Research progress on the role of cytochrome P450 in plant sesquiterpene biosynthesis / 药学学报

Sesquiterpenes are natural terpenoids with 15 carbon atoms in the basic skeleton, which mainly exist in plant volatile oil and have important physiological and medicinal value. Cytochrome P450 (CYP450) is a kind of monooxygenase encoded by supergene family, which is one of the largest gene families in plants. It is involved in the synthesis and metabolism of terpenoids, alkaloids and other secondary metabolites. In the process of terpene biosynthesis, CYP450 participates in the post-modification stage of terpenes by introducing functional groups such as hydroxyl, carboxyl and carbonyl, which plays an important role in enriching the diversity of terpenes. The CYP450 enzymes involved in sesquiterpene synthesis and their substrate catalytic specificity mechanisms have been partially investigated. In this paper, the biosynthetic pathway of plant sesquiterpenes, the structure and classification of CYP450 enzymes were briefly introduced, and the CYP450 enzymes involved in sesquiterpene biosynthesis were summarized, in order to provide a reference for intensive study of the role of CYP450 enzymes in the synthesis of sesquiterpenoids.

Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis (Lour.) Gilg / 药学学报

Cytochrome P450 (CYP450) is a kind of superfamily oxidase containing heme, which is distributed in various aerobic organisms. They are widely involved in the biosynthesis of terpenoids, alkaloids, flavonoids, fatty acids, etc. In this study, the full-length cDNA sequence of a P450 was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, with the specific primers that designed according to the sequence of a transcript annotated as P450 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The tissue expression and subcellular localization were also studied. The full-length cDNA of the cloned P450 gene is 1 920 bp, with 88 bp 5′-untranslated region (UTR), 344 bp 3′-UTR and a 21 bp polyA tail, and 1 488 bp open reading frame (ORF), encoding 495 amino acids. Sequence alignment revealed that the protein belonged to CYP71D family of cytochrome P450 family, and named AsCYP71D1. Tissue expression analysis indicated that AsCYP71D1 was mainly expressed in stem. Further subcellular localization of onion epidermis showed that AsCYP71D1 was expressed in cytoplasm, nucleus and cell membrane. This study will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Targeted MR Imaging Adopting T1-Weighted Ultra-Small Iron Oxide Nanoparticles for Early Hepatocellular Carcinoma: An and Study / 中国医学科学杂志(英文版)

Objective The purpose of this study was to produce an arginylglycylaspartic acid (RGD) peptide-modified ultra-small superparamagnetic iron oxide (FeO) nanoparticles (NPs) for targeted magnetic resonance (MR) imaging of hepatocellular carcinoma (HCC) cells and verify its utility as a T1 positive MRI imaging contrast agent and .Methods The carboxylated FeO NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD nanoparticles to form a novel target contrast agent FeO-PEG-RGD NPs. The specificity of FeO-PEG-RGD to bind RGD receptor was investigated by HepG2 cellular uptake and cell MR imaging, and by MR imaging of subcutaneous HepG2 tumors of nude mice.Results The formed FeO-PEG-RGD NPs displayed good biocompatibility, and the ultrahigh r1 relaxivity was 1.37 mM S . The synthesized FeO-PEG-RGD NPs were demonstrated spherical-like with an approximate diameter of 2.7 nm in similar size. The targeting effect to HepG2 cells was confirmed by cellular uptake and cell MR imaging. The MR imaging of nude mice demonstrated that the MR signal intensity enhancement of HepG2 tumor in FeO-PEG-RGD NPs treated mice was significantly higher than in mice treated with non-targeted FeO-mPEG NPs at the same post-administration time point. Conclusion The results indicate that the FeO-PEG-RGD particles have potential utility as T1 positive contrast agent in targeted MR imaging.

Prokaryotic expression of Aquilaria sinensis AsMAPK3 gene and its localization in transient expression system in onion epidermis / 中草药

Objective To construct a prokaryotic vector of AsMAPK3 gene from Aquilaria sinensis, the original plant of agarwood, and induce the recombinant proteins expression so as to study the subcellular localization of AsMAPK3. This work will prepare materials for antibody preparation and lay a foundation for screening the interaction proteins and further studying their functions. Methods Partial cDNA sequence was amplified by PCR and recombined to pET-28a vector to construct a prokaryotic expression vector pET-28a-AsMAPK3, and induced the expression of the fusion protein. The full-length cDNA of AsMAPK3 was amplified and subcloned to pAN580 vector to construct a pAN580-AsMAPK3 transient expression vector. The recombinant plasmid of pAN580-AsMAPK3 was introduced into the onion epidermis by gold particle bombardment, and GFP fluorescence was observed by luorescence microscope. Results The Escherichia coli BL21 (DE3) containing the recombinant plasmid was induced with 0.5 mmol/L isopropyl-β-D-galactoside (IPTG) at 37 ℃ for 4 h, and a fusion protein about 39 000 was obtained which was expressed in supernatant and inclusion bodies. The results of GFP fluorescence observation of transient transformed onion epidermis showed that AsMAPK3 was mainly expressed in the nucleus and plasma membrane. Conclusion The expression and purification of AsMAPK3 in vitro were successfully carried out, and the subcellular localization of AsMAPK3 gene was confirmed. This work provides a substantial foundation for follow-up function study of AsMAPK3.

Construction and expression of prokaryotic vector of AsJAZ1 gene from Aquilaria sinensis / 中国中药杂志

The full-length coding sequence (cds) of jasmonate-zim-domain protein (AsJAZ1) gene was cloned from Aquilaria sinensis, the prokaryotic vector was constructed and the recombinant proteins expression was induced to provide the basic material for interactive proteins screen and gene function research. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length cds of AsJAZ1 gene was amplified using RT-PCR method and subcloned into pET-28a vector. The recombinant plasmid identified by restriction enzyme digestion and nucleotide sequencing was transformed into E. coli BL21(DE3). Inducing with 0.5 mmol•L⁻¹ IPTG at 37 ℃ for 4 hours, a fusion protein about 39 kDa was maximumly obtained. AsJAZ1 fusion protein had been expressed successfully mainly in the form of inclusion bodies and only a very small amount was secreted into the cytoplasm in the supernatant.

Prokaryotic expression and purification of Aquilaria sinensis (Lour.) Gilg AsMYC2 protein / 药学学报

The MYC2 transcription factor is a member of the important plant bHLH transcription factor families, and it is also the core regulatory elements in jasmonate (JA) signaling pathway. However, there is a little information about AsMYC2 gene in Aquilaria sinensis. In this study, with the total RNA isolated from A. sinensis leave as template, the full-length coding sequence (CDS) of AsMYC2 gene was amplified using RT-PCR method and subcloned into pGEX-4T-1 vector by the gene recombination technique. The recombinant vector pGEX-4T-1-AsMYC2 was verified by restriction enzyme digestion and nucleotide sequencing, and was transformed into E. coli BL21(DE3) to express the protein. A maximum expression of soluble protein was observed with induction by 0.1 mmol·L-1 IPTG at 37℃ for 4 hours. The fusion protein was purified through a Sepharose-Glutathione column, and verified by SDS-PAGE and Western blotting using an anti-GST polyclonal antibody. We successfully constructed the GST-AsMYC2 plasmid, produced and purified the GST-AsMYC2 fusion protein, which would provide the basic material for polyclonal antibody preparation, interactive factors screening and gene function research. According to the tissue-specific expression pattern analysis by qRT-PCR method, the AsMYC2 gene in A. sinensis tissues is mainly expressed in roots and stems, the main agarwood formation parts, and lowest expressed in leaves. These results indicate that AsMYC2 gene likely play some roles in agarwood formation in A. sinensis.

Establishment of a cell suspension culture system of endangered Aquilaria sinensis (Lour.) Gilg / 药学学报

Aquilaria sinensis callus induced by stem tips were used to establish the suspension cell system. The results showed that the most suitable medium for callus induction and subculture is MS + 2.0 mg x L(-1) NAA + 1.0 mg x L(-1) 6-BA. After 12 times of subculture, the energetic and loose callus, which were appropriate for cell suspension culture, were cultured and shook in liquid medium MS + 2.0 mg x L(-1) NAA + 1.0 mg x L(-1) 6-BA + 500.0 mg x L(-1) casein hydrolysate (CH) to establish the suspension cell system. The growth curve of suspension cells showed a "S" type. At the beginning of the culture, cell density increased slowly; during 4 to 6 days, suspension cells reached logarithmic growth period; during 7 to 12 days, suspension cells were in the platform period; but after 12 days, cell density and activity went down obviously. Agarwood sesquiterpenes were not detected in the suspension cells during the growth period, however, they could be detected in MeJA treated suspension cells. In this study, a stable and active growing suspension cell system was established, which was a proper system to study the mechanism of agarwood sesquiterpene formation, and additionally provided a potential way to generate agarwood sesquiterpenes through application of cell culture.

Cloning, prokaryotic expression, and functional identification of a sesquiterpene synthase gene (AsSS4) from Aquilaria sinensis / 药学学报

A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1,698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis

Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis / 中国中药杂志

The study aimed to clone the open reading frame of cinnamate 4-hydroxylase (C4H) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. One unique sequence containing C4H domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of C4H was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. C4H expression profiles in responds to MeJA (methyl jasmonate) application were analyzed by real-time PCR. The length of C4H open reading frame (ORF) was 1 515 bp, encoding 514 amino acids. The GenBank accession number is KF134783. Inducible-experiments showed that the genes were induced by mechanical wound as well as MeJA induction, and reached the highest expression level at 8 h and 20 h, respectively. The full-length cDNA of C4H and its expression patterns will provide a foundation for further research on its function in the molecular mechanisms of aromatic compounds and flavonoids biosynthesis.

Cloning and gene expression of acetyl-CoA C-acetyl transferase gene (AsAACT) from Aquilaria sinensis / 中国中药杂志

<p><b>OBJECTIVE</b>This study aimed to clone the acetyl-CoA C-acetyl transferase (AACT) gene from Aquilaria sinensis and analyze the bioinformatics and expression of the gene.</p><p><b>METHOD</b>One unique sequence containing partly AACT gene sequence was discovered in our previous transcriptome dataset of A. sinensis. AACT gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from A. sinensis stem. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsAACT expression in calli was analyzed with GADPH gene as an internal control gene in wounded condition by qRT-PCR technique.</p><p><b>RESULT</b>One unique sequence of AACT, named as AsAACT, was cloned from A. sinensis. The full length of AsAACT cDNA was containing a 1 236 bp ORF that encoded 411 amino acids. The result of qRT-PCR displayed that the highest expression level was at 4 h. which indicated that it was possibly involved in early-stage response to wound.</p><p><b>CONCLUSION</b>Cloning and analyzing AsAACT gene from A. sinensis provided basic information for study the function and expression regulation of AsAACT in terpenoid biosynthesis.</p>

Study of production of sesquiterpenes of Aquilaria senensis stimulated by Lasiodiplodia theobromae / 中国中药杂志

To investigate the mechanism of agarwood formation in Aquilaria sinensis induced by Lasiodiplodia theobromae, the fermentation liquor of L. theobromae was analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry (GC-MS). JAs were detected in the fermentation liquor. The effect of the fermentation liquor on the abundance of sesquiterpenes in the callus of A. sinensis was analyzed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). And the fermentation liquor stimulated alpha-guaiene, alpha-humulene and delta-guaiene biosynthesis in calli. It was inferred that L. theobromae produced JAs, which resulted in a significant increase of sesquiterpenes in A. sinensis.

Cloning and expression analysis on 3-hydroxy-3-methylglutaryl-coenzyme A reductase from aquilaria sinensis / 中草药·英文版

Objective: To clone the full-length cDNA of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) from Aquilaria sinensis (AsHMGR1) and to analyze its expression profile in different tissues and in response to different treatments. HMGR is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate pathway. Methods: RT-PCR and RACE were used to clone the full-length cDNA of HMGR from A. sinensis based on the conserved HMGR gene fragments. The bioinformatic analysis was performed on its nucleic acid and protein sequence. The expression profile of AsHMGR1 in different tissues and in response to different treatments was analyzed by quantitative RT-PCR. Results: The full-length AsHMGR1 cDNA was 2026 bp, containing a 1719 bp open reading frame which encoded a protein of 572 amino acids. Amino acid sequence homology alignment and phylogenetic analysis demonstrated that AsHMGR1 belonged to the HMGR gene family. The detection of tissue expression patterns showed that AsHMGR1 was mainly expressed in the stem, followed by roots and branches. AsHMGR1 could be stimulated by methyl jasmonate and H2O2 to varying degrees in a time-dependent manner. Conclusion: These data will provide a foundation for further investigation on AsHMGR1 functions and regulatory mechanisms in sesquiterpene synthesis in A. sinensis. © 2013 Tianjin Press of Chinese Herbal Medicines.

Cloning and expression analysis of farnesyl pyrophosphate synthase from Aquilaria sinensis / 中国中药杂志

Farnesyl diphosphate synthase (FPS) is one of the key rate-limiting enzymes in the sesquiterpene metabolic pathways. In this study, the open reading frame (ORF) of FPS was cloned by PCR based on the transcript sequence of AsFPS1 from the Aquilaria sinensis transcriptome database and sequenced. Total RNA was extracted from the root, stem and leaves of three-year-old A. sinensis, and from healthy and wounded A. sinensis calli, and then reverse-transcribed into single-stranded cDNA as a template for real-time PCR, to detect the expression specificity of AsFPSI in different tissues and its expression profile in responding to different treatments. The result showed that the full length of AsFPS1 was 1 342 bp with the ORF 1 029 bp, encoding 342 amino acids. Tissue expression analysis indicated that AsFPS1 was mainly expressed in root and stem, and was lower in leaves. Inducible-experiments showed that the genes was induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 12 h, respectively. The full-length cDNA clone of AsFPSI and its expression patterns analysis will provide a foundation for follow-up study on its biological function and agarwood sesquiterpene biosynthesis mechanism.

Expression analysis of glycosyltransferase BcUGT1 from Bupleurum chinense DC. and its expression in E. coli and the target protein purification / 药学学报

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.

Cloning and expression analysis of HMG-CoA reductase from Aquilaria sinensis (Lour.) Gilg / 药学学报

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate (MVA) pathway. The specific primers were designed according to the transcript sequence of AsHMGR2 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The full-length cDNA of AsHMGR2 was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) technology, and was analyzed at bioinformatics levels; AsHMGR2 expression profiles in different tissues and in responds to different treatments were analyzed by real-time PCR. The length of AsHMGR2 Open Reading Frame (ORF) was 1 749 bp, encoding 582 amino acids. The GenBank accession number is KC140287. Tissue expression analysis indicated that AsHMGR2 was mainly expressed in root and shoot tips, followed by stem, and was lowest in leaves. Inducible-experiments showed that the genes were induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 8 h, separately. The full-length cDNA of AsHMGR2 and its expression patterns will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Cloning and bioinformatics analysis of chalcone synthase (AsCHS1) gene in Aquilaria sinensis / 中国中药杂志

<p><b>OBJECTIVE</b>The study aimed to clone the open reading frame of chalcone synthase (CHS) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene.</p><p><b>METHOD</b>One unique sequence containing CHS domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of CHS was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsCHS1 expression in calli was analyzed with histone gene as an internal control gene under wound condition by qRT-PCR technique.</p><p><b>RESULT</b>One unique sequence of CHS, named as AsCHS1, was cloned from A. sinensis. The full length of AsCHS1 cDNA was containing a 1 192 bp ORF that encoded 397 amino acids. The result of qRT-PCR displayed that the highest expression level was at 12 h, which indicated that it was possibly involved in early-stage response to wound.</p><p><b>CONCLUSION</b>Cloning and analyzing AsCHS1 gene from A. sinensis provided basic information for study the function and expression regulation of AsCHS1 in the flavonoids biosynthesis.</p>

The mechanical wound transcriptome of three-year-old Aquilaria sinensis / 药学学报

Chinese agarwood is formed in the aromatic resinous wood formed in Aquilaria sinensis (Lour.) Gilg (botanical family: Thymelaeaceae). Only when suffering stress of wound, etc, can A. sinensis produce sesquiterpenes etc. compounds of agarwood around wounds. However, little is known about how wound induced the biosynthesis pathway of sesquiterpenes. To reveal the molecular mechanism of wound-induced agarwood formation, RNA sequencing (RNA-seq) technology was used to investigate the profile of gene expression in A. sinensis treated by mechanical wounding and elucidate its functional gene. A total of 40,295 ESTs with an average read length of 305 bp were generated and 22 095 unigenes were formed by initial gene splicing. 61.6% of these unigenes (13 611) were annotated using BLAST searches against the SwissProt, KEGG, Nr and Nt databases. Twenty-six unigenes (encoding 7 enzymes) were found to be involved in sesquiterpene of agarwood biosynthesis by bioinformatic tools of Gene Ontology and KEGG. Novel genes that are potentially involved in sesquiterpenes biosynthesis were identified in A. sinensis, providing data for further sesquiterpenes biosynthesis pathway by molecular methods and the EST data establish a foundation for future studies in the molecular mechanisms of wound-induce agarwood formation in A. sinensis.

Evaluation of 131I treatment efficacy and prognostication for bone metastases from differentiated thyroid cancer / 中华核医学杂志

Objective To evaluate the efficacy of 131I treatment for bone metastases from DTC and analyze the survival rates after 131I treatment and prognostic factors. Methods One hundred and six DTC patients with bone metastases treated by 131I during January 1991 and January 2009 were retrospectively analyzed. Treatment efficacy was assessed based on serum Tg change, bone pain palliation and changes on medical imaging. Univariate analysis was performed for defining the factors affecting 131I treatment efficacy. Survival curves were estimated using the life table method. Survival analysis was performed using Kaplan-Meier method. Results Serum Tg decreased dramatically in 37/106 (34.9%) patients treated with131I. Thirty-nine of 61 patients (63.9%) with bone pain had pain relief. Age, tumor subtype and presence of non-osseous distant metastases were significant factors affecting 131I treatment efficacy based on serum Tg change (χ2=6.443, 11.455, 6.756, all P0.05). There were no imaging changes of bone metastases in 77.4% of patients after 131I treatment. The overall 5-year and 10-year survival rates from initial diagnosis of bone metastases was 86.47% and 57.90%, respectively. Univariate analysis showed that number of metastases, presence of non-osseous distant metastases and pre-131I treatment surgery were significant factors for survival (Log-rank values were 4.05, 5.98, 4.22, all P<0.05). Conclusions 131I treatment for bone metastases from DTC is effective for lowering serum Tg and palliation of bone pain. Single metastasis, absence of non-osseous distant metastases and pre-131I therapy surgery are favorable predictors of prognosis.