| The bryophyte that placed between algae and pteridophytes is an important plant taxa. There are over23,000species at present including6000species of liverworts (Hepaticae),14000species of mosses (Musci) and300species of hornworts (Anthocerotae). The natural products in liverworts are with multiplicity structure and bioactivity such as the terpenoids and phenols with antifungal, antitumor, insect antifcedant, antioxidant ct al. It is said that liverworts are the treasury of bioactive natural products. But it is difficult to collect sufficient biomass of taxonomic clean entities for investigation as the liverworts are generally small and grow together and the special habitats of liverworts. Modern biotechnology provides a new approach to solve such difficulties.Tissue culture is a practical way to mitigate the problem of limited natural sources of liverworts. It will be possible to provide sufficiently biomass of taxonomic clean entities by inducing callus from cell, tissue and organ in an aseptic condition, and by culturing them in a controllable, automatized way. In this paper, the rapid growth callus were induced on the MSK2medium through the surface sterilization of the M. paleacea mature sporangia. After two weeks growth, the biomass of the callus was approximately15times of the callus that just subcultured. The callus were phytochemically investigated. Eleven known compounds were isolated and they were: β-sitosterol1, Daucosterine2, Dibutyl phthalate3, Pubinernoid A4, Machantin C5, Neomarchantin A6, Lunularin7, Riccardin C8, Perrottetin E9, Apigenin10, Lunularic acid11. Their structures were elucidated on the basis of their spectral data (ESI-MS,1H NMR,13C NMR,2D NMR) and by comparison with literature values. The allelopathy effect of bibenzyls that isolated from the callus of M. paleacea on Arabidopsis thaliana was studied. The root growth of A.thaliana was inhibited after the treatment of different bibenzyls in a dose dependent manner.In addition, it is crucial to investigate the biosynthetic pathway of the bioactive natural products. It is possible to obtain bioactive compounds by metabolic engineering such as constructing the key enzyme of the bibenzyls biosynthetic pathway to engineered bacteria for protein expression. Then increase the products by enzymatic reaction in vitro or in vivo. In this paper, the cDNA library of the Plagiochasma appendiculatum were constructed, and we got a gene annotated as stilbenecarboxylate synthase (STCS). In addition, we also cloned two types of STCS. They were constructed to prokaryotic expression vector and then transformed to BL21for protein expression. In vitro activities were tested with the purified protein. The result revealed that STCS2was a bifunctional enzyme which could catalyze the p-coumaroyl-CoA and malonyl CoA to generate naringenin and could also accept the dihydro-p-coumaroyl-CoA as substrate to generate5-hydroxylunularic acid, dihydro-4-coumaroyltriacetic acid lactone (dihydro-CTAL), dihydrobisnoryangoni (dihydro-BNY). STCS1could also catalyze the dihydro-p-coumaroyl-CoA and malonyl CoA to generate the three products above. But it could not accept the p-coumaroyl-CoA as starter CoA. In other words, STCS1did not have chalcone synthase activity which was consistent with the literature.PaSTCS2and PaSTCS1were also inserted into plant expression vector and then transformed to Agrobacterium GV3101, which were transformed into the callus of M. paleacea and tt4mutants of A.thaliana by Agrobacterium-mediated genetic transformation. Gene expression and the chemical composition of transgenic lines were analyzed. The result revealed that content of lunularic acid was increased in PaSTCS2callus overexpression PaSTCS2which indicated that PaSTCS2is related with bibenzyls biosynthesis. The seed coat color of tt4mutants was partially restored by overexpress PaSTCS2. PaSTCS1transgenic lines were in further screening and analysis. |
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