The Contents Of Huperzine, Cloning And Funational Characteirzation Of LDC Gene Involved In The Biosynthesis Of Huperzine A Of Huperzia Serrata From Western Hunan

Huperzia serrata(Thunb)Trev belonging to the HuperziaBernh of the Huperiaceaefamily, is a traditional Chinese medicine, and the main medicine resource plant ofhuperzine A currently. There are high-quality and rich resources of Huperzia serrata inwestern Hunan, but lack of systematic research on haperzine contents and biosynthesis.In this study, Huperzia serrata collected from western Hunan as experimental materials,research works are as follows:(1) For determining the contents of huperzine A, B and C in the whole herb anddifferent parts of huperzia serrata from western Hunan, total alkaloids of samples wereextracted by ultrasound with2%tartaric acid and then analyzed by HPLC. The resultsshowed that the contents of huperzine A, huperzine B and huperzine C in huperziaserrata from3sample plots were similar, and up to0.5‰,0.3‰and0.04‰respectively. The contents of huperzine A, B and C in the different parts of huperziaserrata had significant difference, in which the distribution of huperzine A and B wasroot <stem <leaf, but huperzine C was stem <leaf and root. In conclusion, there aresuperior resources of huperzia serrata for huperzine production in western Hunan, thedistribution of huperzine in different parts of huperzia serrata has high selectivity, andthe HPLC method to measure three kinds of huperzine simultaneously was simple,accurate and reliable.(2) The full length cDNA of L-lysine decarboxylase genes were obtained fromhuperzia serrata by RT-PCR, cloned into T-vector and sequenced, and bioinformationanalysis of L-lysine decarboxylase genes was carried out by ExPASy software. In thisstudy two L-lysine decarboxylase genes were obtained from huperzia serrata, namedLDC1and LDC2with open reading frames of636bp and606bp respectively, andGeneBank accession number were JQ308624.1and JQ308625.1respectively; Based onbioinformation analysis, LDC1and LDC2could code corresponding proteins with212aa and202aa, named LDC1and LDC2respectively, they belonged to lysinedecarboxylase family, with the same physicochemical properties and3d proteinstructure basically.(3) The full length cDNA of LDC1and LDC2were cloned into pET-32a(+) toconstruct recombinant vector respectively, and transferred into Escherichia coliBL21(DE3) to express. The activity of the recombinant LDC1and LDC2proteins wereanalyzed by thin layer chromatography (TLC). The result of TLC detection suggestedthat the recombinant LDC1and LDC2proteins could catalyze the L-lysinedecarboxylation to cadaverine. The result showed that LDC1and LDC2all had lysinedecarboxylase activity, LDC1and LDC2could be L-lysine decarboxylase genes ofhuperzia serrata. (4) The content of huperzine A in the root, stem and leaf of huperzia serrata wasdetermined by HPLC, and the expression level of lysine decarboxylase gene in thedifferent parts of huperzia serrata was analyzed by semi-quantitative RT-PCR, withβ-actin gene used as internal reference, for analyzing the relationship between thecontents of huperzine A and the expression of lysine decarboxylase gene in the differentparts of huperzia serrata. The results showed that the content of huperzine A in the root,stem and leaf of huperzia serrata was (0.201±0.008)‰,(0.455±0.002)‰and(0.579±0.009)‰, respectively, with significant difference. Semi-quantitative RT-PCRresults showed that lysine decarboxylase gene had nearly identical expression amongthe root, stem and leaf of huperzia serrata. We speculated that lysine decarboxylasemight be not one key enzyme to regulate the biosynthesis of huperzine A.