Cloning And Analysis Of Related Genes EhAACT And EhMDC Involved In The Terpenoid Biosynthesis Of Euphorbia Helioscopia L.

Terpenoid biosynthesis pathway is one of the important metabolic pathways in plant. Terpenoid not only plays significant roles in maintaining the life activities of plants and regu-lating the relationship between environment and plants, but also has significant commercial value, which is widely used in industrial manufacture and medicine and health. Euphorbia helioscopia L. is a herbaceous species of Euphorbia (Euphorbiaceae) and distributes in most areas of China. As an ancient folk herbal medicine, E. helioscopia has the efficacy of reducing phlegm, eliminating edema, reducing swelling and itching, and so on. The most effective me-dicinal component is terpenoid.The researches for some genes involved in the terpenoid bio-synthesis from plants have been carried out. However, there have been controversies about the terpenoid biosynthesis site, terpenoid transfer and accumulation pathway. In previous studies, we identified two key enzymes involved in the terpenoid biosynthesis from E. helioscopia latex, which is acetyl-CoA acetyltransferase (AACT) and diphosphomevalonate decarbox-ylase (MDC).AACT is a starting enzyme of MVA pathway. MDC is the last rate-limiting en-zyme of generating the isopentenyl pyrophosphate (IPP) precursor of terpenoid in MVA pathway. Therefore, in this paper, two genes, acetyl-CoA acetyltransferase and diphos-phomevalonate decarboxylase from E. helioscopia involved in the terpenoid biosynthesis were cloned using RT-PCR and RACE methods, and bioinfonnatic analyses and prokaryotic expressions were also carried out. All of these studies would lay the foundation for deeper understanding of terpenoid biosynthesis site, terpenoid transfer and accumulation pathway in laticifers of E. helioscopia, and also provide theoretical basis for the regulating and control-ling of terpenoid biosynthesis and laticifer development. The main results were as follows:1. The gene of acetyl-CoA acetyltransferase was cloned successfully from E. helioscopia, named as EhAACT (accession number:KP995935), the full length cDNA of EhAACT is 1847 bp, it contains an open reading frame of 1173 bp nucleotides, a 5’untranslated region of 207 bp and a 3’untranslated region of 401 bp. The protein of acetyl-CoA acetyltransferase is en-coded by this gene contains 413 amino acids, bioinformatic analyses show that its predicted molecular weight is 42.606 kDa, theoretical pI is 8.7, it belongs to hydrophobic protein, it has not transmembrane domain and signal peptide but has 22 phosphorylation sites, it is located in the cytoplasm and belongs to the membership of thiolase family in cond_enzymes superfami-ly. Phylogenetic analysis reveals that the protein of E. helioscopia AACT has the highest identity and the closest genetic relationship with Ricinus communis AACT. Tertiary structure homology modeling reveals that E. helioscopia AACT and Homo sapiens mitochondrial AACT have the similar crystal structure, they are all homo-tetramer proteins. Recombinant protein of E. helioscopia AACT was expressed by procaryotic expression system, its molecu-lar weight is about 43 kDa, which is consistent with the result predicted by software.2. The gene of diphosphomevalonate decarboxylase was cloned successfully from Eu-phorbia helioscopia L., named as EhMDC (accession number:KP995936), the full length cDNA of EhMDC is 1653 bp, it contains an open reading frame of 1245 bp nucleotides, a 5’ untranslated region of 184 bp and a 3’untranslated region of 224 bp. The protein of diphos-phomevalonate decarboxylase is encoded by this gene contains 415 amino acids, bioinfor-matic analyses show that predicted molecular weight is 45.971 kDa, its theoretical pI is 5.77, it belongs to hydrophilic protein, it has not transmembrane domain and signal peptide, it has 19 predicted phosphorylation sites, it is located in the cytoplasm and belongs to the member-ship of GHMP kinases superfamily. Homology sequence analysis shows that the amino acid sequence of E. helioscopia MDC and R. communis MDC have the highest identity of 88%. MDC of E. helioscopia, Hevea brasiliensis, Jatropha carcas, Populus euphratica and R. communis are classified as a class by phylogenetic analysis. Tertiary structure homology modeling reveals that E. helioscopia MDC and Mus musculus muscle MDC have the similar crystal structure, they are all homo-dimer proteins. The partial protein of MDC was expressed by procaryotic expression system, its molecular weight is about 18.5 kDa, it is consistent with the weight of using protein fragment.