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Radix Ginseng Rubra (RGR), a well-known edible-medicinal herb, has been widely used to attenuate heart failure (HF). However, the active ingredients and mechanism of RGR for attenuating HF have not been illuminated. Thus, the metabolic profile in vivo of RGR was investigated by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and further the compounds identified in serum and heart were employed to search for the potential active compounds and targets via network pharmacology and molecular docking. 41 chemical compounds were identified in RGR aqueous extract. 10 prototypes and 8 metabolites were identified or tentatively characterized in vivo. Additionally, two compounds with 51 corresponding targets had been speculated to improve HF, and the role of ginsenoside Rh4 (P8) in RGR for attenuating HF by interfering with MAP2K1 and SRC targets was verified by molecular docking. Therefore, the potential active ingredients and targets were expected to be the basis of RGR therapy for HF.

The flammability of epoxy resins and knowing how to achieve curing are particularly important factors during use. A novel approach for enhancing the fire resistance and reducing the smoke emission of epoxy resin during the curing process is suggested, which involves the utilization of a three-source integrated polymerization intumescent flame-retardant. In this study, the synthesis of poly 4,4-diaminodiphenylsulfone spirocyclic pentaerythritol bisphosphonate (PCS) is achieved through using solution polymerization, utilizing 4,4′-diaminodiphenylsulfone (DDS) and spirocyclic pentaerythritol bisphosphorate disphosphoryl chloride (SPDPC) as initial components. Following that, the EP underwent the inclusion of PCS to examine its resistance to heat, its ability to prevent flames, its effectiveness in reducing smoke and its curing effect. Compared to the unmodified epoxy resin, the addition of PCS can not only cure the epoxy resin, but also decompose before the epoxy resin and has a good carbonization effect. With the addition of 7 wt.% PCS, the LOI value can achieve 31.2% and successfully pass the UL-94 test with a V-0 rating. Moreover, the cone calorimeter experiment demonstrated a noteworthy decline of 59.7% in the maximum heat release rate (pHRR), 63.7% in overall heat release (THR), and 42.3% in total smoke generation (TSP). Based on the examination of TG-FTIR and SEM findings, there is ample evidence to suggest that PCS, functioning as a phosphorus-nitrogen intumescent flame-retardant that combines three origins, has the potential to exhibit a favorable flame-retardant impact in both its gas and condensed phases.

Graphene has garnered widespread attention, and its use is being explored for various electronic devices due to its exceptional material properties. However, the use of polymers (PMMA, photoresists, etc.) during graphene transfer and patterning processes inevitably leaves residues on graphene surface, which can decrease the performance and yield of graphene-based devices. This paper proposes a new transfer and patterning process that utilizes an Al intermediate layer to separate graphene from polymers. Through DFT calculations, the binding energy of graphene–Al was found to be only −0.48 eV, much lower than that of PMMA and photoresist with graphene, making it easier to remove Al from graphene. Subsequently, this was confirmed through XPS analysis. A morphological characterization demonstrated that the graphene patterns prepared using the Al intermediate layer process exhibited higher surface quality, with significantly reduced roughness. It is noteworthy that the devices obtained with the proposed method exhibited a notable enhancement in both consistency and sensitivity during electrical testing (increase of 67.14% in temperature sensitivity). The low-cost and pollution-free graphene-processing method proposed in this study will facilitate the further commercialization of graphene-based devices.

Background: Cuproptosis has been found as a novel cell death mode significantly associated with mitochondrial metabolism, which may be significantly associated with the occurrence and growth of tumors. LncRNAs take on critical significance in regulating the development of kidney renal clear cell carcinoma (KIRC), whereas the correlation between cuproptosis-related LncRNAs (CRLs) and KIRC is not clear at present. Therefore, this study built a prognosis signature based on CRLs, which can achieve accurate prediction of the outcome of KIRC patients.Methods: The TCGA database provided the expression profile information and relevant clinical information of KIRC patients. Univariate Cox, Lasso, and multivariate Cox were employed for building a risk signature based on CRLs. Kaplan-Meier (K-M) survival analysis and time-dependent receiver operating characteristic (ROC) curve were employed for the verification and evaluation of the reliability and accuracy of risk signature. Then, qRT-PCR analysis of risk LncRNAs was conducted. Finally, the possible effect of the developed risk signature on the microenvironment for tumor immunization was speculated in accordance with ssGSEA and ESTIMATE algorithms.Results: A prognosis signature composed of APCDD1L-DT, MINCR, AL161782.1, and AC026401.3 was built based on CRLs. As revealed by the results of the K-M survival study, the OS rate and progression-free survival rate of highrisk KIRC patients were lower than those of lowrisk KIRC patients, and the areas under ROC curves of 1, 3, and 5 years were 0.828, 0.780, and 0.794, separately. The results of the immune analysis showed that there were significant differences in the status of immunization and the microenvironment of tumor between groups at low-risk and at high-risk. The qRT-PCR results showed that the relative expression level of MINCR and APCDD1L-DT were higher in 786-O and 769-P tumor cells than in HK-2 cells, which were normal renal tubular epithelial cells.Conclusion: The developed risk signature takes on critical significance in the prediction of the prognosis of patients with KIRC, and it can bring a novel direction for immunotherapy and clinical drug treatment of KIRC. In addition, 4 identified risk LncRNAs (especially APCDD1L-DT and MINCR) can be novel targets for immunotherapy of KIRC patients.

Abstract Iris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of RT-qPCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and IgTUB and IgUBC showed stable expression in ‘2010200’. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

Licorice is well known for its ability to reduce the toxicity of the whole prescription in traditional Chinese medicine theory. However, honey-fired licorice (ZGC for short), which is made of licorice after being stir-fried with honey water, is more commonly used for clinical practice. The metabolism in vivo and detoxification-related compounds of ZGC have not been fully elucidated. In this work, the chemical constituents in ZGC and its metabolic profile in rats were both identified by high ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The network pharmacology was applied to predict the potential detoxifying ingredients of ZGC. As a result, a total of 115 chemical compounds were identified or tentatively characterized in ZGC aqueous extract, and 232 xenobiotics (70 prototypes and 162 metabolites) were identified in serum, heart, liver, kidneys, feces, and urine. Furthermore, 41 compounds absorbed in serum, heart, liver, and kidneys were employed for exploring the detoxification of ZGC by network pharmacology. Ultimately, 13 compounds (five prototypes including P5, P24, P30, P41 and P44, and 8 phase Ⅰ metabolites including M23, M47, M53, M93, M100, M106, M118, and M134) and nine targets were anticipated to be potential mediums regulating detoxification actions. The network pharmacology analysis had shown that the ZGC could detoxify mainly through regulating the related targets of cytochrome P450 and glutathione. In summary, this study would help reveal potential active ingredients in vivo for detoxification of ZGC and provided practical evidence for explaining the theory of traditional Chinese medicine with modern technology.

Zeolitic Imidazole Frameworks (ZIFs) are widely applied in nanomedicine for their high drug loading, suitable pore size, pH-responsive drug release, and so on. However, fast drug release during circulation, unexpected toxicity to mice major organs, undesirable long-term accumulation in the lung and even death currently hinder their in vivo biomedical applications. Herein, we report an amorphous ZIF-8 (aZIF-8) with high loading of 5-Fu through pressure-induced amorphization. This nano-system avoids early drug release during circulation and provides tumor microenvironment-responsive drug release with improved in vitro cell viability, and survival rate in in vivo evaluations as compared to ZIF-8. Furthermore, aZIF-8 shows longer blood circulation and lower lung accumulation than ZIF-8 at same injected doses. Less drug release during circulation, longer blood circulation, and better biocompatibility of aZIF-8/5-Fu significantly improves its therapeutic efficacy in ECA-109 tumor-bearing mouse, and result in 100% survival rate over 50 days after treatment. Therefore, aZIF-8 with favorable biocompatibility and long blood circulation is expected to be a promising nano-system for efficacious cancer therapy in vivo.

Esophageal squamous cell carcinoma (ESCC) is one of the most common and lethal malignant tumors. The incidence of malignant transformation of esophageal mucosa increases greatly due to long-term exposure to factors such as smoking, drinking, and poor eating habits. Furthermore, multiple primary tumors could occur synchronously or asynchronously in the upper aerodigestive tract, especially in the esophagus, adding difficulty to the treatment of ESCC. Genetic mutations are important during the malignant transformation from normal mucosa to esophageal cancer, but the underlying mechanism has not been fully elucidated. In this study, we used whole-exome sequencing (WES) to profile genetic variations in physiologically normal mucosa (PNM) and ESCC tumors, as well as PNM of non-ESCC subjects. We found significant differences in mutation frequencies of NOTCH1 and NOTCH2, copy number variations (CNVs) at both gene and chromosomal arm levels, and cancer-related HIPPO, WNT, and NRF2 signaling pathways between ESCC tumors and normal mucosa. Our analysis of both primary tumors and paired PNM in bifocal ESCC revealed three different primary tumor evolution modes, and the most common mode exhibited a complete genomic divergence in all the samples from the same patient. Furthermore, the mutation frequency of TP53 was significantly higher in ESCC cases than that in non-ESCC cases. Overall, our results provide important evidence for further elucidating the mechanisms of genetic mutations underlying the cause of ESCC.

White rust caused by Puccinia horiana is a destructive disease of chrysanthemum plants. To better understand the resistance mechanisms of composite species to this disease, the leaf cuticular traits, antioxidant and defensive enzymes activities of immune (Chrysanthemum makinoi var. wakasaense) and highly susceptible (Ajania shiwogiku var. kinokuniense) species were compared. Trichome density of two species was markedly different, negatively associated with plant resistance to P. horiana. Total wax load in C. makinoi var. wakasaense was two times more than that in A. shiwogiku var. kinokuniense. The wax composition in immune one was abundant in esters and primary alcohols. Superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), polyphenoloxidase (PPO, EC 1.14.18.1 or EC 1.10.3.2) and phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activities were investigated. In C. makinoi var. wakasaense, the activity of SOD and POD increased rapidly after inoculation, which might be non-host induced reactive oxygen species (ROS) activated antioxidant enzymes, however SOD and POD remained a low and steady level in the highly susceptible one after inoculation. Quick increase in PPO activities after inoculation was observed in both species, however it remained higher in C. makinoi var. wakasaense at the late period of inoculation. PAL in C. makinoi var. wakasaense was induced after pathogen inoculation, but not in A. shiwogiku var. kinokuniense, suggesting that these two enzymes might contribute to the resistance to P. horiana.

Abstract MYB transcription factors are widely involved in the development of and physiological processes in plants. Here, we isolated the chrysanthemum R2R3-MYB family transcription factor CmMYB15, a homologous gene of AtMYB15. It was demonstrated that CmMYB15 expression was induced by aphids and that CmMYB15 could bind to AC elements, which usually exist in the promoter of lignin biosynthesis genes. Overexpression of CmMYB15 in chrysanthemum enhanced the resistance of aphids. Additionally, the content of lignin and the expression of several lignin biosynthesis genes increased. In summary, the results indicate that CmMYB15 regulates lignin biosynthesis genes that enhance the resistance of chrysanthemum to aphids.

Abstract Zeolitic imidazolate frameworks (ZIFs) as smart drug delivery systems with microenvironment-triggered release have attracted much attention for tumor therapy. However, the exploration of ZIFs in biomedicine still encounters many issues, such as inconvenient surface modification, fast drug release during blood circulation, undesired damage to major organs, and severe in vivo toxicity. To address the above issues, we developed an Mn-ZIF-90 nanosystem functionalized with an originally designed active-targeting and pH-responsive magnetic resonance imaging (MRI) Y1 receptor ligand [Asn28, Pro30, Trp32]-NPY (25–36) for imaging-guided tumor therapy. After Y1 receptor ligand modification, the Mn-ZIF-90 nanosystem exhibited high drug loading, better blood circulation stability, and dual breast cancer cell membrane and mitochondria targetability, further favoring specific microenvironment-triggered tumor therapy. Meanwhile, this nanosystem showed promising T1-weighted magnetic resonance imaging contrast in vivo in the tumor sites. Especially, this nanosystem with fast clean-up had almost no obvious toxicity and no damage occurred to the major organs in mice. Therefore, this nanosystem shows potential for use in imaging-guided tumor therapy.

Ilex × attenuata ‘Fosteri’ is an important ornamental plant widely distributed in mid-southern China and south-eastern United States. In this study, we assembled the complete chloroplast (cp) genome of I. attenuata by high-throughput sequencing and bioinformatics. The full length of cp genome was 157,833 bp with 37.63% overall GC content, which contained two inverted repeats (IR) of 26,093 bp separated by a large single-copy (LSC) and a small single copy (SSC) of 87,188 bp and 18,459 bp, respectively. The cp genome contained 135 genes, including 88 protein-coding genes, 8 rRNA genes and 39 tRNA genes. Phylogenetic tree showed that the close relationship of three species of Ilex (I. attenuata, I. viridis and I. szechwanensis) in the Aquifoliaceae family.

Abstract Background Chrysanthemum is among the top ten traditional flowers in China, and one of the four major cut flowers in the world, but the growth of chrysanthemum is severely restricted by high temperatures which retard growth and cause defects in flowers. DREB (dehydration-responsive element-binding) transcription factors play important roles in the response to abiotic and biotic stresses. However, whether the DREB A-6 subgroup is involved in heat tolerance has not been reported conclusively. Result In the present study, CmDREB6 was cloned from chrysanthemum (Chrysanthemum morifolium) ‘Jinba’. CmDREB6, containing a typical AP2/ERF domain, was classed into the DREB A-6 subgroup and shared highest homology with Cichorium intybus L. CiDREB6 (73%). CmDREB6 was expressed at its highest levels in the leaf. The CmDREB6 protein localized to the nucleus. Based on the yeast one hybrid assay, CmDREB6 showed transcription activation activity in yeast, and the transcriptional activation domain was located in the 3 ‘end ranging from 230 to 289 amino acids residues. CmDREB6 overexpression enhanced the tolerance of chrysanthemum to heat. The survival rate of two transgenic lines was as high as 85%, 50%, respectively, in contrast to 3.8% of wild-type (WT). Over-expression of CmDREB6 promoted the expression of CmHsfA4, CmHSP90, and the active oxygen scavenging genes CmSOD and CmCAT. Conclusion In this study, DREB A-6 subgroup gene CmDREB6 was cloned from chrysanthemum ‘Jinba’. Overexpression of CmDREB6 enhanced heat tolerance of chrysanthemum by regulating genes involved in the heat shock response and ROS homeogenesis.

Abstract Background Staphylococcus aureus (S. aureus) internalization into bovine mammary epithelial cells (bMECs) is considered an important pathogenic mechanism for the establishment of mastitis. Given the interesting link between selenium (Se) status and mastitis, our objective was to prove that Se was essential to suppress pro-inflammatory mediators, in part, by modulation of Toll-like receptor2 (TLR2), nuclear factor kappaB (NF-κB) and mitogen activated protein kinase (MAPK) signal transduction pathway in bMECs. Results Results showed that Se (0~ 16 μM) did not affect the growth of bMECs. The mRNA expression of TLR2, Myeloid differentiation factor 88 (Myd88), Interleukin-1 receptor-associated kinase4 (Irak4), Interleukin-1 receptor-associated kinase1 (Irak1) and TNF receptor-associated factor6 (Traf6) in TLR2 signal pathway were increased or significantly increased by S. aureus. Se played an important role in regulating the genes expression of TLR2, Myd88, Traf6 but not in controlling the expression of Irak4 and Irak1. In addition, Se exerted strong inhibitory effects on the genes expression of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) induced by S. aureus. To further investigate the possible signaling mechanisms involved in the processes, we analyzed the role of MAPK and NF-κB signaling pathway in inflammation response in S. aureus-stimulated bMECs in vitro. Results showed that the phosphorylation of inhibitory kappaB alpha (IκBα), p65, p38 and extracellular regulated protein kinase (Erk) were significantly increased in S. aureus-stimulated bMECs. It indicated that S. aureus activated NF-κB and MAPK signaling pathway. We also examined the effects of Se on the phosphorylation of IκBα, p65, p38 and Erk in NF-κB and MAPK signaling pathway, which have well been proved to control the synthesis and release of pro-inflammatory mediators during inflammation. The findings are exciting, that pretreatment with Se (4, 8 μM) significantly suppressed the phosphorylation of IκBα, p65, p38 and Erk. Conclusions These results suggest that Se down-regulates inflammatory mediators TNF-α, IL-1β and IL-6 gene expressions via TLR2, NF-κB and MAPK signaling pathway in S. aureus-stimulated bMECs, which may be responsible for the anti-inflammatory effect of Se.

The family of B-box (BBX) transcription factors contains one or two B-BOX domains and sometimes also features a highly conserved CCT domain, which plays important roles in plant growth, development and stress response. Nevertheless, no systematic study of the BBX gene family in Iris germanica L. has been undertaken. In this study, a set of six BBX TF family genes from I. germanica was identified based on transcriptomic sequences, and clustered into three clades according to phylogenetic analysis. A transient expression analysis revealed that all six BBX proteins were localized in the nucleus. A yeast one-hybrid assay demonstrated that IgBBX3 has transactivational activity, while IgBBX1, IgBBX2, IgBBX4, and IgBBX5 have no transcriptional activation ability. The transcript abundance of IgBBXs in different tissues was divided into two major groups. The expression of IgBBX1, IgBBX2, IgBBX3 and IgBBX5 was higher in leaves, whereas IgBBX4 and IgBBX6 was higher in roots. The stress response patterns of six IgBBX were detected under phytohormone treatments and abiotic stresses. The results of this study lay the basis for further research on the functions of BBX gene family members in plant hormone and stress responses, which will promote their application in I. germanica breeding.

Developing superior properties of epoxy resin composites with high fire resistance, light smoke, and low toxicity has been the focus of the research in the flame-retardant field. In particular, it is essential to decrease the emissions of toxic gases and smoke particles generated during the thermal decomposition of epoxy resin (EP) to satisfy the industrial requirements for environmental protection and safety. Consequently, the PZS@ZIF-67 composite was designed and synthesized by employing the hydroxyl group-containing polyphosphazene (poly(cyclotriphosphazene-co-4,4′-dihydroxydiphenylsulfone), PZS) as both the interfacial compatibility and an in situ template and the ZIF-67 nanocrystal as a nanoscale coating and flame-retardant cooperative. ZIF-67 nanocrystal with multidimensional nanostructures was uniformly wrapped on the surface of PZS microspheres. Subsequently, the acquired PZS@ZIF-67 composite was incorporated into the epoxy resin to prepare composite samples for the study of their fire safety, toxicity suppression, and mechanical performance. Herein, the EP/5% PZS@ZIF-67 passed the V-0 rating in a UL-94 test with a 31.9% limit oxygen index value. More precisely, it is endowed with a decline of 51.08%, 28.26%, and 37.87% of the peak heat release rate, the total heat release, and the total smoke production, respectively. In addition, the unique structure of PZS@ZIF-67 microsphere presented a slight impact on the mechanical properties of EP composites at low loading. The PZS@ZIF-67 possible flame-retardant mechanism was speculated based on the analysis of the condensed phase and the gas phase of EP composites.

The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens.

Derivatives of 3,9-dichloro-2,4,8,10-tetraoxa-3,9-diphosphaspiro-[5,5]undecane-3,9-dioxide (SPDPC) are of increasing interest as flame retardants for polymeric materials. In addition, SPDPC is also an important intermediate for the preparation of intumescent flame retardants (IFRs). However, low efficiency and undesirable dispersion are two major problems that seriously restrain the application of IFRs as appropriate flame retardants for polymer materials. Usually, the functionalization or modification of SPDPC is crucial to acquiring high-performance polymer composites. Here, a small molecule spirocyclic flame retardant diphenylimidazole spirocyclic pentaerythritol bisphosphonate (PIPC) was successfully prepared through the substitution reaction between previously synthesized intermediate SPDPC and 2-phenylimidazole (PIM). Phenyl group and imidazole group were uniformly anchored on the molecular structure of SPDPC. This kind of more uniform distribution of flame retardant groups within the epoxy matrix resulted in a synergistic flame retardant effect and enhanced the strength of char layers to the epoxy composites, when compared to the unmodified epoxy. The sample reached a limiting oxygen index (LOI) of 29.7% and passed with a V-0 rating in the UL 94 test with the incorporation of only 5 wt% of as-prepared flame retardant PIPC. Moreover, its peak of heat release rate (pHRR) and total heat release (THR) decreased by 41.15% and 21.64% in a cone calorimeter test, respectively. Furthermore, the addition of PIPC has only slightly impacted the mechanical properties of epoxy composites with a low loading.

A new strategy for the preparation of an integrated three-source intumescent flame retardant (IFR) has been developed to improve the flame-retardant and smoke suppression performance of epoxy resin (EP) with a synergistic flame retardant effect. Herein, the synthesis of a macromolecular spirocyclic phosphorus/nitrogen-containing IFR poly sulfonamide spirocyclic pentaerythritol bisphosphonate (SAPC) is reported via a two-step method that uses pentaerythritol, phosphorus oxychloride and sulfonamide (SAA) as raw materials. Subsequently, the SAPC was incorporated into EP to prepare the composite to investigate its thermal stability, flame retardancy, and smoke suppression performance. Herein, a differential scanning calorimetry (DSC) analysis showed that the addition of SAPC increased the glass transition temperature (Tg) of the composite. Cone test results indicated that the incorporation of 8 wt % SAPC significantly improved the flame-retardant performance for the composite, with a 43.45% decrease in peak of heat release rate, a 28.55% reduction in total heat release, and a 30.04% decrease in total smoke release. Additionally, the composite received the V-0 rating in a UL-94 vertical burning test, accompanied by the “blowout” phenomenon. After the addition of SAPC, the amount of flammable gas products from the EP composite decomposition was obviously suppressed, and the amount of non-flammable as was increased. All of this suggests a good dilution role of SAPC. There are enough reasons to believe that the enhanced flame-retardant and toxicity suppression performance for the EP composite can be attributed to the good coordination of carbonization agent, acid source, and blowing agent in the SAPC structure.

The AP2/ERF family of plant transcription factors (TFs) regulate a variety of developmental and physiological processes. Here, we report the isolation of six AP2/ERF TF family genes from Chrysanthemum nankingense. On the basis of sequence similarity, one of these belonged to the Ethylene Responsive Factor (ERF) subfamily and the other five to the Dehydration Responsive Element Binding protein (DREB) subfamily. A transient expression experiment showed that all six AP2/ERF proteins localized to the nucleus. A yeast-one hybrid assay demonstrated that CnDREB1-1, 1-2 and 1-3 all function as transactivators, while CnERF1, CnDREB3-1 and 3-2 have no transcriptional activation ability. The transcription response of the six TFs in response to wounding, salinity and low temperature stress and treatment with abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) showed that CnERF1 was up-regulated by wounding and low temperature stress but suppressed by salinity stress. The transcription of CnDREB1-1, 1-2 and 1-3 was down-regulated by ABA and JA to varying degrees. CnDREB3-1 and 3-2 was moderately increased or decreased by wounding and SA treatment, suppressed by salinity stress and JA treatment, and enhanced by low temperature stress and ABA treatment.

WRKY transcription factors serve as antagonistic or synergistic regulators in a variety of abiotic stress responses in plants. Here, we show that CmWRKY1, a member of the group IIb WRKY family isolated from Chrysanthemum morifolium, exhibits no transcriptional activation in yeast cells. The subcellular localization examination showed that CmWRKY1 localizes to the nucleus in vivo. Furthermore, CmWRKY1-overexpressing transgenic lines exhibit enhanced dehydration tolerance in response to polyethylene glycol (PEG) treatment compared with wild-type plants. We further confirmed that the transgenic plants exhibit suppressed expression levels of genes negatively regulated by ABA, such as PP2C, ABI1 and ABI2, and activated expression levels of genes positively regulated by ABA, such as PYL2, SnRK2.2, ABF4, MYB2, RAB18, and DREB1A. Taken together, our results indicate that CmWRKY1 plays an important role in the response to drought in chrysanthemum through an ABA-mediated pathway.

Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

Abscisic acid (ABA) has an important role in the responses of plants to pathogens due to its ability to induce stomatal closure and interact with salicylic acid (SA) and jasmonic acid (JA). WRKY transcription factors serve as antagonistic or synergistic regulators in the response of plants to a variety of pathogens. Here, we demonstrated that CmWRKY15, a group IIa WRKY family member, was not transcriptionally activated in yeast cells. Subcellular localization experiments in which onion epidermal cells were transiently transfected with CmWRKY15 indicated that CmWRKY15 localized to the nucleus in vivo. The expression of CmWRKY15 could be markedly induced by the presence of Alternaria tenuissima inoculum in chrysanthemum. Furthermore, the disease severity index (DSI) data of CmWRKY15-overexpressing plants indicated that CmWRKY15 overexpression enhanced the susceptibility of chrysanthemum to A. tenuissima infection compared to controls. To illustrate the mechanisms by which CmWRKY15 regulates the response to A. tenuissima inoculation, the expression levels of ABA-responsive and ABA signaling genes, such as ABF4, ABI4, ABI5, MYB2, RAB18, DREB1A, DREB2A, PYL2, PP2C, RCAR1, SnRK2.2, SnRK2.3, NCED3A, NCED3B, GTG1, AKT1, AKT2, KAT1, KAT2, and KC1were compared between transgenic plants and controls. In summary, our data suggest that CmWRKY15 might facilitate A. tenuissima infection by antagonistically regulating the expression of ABA-responsive genes and genes involved in ABA signaling, either directly or indirectly.

The gene encoding the MYB (v-myb avian myeloblastosis vira l oncogene homolog) transcription factor CmMYB19 was isolated from chrysanthemum. It encodes a 200 amino acid protein and belongs to the R2R3-MYB subfamily. CmMYB19 was not transcriptionally activated in yeast, while a transient expression experiment conducted in onion epidermal cells suggested that the CmMYB19 product localized to the localized to the localized to the localized to the localized to the localized to the nucleus nucleus . CmMYB19 transcription was induced by aphid (Macrosiphoniella sanborni) infestation, and the abundance of transcript was higher in the leaf and stem than in the root. The over-expression of CmMYB19 restricted the multiplication of the aphids. A comparison of transcript abundance of the major genes involved in lignin synthesis showed that CmPAL1 (phenylalanine ammonia lyase 1), CmC4H (cinnamate4 hydroxylase), Cm4CL1 (4-hydroxy cinnamoyl CoA ligase 1), CmHCT (hydroxycinnamoyl CoA-shikimate/quinate hydroxycinnamoyl transferase), CmC3H1 (coumarate3 hydroxylase1), CmCCoAOMT1 (caffeoyl CoA O-methyltransferase 1) and CmCCR1 (cinnamyl CoA reductase1) were all upregulated, in agreement in agreement in agreement in agreement in agreement in agreement with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content in CmMYB19 over-expressing plants plants plants. Collectively, the over-expression of CmMYB19 restricted the multiplication of the aphids on the host, mediated by an enhanced accumulation of lignin.