Transcriptome analysis of transcription factors and enzymes involved in monoterpenoid biosynthesis in different chemotypes of Mentha haplocalyx Briq.

Background: The main active ingredients of Mentha haplocalyx Briq. essential oils are monoterpenes. According to the component of essential oils, M. haplocalyx can be divided into different chemotypes. Chemotype variation is widespread in Mentha plants but its formation mechanism is unclear. Methods: We selected the stable chemotype l-menthol, pulegone, and carvone of M. haplocalyx for transcriptome sequencing. To further investigate the variation of chemotypes, we analyzed the correlation between differential transcription factors (TFs) and key enzymes. Results: Fourteen unigenes related to monoterpenoid biosynthesis were identified, among which (+)-pulegone reductase (PR) and (-)-menthol dehydrogenase (MD) were significantly upregulated in l-menthol chemotype and (-)-limonene 6-hydroxylase was significantly upregulated in carvone chemotype. In addition, 2,599 TFs from 66 families were identified from transcriptome data and the differential TFs included 113 TFs from 34 families. The families of bHLH, bZIP, AP2/ERF, MYB, and WRKY were highly correlated with the key enzymes PR, MD, and (-)-limonene 3-hydroxylase (L3OH) in different M. haplocalyx chemotypes (r > 0.85). The results indicate that these TFs regulate the variation of different chemotypes by regulating the expression patterns of PR, MD, and L3OH. The results of this study provide a basis for revealing the molecular mechanism of the formation of different chemotypes and offer strategies for effective breeding and metabolic engineering of different chemotypes in M. haplocalyx.

Comparative analysis of chemical constituents by HPLC-ESI-MSn and antioxidant activities of Dendrobium huoshanense and Dendrobium officinale.

Dendrobium huoshanense is a Chinese medicinal herb that has high quality and excellent efficacy. However, the chemical basis of its activity is still unclear. Of note, Dendrobium officinale is the most widely utilized among the Dendrobium species. Therefore, the current study systematically investigated the chemical constituents of methanolic extracts and different polar fractions of aqueous extracts from the two herbs by HPLC-ESI-MSn , and then compared in vitro antioxidant activities of their five different polar extracts. Consequently, 61 and 49 compounds were identified from D. huoshanense and D. officinale, respectively, of which 43 compounds were common to both species. In addition, 17 out of 22 different compounds were identified only in D. huoshanense. Moreover, the peak areas of some shared identical compounds of D. huoshanense were significantly larger than that of D. officinale. In vitro antioxidant evaluation results showed that the n-BuOH-soluble fraction of the two herbs exhibited remarkable antioxidant activities. Furthermore, the antioxidant activities of different fractions of D. huoshanense were separately superior to that of D. officinale, which may be attributed to its variable and high contents of flavonoids, bibenzyls and phenanthrenes. These results provide the evidence for the high quality and efficacy of D. huoshanense.

In Vivo Functional Verification of Four Related Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves.

The alkaloid 1-deoxynojirimycin (DNJ) is one of the major bioactive compounds in mulberry leaves (Morus alba L.). Previously, we discovered four key genes involved in the pathway from lysine to piperidine in the biosynthesis of DNJ in mulberry leaves, MaLDC (MG727866), MaCAO (MH205733), MaSDR1 (MT989445), and MaSDR2 (MT989446), which encoded lysine decarboxylase, copper amine oxidase, and short-chain dehydrogenase/reductase 1 and 2, respectively. However, the in vivo functions of these four genes have not been verified yet. Here, these four genes were successfully cloned and used for the establishment of C58C1 Agrobacterium rhizogenes mediated overexpression genetic transformation systems and GV3101 Agrobacterium-mediated virus-induced gene silencing transformation systems in order to verify the influence of these four genes on the biosynthetic content of DNJ in mulberry leaves. The results showed that the content of DNJ increased after the four genes were overexpressed. When these four genes were silenced, the gene expression was blocked, which affected the biosynthesis of DNJ, and the DNJ content decreased. The above results indicated that these four genes participated in DNJ biosynthesis. This study provided a foundation for further elucidating the regulatory mechanisms of DNJ biosynthesis in mulberry leaves.

[Systematic analysis on chemical constituents of Mori Cortex, mulberry root bark and its phellem layer based on HPLC-ESI-MS].

In this study, HPLC-ESI-MS and HPLC methods were established to explore the differences in the main chemical components and content of Mori Cortex with(mulberry root bark) and without(Mori Cortex) the phellem layer from both qualitative and quantitative aspects. The HPLC-ESI-MS method was used for quality analysis in positive and negative ion modes, and 33 compounds were identified in mulberry root bark, 22 compounds in Mori Cortex, and 26 compounds in phellem layer; mulberry root bark and Mori Cortex shared 22 components, and mulberry root bark has 11 unique compounds; Mori Cortex and its phellem layer shared 15 components, while Mori Cortex has 7 unique compounds. HPLC method was used to simultaneously determine 7 major constituents, including mulberroside A, chlorogenic acid, dihydromorin, oxyresveratrol, moracin O, kuwanon G, and kuwanon H, and the developed method showed good linearity(r>0.998 9) within the concentration range and the recoveries varied from 99.88% to 103.0%, and the RSD was 1.7%-2.9%. The HPLC results showed that the contents of the 7 compounds have great differences in 13 batches samples, compared with mulberry root bark, the contents of mulberroside A, chlorogenic acid, dihydromorin and moracin O of Mori Cortex were increased, while the contents of oxyresveratrol, kuwanon G and kuwanon H were decreased after peeling process. These results can provide a basis for the rationality and quality control of Mori Cortex required to remove the phellem layer.

Comparative Transcriptome Analysis of Key Reductase Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves and Cloning, Prokaryotic Expression, and Functional Analysis of MaSDR1 and MaSDR2.

The alkaloid 1-deoxynojirimycin (DNJ) is the main bioactive ingredient in the hypoglycemic action of mulberry leaves (Morus alba L.). Our previous research clarified the upstream pathway from lysine to Δ1-piperideine in the biosynthesis of DNJ in mulberry leaves, but the pathway and related reductase genes from Δ1-piperideine to piperidine are still unclear. Here, a comparative transcriptome was used to analyze the transcriptome data of two samples (July and November) of mulberry leaves with significant differences in the content of DNJ and screen-related reductase genes. Results showed that expression levels of MaSDR1 and MaSDR2 were significantly and positively correlated with the content of DNJ (P < 0.05) in different seasons. MaSDR1 (GenBank accession no. MT989445) and MaSDR2 (GenBank accession no. MT989446) were successfully cloned and used for prokaryotic expression and functional analysis in vitro. MaSDR1 and MaSDR2 could catalyze the reaction of Δ1-piperideine with the coenzyme NADPH to generate piperidine. The kinetic parameters of MaSDR1 and MaSDR2 indicated that MaSDR2 had a higher binding ability to Δ1-piperideine than MaSDR1. This study provided insights into the biosynthesis of DNJ in mulberry leaves.

Transcriptomic analysis of key genes involved in chlorogenic acid biosynthetic pathway and characterization of MaHCT from Morus alba L.

Mulberry leaves (Morus alba L.) are of high medicinal value in traditional Chinese medicine with chlorogenic acid (CGA) as its major biologically active constituent. Mulberry leaves require that they be harvested after frost; previous studies have shown CGA accumulation significantly increased after frost. However, the molecular mechanism of how frost changes the CGA content in mulberry leaves is unclear. Additionally, the mechanism of CGA biosynthesis and key genes in mulberry leaves are not well-understood. In this study, transcriptome sequencing was performed on two mulberry leaf samples with different CGA contents (before and after frost). Fifty-eight genes were annotated in the CGA biosynthetic pathway. Compared to those in pre-frost mulberry leaves, 12 and 5 genes were upregulated and downregulated, respectively, in post-frost leaves. Correlation analysis showed that the expression levels of four genes were significantly positively correlated with CGA content, including those encoding phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), and coumaroyl quinate/shikimate 3'-hydroxylase, and may be key genes in the CGA biosynthetic pathway. We cloned MaHCT4 (GenBank accession no. MH476577) from mulberry leaves. Multiple sequence alignment suggested that MaHCT4 contains the conserved domains HXXXD and DFGWG. Enzymatic assays indicated that MaHCT4 catalyzes the formation of p-coumaroyl shikimic acid, p-coumaroyl quinic acid, and CGA. The Km values of quinic acid and shikimic acid were 10 ±â€¯1.0 and 31 ±â€¯1.7 µM, respectively, suggesting that MaHCT4 favored quinic acid over shikimic acid as its acyl acceptor. Using quinic acid as an acyl acceptor, MaHCT4 showed a preference for p-coumaroyl-CoA over caffeoyl-CoA. Our results provide insight into the molecular mechanism of how frost alters the CGA content and roles of key genes involved in the CGA biosynthetic pathway in mulberry leaves.

Characterization and anti-tumor activity of a polysaccharide isolated from Dendrobium officinale grown in the Huoshan County.

BACKGROUND: Polysaccharides are carbohydrate chains composed of linked monosaccharide units. Accumulating studies report that polysaccharides isolated from Dendrobium officinale have a variety of functions. However, the composition and anti-tumor activity of D. officinale grown in the Huoshan area are largely unknown. METHODS: A polysaccharide (DOPA-1) was isolated from D. officinale by hot water extraction and ethanol precipitation, followed by purification via DEAE-cellulose and Sephadex G-100 chromatography. DOPA-1 was analyzed by infrared and nuclear magnetic resonance and then characterized by periodate oxidation and Smith degradation. The anti-tumor activity of DOPA-1 was then tested in HepG-2 cells. RESULTS: Our results show that DOPA-1 is mainly comprised of mannose, glucose, and galactose at a molar ratio of 1:0.42:0.27 and has an average molecular weight of 2.29 × 105 Da. Additionally, DOPA-1 inhibited HepG-2 cell growth in a dose-dependent manner. DOPA-1-treated HepG-2 cells also had increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential. Furthermore, apoptosis was observed in DOPA-1-treated HepG-2 cells along with Bcl-2 downregulation and Bax upregulation at the protein level. CONCLUSIONS: Our findings suggest that DOPA-1 induces apoptosis in tumor cells via altered mitochondrial function, ROS production, and altered apoptosis-related protein expression. This bioactive polysaccharide could, therefore, potentially be further developed as an anti-tumor adjuvant drug.

Transcriptional and proteolytic regulation of the toxin-antitoxin locus vapBC10 (ssr2962/slr1767) on the chromosome of Synechocystis sp. PCC 6803.

VapBC toxin-antitoxin (TA) systems are defined by the association of a PIN-domain toxin with a DNA-binding antitoxin, and are thought to play important physiological roles in bacteria and archaea. Recently, the PIN-associated gene pair PIN-COG2442 was proposed to encode VapBC-family TA system and found to be abundant in cyanobacteria. However, the features of these predicted TA loci remain under investigation. We here report characterization of the PIN-COG2442 locus vapBC10 (ssr2962/slr1767) on the chromosome of Synechocystis sp. PCC 6803. RT-PCR analysis revealed that the vapBC10 genes were co-transcribed under normal growth conditions. Ectopic expression of the PIN-domain protein VapC10 caused growth arrest of Escherichia coli that does not possess vapBC TA locus. Coincidentally, this growth-inhibition effect could be neutralized by either simultaneous or subsequent production of the COG2442-domain protein VapB10 through formation of the TA complex VapBC10 in vivo. In contrast to the transcription repression activity of the well-studied antitoxins, VapB10 positively auto-regulated the transcription of its own operon via specific binding to the promoter region. Furthermore, in vivo experiments in E. coli demonstrated that the Synechocystis protease ClpXP2s, rather than Lons, could cleave VapB10 and proteolytically activate the VapC10 toxicity. Our results show that the PIN-COG2442 locus vapBC10 encodes a functional VapBC TA system with an alternative mechanism for the transcriptional auto-regulation of its own operon.

Role of the all1549 (ana-rsh) gene, a relA/spoT homolog, of the Cyanobacterium Anabaena sp. PCC7120.

The role of a single relA/spoT homolog all1549 (designated hereafter as ana-rsh) of the cyanobacterium Anabaena sp. PCC7120 was investigated. The complementation test in Escherichia coli showed that the protein encoded by ana-rsh possesses guanosine tetraphosphate (p)ppGpp-synthase/hydrolase activity. Under laboratory growth conditions, a low level of ppGpp was detected in Anabaena sp. PCC7120 and the loss of ana-rsh was lethal. Amino acid starvation induced ppGpp accumulation to an appropriate level, and nitrogen deficiency did not alter the ppGpp concentration in Anabaena cells. These data suggest that ana-rsh is required for cell viability under normal growth conditions and involved in the (p)ppGpp-related stringent response to amino acid deprivation, but not related to heterocyst formation and nitrogen fixation of Anabaena sp. PCC7120.

Construction of secretory expression system suitable to express glucagon under the control of PL promoter.

It was reported that PL promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and [Des-His1] glucagon in E. coli BL21 herein. Expression studies showed these two peptides could be expressed and secreted into the culture medium. The expression yield of recombinant glucagon reached 3.46 mg/L/OD600 unit of cells in shake flask. The yield of [Des-His1] glucagon was found to be higher than that of glucagon. In addition, some factors involved in secretion were studied too.

[Secretory Expression of the Superactive [Lys(17,18),Glu(21)]-Glucagon in E. coli].

One of the most important findings in structure-function studies on glucagon by means of chemical synthesis is the discovery that [Lys(17,18),Glu(21)]-glucagon had higher biological activity than native glucagon. This mutant of glucagon was called superactive glucagon (SA-glucagon). In the present work, the possibility to obtain SA-glucagon by means of genetic engineering was studied. The gene of SA-glucagon (SAG) was obtained by PCR from a constructed recombinant glucagon plasmid, pAGluT. A secretory expression vector harboring SAG, pBLSG7, containing P(L) promoter and the gene of phoA signal peptide was constructed. In expression studies after transformation of pBLSG7 into E. coli BL21, it was found that the expression yield of SA-glucagon reached 3.65 mg/L(A(600)=1), about 19.5% of total proteins in the culture medium under shaken flask conditions. In addition, the influence of induction temperature and of E. coli strain on the expression yield of SA-glucagon was also studied.