Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora / 中国中药杂志

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Transcriptome analysis to identify genes involved in the biosynthesis of aconitines in Aconitum pendulum / 药学学报

italic>Aconitum pendulum is a Tibetan medicine that is rich in bioactive compounds such as aconitine-type C19-diterpenoid alkaloids. To investigate the key enzymes in the aconitine biosynthesis pathway, roots, leaves and flowers of Aconitum pendulum were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM2000. Trinity de novo assembly yielded 47 264 unigenes with an average length of 1 140 bp and N50 of 1 678 bp, of which 30 231 unigenes (63.96%) were annotated. In the KEGG database, 542 unigenes were implicated in 17 secondary metabolic pathways; the analysis showed that 44 genes encoded 20 key enzymes in the diterpene skeleton of aconitine biosynthesis and 12 BAHD acyltransferase genes were related to the acetylation modification, with differential expression among three organs. For example, ApTPS8 was the only committed enzyme in the upstream aconitine biosynthetic pathway. The high expression level of ApTPS8 in root indicated that it is the main tissue for the production of precursors of diterpene alkaloids. Consistent with the accumulation of aconitine, we propose that ApBAHD1/2/8 is involved in the biosynthesis of 2-hydroxyaconitine, dehydrated 14-benzoylaconitine, 8-O-methyl-14-benzoylaconine, benzoyldeoxyaconitine and benzoylaconitine, and ApBAHD10 is involved in the biosynthesis of acontine, lucidusculine, 14-O-acetylneoline and 14-O-acetylvirescenin. Comparative transcriptome analysis of A. pendulum and A. carmichaeli indicates significant gene loss in the family of diterpene synthases and acyltransferases in A. pendulum, which is in accordance with the significantly fewer type and quantity of aconitine compounds in this species. Therefore, A. pendulum has proved to be an ideal material for the study of the aconitine biosynthesis pathway. This work provides basic scientific data for further study of aconitine biosynthesis, the discussion of molecular mechanisms of toxicity, and the synthesis of genuine medicinal materials.

Study on tanshinones regulating root-associated microbiomes of Salvia miltiorrhiza / 中国中药杂志

The plant root-associated microbiomes include root microbiome and rhizosphere microbiome, which are closely related to plant life activities. Nearly 30% of photosynthesis products of plants are used to synthesize root compounds, there is evidence that root compounds regulate and significantly affect the root microbiome Tanshinones are the main hydrophobic components in Salvia miltiorrhiza. In order to study whether these compounds can regulate the root-associated microbiomes of S. miltiorrhiza, our study first identified a white root S. miltiorrhiza(BG) which contains little tanshinones. Retain of the fifth intron of tanshinones synthesis key enzyme gene SmCPS1 leading to the early termination of the SmCPS1 gene, and a stable white root phenotype. Further, wild type(WT) and BG were planted in greenhouse with nutrient soil(Pindstrup, Denmark) and Shandong soil(collected from the S. miltiorrhiza base in Weifang, Shandong), then high-throughput sequencing was used to analyze the root-associated microbiomes. The results showed that the tanshinones significantly affected the root-associated microbiomes of S. miltiorrhiza, and the impact on root microbiomes was more significant. There are significant differences between WT and BG root microbiomes in species richness, dominant strains and co-occurrence network. Tanshinones have a certain repelling effect on Bacilli which belongs to Gram-positive, while specifically attract some Gram-negative bacteria such as Betaproteobacteria and some specific genus of Alphaproteobacteria. This study determined the important role of tanshinones in regulating the structure of root-associated microbiomes from multiple angles, and shed a light for further improving the quality and yield of S. miltiorrhiza through microenvironment regulation.

Research progress of natural borneol resources / 中国中药杂志

Natural borneol is an important traditional Chinese medicine herb with resuscitation-inducing, antipyretic and analgesic effects, and has been widely used in the fields of medicine, perfume and chemical industry. At present, natural borneol is short supply, with promising market development prospects. This paper summarized the distribution of borneol plant resources, cultivation status and molecular biological research progress, in the expectation of providing basis and ideas for the research and application of natural borneol.