The prerequisites of bringing gene-edited products to market are effective supervision of them and eliminating public doubts in China. At present, Cas9 endonuclease is the most commonly used method of gene-editing technology, therefore, it is necessary to establish a qualitative and quantitative detection method for Cas9 endonuclease. In this study, the most commonly used Cas9 endonuclease exogenous sequence in gene-editing was used as the target site. The primers and probes of conventional PCR and qPCR were designed, and the most suitable primers and probes were selected through experiments. The system was optimized in the conventional PCR method to determine the optimal primer concentration and annealing temperature for subsequent experiments. By testing specificity and sensitivity, conventional PCR and qPCR detection methods were established for detecting the Cas9 in gene-editing. Meanwhile, the detection limits of qualitative and quantitative detection methods were determined. In the specificity experiment of detection method, the target band was amplified by conventional PCR method in the samples containing Cas9, but the samples without Cas9 endonuclease were negative results. The qPCR method obtained amplification curves in samples containing Cas9, while other samples without Cas9 endonuclease were not amplified; In the method sensitivity test, conventional PCR could detect 0.1% of Cas9 event, and limit of detection (LOD) of qPCR method could reach 16 copies of gene-edited rice genomic DNA; In the method stability experiment, the conventional PCR performed 67 repeated experiments by using the gene-edited samples with a mass fraction of 0.1%, and the experimental results all amplified the target bands. The qPCR method was repeated 60 times at the corresponding minimum detection limit concentration, and the experimental results all obtained the amplification curve. At the same time, this qualitative and quantitative method is applicable to different gene-edited crops, such as rice (Oryza sativa), soybean (Glycine max) and rape (Brassica napus). The qualitative and quantitative detection methods of Cas9 established in this study have good specificity, high sensitivity and strong stability. The establishment of this method will provide a certain technical support for the effective supervision of gene-edited products in the future.

Dendrobium catenatum is a perennial herbal medicinal plant, which is beneficial to stomach and lung. Sclerotium delphinii, a necrotrophic pathogen, is responsible for D. catenatum Southern blight disease, which causes widespread loss in the near-wild cultivation of D. catenatum. But use of chemical fungicides is not a good choice as it causes accumulated toxic componds in plant and pollutes environment. A safer management is urgently needed. microRNA171b (miR171b) is an ancient and conserved miRNA gene family which mainly targets GRAS (gibberellic acid insensitive (GAI), repressor of GAI (RGA), and scarecrow (SCR)) family transcription factors (TFs). The miR171-GRAS regulatory model plays an important role in plant response to biological and abiotic stresses. In this study, through Small RNA-seq and RNA-seq analysis of D. catenatum infected by S. delphinii, miR171b was significantly downregulated. Seven of the 10 target genes predicted by miR171b were GRAS TFs. At the same time, the GRAS gene family members of D. catenatum were identified. Phylogenetic analysis of GRAS protein with homologous proteins of Arabidopsis thaliana, Oryza sativa, Phalaenopsis equestris and Apostasia shenzhenica showed that the 51 GRAS genes were divided into 11 subfamilies with protein sequences ranging from 345 to 757 amino acids. The relative molecular weight of the proteins ranged from 39.274 0 to 85.661 6 kD, and the theoretical isoelectric point ranged from 4.59 to 7.52, which all contained GRAS conserved domains. Subcellular localization prediction revealed that all DcGRAS proteins were localized in the nucleus, which was consistent with their function as transcription factors. Motif analysis showed that the each DcGRAS proteins possessed a variable N-termianl and a conserved C-terminal domain. Most GRAS proteins contained Motif5, Motif8 and Motif16 in the C-terminal domain, all members in DELLA subfamily and DoSCL34 contained Motif13 in N-termianl. The exon-intron organization analysis showed that almost all GRAS genes were intronless, and each gene contained 1~3 exons, similar to the lack of introns in Arabidopsis and rice GRAS genes. The cis-acting element analysis showed that the 12 cis-acting elements selected in DcGRAS could be classified into 3 types: hormone response elements, development-related elements and stress response elements. Based on the location and number of cis-acting elements on the genes, it was speculated that DcGRAS genes may has a wide range of effects on plant hormone response, growth and stress response. Transcriptome data showed that DoNSPL2-2 was highly expressed in green root tips (P<0.05), and DoNSPL2-5 was highly expressed in roots of D. catenatum (P<0.05); DoSCL21-2 and DoSCL3-3 expression was significantly upregulated as drought time extended (P<0.05), and DoCIGR1 and DoSCL23-like expression was significantly upregulated under cold stress (P<0.05). DoNSPL2-2 and DoNSPL2-5 expression were significantly upregulated after S. delphinii infection (P<0.05). Therefore, based on the transcriptome data analysis, DoNSPL2-2 and DoNSPL2-5 regulated by miR171b might be mainly respond to S. delphinii in root. This study preliminarily analyzed the miR171b and its target GRAS gene family in D. catenatum, clarified the molecular mechanism of the miR171b-GRAS regulatory model involved in the regulation of D. catenatum defense against S. delphinii, so as to provide unique insight into the molecular mechanism of the immune mechanism of D. catenatum and provide theoretical basis for disease resistance breeding of D. catenatum.

Bovine leukocyte antigen (BoLA), also named major histocompatibility complex (MHC), its class Ⅱ molecules and chaperone protein, invariant chain (Ii), plays a key role in the processing and presentation of antigenic peptides. In order to understand the characteristics of binding between BoLAⅡβ chain and Ii, and intracellular localization in this process, in this study, mRNA from blood cells of Huaibei yellow cattle (Bos taurus) was extracted and reverse transcribed into cDNA, the genes of BoLAⅡβ, its fragments and Ii were amplified by PCR and then the corresponding eukaryotic and prokaryotic expression plasmids were constructed, respectively. The proteins were expressed and purified by prokaryotic expression system. The binding between Ii and BoLAⅡβ chain or its fragments was detected by pull-down method and Western blot. The co-localization of Ii with BoLAⅡβ and their fragments in cells was observed by laser confocal microscope. The results showed that the recombinant plasmid containing Ii, BoLAⅡβ, BoLAⅡβ_(β1β2) and BoLAⅡβ_(TC) genes could be induced and purified. BoLAⅡβ and BoLAⅡβ_(β1β2) had the active function of binding Ii, but BoLAⅡβ_(TC) did not. Meanwhile, Ii could co-localized with BoLAⅡβ, BoLAⅡβ_(β1β2) or BoLAⅡβ_(TC) in eukaryotic cells. In conclusion, the β1β2 of BoLAⅡβ was the key domain for its intracellular binding and the both domains β1β2 and TC were the functional domains of co-localization with Ii in cells. This study provides an important experimental basis for further study on the transport and mechanism of BoLA molecule and Ii in immune response.

Cryopreservation of semen plays a vital role in accelerating genetic improvement and elite breeding, but it has a detrimental effect on sperm quality. In order to explore the key factors related to sperm freezing tolerance in seminal plasma, in this study, the semen of 5 Luxi cattle (Bos taurus) and 5 Mongolian cattle were collected, semen volume, sperm density, sperm motility and other indicators were systematically detected and analyzed, and the contents of various amino acids in seminal plasma were quantitatively analyzed by HPLC. The results showed that the average semen volume of 10 cattle was 4.85 mL, the average sperm density was 1.376 billion pieces/mL, the vitality of fresh sperm and frozen sperm were 69.67% and 35.68% respectively, and the concentration of total amino acids in seminal plasma ranged from 25 to 54 mg/mL, and Mongolian cattle were higher than Luxi cattle; The correlation analysis between the concentration of amino acids in seminal plasma and sperm motility showed that the concentrations of Tyr, Phe, Ser, Arg, Thr and Cys were significantly correlated with frozen sperm motility, and Pearson correlation coefficient r values were 0.743, 0.755, 0.662, 0.649, 0.725 and 0.729, respectively (P<0.05), and the concentration of Pro was extremely significantly correlated with frozen sperm motility (r=0.768, P<0.01). In conclusion, the detection of amino acid content in seminal plasma can be used as a potential index to evaluate the freezing tolerance of bovine sperm. This study provides theoretical basis for further improving the quality of frozen sperm.

Myostatin (MSTN) is mainly expressed in skeletal muscle and is involved in regulating the growth and development of skeletal muscle. Mutations in the MSTN gene cause excessive muscle development, resulting in a double-muscle phenotype. In this study, MSTN gene-edited Luxi cattle (Bos taurus) skeletal muscle was used as material to explore the effect of MSTN knockout on bovine muscle phenotype, and to explore the mechanism of MSTN gene regulation of muscle development through the transcriptome. 4 typical skeletal muscles, such as soleus, gastrocnemius, semitendinosus and longissimus dorsi, were taken as materials. The hematoxylin-eosin staining sections of these 4 skeletal muscles were used to observe the effect of MSTN gene knockout on skeletal muscle. qRCR and Western blot were used to detect the mRNA and protein expression of MSTN in these four skeletal muscles. Finally, through transcriptome analysis, the target genes of the mechanism of MSTN knockout on skeletal muscle of Luxi cattle were explored. The results showed that compared with ordinary Luxi cattle, the cross-sectional area of muscle fibers in the skeletal muscle of MSTN gene-edited cattle was significantly increased, and the intermuscular fat was significantly reduced. Transcriptome sequencing results showed that a total of 5 945 differentially expressed genes were identified between the skeletal muscle of MSTN gene-edited cattle and ordinary cattle (FC>1.5, P<0.05), of which the expression was up-regulated in the skeletal muscle of MSTN gene-edited cattle. There were 672 genes and 5 273 down-regulated genes; these differential genes were mainly concentrated in the signaling pathways such as PI3K-Akt, MAPK, p53 and AMPK, and were involved in the developmental processes of muscle satellite cell proliferation and differentiation. Studies have shown that MSTN gene deletion promotes the expression and regulation of genes related to muscle development and differentiation, and increases the cross-sectional area of skeletal muscle fibers, resulting in a muscle hypertrophy phenotype. These results add a little more insight to bovine skeletal muscle research.

Long non-coding RNA (lncRNA) is known to be involved in adipogenesis, but the exact molecular mechanisms remain largely unknown. In order to further explore the potential role of lncRNAs in fat deposition and provide theoretical basis for beef cattle (Bos taurus) improvement, a total of 3 040 lncRNAs was identified in Nanyang and Pinan cattle back fat using high-throughput RNA sequencing technology, among which 3 008 (98.9%) lncRNAs were co-expressed by the two cattle. Characteristic analysis results showed that most (66.02%) lncRNAs were concentrated in length from 1 000 to 2 500 bp and distributed in all chromosomes. Differential expression analysis showed that there were 67 differentially expressed lncRNAs in Pinan cattle compared with Nanyang cattle, among which 32 were up-regulated and 35 were down-regulated. GO analysis results showed that differentially expressed lncRNAs were mainly enriched in lipids transport process, membrane composition and lipid antigen binding and other related biological processes. KEGG pathway analysis showed that these differentially expressed lncRNAs genes were mainly enriched in glycerophospholipid metabolism, ether lipid metabolism, triglyceride metabolism, carbohydrate digestion and absorption pathways. In order to explore the function mechanism of lncRNA, lncRNA-miRNA-mRNA control network was constructed. It was found that LOC104972839/LOC107131703-miR-27b-phospholipase A2 group IVF (PLA2G4F)/ major facilitator superfamily domain containing 2A (MFSD2A) might play an important role in fatty acid degradation and fat deposition. It can be used as a candidate target to study the mechanism of fat development. To test the reliability of sequencing results, qRT-PCR was used for expression level identification, and online software was used for coding ability prediction. The results showed that the expression level of differentially expressed lncRNAs was consistent with the RNA-sequencing results, and the coding ability was very low. In conclusion, this study provides new insights into the discovery and annotation of lncRNA related to fat deposition, and provides basis for further elucidation of the molecular regulatory mechanism of fat deposition in cattle.

Heat stress leads to casein reduction in milk which adversely affects milk quality. The study was aimed to identify the casein derived circular RNA (circRNA) involved in heat stress response and to provide new insight for further elucidation of the molecular mechanism of heat stress. In the previous study of this research group, we obtained the genome wide circRNA expression data of mammary gland of cows (Bos taurus) by RNA-seq. Four circRNAs were randomly selected and the back splice junction sites were confirmed by Sanger sequencing. The relative expression level of circRNAs were verified by quantitative real-time PCR, and the results were consistent with that of RNA sequencing. The casein coding genes include casein alpha s1 (CSN1S1), CSN1S2, casein beta (CSN2) and casein kappa (CSN3). The host genes of circRNAs were analyzed and casein derived circRNAs were identified. Moreover, the expression patterns of these circRNAs in different samples and groups were shown by clustering heat map and Venn diagram. Finally, the relative expression level of casein derived circRNAs were analyzed according to the transcripts per million (TPM) data obtained from RNA sequencing. 52 casein derived circRNAs were identified, of which 29, 16 and 7 were derived from CSN1S1, CSN1S2 and CSN2, respectively. No circRNA derived from CSN3 was detected. Compared with the non heat stress group, 5 circRNAs were significantly up-regulated, 3 of which were only expressed in heat stress condition. The casein derived circRNAs identified in this study were significantly differentially expressed in heat stress group compared to the non heat stressed group, which may be involved in the regulation of milk protein synthesis in heat stress response. The study identified the differentially expressed casein derived circRNAs in heat stressed cows, which may contribute to the understanding of the mechanism of casein reduction casused by heat stress.

Pyometra is a common reproductive disease of the female dogs (Canis lupus familiaris). Hormonal disorder and bacterial infections are main predisposing factors. The endometrium is the target organ for hormone response. A large amount of inflammatory cytokines will produce when endometrial cells are abnormally activated. However, the immunoregulatory effects of steroid hormones on canine endometrial stromal cells (ESCs) in an inflammatory environment are still poorly understood. To study the regulation of estradiol (E₂) and progesterone (P₄) on the secretion of Toll like receptor 4 (TLR4) and related inflammatory cytokines in canine ESCs in vitro. The peripheral blood serum of pyometra and healthy canine were collected, and the concentration of E₂ and P₄ were detected by ELISA; canine endometrial ESCs were isolated by collagenase enzymolysis and identified by immunofluorescence. The detected E₂ (0.2 nmol/L) and P₄ (0.1 μmol/L) were added to the cell culture medium, then the effects of E₂ and P₄ on cell growth in vitro were determined by the method of 3- (4,5-dimethylthiazol-2-yl) - 2,5-diphenyltetrazolium bromide (MTT); then the cells were treated with 1 μg/mL lipopolysaccharide (LPS), and qPCR was performed to detect the gene expression of TLR4 and inflammatory cytokines in canine ESCs induced by LPS at 6, 12, and 24 h. The results showed that the cells isolated were ESCs with a high purity rate after immunofluorescence identification. E₂ had no significant effect on the ESCs, while P₄ and combination of E₂ and P₄ could significantly stimulate proliferation of ESCs in vitro. Compared with the control group, the gene expression of TLR4 and inflammatory cytokines were significantly up-regulated after LPS stimulation (P<0.05). Compared with the LPS group, P₄ alone and the combination of two hormones significantly down-regulated the gene expression of TLR4 and inflammatory cytokines caused by LPS stimulation (P<0.05). This experiment proves that E₂ and P₄ play an important role in ESCs. This study provides basic data for clarifying the molecular mechanism of pyometra and the effects of E₂ and P₄ on endometrial innate mucosal immunity.

Immunoglobulin A (IgA) Fc receptor (FcαR) can mediate the immune activation or immunosuppression of cells, which plays an important role in maintaining the homeostasis of immune response. The spleen of bactrian camel (Camelus bactrianus) is one of the main expression sites of FcαR, which can prompt corresponding immune response. In order to promote the research progress of bactrian camel immunology. In this study, the expression of FcαR positive cells in the red pulp, white pulp and marginal region of bactrian camel spleen was studied by preparing FcαR polyclonal antibody and using the spleen of young bactrian camel (1~2 years old), pubertal bactrian camel (3~5 years old) and adult bactrian camel (6~8 years old). The gene sequence of FcαR extracellular region in bactrian camel was firstly isolated and synthesized to construct the recombinant plasmid, which was transferred into Escherichia coli Rosetta for induction expression, and the induction conditions were optimized. The rabbit (Oryctolagus cuniculus) was next immunized with purified FcαR protein to produce a polyclonal antibody. After that, ELISA and Western blot were used to determine the titer and specificity of antibody serum. Finally, immunohistochemical staining and hematoxylin-eosin staining (HE) staining were used to analyze the distribution characteristics and the density of FcαR positive cells in different areas of spleen of bactrian camel in different age groups. The results showed the FcαR plasmid was expressed as inclusion bodies, and the optimal induction conditions were 0.7 mmol/L isopropyl β-D-thiogalactoside (IPTG) 5 h. The optimal elution concentration of hybrid protein was 5 mmol/L imidazole, and the antibody serum titer reached 1∶64 000, which could specifically recognize the recombinant protein FcαR. The distribution of FcαR positive cells was similar in the spleen of young, pubertal and adult bactrian camels, all of which were concentrated around the walls of sinuses and central arterial ducts in the red pulp region, and the positive cell type was dominated by monocytes/macrophages, with expression also observed on neutrophils and dendritic cells. In addition, the density of FcαR positive cells in the spleen of bactrian camels showed an obvious increasing trend from young to adult. The results suggested that the site of expression of FcαR positive cells in bimodal hump spleens was not affected by age, and that the increased number of positive cells in spleens with age provided some physiological basis for the refinement of their immune functions. This study provides reference for further exploring of immunological functions of bactrian camels.

Polyamines are involved in regulating the development and regeneration of nervous system.To study the developmental changes of polyamine metabolism in cerebrum and cerebellum tissues from Nonghua ducks (Anas platyrhynchos) at different day-age, polyamine content in cerebrum and cerebellum tissues from ducks at 0~120 day-age were determined by HPLC. The relative abundance of genes related to polyamine metabolism were determined by qPCR. The result showed that contents of spermidine and spermine in cerebrum and cerebellum tissues were significantly higher than putrescine (P<0.05), and the contents of polyamine decreased with the increase of day-age. Putrescine content (30.80 mg/kg) in cerebrum tissues at 60 day-age was significantly higher than others (P<0.05). Spermidine content (643.26 mg/kg) in cerebrum tissues at 0 day-age was significantly higher than others (P<0.05). Spermine content in cerebrum tissues at 0 day-age (1665.95 mg/kg) and 120 day-age (1623.66 mg/kg) were significantly higher than others (P<0.05). Spermidine (54.09 mg/kg) and spermine (413.5 mg/kg) contents in cerebellum tissues at 0 day-age were significantly higher than others (P<0.05). Putrescine content in cerebellum tissues at 0 day-age (26.46 mg/kg) and 120 day-age (22.03 mg/kg) were significantly higher than others (P<0.05). The expression levels of key genes related to polyamine metabolism decreased in cerebrum tissues of ducks at 30 day-age, increased at 60 day-age, decreased at 90 day-age, and then increased at 120 day-age. The expression levels of key genes related to polyamine metabolism in cerebellum tissues of ducks at 0 day-age were relatively higher than that of other ducks. The trend in gene expression levels was different. This study reveals the changes of polyamine content and the expression levels of key genes related to polyamine metabolism in brain tissues from different age ducks, and provides basic data for polyamine regulating brain growth and development in ducks.

The key enzymes of glycolysis pathway play an important role in regulation and adaptation of echinoderms to the changes of marine environment.In order to clarify the sequence information and expression pattern of phosphofructokinase (PFK) in Strongylocentrotus intermedius, as well as, to understand the effect of "acidification-high temperature" stress on its expression and biological activity, in this study, the rapid-amplification of cDNA ends (RACE) technique was used to clone the full-length cDNA sequence of PFK gene in S. intermedius (SiPFK)(GenBank No. OM780114), and then, the characteristics of SiPFK were analyzed by bioinformatics software, at last, the relative expression pattern of SiPFK and enzyme activity of SiPFK in intestines and gonads of S. intermedius under "acidification-high temperature" stress were investigated. The results showed that, the full- length cDNA of SiPFK gene was 2 787 bp, encoding 840 amino acids, the theoretical isoelectric point of SiPFK protein was 7.48 with a predicted molecular weight of 92.01 kD; The amino acid sequence of SiPFK protein was the most similar to that from S. purpuratus (similarity: 96.20 %); The results of qPCR showed that SiPFK gene specificly expressed in all examined tissue, the relative expression level of SiPFK and total SiPFK enzyme activities in intestines and gonads of S. intermedius were changed after 60 d of "acidification-high temperature" stress, suggesting that "acidification-high temperature" might affect the metabolic process of sea urchins by regulating the expression and activity of key enzymes of glucose metabolism. This study provides an important reference basis for exploring the response of echinoderms to future marine environmental changes.

Protein kinase A (PKA) is the core element of cAMP signal transduction pathway in eukaryotie, which regulates the growth and development of filamentous fungus through phosphorylation of a variety of active proteins. In order to clarify the possible interaction protein of PKA in Setosphaeria turcica, and further analyze the molecular mechanism of its acts, in this study, the complete coding region of StPKA-C1/2 was amplified from the total complementary DNA of S. turcica to construct the expression vector pGST-StPKA-C1/2, which was introduced into Eschaeria coli. The fusion protein GST-StPKA-C1/C2 was induced in E. coli by isopropyl β-D-thiogalactopyranoside and then purfied by affinity chromatography. The GST pull-down strategy was used to screen the interacting proteins of StPKA-C1/2. The interacting proteins were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that 69 StPKA-C1 interacting proteins and 70 StPKA-C2 interacting proteins were identified. The functional annotations of proteins included catalytically active protein, enzyme regulating active protein, transport electron carrier protein, molecular chaperone protein, structural molecular protein, metabolic enzyme, etc. Venn analysis found that StPKA-C1 shared 52 interacting proteins with StPKA-C2, with only 17 proteins interacting with StPKA-C1, and only 18 proteins interacting with StPKA-C2. The target protein SETTUDRAFT_ 168881, as a member of the elongation factor G (EFG) family, may regulate hyphal growth. The target protein SETTUDRAF_37808 belonged to the Fimbrin protein family of microfilament-binding proteins and may be involved in the regulation of the actin cytoskeleton, oxidative stress response, and morphogenesis. It was suggested that StPKA-C1/2 might play important biological functions by interacting with these target proteins.The above results preliminarily clarified the potential interaction protein of StPKA-C1/2. This study provides a theoretical basis for further analyzing the molecular mechanism of PKA.

The attenuated bacterial pathogen Listeria monocytogenes (LM) have been widely studied for cancer immunotherapy as a safe vaccine via presenting tumor-associated antigens. The most common attenuation strategy is to delete the virulence-associated genes. The Listeria attenuated double genes deletion strain ΔactAΔinlB (ΔAB) has become the ideal platform for cancer vaccine research and development. This study aim to develop a ΔAB-based vaccine candidate that can efficiently express and present the cervical cancer-associated E7 antigen (ΔAB-E7) to evaluate the immunotherapeutic efficacy in the mice (Mus musculus) cervical cancer model. The bacterial genetic methods were employed to construct the ΔAB and ΔAB-E7 by introducing E7 with in-frame fusion to the virulence factor listeriolysin O (LLO). The ΔAB-E7 was verified whether the E7 antigen could be expressed in fusion with LLO by Western blot. The infection characteristics of these strains were studied, including in vitro and intracellular growth, bacterial proliferation, and pathogenicity in mice. Then, the immunotherapy efficacy of ΔAB-E7 was evaluated on the mice cervical cancer model by tracking tumor growth. The results showed that ΔAB-E7 could efficiently express and secrete LLO-fused E7, the in vitro and intracellular growth abilities of ΔAB and ΔAB-E7 were comparable to those of wild-type strain, while the virulence was significantly attenuated in mice with the highly-reduced proliferation ability in mice organs. Importantly, vaccination of ΔAB-E7 could inhibit cervical cancer growth in mice, eliciting promising anti-tumor efficacy. Collectively, this study present a favorable Listeria-based vaccine candidate against cervical cancer, which provides new strategies for future research and applications of immunotherapies for human cervical cancer and other cancers.

Excess nitrogen in the aquaculture waterbodies has caused deterioration of the aquaculture environment, and seriously affects the benefits of aquaculture. Microbial denitrification is one of the most economical and effective measures for the treatment of nitrogenous pollutants. In this study, a high-efficiency aerobic denitrifying bacteria DS2 (GenBank No. PRJNA838789) was isolated from the biofilter filler of circulating water aquaculture system. Strain DS2 was identified as Pseudomonas putida based on physiological and biochemical characteristics, and analysis of 16S rRNA gene sequence. Single-factor experiment, denitrification experiment, and whole-genome sequencing technology were used to analyze the optimal growth conditions, denitrification performance and mechanism of strain DS2. The results showed that the strain grew fastest under the condition of 30 ℃, pH7, 250 r/min, and salinity of 10‰~20‰. After 24 h of cultivation in heterotrophic nitrification medium (HNM), short-cut denitrification medium (SDM), denitrification medium (DM), and simultaneous nitrification and denitrification medium (SNDM), the total nitrogen (TN) removal efficiency of strain DS2 was 93.50%, 55.60%, 65.98%, 90.10%, respectively. Moreover, the functional genes related to nitrogen metabolism were defined in the genome of strain DS2, such as narB, nirK, nirA, norB, and nirB. Finally, in aquaculture wastewater treatment experiment, strain DS2 obtained 99.72% removal rate of nitrate and 89.52% removal rate of TN in 12 h. The present study indicates that Pseudomonas putida strain DS2 is a high efficiency aerobic denitrifying bacterium, and possesses great potential for application in aquaculture wastewater treatment.

The endoplasmic reticulum (ER) is a well-orchestrated organelle for protein folding and highly sensitive to changes of cellular homeostasis. Alterations in the protein-folding environment cause accumulation of unfolded/misfolded proteins in the ER which affect a variety of cellular signal transduction pathways, including calcium and redox homeostasis, inflammation, and apoptosis. Upon ER stress, a series of adaptive mechanisms known as the unfolded protein response (UPR) are activated to adapt to protein-folding alterations and restore homeostasis, and failure to adapt to ER stress leads to apoptosis. Production of reactive oxygen species (ROS) has been linked to ER stress and the UPR. ROS has been shown to be toxic but also function as signaling molecules. It can be produced in the cytosol and several organelles, including the ER and mitochondria. Altered redox homeostasis in the ER is sufficient to cause ER stress, which in turn, can induce the production of ROS in the ER and mitochondria. Unremitting oxidative stress and ER stress will initiate apoptosis. In this review, we summarize the mechanism of ER stress and oxidative stress, the intertwined relationship between them, and the emerging roles of the UPR and oxidative stress in viral infections.

Deubiquitination is the reverse process of ubiquitination. Deubiquitinase (DUB) removed the ubiquitination of substrates, thereby regulating various life processes. In recent years, more and more reports have shown that deubiquitination could regulate viral infection. In this paper, the current research on how deubiquitination regulates the infection of plant virus and animal virus is reviewed from four aspects including types, function characteristics, regulatory roles in vivo, and regulatory roles in viral infection of DUBs, Some suggestions are made for further study on how deubiquitination regulate viral infection.

CD163 is a macrophage-specific protein in the cysteine superfamily rich in scavenger receptors and is the most important cellular receptor of Porcine reproductive and respiratory syndrome virus (PRRSV). Induced pluripotent stem cells (iPSCs) are pluripotent stem cells with biological characteristics of embryonic stem cells obtained by reprogramming after introducing foreign transcription factors into somatic cells. Firstly, porcine (Sus scrofa) CD163 gene was transferred into porcine induced pluripotent stem cells (piPSCs) by lentiviral vector system, and an induced pluripotent stem cell line stably expressing CD163 was established, which was named CD163OE-piPSC. The susceptibility of the cell line to PRRSV was verified by virus infection, and the pluripotency and proliferation of iPSCs were not affected by alkaline phosphatase staining, cell population doubling time test and 5-ethynyl-2'- deoxyuridine (EdU) cell proliferation test.The cell lines obtained in this study could be effectively used to study the pathogenesis of PRRSV-host interaction and screen the target molecules of viral diseases.

Stephylium solani is an important plant pathogenic fungus with a wide range of hosts. Loop-mediated isothermal amplification (LAMP) technology has been widely used in many fields such as plant protection because of its strong specificity, high sensitivity, rapid response and low cost. To establish a rapid, efficient and accurate detection technology for Lycopersicon esculentum petiole leaf spot, SYBR GreenⅠwas used as the indicator and the glyceraldehyde-2-phosphate dehydrogenase (gpd) gene was used as the target. 6 primers (2 inner primers FIP/BIP, 2 outer primers F3/B3 and loop primer LB25/LF25) were designed for LAMP amplification. At the same time, a set of rapid LAMP detection system for S. solani was established by accelerating the reaction with loop primers. The results of specificity verification showed that only 16 strains of S. solani had positive amplification and had high specificity. The sensitivity results showed that the LAMP detection sensitivity of the system was 2.4×10³ fg/μL, significantly higher than the PCR detection technology. The results showed that the addition of ring primers could shorten the reaction time to 27 min, and effectively accelerate the LAMP reaction. Through the color reaction of calcein, under natural light, only the reaction solution with the template of S. solani turned green, and the other reaction solutions were orange. The system was used to detect the dynamic changes of pathogen content in tomato leaves after inoculation with S. solani. 12 h after inoculation, the LAMP detection had a positive color reaction, and the color gradually became darker with time, which was consistent with the severity of tomato leaves after artificial inoculation. The reaction results can be directly observed. The loop primer accelerates the reaction to further shorten the detection time to 27 min, which can quickly and accurately reflect the pathogen content. The LAMP detection system established in this study has strong advantages in the rapid detection of S. solani. It can be used for the rapid detection of latent and non symptomatic stages of S. solani. It is of great significance for the timely and scientific control of tomato petioles leaf spots.

Rabbit hemorrhagic disease (RHD) is an acute hemorrhagic infectious disease caused by Rabbit hemorrhagic disease virus (RHDV). RHDV2 VP60 protein (pET-32a-R2VP60 protein) and RHDV VP60 protein (pET-32a-R1VP60 protein) were expressed and purified by prokaryotic expression in order to prepare specific monoclonal antibody (McAb) of RHDV2 and explore the differential diagnosis technology between RHDV2 and classical rabbit hemorrhagic disease virus (RHDV). BALB/ C mice were immunized with pET-32a-R2VP60 protein, and specific monoclonal antibodies against RHDV2 were screened by indirect ELISA using pET-32a-R1VP60 protein and pET-32a-R2VP60 protein, respectively. Conventional hybridoma cell fusion technique was used. By cloning, subcloning, ascites preparation, titer determination, Western blot analysis, hemagglutination - hemagglutination inhibition test and subtype identification, a hybridoma cell strain resistant to RHDV2 was successfully screened out, named 2A1. The results showed that 2A1 fusion cell had about 100 chromosomes, secreted IgG3 and Kappa subtype McAb. Hybridoma 2A1 antibody secretion was stable, ascites purification McAb titer was high, good specificity, indirect ELISA titer was up to 1∶128 000. Western blot analysis showed that McAb 2A1 reacted to RHDV2 virion and pET-32A-R2VP60 protein, but negatively with RHDV1 virion and pET-32a-R1VP60 protein. Hemagglutination and hemagglutination inhibition tests showed that 2A1 purified McAb could effectively inhibit hemagglutination of RHDV2 with HI value up to 10lg2. This study provides scientific materials and lays a foundation for the establishment of specific and rapid serological diagnosis methods and kit development of RHDV2-specific McAb 2A1.