A chromosome-level genome assembly for Dracaena cochinchinensis reveals the molecular basis of its longevity and formation of dragon's blood.

Dracaena, a remarkably long-lived and slowly maturing species of plant, is world famous for its ability to produce dragon's blood, a precious traditional medicine used by different cultures since ancient times. However, there is no detailed and high-quality genome available for this species at present; thus, the molecular mechanisms that underlie its important traits are largely unknown. These factors seriously limit the protection and regeneration of this rare and endangered plant resource. Here, we sequenced and assembled the genome of Dracaena cochinchinensis at the chromosome level. The D. cochinchinensis genome covers 1.21 Gb with a scaffold N50 of 50.06 Mb and encodes 31 619 predicted protein-coding genes. Analysis showed that D. cochinchinensis has undergone two whole-genome duplications and two bursts of long terminal repeat insertions. The expansion of two gene classes, cis-zeatin O-glucosyltransferase and small auxin upregulated RNA, were found to account for its longevity and slow growth. Two transcription factors (bHLH and MYB) were found to be core regulators of the flavonoid biosynthesis pathway, and reactive oxygen species were identified as the specific signaling molecules responsible for the injury-induced formation of dragon's blood. Our study provides high-quality genomic information relating to D. cochinchinensis and significant insight into the molecular mechanisms responsible for its longevity and formation of dragon's blood. These findings will facilitate resource protection and sustainable utilization of Dracaena.

Comparative transcriptome analysis reveals different defence responses during the early stage of wounding stress in Chi-Nan germplasm and ordinary Aquilaria sinensis.

BACKGROUND: Agarwood is a valuable Chinese medicinal herb and spice that is produced from wounded Aquilaria spp., is widely used in Southeast Asia and is highly traded on the market. The lack of highly responsive Aquilaria lines has seriously restricted agarwood yield and the development of its industry. In this article, a comparative transcriptome analysis was carried out between ordinary A. sinensis and Chi-Nan germplasm, which is a kind of A. sinensis tree with high agarwood-producing capacity in response to wounding stress, to elucidate the molecular mechanism underlying wounding stress in different A. sinensis germplasm resources and to help identify and breed high agarwood-producing strains. RESULTS: A total of 2427 and 1153 differentially expressed genes (DEGs) were detected in wounded ordinary A. sinensis and Chi-Nan germplasm compared with the control groups, respectively. KEGG enrichment analysis revealed that genes participating in starch metabolism, secondary metabolism and plant hormone signal transduction might play major roles in the early regulation of wound stress. 86 DEGs related to oxygen metabolism, JA pathway and sesquiterpene biosynthesis were identified. The majority of the expression of these genes was differentially induced between two germplasm resources under wounding stress. 13 candidate genes related to defence and sesquiterpene biosynthesis were obtained by WGCNA. Furthermore, the expression pattern of genes were verified by qRT-PCR. The candidate genes expression levels were higher in Chi-Nan germplasm than that in ordinary A. sinensis during early stage of wounding stress, which may play important roles in regulating high agarwood-producing capacity in Chi-Nan germplasm. CONCLUSIONS: Compared with A. sinensis, Chi-Nan germplasm invoked different biological processes in response to wounding stress. The genes related to defence signals and sesquiterepene biosynthesis pathway were induced to expression differentially between two germplasm resources. A total of 13 candidate genes were identified, which may correlate with high agarwood-producting capacity in Chi-Nan germplasm during the early stage of wounding stress. These genes will contribute to the development of functional molecular markers and the rapid breeding highly of responsive Aquilaria lines.

Genetic relationship and source species identification of 58 Qi-Nan germplasms of Aquilaria species in China that easily form agarwood.

Recently, Qi-Nan germplasm, the germplasm of Aquilaria species that easily forms agarwood, has been widely cultivated in Guangdong and Hainan Provinces in China. Since the morphological characteristics of Qi-Nan germplasm are similar to those of Aquilaria species and germplasm is bred by grafting, it is difficult to determine the source species of this germplasm by traditional taxonomic characteristics. In this study, we performed a DNA barcoding analysis of 58 major Qi-Nan germplasms as well as Aquilaria sinensis, A. yunnanensis, A. crassna, A. malaccensis and A. hirta with 5 primers (nuclear gene internal transcribed spacer 2 (ITS2) and the chloroplast genes matK, trnH-psbA, rbcL and trnL-trnF). This field survey in the Qi-Nan germplasm plantations in Guangdong and Hainan Provinces aimed to accurately identify the source species of Qi-Nan germplasm. According to the results, ITS2 and matK showed the most variability and the highest divergence at all genetic distances. This ITS2+matK combination, screened for with TaxonDNA analysis, showed the highest success rate in species identification of the Qi-Nan germplasm. Clustering in the phylogenetic trees constructed with Bayesian inference and maximum likelihood indicated that the Qi-Nan germplasm was most closely related to A. sinensis and more distantly related to A. yunnanensis, A. crassna, A. malaccensis and A. hirta. Therefore, this study determined that the source species of the Qi-Nan germplasm is A. sinensis.

Effects of Different Sources of Calcium in the Diet on Growth Performance, Blood Metabolic Parameters, and Intestinal Bacterial Community and Function of Weaned Piglets.

Despite a well-documented effect of calcium on the piglet's intestinal microbiota composition, it is less known about changes in microbial function or the effect of different sources of calcium. The experiment was designed to study the effects of dietary calcium from different sources on production, immune indexes, antioxidant capacity, serum biochemical indexes, and intestinal microflora of weaning piglets. A total of 1,000 piglets were randomly assigned to five groups (10 replicate pens per treatment with 20 pigs per pen) and fed diets supplemented with calcium carbonate, calcium citrate, multiple calcium, organic trace minerals, and different concentrations of acidifier. The results showed that the replacement of calcium carbonate with calcium citrate and multiple calcium had almost no significant difference in the growth performance of pigs compared with the control group, and only the diet of multiple calcium dramatically decreased the average daily feed intake (ADFI) compared to the calcium citrate diet on days 15-28 (p < 0.05). The five groups did not change the content of MDA, SOD, and GSH-Px (p > 0.10). A similar situation occurs in the immune function of the blood. There was no significant effect in immune indexes (IgA, IgG, and IgM) among different treatments after weaning at 6 weeks for piglets (p > 0.10). The 16S rRNA sequencing of ileal and cecal microbiota revealed that only the relative abundance of Actinobacteriota at the phyla level was significantly greater in the ileum of the A group compared to the other treatments (p < 0.05). There was a clear effect on seven bacteria in the top 30 genera of ileum and cecum for five groups (p < 0.05). The result of PICRUSt predicted that the intestinal microbe was mainly involved in carbohydrate and amino acid metabolism, membrane transport, and metabolism of cofactors and vitamins. Besides, adding calcium citrate to a weaned piglet diet is better than other choices from the third week to the fourth week. In conclusion, diets with different calcium sources changed ADFI and some intestinal microbial composition of weaned piglets but had little effect on intestinal microbial function.

Programmed cell death might involve in progress of wounding induced agarwood formation in stems of Aquilaria sinensis.

Agarwood, a non-timber fragrant wood, is derived from wounded Aquilaria trees (Thymelaeaceae) and is widely used in traditional medicine, incense and perfume. Agarwood-like substances and programmed cell death (PCD) can be induced by wounding signals in the suspension cells and aerial roots of Aquilaria sinensis. In this study, the dynamic process of wound-induced agarwood formation in stems of A. sinensis was observed, and the occurrence of PCD was synchronously detected using techniques such as 4',6-diamidino-2-phenylindole and dUTP nick-end labeling staining. The results showed that the wounding was a induce signal for agarwood resin formation, meanwhile might induce PCD. Interxylary phloem and xylem ray were the main sites of agarwood resin formation and PCD occurrence. There might be a relationship between the spatiotemporal pattern of PCD and agarwood resin formation: more severe PCD corresponded to a higher rate of resin formation but a shorter resin formation time; conversely, slower PCD progression corresponded to a lower rate of resin formation but a longer resin formation time. Our findings are the first to demonstrate that PCD might occur in the process of wound-induced agarwood formation at the tree level, and the spatiotemporal pattern is closely related to the formation of agarwood resin. This study provides valuable insight for further studies on the relationship between PCD and agarwood formation. HIGHLIGHTS: Programmed cell death (PCD) might occur in the process of wound-induced agarwood formation at the tree level. Interxylary phloem and xylem ray were the main sites of agarwood resin formation and PCD occurrence. Spatiotemporal pattern of PCD might have a strong impact on agarwood resin formation.

Exploration of the Mechanism of the Control of Coccidiosis in Chickens Based on Network Pharmacology and Molecular Docking With the Addition of Modified Gegen Qinlian Decoction.

Gegen Qinlian Decoction is a long-established Chinese herbal compound for the treatment of diarrhea and dysentery, while Magnolia officinalis has been demonstrated to have some anthelmintic activity. The preliminary screening of this study showed that the addition of Modified Gegen Qinlian Decoction has some effective on the prevention and treatment of coccidiosis in chickens. However, the mechanism of its treatment of chicken coccidiosis is not clear. The network pharmacology study was based on the screening of chemical components and related targets from TCMSP and PharmMapper server databases. Genes related to chicken coccidiosis were obtained from the SRA database, and those genes that intersected with the target genes of Modified Gegen Qinlian Decoction were screened. By exploring the target interactions through the String system and enrichment analysis by the Metascape system, the mechanism of action of Modified Gegen Qinlian Decoction in chicken coccidiosis was identified. Using real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the mRNA levels of the relevant factors in chicken coccidiosis, molecular docking was used to reveal the extent of binding of the key target genes predicted in the network pharmacology by the action of Modified Gegen Qinlian Decoction. Compound and target screening suggested that the 99 chemical targets of Modified Gegen Qinlian Decoction were involved in chicken coccidiosis, and the enrichment results of KEGG pathway suggested that Modified Gegen Qinlian Decoction was significantly associated with PI3K/AKT signaling pathway in chicken coccidiosis. The Hubba gene module in Cytoscape_v3.7.1 software was used to analyze the network topology to obtain the Hubba gene SRC, STAT3, and PPARG, etc. The molecular docking results showed that SRC, STAT3, and PPARG were key targets in the treatment of coccidiosis in chickens by Modified Gegen Qinlian Decoction, which was in agreement with the RT-qPCR results. Through network pharmacology, molecular docking and in vitro experiments, it was confirmed that Modified Gegen Qinlian Decoction fights against chicken coccidiosis through key targets such as SRC, STAT3, and PPARG.

Dietary supplementation with Piper sarmentosum extract on gut health of chickens infected with Eimeria tenella.

To explore the potential alternative of anti-coccidials, we investigated the therapeutic efficacy of dietary Piper sarmentosum extract (PSE) on induced coccidia infection in chickens. A total of 96-day-old chickens were randomly distributed to 1 of 3 treatment groups, including (1) control negative untreated uninfected (CN), (2) control positive untreated infected (CP), and (3) Piper sarmentosum (P. sarmentosum) extract-treated infected group (PSE). Our results demonstrated that E. tenella challenged untreated group showed a reduction (P < 0.05) in post-infection (PI) body weight compared to control negative group. However, supplementation of P. sarmentosum extract had no significant effects on body weight and cecal lesions compared with control positive group. Infected chickens fed PSE diet decreased (P < 0.05) the bloody diarrhea scores and oocyst shedding (during the day 5 to 8 post-infection) than that of CP chickens. E. tenella-challenged chickens upregulated (P < 0.05) the mRNA expression of IL-8 and Bcl-2 compared to PSE chickens, while IFN-γ compared to CN chickens. On the other hand, treatment of P. sarmentosum extract tended to increase (P < 0.05) the transcription patterns of IL-4, IL-10, CLDN 1, SOD 1, and Bax with the comparison of control positive group; however, there were no significant effects on IL-8, ZO 1, and CAT expression between the PSE and CP groups. Collectively, these findings elaborated that dietary P. sarmentosum extract exhibit potential anti-coccidial effects in controlling the coccidia infection in chickens.

Main active components of Jiawei Gegen Qinlian decoction protects against ulcerative colitis under different dietary environments in a gut microbiota-dependent manner.

As an effective drug against acute enteritis diarrhea, Gegen Qinlian decoction (GQD) has a history of 2000 years. However, the potential molecular mechanism through which GQD could protect intestinal barrier from ulcerative colitis (UC) still remains undefined. As an important part of the homeostasis of the colon, gut microbiota is closely related to the dynamic evolution of the surrounding environment and the adjustment of dietary structure. At present, the effectiveness and mechanism of Jiawei Gegen Qinlian decoction against UC in different dietary environments are not clear. Here, the main active components of Jiawei Gegen Qinlian Decoction (PBM), were selected to construct a reasonable and effective compound scheme. We adopted "5% dextran sulfate sodium (DSS)" and "high temperature and humidity + high sugar and high fat + alcohol + 5%DSS" to induce UC rat models in general environment and UC rat models in Lingnan area, respectively. Then, we examined the therapeutic effects of PBM (89.96 mg/kg and 179.92 mg/kg) on two kinds of UC rats. The role of gut microbiota in the anti-UC effect of PBM was identified by intestinal flora consumption and fecal microbiota transplantation (FMT) experiments. Subsequently, we monitored the alterations of gut microbiota and fecal metabolism in the rat colon by 16Sr DNA technique and targeted metabonomics, respectively. The colon inflammation of the PBM-treated and the FMT-treated rats both showed significant relief, as evidenced by a reduction in body weight loss, bloody stool, diarrhea, disease activity index (DAI) score, shortening of colon length as well as decreased colon histology damage. Interestingly enough, the depletion of intestinal flora took away the protective effect of PBM, confirming the importance of intestinal flora in the anti-UC effect of PBM. Then our findings suggested that PBM could not only regulate the gut microbiota by increasing Akkermansia and Romboutsia but also decrease Escherichia-Shigella. More importantly, PBM could increase the production of propionate and total short-chain fatty acids (SCFAs) in colitis rats, regulate medium and long chain fatty acids (M-LCFAs), maintain bile acids (BAs) homeostasis, and regulate amino acids (AAs) metabolism. The transformation of intestinal environment might be related to the upregulation of anti-inflammation, anti-oxidation and tight junction protein expression in colonic mucosa. In summary, PBM showed potential for anti-UC activity through gut microbiota dependence and was expected to be a complementary and alternative medicine herb therapy.

Effects of Baihui electroacupuncture in a rat model of depression.

BACKGROUND: This study aimed to investigate the effect of electroacupuncture (EA) on behavior in a rat model of chronic unpredictable mild stress (CUMS) and to explore the underlying molecular mechanisms. METHODS: A total of 45 adult male Sprague-Dawley rats were randomly divided into three groups: the control, CUMS, and CUMS plus EA groups. Rats in the CUMS and EA groups were subjected to a 3-week CUMS condition, while rats in the EA group received EA at the Baihui (GV 20) acupoint (2 Hz, 0.6 mA) for 10 min once daily before being subjected to the CUMS condition. The sucrose preference test (SPT) was used as a measure to infer activation of the pleasure response to depression-like behaviour. After the behavioral test, 5-bromodeoxyuridine (BrdU) was intraperitoneally injected (100 mg/kg) and brain samples were collected 24 h later for the detection of hippocampal BrdU. Cell proliferation was determined according to the proportion of BrdU-positive cells. Brain-derived neurotrophic factor (BDNF) expression was detected. RESULTS: The severity of anhedonia, BDNF+ cells, and BrdU+ neurons in DG significantly decreased in CUMS rats, and was accompanied by a reduced BDNF and BrdU+ expression (P<0.05). After EA, the low levels of BDNF+ cells and BrdU+ expression and the depression-like behavior increased markedly (P<0.05). CONCLUSIONS: EA contributes to neuroprotection against CUMS by enhancing BDNF expression and improving hippocampal neurogenesis.

WRKY44 represses expression of the wound-induced sesquiterpene biosynthetic gene ASS1 in Aquilaria sinensis.

Agarwood is derived from wounds in Aquilaria trees and is widely used in traditional medicine, incense, and perfume. Sesquiterpenes are one of the main active components in agarwood and are known to be induced by wounding or injury; However, the molecular mechanisms by which wounding leads to sesquiterpene formation remain largely unknown. Agarwood sesquiterpene synthase 1 (ASS1) is one of key enzymes responsible for the biosynthesis of sesquiterpenes and is a crucial jasmonate (JA)-responsive wound-inducible synthase. However, it is not known why ASS1 is not expressed in healthy trees and how its expression is induced as a result of wounding. Here, we report that ASS1 is a wound-induced gene with a promoter in which a 242-bp region (-973 to -731bp) is identified as the core sequence for responding to wound signals. AsWRKY44 binds directly to this region and represses ASS1 promoter activity. Down-regulation or disruption of AsWRKY44 can relieve the inhibition and activate ASS1 expression. In addition, AsWRKY44 is degraded and the expression of ASS1 is significantly up-regulated in response to exogenous application of methyl jasmonate. Thus, AsWRKY44 is a crucial negative regulator of wound-induced ASS1 transcription, and is central to the mechanism of sesquiterpene biosynthesis in agarwood.