The Research On Pathway Involved In Huperzine Biosynthesis And Development Regulation

To establish the possible way of biosynthesis and metabolic regulation of Huperzine A in Huperziaceae plants has attracted widespread interest in the field of metabolic engineering. Hup A as a potent and highly selective inhibitor of acetylcholinesterase, can be used as Alzheimer’s cure drug or developed into functional foods. Its medicinal value has become increasingly prominent. While these species are difficult to obtain and are rare in nature, and grows very slowly, making them even less desirable candidates as natural sources for Hup A. Nevertheless, the total synthesize Hup A has been reported is not yet industrialized synthesis. So far, no successful commercial cultivation has been reported for Huperziaceae because it is extremely difficult to get sterilized explants. Therefore, The Hup A obtained still all depends on the whole Huperziaceae plants extraction. it is particularly essential to expound Hup A biosynthetic and metabolic processes and analyze the key enzyme and its molecular mechanism involved in these processes, for providing an invaluable resource for production of Hup A to effective protect the Huperziaceae plant from harvest practices.The purpose of this paper is to establish the Hup A biosynthetic pathways and metabolic regulation system. We studied the effects of concentrations of Mg2+, dNTP mix(10 mM each), primer, template cDNA and Taq polymerase on L-lysine decarboxylase gene, Cytochrome P450 gene, dioxygenase gene, and methyltransferase gene-PCR amplification based on cDNA reverse transcribed by total RNA extracted from 4 Huperziaceae plant leaf using orthogonal design. The amplification gene sequences were confirmed by comparison the nucleic acid sequence and amino acid sequence with NCBI database.Real-time quantitative PCR analysis showed that the key enzyme genes involved in biosynthetic pathway have different expression between phlegariurus austrosinicus (ching) L. B. Zhang and phlegariurus petiolatus (C.B.Clarke) H.S.Kung et L. B. Zhang. It is worth noting that the dioxygenase gene did not detected expression in phlegariurus austrosinicus (ching)L. B. Zhang, therefore assume that the encoded putative protein may a key rate-limiting enzyme. Further, with highly expressed genes in phlegariurus petiolatus (C.B.Clarke) H.S.Kung et L. B. Zhang were L-lysine decarboxylase gene, Cytochrome P450 71A1 and Cytochrome P450 72A1. While, the amount of Methyltransferase gene expression in the two materials was close. These genes encoding the protein were active in the the Huperzine metabolic pathways.The full-length cDNA of L-lysine decarboxylase gene sequence of the Huperzia serrata (Thunb. ex Murray) Trev. var. longipetiolata (Spring) H. M. Chang was obtained using RACE technology, which was 1266 bp with a molecular weight of 780.41 kDa. The content of the four bases in gene were:29.5%A; 19.4%C; 24.4%G; 26.7%T. the putative protein was 403 amino acid which has 34.49% α-helix,19.35% Extended strand,46.15% random coil of the secondary structure. Swiss-model analysis of protein tertiary structure, compared with existing lysine decarboxylase protein tertiary structure revealed that it has same conserved regions and related functional domains.The possible pathway involved in Huperzine (A) biosynthesis and development regulation can be describe as follow. In the first, L-lysine forms △1-piperideine after a series of chemical reactions. And the subsequent reaction is integrating △1-piperideine and malony-coa to pelletierine and 4PAA. Next, the general intermediate of all Huperzine (A) is phlegmarine, which originates from the coupling of the 4PAA and the pelletierine. Then the closed-loop phlegmariurus is lycodane. Thus, they produce a series of precursors of Huperzine (A).All in all, Huperzine biosynthetic pathway is complex, and a wide range types of enzymes involved in the regulation. There are still many uncertain intermediate product exist to carry out. this paper is a domestic and international Huperzine metabolic pathways research frontier, providing important guidance value and a new way of thinking.