[Bioinformatics analysis of geranylgeranyl pyrophosphate synthase coding gene and amino acid sequence in Lamiaceae].

Geranylgeranyl pyrophosphate synthase enzyme is one of the key enzymes in the synthesis pathway of diterpenoid. Nine Lamiaceae genus GGPS synthase in Genebank was analyzed in this article. GGPS synthase the nucleic acid sequences and amino acid sequences, physicochemical properties, the signal peptide, leader peptides, transmembrane topological structure, hydrophobic, hydrophilic, subcellular localization, secondary structure, function domain, tertiary structure and evolutional relationship were predicted by using bioinformatics methods.Phylogenetic tree was constructed for the geranylgeranyl pyrophosphate synthase enzyme protein family. The results showed that GGPS amino acid sequence of the physical and chemical properties were basically identical, mainly hydrophilic protein, there existed chloroplast transit peptide, and no signal peptide and membrane structure domain, which mainly located in the chloroplast, the minor part located in mitochondria. The main secondary structures of the proteins are alpha helix and random coil. All these proteins have catalytic residues, aspartate-rich region, active site lid residues, substrate-Mg2+ binding site. The results provide theoretical reference for study on both the enzymatic characteristics of GGPS and the biosynthesis pathway of diterpenoid.

The Biosynthetic Pathways of Tanshinones and Phenolic Acids in Salvia miltiorrhiza.

Secondary metabolites from plants play key roles in human medicine and chemical industries. Due to limited accumulation of secondary metabolites in plants and their important roles, characterization of key enzymes involved in biosynthetic pathway will enable metabolic engineering or synthetic biology to improve or produce the compounds in plants or microorganisms, which provides an alternative for production of these valuable compounds. Salvia miltiorrhiza, containing tanshinones and phenolic acids as its active compounds, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. The biosynthetic analysis of secondary metabolites in S. miltiorrhiza has made great progress due to the successful genetic transformation system, simplified hairy roots system, and high-throughput sequencing. The cloned genes in S. miltiorrhiza had provided references for functional characterization of the post-modification steps involved in biosynthesis of tanshinones and phenolic acids, and further utilization of these steps in metabolic engineering. The strategies used in these studies could provide solid foundation for elucidation of biosynthetic pathways of diterpenoids and phenolic acids in other species. The present review systematically summarizes recent advances in biosynthetic pathway analysis of tanshinones and phenolic acids as well as synthetic biology and metabolic engineering applications of the rate-limiting genes involved in the secondary metabolism in S. miltiorrhiza.

[Cloning and prokaryotic expression analysis of squalene synthase 2 (SQS2) from Salvia miltiorrhiza f. alba].

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, a full-length cDNA sequence of SQS2 from S. miltiorrhiza f. alba was cloned by the method of reverse transcription polymerase chain reaction (RT-PCR). The SmSQS2 cDNA sequence was obtained, this sequence is named SmSQS2 and its GenBank registration number is KM244731. The full length of SmSQS2 cDNA was 1245 bp, encoding 414 amino acids including 5'UTR 115 bp and 3'UTR 237 bp. Sequence alignment and phylogenetic analysis demonstrated that SmSQS2 had relative close relationship to the SQS2 of S. miltiorrhiza. The induction of E. coli [pET28-SQS2] in different temperature, induction time, IPTG concentrations and density of inducing host bacterium (A600) were performed, Shaking the culture at 30 degrees C until the A600 is approximately 0.6 and add IPTG to final concentration of 0.2 mmol x L(-1), and then the optimal expression of SmSQS2 recombinant protein were accumulated after the induction time of 20 h. The research provided important base for the study of sterol and terpene biosynthesis of SQS2 in S. miltiorrhiza f. alba.

[Cloning and prokaryotic expression analysis of HDS from Salvia miltiorrhiza bge.f.alba].

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction (RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium (A600). The optimal expression conditions of SmHDS were 30 degrees C until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol x L(-1), and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.

[Construction of RNAi vectors for SmNAC1 transcription factors of Salvia miltiorrhiza using Gateway cloning technology].

NAC transcription factors involved in plant growth and development, as well as responses to biotic and abiotic stress. RNAi Vectors for SmNAC transcription factors of Salvia miltiorrhiza was constructed by using Gateway cloning technology, in order to further study the function of SmNAC1 transcription factor. According to Gateway cloning technology, the specific fragments of SmNAC1 containing attB adapter was amplified by PCR using ultra-fideling phusion polymerase of NEB. By the BP recombination reaction, the PCR product containing attB was transferred to an donor vector (pENTR/SD/D-TOPO). Finally, SmNACi specific gene was cloned into pK7GWIWG2D plant expression vectors by LR recombination reaction. Experimental results showed that Gateway cloning technology provide a rapid and highly efficient way to clone the interested gene.

[Optimization of expression and purification of recombinant Salvia miltiorrhiza WRKY1 protein in Escherichia coli].

WRKY transcription factor is one of the Zinc finger proteins which contains a highly conserved WRKY domain and is a family of the plant-specific transcription factor. The plasmid pET28a-SmWRKY1 harboring Salvia miltiorrhiza WRKY1 (SmWRKY1) gene was successfully transformed and expressed in Escherichia coli BL21 (DE3). The conditions on protein expression of SmWRKY1 in E. coli, including induction duration, temperature, IPTG concentration and the E. coli concentration were optimized. The results showed that the highest protein expression of SmWRKY1 was obtained at 24 hours after the E. coli was cultured in the presence of 0.2 mol x L(-1) IPTG at 20 degrees C with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g x L(-1) as inclusion body, which was first extracted using urea, and then purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The expression of SmWRKY1 in E. coli was further confirmed by western blotting analysis.

Mechanisms of co-modified liver-targeting liposomes as gene delivery carriers based on cellular uptake and antigens inhibition effect.

In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, the liver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor (ASGPR), beta-sitosterol-beta-d-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linked immunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strong antigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different steps during the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated through antigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiency seemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis, the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfection but no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effective carrier to transfer asODN into hepatocytes infected with HBV orientedly.

[Content deternmination of glycyrrhizic acid in cortex glycyrrhizae].

Glycyrrhizic acid content in Fen Gancao (barked licorice root) and its rough bark (Cortex Glycyrrhizae) was determined by HPLC. The result showed that at least three unknown ingredients were detected in Cortex Glycyrrhizae which were not in Fen Gancao, and glycyrrhizic acid content in the Cortex Glycyrrhizae is higher than that in Fen Gancao. It suggests that Cortex Glycyrrhizae can be used as the material not only to extract glycyrrhizic acid but also for making additives. Furtheronore, Fen Gancao should be further studied in order to reveal the differences of pharmacological effects between Fen Gancao and Licorice Root (Radix Glycyrrhizae).