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A Comparative Study of Mrna Level, Protein Expression and Metabolite Production in Eschscholtzia Californica with Methyl Jasmonate, Salicylic Acid and Their Combination

Hwa-Young Cho1, Hong Soon Rhee2, Sung-Yong H. Yoon1, Carolyn W. T. Lee-Parsons3, Jeong Jin Park2, Seok-Young Son1, and Jong Moon Park2. (1) POSTECH, POSTECH, San 31, Pohang, 790784, South Korea, (2) Division of Molecular and life science, POSTECH, POSTECH, Pohang, Kyungbuk 790-784, Korea, (3) Northeastern University, 342 Snell Engineering Center, 360 Huntington Avenue, Boston, MA 02115-5000

A comparative analysis was performed on Eschscholtzia californica cells with signal elicitors; methyl jasmonate, salicylic acid and their combination. Elicitor dosage as well as addition timing was optimized and combination of optimized elicitors effects on the benzophenanthridine alkaloid production in E. californica. 0.5 mg g-FCW-1 MJ and 0.02 mg g-FCW-1 SA increased the total alkaloid production and the optimum growth stage for alkaloid overproduction with MJ and SA was on day 7 after subculture. Highest sanguinarine production reached 24.4 0.4 mg g-DCW-1, which was 9.8 times higher than that in un-elicited cell culture and it was obtained with combination of 0.5 mg g-FCW-1 MJ and 0.02 mg g-FCW-1 SA. Protein expressions and mRNA levels of (S)-norcoclaurine-6-O-methyltransferase (6OMT), (S)-coclaurine-N-methyltransferase (CNMT), (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), 3'-hydroxy-(S)-N-methyl-coclaurine-4'-O-methyl-transferase (4'OMT), berberine bridge enzyme (BBE) and dihydrobenzophenanthridine oxidase (DHBO) were quantitated by western blotting and real-time PCR analysis. The combination of elicitors with different signal transduction pathway could give a synergistic effect of benzophenanthridine alkaloid and benzophenanthridine alkaloid production results from both an increase in the enzyme expression and more efficient transcription of each gene.