Anti-Inflammatory Activity of Mulberry Leaf Flavonoids In Vitro and In Vivo.

Mulberry (Morus alba L.) is a flowering tree traditionally used in Chinese herbal medicine. Mulberry leaf flavonoids (MLFs) have been reported to exert important anti-inflammatory and antioxidant properties. The purpose of this study was to select the MLF with the best anti-inflammatory and antioxidative activities from MLFs eluted by different ethanol concentrations (30%, 50%, and 75%) and explore its pharmacological properties. Three types of MLFs inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 cells. All MLFs boosted the antioxidative capacity by decreasing the reactive oxygen species (ROS) production and the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and improving the metal ion chelating activity and reducing power. The results revealed that the MLFs eluted by 30% ethanol exhibited the best anti-inflammatory and antioxidative activities. A nontargeted metabolomic analysis was used to analyze 24 types of differential flavonoids between the MLFs. Quercetin, kaempferol, and their derivatives in 30%MLF were more abundant than the other two MLFs. Furthermore, we evaluated the pharmacological activities of 30%MLF in dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) mice. The 30%MLF could alleviate the clinical symptoms, reduce the secretion of inflammatory cytokines, and inhibit the activation of the inflammatory pathway in DSS-induced colitis mice. This study will provide valuable information for the development of MLFs eluted by 30% ethanol as a functional food.

Comparative Proteomic Analysis of Tolerant and Sensitive Varieties Reveals That Phenylpropanoid Biosynthesis Contributes to Salt Tolerance in Mulberry.

Mulberry, an important woody tree, has strong tolerance to environmental stresses, including salinity, drought, and heavy metal stress. However, the current research on mulberry resistance focuses mainly on the selection of resistant resources and the determination of physiological indicators. In order to clarify the molecular mechanism of salt tolerance in mulberry, the physiological changes and proteomic profiles were comprehensively analyzed in salt-tolerant (Jisang3) and salt-sensitive (Guisangyou12) mulberry varieties. After salt treatment, the malondialdehyde (MDA) content and proline content were significantly increased compared to control, and the MDA and proline content in G12 was significantly lower than in Jisang3 under salt stress. The calcium content was significantly reduced in the salt-sensitive mulberry varieties Guisangyou12 (G12), while sodium content was significantly increased in both mulberry varieties. Although the Jisang3 is salt-tolerant, salt stress caused more reductions of photosynthetic rate in Jisang3 than Guisangyou12. Using tandem mass tags (TMT)-based proteomics, the changes of mulberry proteome levels were analyzed in salt-tolerant and salt-sensitive mulberry varieties under salt stress. Combined with GO and KEGG databases, the differentially expressed proteins were significantly enriched in the GO terms of amino acid transport and metabolism and posttranslational modification, protein turnover up-classified in Guisangyou12 while down-classified in Jisang3. Through the comparison of proteomic level, we identified the phenylpropanoid biosynthesis may play an important role in salt tolerance of mulberry. We clarified the molecular mechanism of mulberry salt tolerance, which is of great significance for the selection of excellent candidate genes for saline-alkali soil management and mulberry stress resistance genetic engineering.

Integrated Transcriptomic and Un-Targeted Metabolomics Analysis Reveals Mulberry Fruit (Morus atropurpurea) in Response to Sclerotiniose Pathogen Ciboria shiraiana Infection.

Mulberry sclerotiniose caused by Ciboria shiraiana is a devastating disease of mulberry (Morus alba L.) fruit in Northwest China. At present, no disease-resistant varieties are used in production, as the molecular mechanisms of this disease are not well understood. In this study, to explore new prevention methods and provide direction for molecular breeding, transcriptomic sequencing and un-targeted metabolomics were performed on healthy (CK), early-stage diseased (HB1), and middle-stage diseased (HB2) mulberry fruits. Functional annotation, gene ontology, a Kyoto encyclopedia of genes and genomes (KEGG) analysis, and a Mapman analysis of the differentially expressed genes revealed differential regulation of genes related to plant hormone signal transduction, transcription factors, and phenylpropanoid biosynthesis. A correspondence between the transcript pattern and metabolite profile was observed in the phenylpropanoid biosynthesis pathway. It should be noted that the log2 ratio of eugenol (isoeugenol) in HB1 and HB2 are 85 times and 23 times higher than CK, respectively. Our study shows that phenylpropanoid biosynthesis may play an essential role in response to sclerotiniose pathogen infection and eugenol(isoeugenol) enrichment in mulberry fruit, which may provide a novel method for mulberry sclerotiniose control.

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori.

Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus.

Mulberry leaves supplementation alters lipid metabolism and promotes fatty acid ß oxidation in growing mutton sheep.

Mulberry leaves (MLs) are an unconventional feed with fiber and various active ingredients, and are acknowledged as likely to regulate lipid metabolism, while the molecular mechanism remains undefined. Therefore, our objective was to define the role of MLs on the overall lipid metabolism. We conducted a feeding experiment of three groups on growing mutton sheep fed with dried mulberry leaves (DMLs), with fermented mulberry leaves (FMLs), or without MLs (as control). Analyses of transcriptome and widely target lipids demonstrated the addition of MLs triggered big perturbations in genes and metabolites related to glycerolipid, phospholipid, ether lipid, and sphingolipid metabolism. Additionally, the variations of the above lipids in the treatment of MLs possibly facilitate immunity enhancement of growing mutton sheep via the activation of complement and coagulation cascades. Furthermore, treatments with MLs could expedite proceedings of lipid degradation and fatty acid ß oxidation in mitochondria, thereby to achieve the effect of lipid reduction. Besides, added DMLs also fuel fatty acid ß-oxidation in peroxisomes and own much stronger lipolysis than added FMLs, possibly attributed to high fiber content in DMLs. These findings establish the novel lipid-lowering role and immune protection of MLs, which lays the foundation for the medicinal application of MLs.

Mulberry leaves (MLs) are rich in a wide variety of active ingredients and are also a kind of traditional Chinese medicine with the same origin as medicine and food. Previous studies have found that MLs may regulate lipid metabolism. But the exact mechanism remains unclear. Our study reveals that ML supplement not only alters lipid metabolism including glycerol phospholipid, ether lipid as well as sphingolipid metabolism, which may help to improve immunity but also promote fatty acid degradation as well as ß oxidation to achieve the effect of fat reduction.

Metabolomic and transcriptomic analysis of flavonoids biosynthesis mechanisms in mulberry fruit (Hongguo 2) under exogenous hormone treatments.

The mulberry fruit is prized for its superior nutrition value and abundant color due to its high flavone content. To enhance comprehension of flavone biogenesis induced by external hormones, we sprayed exogenous ethylene (ETH), indoleacetic acid (IAA) and spermine (SPM) on mulberry fruit (Hongguo 2) during its color-changed period. The levels of anthocyanin, titratable acid, soluble sugar and endogenous hormones were determined after hormone treatment, integrated transcriptome and metabolome analysis were performed for mechanism exploration. Our results indicated that exogenous ETH, SPM, and IAA play important roles in mulberry ripening, including acid reduction, sugar increase and flavonoid synthesis.

Quantitative Proteomics and Functional Characterization Reveal That Glutathione Peroxidases Act as Important Antioxidant Regulators in Mulberry Response to Drought Stress.

Mulberry (Morus alba L.) has been an economically important food crop for the domesticated silkworm, Bombyx mori, in China for more than 5000 years. However, little is known about the mechanism underlying mulberry response to environmental stress. In this study, quantitative proteomics was applied to elucidate the molecular mechanism of drought response in mulberry. A total of 604 differentially expressed proteins (DEPs) were identified via LC-MS/MS. The proteomic profiles associated with antioxidant enzymes, especially five glutathione peroxidase (GPX) isoforms, as a scavenger of reactive oxygen species (ROS), were systematically increased in the drought-stressed mulberry. This was further confirmed by gene expression and enzymatic activity. Furthermore, overexpression of the GPX isoforms led to enhancements in both antioxidant system and ROS-scavenging capacity, and greater tolerance to drought stress in transgenic plants. Taken together, these results indicated that GPX-based antioxidant enzymes play an important role in modulating mulberry response to drought stress, and higher levels of GPX can improve drought tolerance through enhancing the capacity of the antioxidant system for ROS scavenging.

Transcriptomics and metabolomics analysis reveal the anti-oxidation and immune boosting effects of mulberry leaves in growing mutton sheep.

Introduction: Currently, the anti-oxidation of active ingredients in mulberry leaves (MLs) and their forage utilization is receiving increasing attention. Here, we propose that MLs supplementation improves oxidative resistance and immunity. Methods: We conducted a trial including three groups of growing mutton sheep, each receiving fermented mulberry leaves (FMLs) feeding, dried mulberry leaves (DMLs) feeding or normal control feeding without MLs. Results: Transcriptomic and metabolomic analyses revealed that promoting anti-oxidation and enhancing disease resistance of MLs is attributed to improved tryptophan metabolic pathways and reduced peroxidation of polyunsaturated fatty acids (PUFAs). Furthermore, immunity was markedly increased after FMLs treatment by regulating glycolysis and mannose-6-phosphate pathways. Additionally, there was better average daily gain in the MLs treatment groups. Conclusion: These findings provide new insights for understanding the beneficial effects of MLs in animal husbandry and provide a theoretical support for extensive application of MLs in improving nutrition and health care values.

Optimized Ensiling Conditions and Microbial Community in Mulberry Leaves Silage With Inoculants.

Mulberry leaves (ML) are a promising alternative fodder source due to their high protein content and the abundance of active components. A test of three inoculants in various combinations revealed that high-quality ML silage was produced at an inoculum ratio of 1:1:0 (50% Saccharomyces cerevisiae, 50% Lactobacillus plantarum, and 0% Bacillus subtilis). Using dry matter (DM) loss, pH, ammonia-N and amino acid contents, total antioxidant activity, and total flavonoids content to evaluate silage quality, this inoculant mixture was shown to produce high-quality silage within a range of inoculum size (5-15%), moisture contents (50-67%), ensiling temperatures (27-30°C), and ensiling duration (14-30 days). A third trial comparing silages produced after 30 days at 28°C and 50% moisture content revealed that silage E, prepared using an L. plantarum inoculant alone, displayed the lowest DM loss and pH, and low bacterial diversity, and it was dominated by Lactobacillus (88.6%), with low abundance of Enterobacter (6.17%). In contrast, silage B5, prepared with equal ratios of L. plantarum and S. cerevisiae, was dominated by Enterococcus (67.16%) and Lactobacillus (26.94%), with less marked yeast persistence, and reducing the DM content from 50 to 40% altered these relative abundances to 5.47 and 60.61, respectively. Control silages produced without an inoculant had the highest pH and ammonia-N content (indicative of poor quality), had the lowest antioxidant activity, had higher bacterial diversity, and were dominated by Carnobacterium (74.28%) and Enterococcus (17.3%). In summary, ensiling of ML conditions with proper inoculants yielded high-quality silage with a favorable microbial community composition.

Secondary Metabolism and Hormone Response Reveal the Molecular Mechanism of Triploid Mulberry (Morus Alba L.) Trees Against Drought.

The improvement of a plant's tolerance to drought is a major endeavor in agriculture. Polyploid plants often exhibit enhanced stress tolerance relative to their diploid progenitor, but the matching stress tolerance is still little understood. Own-rooted stem cuttings of mulberry (Morus alba L.) cultivar Shinichinose (2n = 2x = 28) and Shaansang-305 (2n = 3x = 42) were used in this study, of which the latter (triploid) has more production and application purposes. The responses of triploid Shaansang-305 and diploid progenitor ShinIchinose under drought stress were compared through an investigation of their physiological traits, RNA-seq, and secondary metabolome analysis. The results showed that the triploid exhibited an augmented abscisic acid (ABA) content and a better stress tolerance phenotype under severe drought stress. Further, in the triploid plant some genes (TSPO, NCED3, and LOC21398866) and ATG gene related to ABA signaling showed significantly upregulated expression. Interestingly, the triploid accumulated higher levels of RWC and SOD activity, as well as more wax on the leaf surface, but with less reductive flavonoid than in diploid. Our results suggest triploid plants may better adapt to with drought events. Furthermore, the flavonoid metabolism involved in drought resistance identified here may be of great value to medicinal usage of mulberry. The findings presented here could have substantial implications for future studies of crop breeding.

MaCDSP32 From Mulberry Enhances Resilience Post-drought by Regulating Antioxidant Activity and the Osmotic Content in Transgenic Tobacco.

Desiccation tolerance is a complex phenomenon that depends on the regulated expression of numerous genes during dehydration and subsequent rehydration. Our previous study identified a chloroplast drought-induced stress protein (MaCDSP32) in mulberry, a thioredoxin (Trx) that is upregulated under drought conditions and is likely to confer drought tolerance to transgenic plants. Mulberry (Morus spp.) is an ecologically and economically important perennial woody plant that is widely used in forest management to combat desertification. However, its stress tolerance physiology is not well understood. In this study, the functions of MaCDSP32 gene were investigated. The expression of MaCDSP32 exhibited a circadian rhythm and was induced by mild and severe water deficits. Under abiotic stress, MaCDSP32-overexpressing plants exhibited increased stress sensitivity with lower water retention capacity and more severe lipid peroxidation than the wild-type (WT) plants. Furthermore, the activity of superoxide dismutase (SOD), the contents of proline and soluble sugars and the expression of stress-related transcription factors were lower in the MaCDSP32-overexpressing plants than in the WT plants. However, the MaCDSP32-overexpressing lines exhibited stronger recovery capability after rewatering post-drought. Moreover, the SOD enzyme activity, proline content, and soluble sugar content were higher in the transgenic plants after rewatering than in the WT plants. The production of the reactive oxygen species (ROS) H2O2 and O2 - was significantly lower in the transgenic plants than in the WT plants. In addition, under abiotic stress, the MaCDSP32-overexpressing lines exhibited improved seed germination and seedling growth, these effects were regulated by a positive redox reaction involving MaCDSP32 and one of its targets. In summary, this study indicated that MaCDSP32 from mulberry regulates plant drought tolerance and ROS homeostasis mainly by controlling SOD enzyme activity and proline and soluble sugar concentrations and that this control might trigger the stress response during seed germination and plant growth. Overall, MaCDSP32 exerts pleiotropic effects on the stress response and stress recovery in plants.

Chromosome-Level Reference Genome and Population Genomic Analysis Provide Insights into the Evolution and Improvement of Domesticated Mulberry (Morus alba).

Mulberry (Morus spp.) is the sole plant consumed by the domesticated silkworm. However, the genome of domesticated mulberry has not yet been sequenced, and the ploidy level of this species remains unclear. Here, we report a high-quality, chromosome-level domesticated mulberry (Morus alba) genome. Analysis of genomic data and karyotype analyses confirmed that M. alba is a diploid with 28 chromosomes (2n = 2x = 28). Population genomic analysis based on resequencing of 134 mulberry accessions classified domesticated mulberry into three geographical groups, namely, Taihu Basin of southeastern China (Hu mulberry), northern and southwestern China, and Japan. Hu mulberry had the lowest nucleotide diversity among these accessions and demonstrated obvious signatures of selection associated with environmental adaptation. Further phylogenetic analysis supports a previous proposal that multiple domesticated mulberry accessions previously classified as different species actually belong to one species. This study expands our understanding of genome evolution of the genus Morus and population structure of domesticated mulberry, which would facilitate mulberry breeding and improvement.

[The study of multiple RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses].

OBJECTIVE: To establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses. METHODS: Obtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance. RESULTS: Successfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity. CONCLUSION: Successfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.

[Expression and analysis of recombinant chicken IL-18 in Pichia pastoris.].

AIM: Expression and analysis of recombinant chicken IL-18 in Pichia pastoris. METHODS: Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR, and was subcloned into Pichia pastoris expression vector pPICZalphaA to construct the recombinant plasmid pPICZalphaA-ChIL-18. After identified by restriction enzymes digestion analysis, PCR and DNA sequencing, the recombinant plasmid was transformed into Pichia pastoris X-33.Then choosing the multi-copy recombinant strains to be induced for expression.Then the bioactivity of rchIL-18 was analysed by Western blot, ELISA and MTT after purified by Sephadex G-100 column. RESULTS: The chicken IL-18 with the immunogenicity was secreted by Pichia pastoris. It could induce T lymphocytes proliferation and secreting IFN-gamma in vitro. CONCLUSION: The chicken IL-18 with obvious biological activity is secreted by Pichia pastoris X-33.