methyl-jasmonate and 2-3-oxidosqualene

Triterpenoids produced by the secondary metabolism of Betula platyphylla Suk. exhibit important pharmacological activities, such as tumor inhibition, anti-HIV, and defense against pathogens, but the yield of natural synthesis is low, which is insufficient to meet people's needs. In this study, we identified two OSC genes of birch, named as BpCAS and Bpβ-AS, respectively. The expression of BpCAS and Bpβ-AS were higher levels in roots and in stems, respectively, and they induced expression in response to methyl jasmonate (MeJA), gibberellin (GA3), abscisic acid (ABA), ethylene and mechanical damage. The function of the two genes in the triterpene synthesis of birch was identified by reverse genetics. The inhibition of Bpβ-AS gene positively regulates synthesis of betulinic acid. BpCAS interference can significantly promote the upregulation of lupeol synthase gene (BPW) and β-amyrin synthase gene(BPY), and conversion of 2,3-oxidosqualene to the downstream products betulinic acid and oleanolic acid. This study provided a basis for the genetic improvement of triterpenoid synthesis in birch through genetic engineering. The obtained transgenic birch and suspension cells served as material resources for birch triterpenoid applications in further.

Topics: Abscisic Acid; Acetates; Betula; Betulinic Acid; Cyclopentanes; Gene Expression Regulation, Plant; Gibberellins; Intramolecular Transferases; Oleanolic Acid; Oxylipins; Pentacyclic Triterpenes; Squalene; Triterpenes

Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.

Topics: Acetates; Alisma; Animals; Antibodies; Cholestenones; Cloning, Molecular; Cyclopentanes; Escherichia coli; Gene Expression Regulation, Plant; Oxylipins; Plant Proteins; Plant Tubers; Rabbits; Recombinant Proteins; Squalene; Squalene Monooxygenase; Triterpenes

Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro, and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible.

Topics: Acetates; Cyclopentanes; Dioscorea; Gene Expression Regulation, Plant; NADP; NADPH-Ferrihemoprotein Reductase; Oxylipins; Plant Proteins; Recombinant Proteins; Squalene; Squalene Monooxygenase