. Thirty-six independent root clones resistant with kanamycin were evaluated for number of integration of Ri T-DNA genes, GUS histo-chemistry analysis and catharanthine contents. There were three categories of independent hairy root GmMYBZ2+/RolAB+/Ags-(7 clones) and GmMYBZ2+/RolAB+/Ags+(16 clones). PCR-Southern Blotting analysis GmMYBZ2 of 6 clones of GmMYBZ2+/RolAB+/Ags+ and GmMYBZ2+/RolAB+/Ags+(23 clones) were positive. HPLC detection show that catharanthine levels in these lines were low compare to A4 lines, ranging from 0.20 to 0.25 mg/g FW. Relative quantitative real-time PCR analysis show that the largest increase in the mRNA transcripts for gene Cpr, PGGT-I, Tdc, Zctl, Pal, especially Zctl. However, the mRNA levels of key enzymes (ASa, ORCA3, STR) were decreased notable. Meanwhile, GmMYBZ2 transcription factor has a conserved amino acid motif and a predicted zinc finger region at its C-termini. There would contribute to elucidate GmMYBZ2 down-regulate the biosynthesis of catharanthine in C.roseus hairy root.Growth index studies show that hairy root line A4-MYBZ2 grew slowly comp-are to line A4. Relative quantitative real-time PCR analysis show that the largest increase (increase 50%) in the mRNA transcripts for both A type cyclins CYSA and B type cyclins CYMB of hairy root line A4 compare to line A4-MYBZ2. This demon-strates that Ri T-DNA genes and GmMYBZ2 could regulate the cell cycle, and then affect the growth characteristics.The five conditions of hairy root were established:A4 hairy root which served as the control (A4); hairy root which ORCA3 was overexpressed (A4-ORCA3); SNP (10 mmol/L) elicited hairy root (A4+SNP); MeJA (50mg/L) elicited hairy root (A4+Me JA); SNP+MeJA elicited hairy root (A4+SNP+MeJA). The catharanthine content and RNA were evaluated at five different conditions at 0,2,4,6,8 day after the cultures were fed. Relative quantitative real-time PCR analysis demonstrate that elicitation by SNP showed the largest increase in CPR and ZCT1 transcripts, the remaining transcripts of gene of TIAs pathway were decreased notable. However, elicitation by MeJA show the transcripts levels of TIAs pathway genes were opposite from elicitation by SNP. More interesting, elicitation by MeJA and SNP show the transcripts levels in the middle of between SNP and MeJA elicitation. HPLC detection shows that the change trends of catharanthine levels were the same as transcripts levels. In a word, NO and MeJA have been well above demonstrated not only to be involved in TIAs biosynthesis but also to interact in mediating TIAs production by antagonistic. Considering the differences in transcripts levels of these five conditions can give us deeper insights into the TIAs regulate network. Two possible mechanisms may exist in this net work concerning NO and MeJA interaction: Firstly, NO play a negative role in JA biosynthesis; Secondly, NO inhibits the 26S proteasome ubiquitination which induced by MeJA.