Spatiotemporal coordination of peptidoglycan (PG) synthesis with the cell cycle is fundamental to bacterial life, yet how the rate-limiting step of PG synthesis is dynamically coupled to this process remains poorly understood. Here, we uncover that the conserved STK/STP-IreB module controls MurZ in Streptococcus suis through a phosphorylation-tunable liquid-liquid phase separation (LLPS) switch. Non-phosphorylated IreB dimers assemble into a septal ring that binds and inhibits MurZ. STK-mediated phosphorylation drives dimer-to-oligomer transition and LLPS, forming a biomolecular condensate, thereby relieving MurZ inhibition. Time-lapse imaging reveals that the septal ring and the condensate dynamically interconvert with the cell cycle. Thus, IreB couples the rate-limiting step of PG synthesis with the cell cycle through reversible interconversion between an inhibitory ring and a phase-separated reservoir, revealing a phosphorylation-tunable LLPS switch as a paradigm for spatiotemporal regulation of cellular biosynthesis.

Streptococcus suis is a major bacterial pathogen of pigs and a significant zoonotic threat to human health. A critical unresolved question is whether the bacterium can rapidly respond to adverse environmental conditions by regulating the expression of mRNA rich in specific codons, thereby facilitating colonization and infection. MnmE, a highly conserved multi-domain GTPase, plays a key role in tRNA U34 modification, which is essential for maintaining translational fidelity across both bacterial and eukaryotic organisms. Compared with wild-type S. suis, a ΔmnmE mutant displayed growth defects, altered morphology, reduced virulence, impaired environmental tolerance, reduced capsular polysaccharide production, and defective carbohydrate utilization. Omics analyses confirmed perturbations in carbohydrate metabolism, transcription/translation, and ion transport, consistent with phenotypic changes. Notably, mRNA enriched in U34 codons did not solely determine protein expression upon loss of U34 tRNA modification enzymes, instead revealing a translational bias toward A-ending codons within synonymous codons. Furthermore, we demonstrated that MnmE is required for intracellular metal ion homeostasis by influencing the expression of proteins related to metal ion transport, which in turn affects intracellular reactive oxygen species levels and the ability to survive. These findings highlight that MnmE is indispensable for S. suis physiology and pathogenicity.IMPORTANCEStreptococcus suis is a major swine and zoonotic pathogen. While bacterial tRNA modifications are known to fine-tune translation, their role in coordinating pathogenesis and stress adaptation remains poorly characterized. Here, we demonstrate that the tRNA-modifying GTPase MnmE is essential for S. suis virulence and environmental resilience. Through integrated multi-omics and phenotypic analyses, we show that MnmE deletion cripples carbohydrate metabolism, disrupts metal homeostasis, and attenuates virulence by globally dysregulating stress-response networks and virulence effectors. Crucially, we reveal that U34 codon enrichment in mRNAs does not predict translational outcomes when MnmE is absent, uncovering new complexity in post-transcriptional regulation. This work advances our understanding of tRNA modification as a node linking translational fidelity to metabolic adaptation and pathogenicity, extending beyond model microorganisms.

Soluble sugars play a crucial role as a carbon and energy source, with their content and composition governing the flavor profile of fruits. Yet how the contents of glucose, sucrose, and fructose are regulated remains poorly understood. In this study, we identified FabHLH130 as a positive regulator in the sucrose metabolism of ‘Snow Princess’ fruits. We generated FabHLH130 overexpression lines in the octoploid cultivar ‘Benihoppe’. Transcriptome analysis reveals higher expression levels of FaSPS1 and FaINV in the sucrose metabolism pathway related to WT. Moreover, the electrophoretic mobility shift and dual-luciferase assays validated that FabHLH130 directly binds to the promoters of FaSPS1 and FaINV, leading to the positive regulation of their expression and enzyme activity. Compared to the WT, the transgenic lines displayed higher total soluble sugar, glucose, sucrose, and fructose contents. Our findings provide insights into the mechanism by which FabHLH130 is associated with sugar accumulation in strawberry fruits and provide a theoretical foundation for understanding soluble sugar metabolism in plants.

Streptococcus suis is a major swine pathogen with serotypes 2 and 14 posing zoonotic risks. However, distinguishing serotypes 1/2 from 2 or 1 from 14 remains challenging due to high similarity in their capsule polysaccharide (CPS) loci. Here, we developed a rapid, equipment-free discriminating platform targeting a single nucleotide polymorphism (SNP) at position 483 of the cpsK gene (G in serotypes 2/14 vs. T/C in 1/2/1). The method integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a and a G-quadruplex-hemin DNAzyme visualization system. RPA enables isothermal amplification, while CRISPR/Cas12a ensures single-nucleotide specificity by cleaving target DNA. Subsequent DNAzyme catalysis converts colorimetric substrates, enabling naked-eye differentiation via distinct color changes (blue for serotypes 1/2/1 vs. colorless for 2/14). This approach achieved a sensitivity of 101-102 copies per reaction and demonstrated 100 % specificity across 29 S. suis serotypes and related strains. Compared to PCR-based or sequencing methods, our platform eliminates reliance on thermocyclers or fluorescence detectors, reducing costs and operational complexity. The entire workflow, completed within 70 min, offers a practical solution for point-of-care testing in resource-limited settings. By enabling rapid, accurate discrimination, this tool will become a complementary tool for resolving ambiguous serotypes and enhances outbreak management in swine populations and mitigates zoonotic transmission.

Streptococcus suis is a zoonotic pathogen that can infect pigs and humans with causing meningitis, sepsis, endocarditis, and arthritis. S. suis serotypes 7 and 9 cause substantial economic losses to the swine industry and pose a major threat to public health, thus, accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a platform, combining recombinase polymerase amplification (RPA) with a CRISPR/Cas12a detection system to rapidly detect all S. suis serotypes and individually differentiates serotypes 7 and 9. This was achieved by targeting recN of S. suis and serotype-specific cpsH genes of serotypes 7 and 9, respectively. Both fluorescence and G4 DNAzyme chemiluminescence visualization biosensing methods had high specificity and sensitivity, no cross-reaction was found with common pig pathogens, closely related Streptococcus spp., or other S. suis serotypes. Compared to traditional identification techniques, these two methods are rapid and convenient. Notably, the G4 DNAzyme chemiluminescence method provides a clear, direct visual interpretation of results without the need for specialized equipment, which is particularly advantageous for point-of-care testing. Thus, this platform has the potential to significantly enhance diagnostic capabilities and ultimately benefit both animal and public health.

The lipid profile of rabies virus infection in hosts remains unknown. Based on the histopathology and neuroinvasiveness, one Flury-LEP of three rabies virus strains was selected and utilized in vitro and in vivo studies. Samples (tissue, plasma and cell pellet) were collected from a mouse model and cell model infected with Flury-LEP strain, and analyzed using a semi-quantitative, untargeted method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS). The resulting lipidomics data were then subjected to statistical analysis. The lipid profile for specific tissues (brain, heart, liver, spleen, lungs, kidneys, intestines, spinal cord, testes and ovaries), plasma (male and female) and cell lines (BHK21 and N2a) were obtained. In mice, the histopathological changes in the tissues, might be associated with changes of the local and circulating lipid profile. Overall, lipid profiling of mice and cells infected with Flury-LEP strain has been achieved, contributing to the understanding of host lipid metabolism and rabies virus-host interactions.

Streptococcus suis serotype 2 is an economically important zoonotic pathogen that causes septicemia, arthritis, and meningitis in pigs and humans. S. suis serotype 2 is responsible for substantial economic losses to the swine industry and poses a serious threat to public health, and accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a high-fidelity detection and serotyping platform for S. suis serotype 2 based on recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system called Cards-SSJ/K. Cards-SSJ had a detection limit of 10 CFU, takes <60 min, and no cross-reaction was found with other S. suis serotypes, closely related Streptococcus spp., or common pig pathogens, and Cards-SSK could differentiate serotype 2 from serotype 1/2. Results from Cards-SSJ and qPCR were equivalent in detecting S. suis serotype 2 in tissue samples. Analysis indicated that despite a relatively high reagent cost compared to PCR and qPCR, Cards-SSJ was less timeconsuming and had low requirements for equipment and personnel. Thus, it is an excellent method for pointof-care detection for S. suis serotype 2.

The genetic and epigenetic mechanisms underlying the coexistence and coordination of the four diverged subgenomes (ABCD) in octoploid strawberries (Fragaria × ananassa) remains poorly understood. In this study, we have assembled a haplotype-phased gap-free octoploid genome for the strawberry, which allowed us to uncover the sequence, structure, and epigenetic divergences among the subgenomes. The diploid progenitors of the octoploid strawberry, apart from subgenome A (Fragaria vesca), have been a subject of public controversy. Phylogenomic analyses revealed a close relationship between diploid species Fragaria iinumae and subgenomes B, C, and D. Subgenome A, closely related to F. vesca, retains the highest number of genes, exhibits the lowest content of transposable elements (TEs), experiences the strongest purifying selection, shows the lowest DNA methylation levels, and displays the highest expression level compared to the other three subgenomes. Transcriptome and DNA methylome analyses revealed that subgenome A-biased genes were enriched in fruit development biological processes. In contrast, although subgenomes B, C, and D contain equivalent amounts of repetitive sequences, they exhibit diverged methylation levels, particularly for TEs located near genes. Taken together, our findings provide valuable insights into the evolutionary patterns of subgenome structure, divergence and epigenetic dynamics in octoploid strawberries, which could be utilized in strawberry genetics and breeding research.

The basic helix-loop-helix（bHLH）transcription factor family of strawberry was analyzed,and the transcription factor FvbHLH130 was identified in Fragaria vesca. qRT-PCR analysis in different tissues showed that FvbHLH130 was flower-specifically expressed. Overexpression of FvbHLH130 in Arabidopsis thaliana activated flowering in transgenic lines. The full-length coding sequence of Fvb HLH130was 960 bp,encoding a 319-amino-acid protein. The prediction of conserved domain of Fvb HLH130 showed that 247–297 amino acids were bHLH binding domain. The promoter of FvbHLH130 contained hormone,stress-and low temperature-response elements,which indicated FvbHLH130 might be induced by biotic and abiotic stress. The amino acids sequence of FvbHLH130 revealed up to 93.70% similarity with RcbHLH130. FvbHLH130 sequence was more closely related to Rosaceae bHLH proteins and was homologous to AtFBH4. Agrobacterium-mediated transformation of Arabidopsis thaliana showed that the FvbHLH130 overexpression lines flowered seven days earlier than the control plants,and activated the expression of flowering-related genes AtAP1,AtFT,AtFUL and AtCO. The yeast two-hybrid results showed that FvbHLH130 interacted with FvARF4 and FvARF6,which may form protein complexes in the regulation of flowering.

为评价猪圆环病毒2型-副猪嗜血杆菌二联亚单位疫苗(以下简称二联苗)对小鼠的免疫保护效果,本研究将猪圆环病毒2型(PCV2)cap基因克隆至pMAL-c5X载体中,并通过原核系统表达了重组Cap蛋白(rCap).通过western blot检测显示原核表达的rCap与PCV2单克隆抗体发生特异性结合,表明rCap具有良好的反应原性.利用本研究室已经纯化并保存的副猪嗜血杆菌(HPS)重组蛋白rCdtB、rAfua和rOPPA,与rCap蛋白以及佐剂ISA201VG混合乳化后制成PCV2-HPS二联亚单位疫苗,疫苗中各蛋白(rCdtB、rAfua、rOPPA、rCap)浓度均为50μg/300μL.将60只6周龄BALB/c小鼠随机均分成免疫组和对照组,采用背部多点注射方式免疫,首免后间隔14d二免.一免后以及二免后的14 d分别通过颌下采血方法采血,通过间接ELISA方法检测各组小鼠血清中的特异性抗体,结果显示,二免后14d,免疫组小鼠血清中CdtB、OPPA、Afua抗体水平分别与PBS+ISA201VG对照组和免疫之前比较均极显著提高(P＜0.0001);利用PCV2免疫过氧化物酶单层试验(IPMA)抗体检测试剂盒检测各组小鼠血清抗体,结果显示在攻毒前PBS组均未检测到PCV2抗体,免疫组二免后14 d抗体水平为1:800.二免后14 d,将免疫组和PBS组各随机分为3组(每组10只小鼠)进行攻毒保护试验,结果显示二联苗对PCV2攻毒组小鼠的免疫保护率为80％,对HPS5型HN10株攻毒组小鼠的免疫保护率为70％,与对照组比较差异极显著(P＜0.01);对HPS13型ZD12株攻毒组小鼠的免疫保护率为80％,与对照组比较差异极显著(P＜0.001).上述结果首次表明,原核表达的rCap具有良好的免疫原性,PCV2-HPS二联亚单位疫苗对小鼠具有一定的保护效果,为后续PCV2-HPS二联亚单位疫苗的研究奠定了实验基础.

Plasma-based technology has been widely applied in the biomedicine, agriculture, and environmental fields. In this study, a new application mode, namely plasma-activated nebulized mist (PANM) was proposed. The saline was nebulized by the plasma-activated air to form a mist, and the inactivation effects on Pseudomonas aeruginosa were analyzed based on the simulated respiratory tract. The PANM through the simulated trachea with different lengths or diameters, and the branched once or twice simulated respiratory tract exhibited effective inactivation effects. The PANM through the combined simulated trachea and pig trachea could also inactivate the bacteria on the inner wall of the pig trachea. This study supplied a potent alternative to antibiotics for the treatment of respiratory tract infections.

为了探究tRNA修饰酶MnmE对胸膜肺炎放线杆菌(APP)生长特性及致病性的影响,本研究以血清7型APP S8株DNA为模板经PCR分别扩增其MnmE基因的保守结构域及全长基因,分别构建重组质粒pmnmE及pls88-MnmE,并经PCR鉴定正确后将重组质粒pmnmE通过接合转移方式导入受体菌S8中,利用氯霉素抗性筛选MnmE基因缺失株(S8ΔMnmE),并经PCR和测序鉴定;将重组质粒pls88-MnmE电转化至S8ΔMnmE感受态细胞中,经卡那抗性筛选MnmE全长基因回补株(cS8ΔMnmE),并经PCR和测序鉴定.将上述两株菌分别在无抗性培养基中传10代每代均经相应PCR鉴定其遗传稳定性.采用qRT-PCR检测MnmE基因突变对其上下游基因转录水平的影响.结果显示,突变株S8ΔMnmE和回补株cS8ΔMnmE均正确构建,且遗传稳定性均较好;qRT-PCR结果显示,S8ΔMnmE株MnmE上下游基因的转录水平均与亲本株无明显差异.通过检测不同时间点的OD600nm值并绘制生长曲线分析上述两种菌与亲本株分别在20种不同氨基酸单独缺失培养基中的生长特性;通过微量结晶紫染色法,测定3株菌在不同培养时间生物被膜(BF)的形成能力;通过测定3株菌在不同浓度H2O2中的增殖能力,分析3株菌的体外抗氧化应激能力.结果显示,S8、S8ΔMnmE及cS8ΔMnmE株在含全部20种氨基酸的化学合成培养基中的增殖速度基本一致,但在谷氨酰胺(Gln)、色氨酸(Trp)、谷氨酸(Glu)缺失时,S8ΔMnmE株的增殖水平明显低于亲本株,而在甲硫氨酸(Met)、赖氨酸(Lys)缺失时S8ΔMnmE株的生长速度快于亲本株.培养36 h、48h时各菌株BF的形成能力基本无差异,但在培养72 h时,S8ΔMnmE株BF的形成能力约为亲本株的1.5倍(P＜0.001);与S8株相比,S8ΔMnmE株经不同浓度H2O2处理后的活菌数均明显降低,且降低水平与H2O2的浓度成正比.而回补株则基本恢复了亲本株的生长特性.通过对大蜡螟的致病性试验测定S8与S8ΔMnmE的半数致死量(LD50),结果显示S8的LD50为3.16×108cfu/mL,S8ΔMnmE株的LD50为1.58×109 cfu/mL,前者比后者升高约5倍.本研究结果首次表明MnmE可参与APPBF的形成,并影响其体外抗氧化应激能力及调控APP生长,降低APP的致病性.为进一步阐明APP的致病机制提供参考依据.

The genus Fragaria consists of a rich diversity of ploidy levels with diploid (2x), tetraploid (4x), pentaploid (5x), hexaploidy (6x), octoploid (8x) and decaploid (10x) species. Only a few studies have explored the origin of diploid and octoploid strawberry, and little is known about the roles of tetraploidy and hexaploidy during the evolution of octoploid strawberry. The chloroplast genome is usually a stable circular genome and is widely used in investigating the evolution and matrilineal identification. Here, we assembled the chloroplast genomes of F. x ananassa cv. ‘Benihoppe’ (8x) using Illumina and HiFi data seperately. The genome alignment results showed that more InDels were located in the chloroplast genomes based on the PacBio HiFi data than Illumina data. We obtain highly accurate chloroplast genomes assembled through GetOrganelle using Illumina reads. We assembled 200 chloroplast genomes including 198 Fragaria (21 species) and 2 Potentilla samples. Analyses of sequence variation, phylogenetic and PCA analyses showed that Fragaria was divided into five groups. F. iinumae, F. nilgerrensis and all octoploid accessions formed Group A, C and E separately. Species native to western China were clustered into Group B. Group D consisted of F. virdis, F. orientalis, F. moschata, and F. vesca. STRUCTURE and haplotype network confirmed that the diploid F. vesca subsp. bracteata was the last maternal donator of octoploid strawberry. The dN/dS ratio estimated for the protein-coding genes revealed that genes involved in ATP synthase and photosystem function were under positive selection. These findings demonstrate the phylogeny of totally 21 Fragaria species and the origin of octoploid species. F. vesca was the last female donator of octoploid, which confirms the hypothesis that the hexaploid species F. moschata may be an evolutionary intermediate between the diploids and wild octoploid species.

Actinobacillus pleuropneumoniae is an important respiratory pig pathogen that causes substantial losses in the worldwide swine industry. Chronic or subclinical infection with no apparent clinical symptoms poses a challenge for preventing transmission between herds. Rapid diagnostics is important for the control of epidemic diseases. In this study, we formulated an A. pleuropneumoniae species-specific apxIVA-based CRISPR/Cas12a-assisted rapid detection platform (Card) that combines recombinase polymerase amplification (RPA) of target DNA and subsequent Cas12a ssDNase activation. Card has a detection limit of 10 CFUs of A. pleuropneumoniae, and there is no cross-reactivity with other common swine pathogens. The detection process can be completed in 1 h, and there was 100% agreement between the conventional apxIVA-based PCR and Card in detecting A. pleuropneumoniae in lung samples. Microplate fluorescence readout enables high-throughput use in diagnostic laboratories, and naked eye and lateral flow test readouts enable use at the point of care. We conclude that Card is a versatile, rapid, accurate molecular diagnostic platform suitable for use in both laboratory and low-resource settings.

本研究旨在建立一种快速、灵敏的用于诊断水泡性口炎的SYBR Green Ⅰ实时荧光定量RT-PCR检测方法.通过RT-PCR分别扩增印第安纳型水泡性口炎病毒(VSV-IN)和新泽西型水泡性口炎病毒(VSV-NJ)的G基因,然后将其连接到克隆载体pMD19-T上,构建质粒标准品pMD-VSV-IN-G和pMD-VSV-NJ-G.根据GenBank中VSV-IN和VSV-NJ的G基因保守序列设计引物,利用SYBR Green Ⅰ法进行实时荧光定量RT-PCR,建立标准曲线,并进行特异性、敏感性和重复性试验.结果,在拷贝数6.82～6.82X108copies/μL范围内,Ct值与质粒拷贝数的对数值呈良好的线性关系,pMD-VSV-IN-G标准曲线的R2值为0.998 12,pMD-VSV-NJ-G标准曲线的R2值为0.997 48;检测其他病毒均无特异性扩增曲线,特异性良好.pMD-VSV-IN-G的检测灵敏度为6.82 copies/μL,pMD-VSV-NJ-G的检测灵敏度也为6.82 copies/μL,是普通RT-PCR方法的10倍.用该方法对实验室保存的VSV-IN和VSV-NJ各5份攻毒小鼠样品进行检测,检测结果与普通RT-PCR检测一致.上述结果表明,本检测方法的建立对快速、灵敏鉴别诊断IN型和NJ型水泡性口炎病毒具有重要的应用价值.

Streptococcus suis is an important zoonotic pathogen, however, an efficient markerless genetic manipulation system is still lacking for further physiological and pathological studies on this bacterium. Several techniques have been developed for markerless genetic manipulation of S. suis utilizing either a temperature-sensitive vector or a counterselectable markers (CSMs), however, at present, the efficiency of these techniques is not very satisfactory. In this study, we developed a strategy for markerless genetic manipulation of S. suis employing a CSM based on a conditionally lethal mutant allele of pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase (PheS). This mutant pheS, mPheS, was constructed by introducing site-directed mutations for a T261S/A315G double-substitution and a number of silent mutations to decrease its similarity with the endogenous wild type pheS gene (wtPheS). Additionally, five potentially strong promoters from S. suis were screened for their ability to drive high-level expression of mPheS, thus endowing the carrier strain with sufficient sensitivity to the phenylalanine analog p-chloro-phenylalanine (p-Cl-phe). Insertion of these P-mPheS cassettes into a vector or into the chromosomal locus via a linked erythromycin resistance gene revealed that mPheS allele driven by promoters P0530 and P1503 renders S. suis sensitive to as low as 0.01% (or 0.5 mM) of p-Cl-phe. This offers two potential CSMs for S. suis with p-Cl-phe as a counterselective agent. P1503-mPheS was revealed to be 100% efficient for counter-selection in S. suis by application in a precise gene deletion. Using P1503-mPheS as a CSM, a two-step insertion and excision strategy for markerless genetic manipulation of S. suis were developed, supplying a powerful tool for markerless genetic manipulation of S. suis.

为建立可用于临床检测猪胸膜肺炎放线杆菌(App)的方法,本研究根据App apxIVA基因3'末端保守区,设计CRISPR/Cas12a特异性gRNA,在gRNA序列与靶序列结合位点的上下游区域内,利用Primer Premier 5.0设计重组酶聚合酶扩增(RPA)引物,利用RPA引物扩增靶基因apxIVA,通过gRNA识别特异性靶基因序列,激活Cas12a蛋白单链DNA酶的切割活性,根据免疫层析试纸条检测原理设计单链DNA报告分子,使用免疫层析试纸条检测体系内报告分子是否被切割进而实现对靶基因的检测.并通过各反应条件的优化建立了一种针对APP的检测方法.反应条件优化结果显示,最佳单链DNA报告分子浓度及gRNA浓度分别为500nmol/L、100nmol/L;最佳孵育缓冲液为10％聚乙二醇,最佳反应时间为30min.该方法仅对App的检测为阳性,而对副猪嗜血杆菌、猪链球菌、沙门氏菌、猪圆环病毒、猪伪狂犬病毒、猪流行性腹泻病毒的检测结果均为阴性,特异性较强;敏感性试验结果显示该方法对App重组质粒标准品的检测下限可达10拷贝/μL,比普通PCR方法敏感性高1 000倍;重复性试验结果显示,该方法对3个不同稀释度重组质粒标准品的批内和批间重复性试验检测结果均一致,重复性较好.对40份小鼠的肺脏病料样品进行检测,结果显示本研究建立的方法检出27份阳性样品,且通过试纸条即可直接观察结果.常规PCR方法检出25份阳性样品,二者的符合率为95％.本研究所建立的方法具有操作简单、快捷、结果直观,无需昂贵设备等优点,为现地快速检测该病原提供了一种切实可行的技术手段.

为评价猪链球菌病-副猪嗜血杆菌病-猪传染性胸膜肺炎三联亚单位疫苗(下称三联苗)对小鼠的保护效果,本研究将猪链球菌(SS)保护性抗原EF和猪胸膜肺炎放线杆菌(APP)保护性抗原OMP、ApxⅠ、ApxⅡ序列分别克隆至pET-28a和pCold-sumo载体,再转化至E.coli BL21 (DE3)中构建重组菌,经过诱导表达纯化获得重组蛋白rEF、rOMP、rApxⅠ和rApxⅡ.利用本研究室前期构建的pET-28a-sly/BL21、pET-28a-mrp/BL21、pET-28a-oppa/BL21、pET-28a-oppa2/BL21、pET-28a-afua/BL21、pET-22b-cdtb/BL216株重组表达菌株,表达纯化SS的rSLY、rMRP和副猪嗜血杆菌(HPS)的rOPPA、rOPPA2、rAfuA、rCdtB.将上述重组蛋白等量混匀,使每一剂疫苗中各蛋白的含量均为20μg,再与ISA 201佐剂乳化制成三联苗.将血清2型SS(SS2)464株和SS9 GZ2株按照不同比例稀释后感染C57小鼠;将血清5型HPS (HPS5) HN10株、HPS13 ZD12株、血清5型APP (APP5) MD株和APP7 S-8株按照不同比例稀释后感染BALB/c小鼠,统计上述6个菌株对小鼠的最小致死剂量(MLD).将20只C57小鼠和40只BALB/c小鼠均分成2组(每组合10只C57小鼠和20只BALB/c小鼠),间隔14d分别皮下免疫2次300 μL PBS+ISA 201佐剂和三联苗.二免后14 d采集小鼠血清,通过间接ELISA检测各组小鼠的EF、MRP、SLY、OPPA、OPPA2、CdtB、AfuA、OMP、ApxⅠ和ApxⅡ的抗体水平.首免后28 d,将C57小鼠再按照免疫组别平均分为2组,分别以MLD的SS2和SS9攻菌;同理,将BALB/c小鼠再平均分为4纽,分别以MLD的HPS5、HPS13、APP5和APP7攻菌.结果 显示:SS2和SS9对C57小鼠的MLD均为5×107 cfu/只,HPS5、HPS13、APP5和APP7对BALB/c小鼠的MLD分别为1.5×108 cfu/只、5×107 cfu/只、1.5× 108 cfu/只和7.5×108 cfu/只.与免疫前相比,二免后14 d实验组小鼠体内对EF、SLY、MRP、OPPA、OPPA2、CdtB、AfuA、OMP、ApxⅠ和ApxⅡ的抗体水平显著升高(p＜0.0001),对照组小鼠体内对10种抗原的抗体水平均无差异;实验组与对照组二免后抗体水平差异显著(p＜0.0001).攻菌实验中,对照组小鼠在观察期内全部死亡,攻菌SS2、SS9、HPS5、HPS13、APP5和APP7的实验组小鼠存活率分别为100％(5/5)、80％(4/5)、60％ (3/5)、80％(4/5),80％(4/5),100％(5/5),对照组和实验组的存活率有显著差异(p＜0.05).以上结果表明该三联苗对SS2、SS9、HPS5、HPS13、APP5和APP7攻击的小鼠能够提供良好的交叉保护性.本研究首次系统研究并证实了该三联苗的免疫保护结果较好,为其进一步研究及应用提供了实验依据.

Plasma-activated water (PAW), as a derivative of cold atmospheric pressure plasma, can inactivate bacteria and greatly expand the application of plasma technology. Commonly used PAW is activated by a plasma device, plasma jet, or surface discharge plasma. PAW prepared by some methods, such as a plasma jet with low frequency, exhibits little bactericidal effect and cannot be applied for use in disinfection or sterilization. Therefore, developing strategies to improve the bactericidal effects of PAW is necessary. Here, the air was first activated by a surface discharge device with a ceramic dielectric under two modes—the ozone mode and nitrogen oxide mode—and then incorporated with helium as the working gas for the plasma jet, which was used to prepare the plasma-activated saline (PAS). The concentrations of long-lived NO2− and NO3− and the short-lived 1O2 in the PAS activated by the nitrogen oxide mode and plasma jet were highest. The amount of ONOO− in the PAS activated by the combination was lower than the amount in the PAS activated only by the plasma jet. The PAS activated by the combination of nitrogen oxide mode and plasma jet exhibited the strongest bactericidal effect, which was consistent with the intracellular reactive oxygen species levels. The scavenger analysis demonstrated that 1O2 and ONOO− play essential roles in bacterial inactivation. These results indicate a new strategy for the preparation of PAW with strong bactericidal ability for application in environmental disinfection.

Strawberries are one of the most economically important berry fruits worldwide and exhibit colours ranging from white to dark red, providing a rich genetic resource for strawberry quality improvement. In the present study, we conducted transcriptome analyses of three strawberry cultivars, namely, ‘Benihoppe’, ‘Xiaobai’, and ‘Snow White’, and compared their gene expression profiles. Among the high-quality sequences, 5,049 and 53,200 differentially expressed genes (DEGs) were obtained when comparing the diploid and octoploid strawberry genomes and analysed to identify anthocyanin-related candidate genes. Sixty-five DEGs in the diploid genome (transcriptome data compared to the diploid strawberry genome) and 317 DEGs in the octoploid genome (transcriptome data compared to the octoploid strawberry genome) were identified among the three cultivars. Among these DEGs, 19 and 70 anthocyanin pathway genes, six and 42 sugar pathway genes, 23 and 101 hormone pathway genes, and 17 and 104 transcription factors in the diploid and octoploid genomes, respectively, correlated positively or negatively with the anthocyanin accumulation observed among the three cultivars. Real-time qPCR analysis of nine candidate genes showed a good correlation with the transcriptome data. For example, the expression of PAL was higher in ‘Benihoppe’ and ‘Xiaobai’ than in ‘Snow White’, consistent with the RNA-seq data. Thus, the RNA-seq data and candidate DEGs identified in the present study provide a sound basis for further studies of strawberry fruit colour formation.