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.

Identification and analysis of terpene synthase (TPS) gene family in Schizonepeta tenuifolia / 中国中药杂志

Terpenoids are important secondary metabolites of plants that possess both pharmacological activity and economic value. Terpene synthases(TPSs) are key enzymes in the synthesis process of terpenoids. In order to investigate the TPS gene family members and their potential functions in Schizonepeta tenuifolia, this study conducted a systematic analysis of the TPS gene family of S. tenuifolia based on the whole genome data of S. tenuifolia using bioinformatics methods. The results revealed 57 StTPS members identified from the genome database of S. tenuifolia. The StTPS family members encoded 285-819 amino acids, with protein molecular weights ranging from 32.75 to 94.11 kDa, all of which were hydrophilic proteins. The StTPS family members were mainly distributed in the cytoplasm and chloroplasts, exhibiting a random and uneven physical localization pattern. Phylogenetic analysis showed that the StTPS genes family were divided into six subgroups, mainly belonging to the TPS-a and TPS-b subfamilies. Promoter analysis predicted that the TPS gene family members could respond to various stressors such as light, abscisic acid, and methyl jasmonate(MeJA). Transcriptome data analysis revealed that most of the TPS genes were expressed in the roots of S. tenuifolia, and qRT-PCR analysis was conducted on genes with high expression in leaves and low expression in roots. Through the analysis of the TPS gene family of S. tenuifolia, this study identified StTPS5, StTPS18, StTPS32, and StTPS45 as potential genes involved in sesquiterpene synthesis of S. tenuifolia. StTPS45 was cloned for the construction of an prokaryotic expression vector, providing a reference for further investigation of the function and role of the TPS gene family in sesquiterpene synthesis.

Identification of terpene synthase gene family in Gynostemma pentaphyllum and expression pattern analysis under abiotic stresses / 中国中药杂志

The present study aimed to investigate the composition of the terpene synthase(TPS) gene family in Gynostemma pentaphyllum and its role in abiotic stresses. The G. pentaphyllum TPS gene family was identified and analyzed at the genome-wide level using bioinformatics analysis, and the expression patterns of these family members were analyzed in different tissues of G. pentaphyllum as well as under various abiotic stresses. The results showed that there were 24 TPS gene family members in G. pentaphyllum with protein lengths ranging from 294 to 842 aa. All of them were localized in the cytoplasm or chloroplasts and unevenly distributed on the 11 chromosomes of G. pentaphyllum. The results of the phylogenetic tree showed that the G. pentaphyllum TPS gene family members could be divided into five subfamilies. As revealed by the analysis of promoter cis-acting elements, TPS gene family members in G. pentaphyllum were predicted to respond to a variety of abiotic stresses such as salt, low temperature, and dark stress. The analysis of gene expression patterns in different tissues of G. pentaphyllum revealed that nine TPS genes were tissue-specific in expression. The qPCR results showed that GpTPS16, GpTPS17, and GpTPS21 responded to a variety of abiotic stresses. This study is expected to provide references in guiding the further exploration of the biological functions of G. pentaphyllum TPS genes under abiotic stresses.

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.

Expression pattern analysis of terpene synthases of endangered Aquilaria sinensis under different stresses / 中草药

Objective: To analyze the expression of terpene synthases of Aquilaria sinensis, an endangered south medicine, and to predict the influences of the environmental factors and stresses on the synthesis of terpene in A. sinensis. Methods: Two-year old seedlings and calli were treated by different stresses. The gene expression patterns of terpene synthases were analyzed by real-time qPCR. Results: Wounding and cauterizing could induce the transcriptional expression of terpene synthases in the stems, and the effect of cauterizing was more significant. Low temperature inhibited the transcription of terpene synthases. In calli, methyl jasmonate (MeJA) treatment had the most effective induction. Conclusion: Both wounding and cauterizing could induce the synthesis of terpenes in stems, while the cauterizing treatment might have better effect. Low temperature has negative influence on agarwood formation. For calli, different treatments could induce the terpene synthesis, while MeJA has the best efficacy.