[Cloning and induced expression analysis of 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase gene (smHDR) of Salvia miltiorrhiza].

This study reported the obtainment of the full-length cDNA of Salvia miltiorrhiza hairy roots (Abbr: SmHDR, GenBank number: JX233817), via extracting Salvia miltiorrhiza hairy roots total RNA, designing specific primers according to the transcriptome data and using the RACE strategy, and then analyzed it with bioinformatics approaches. On this basis, using the real-time PCR to detect SmHDR gene expression after Ag+ induction, and testing tanshinones contents of corresponding samples by UPLC. SmHDR has 1 647 nucleotides, and an open reading frame (ORF) encoding a protein of 463 amino acid residues. The deduced protein has isoelectric point (pI) of 5.72 and a calculated molecular weight about 51.88 kD. In the secondary structure, the percentage of alpha helix, beta turn and random coil were 35.64%, 20.30% and 44.06%, respectively. Sequence alignment and phylogenetic analysis demonstrated that SmHDR had relative close relationship to the HDR of Picrorhiza kurrooa, similar to HDR from other species of plants. Real time PCR results indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmHDR. At the same time, results of ultra performance liquid chromatography (UPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy roots of Salvia miltiorrhiza were increased dramatically at 12 h after treated with Ag+, and then decreased significantly. This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by Ag+. The content of tanshinones was gradually raised, and it had an obvious increase at 120 h. The bioinformatics analysis and gene expression indicated that SmHDR might be involved in tanshinones biosynthesis, which laid the foundation for further study of secondary metabolic regulation mechanism of tanshinones.

[Cloning and bioinformatics analysis of 2-c-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene in Salvia miltiorrhiza].

OBJECTIVE: To clone and analysis a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (SmMCS) full-length eDNA from Salvia miltiorrhiza hairy roots. METHOD: A full-length eDNA of SmMCS has been cloned by designing specific primers according to the transcriptome database and using the RACE strategy. ORF Finder was used to find the open reading frame of SmMCS cDNA and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5. 1. Real-time quantitative PCR have been applied to detect the transcription level of SmMCS from hairy roots after elicitor Ag+ supplied. RESULT: The SmMCS cDNA sequence was obtained. The full length of SmMCS (DNA was 988 bp encoding 234 amino acids. The deduced protein had isoelectric point (pI) of 8.53 and a calculated molecular weight about 24. 6 kDa. Results of real time PCR indicated that elicitor of Ag+ stimulated the increase of mRNA expression of SmMCS in hairy roots, and were increased dramatically at 12 h. CONCLUSION: The full-length cDNA of SmMCS was cloned from S. miltiorrhiza hairy root,which can provide a gene target for further studies of tanshinones biosynthesis and terpenoid secondary metabolites.

[Research advances of diterpene synthase].

Diterpenes, an important class of natural compounds, are widely distributed in nature. As the valuable diterpenoids continue to be found, diterpene synthase in the course of diterpene synthesis get as much attention as possible. The multiformity of end-product-diterpenoids were also due to the diversity of diterpene synthase. This paper focuses on the advances in recent biosynthesis pathway of diterpene and types, cloning, catalytic mechanism, synthetic biology application.

Dynamic Bioreactor Culture for Infiltration of Bone Mesenchymal Stem Cells within Electrospun Nanofibrous Scaffolds for Annulus Fibrosus Repair.

OBJECTIVE: To compare the ability of three culture strategies of static culture, intermittent centrifugal culture and dynamic bioreactor culture in promoting the infiltration of bone marrow mesenchymal stem cells (BMSCs) throughout electrospun nanoporous aligned nanoyarn scaffold (AYS). METHODS: AYS was constructed by the method of conjugated electrospinning, using the blended solution of poly (L-lactide-co-caprolactone) (P (LLA-CL)) and gelatin. Then the bone marrow mesenchymal stem cells (BMSCs) were transplanted on the scaffolds. Culture the scaffold-cells using three methods of static culture, intermittent centrifugal culture and dynamic bioreactor culture. After 7 and 14 days in culture, the infiltration depth of the cells were observed and measured by hematoxylin and eosin (HE) or 4', 6-diamidino-2-phenylindole (DAPI) staining. RESULT: In the current study, on the 7th day, the BMSCs in the scaffolds of static culture group, intermittent centrifugal culture group, and dynamic bioreactor culture group infiltrated to an average depth of 11.88 ± 1.82 µm, 21.17 ± 13.17 µm, and 26.27 ± 7.42 µm, respectively. There were differences between the bioreactor culture group with the static culture group and the intermittent centrifugal culture group. On the time point of 14 days, the depth of infiltration of BMSCs in dynamic bioreactor culture was the most (115.13 ± 25.44 µm, P < 0.05), and the infiltration of the cells in the intermittent centrifugal culture group was 42.53 ± 13.07 µm, deeper than that of the static culture group (24.53 ± 6.06, P < 0.05). CONCLUSION: Dynamic bioreactor culture may be a preferred method for tissue engineering approaches involving scaffolds with a low porosity, such as those needed for repair of the annulus fibrosus (AF).

The endogenous retrovirus-derived long noncoding RNA TROJAN promotes triple-negative breast cancer progression via ZMYND8 degradation.

Human endogenous retroviruses (HERVs) play pivotal roles in the development of breast cancer. However, the detailed mechanisms of noncoding HERVs remain elusive. Here, our genome-wide transcriptome analysis of HERVs revealed that a primate long noncoding RNA, which we dubbed TROJAN, was highly expressed in human triple-negative breast cancer (TNBC). TROJAN promoted TNBC proliferation and invasion and indicated poor patient outcomes. We further confirmed that TROJAN could bind to ZMYND8, a metastasis-repressing factor, and increase its degradation through the ubiquitin-proteasome pathway by repelling ZNF592. TROJAN also epigenetically up-regulated metastasis-related genes in multiple cell lines. Correlations between TROJAN and ZMYND8 were subsequently confirmed in clinical samples. Furthermore, our study verified that antisense oligonucleotide therapy targeting TROJAN substantially suppressed TNBC progression in vivo. In conclusion, the long noncoding RNA TROJAN promotes TNBC progression and serves as a potential therapeutic target.