Autophagy is a self-defense mechanism under environmental stress in plants. It has been proved that autophagy is involved in salt tolerance of wheat (Triticum aestivum), but the role and regulation mechanism of autophagy in salt tolerance are not clear. In this study, salt-tolerant wheat 'DEKANG961' and salt-intolerant wheat 'LANKAO323' were used as materials, and the methods of lysosomes' fluorescence probe, Western blot and so on were used to detect autophagy level. It was found that salt stress could promote autophagy, but the autophagy level of 'DEKANG961' was higher than that of 'LANKAO323', the gene expression of PYL1 (pyrabactin), PYL2, PYL3 and sucrose non-fermenting related protein kinase 2 (SNRK2), the key genes of abscisic acid (ABA) signaling pathway in 'DEKANG961', were significantly higher than that in 'LANKAO323', and the autophagy level of 'DEKANG961' was significantly higher than that of 'LANKAO323'. It was concluded that ABA signal pathway could promote the autophagy to enhance salt tolerance in wheat. Therefore, this study has certain theoretical and practical significance for further study on salt-tolerant mechanism of wheat and breeding new salt-tolerant wheat varieties.

Plant glycosyltransferases (GTs) play an important role in regulating growth and stress adaptation. To further explore and study the biological functions of new glycosyltransferases in rice (Oryza sativa), in this study, a cold-upregulated glycosyltransferase-like gene 1 (OsCUGT1) was isolated and cloned, which was based on the cold treatment transcriptome library of Guizhou landrace rice 'Pingtang Heinuo' (O. sativa ssp. japonica). The results of qRT-PCR showed that the expression of OsCUGT1 gene was significantly induced by cold treatment. The subcellular localization results showed that it was localized in the chloroplasts. Phylogenetic analysis showed that OsCUGT1 was conserved in monocot plant species. Furthermore, sequence alignment revealed that there were several SNP sites between 'Pingtang Heinuo' and O. sativa ssp. Nipponbare in the OsCUGT1 gene. Among them, the site of 511 bp was changed from C to A, which consequently caused the 170 th amino acid to change from lysine (Q) to glutamine (K). Diversity analysis of the OsCUGT1 gene sequence revealed that there were 12 SNP sites of them were found in the exon region. To further explore the biological function of OsCUGT1, the OsCUGT1 gene was edited by CRISPR/Cas9 technique. The targeted editing mutants of oscugt1 were successfully obtained by optimizing Agrobacterium-mediated transformation method, screening of hygromycin selection and sequencing analysis. The results of this study can provide research materials and theoretical basis for further clarifying the role of OsCUGT1 in rice growth development and stress resistance．

Plants often show significant heterosis at the early developmental stage, and the seedling stage is the most important stage for rice (Oryza sativa) architectural development and yielding ability. In order to investigate the relationship between heterosis performance as well as genes expression levels of hybrid rice at seedling stage and the heterosis of yield in field, and to explore the possibility of yield potential prediction for hybrid rice with their performance at seedling stage, 18 representative hybrid parental lines were obtained to make an incomplete diallel crosses, and 45 hybrids rice were obtained. Heterosis performance of root, shoot, and genes expression pattern at the seedling stage, as well as the relationship between yield and traits mentioned were systematically analyzed. The correlated relationship was detected between the above-ground and underground traits at the same seedling stage, as well as traits in two different periods for the same tissue. 12 pairs of comparison between the trait value were significantly correlated, and two seedling traits were positively correlated with yield (P<0.05). Middle parent heterosis (MPH) heterosis at the early stage of seedling (3DAS) was higher than any other stages. 13 pairs of comparison between MPH values of seedling traits were positively correlated. Root length (RL)-3DAS, RL-14DAS and plant height (PH)-3DAS were correlated with the MPH value of yield (P<0.05). In addition, the expression patterns of the 4 root development related genes were observed at 17 DAS among hybrids and parental lines. Three of them were correlated with the dry root weight and shoot at 17 DAS, and all of them were correlated with the yield in the field (P<0.05). High level of heterosis performances were detected at early seedling stage. The correlations of heterosis value among traits at seedling stage were ubiquitous. Some seedling traits and the expression level of root related genes in hybrids at seedling stage were significantly correlated with the yield of hybrids. This study shed light on the model for heterosis prediction based on the early traits of hybrid rice and also provides a theoretical basis for hybrid rice breeding.

Plants increase their tolerance through accumulation of polyamines under salt stress. S-adenosylmethionine synthetase (SAMS) is an important rate-limiting enzyme involved in the biosynthesis polyamines. In this study, HvSAMS2 gene was isolated from farmhouse two-row barley (Hordeum vulgare), and analyzed for bioinformatics, tissue expression specificity and abiotic stress responses, subcellular localization and salt tolerance in Saccharomyces cerevisiae. The full-length cDNA sequence of HvSAMS2 was 1 297 bp without introns, which contained a 1 185 bp ORF that encoded 394 predicted amino acids, with a molecular weight of 42.83 kD and a hypothetical protein isoelectric point of 5.58. Multiple alignment analysis based on the amino acids indicated that HvSAMS2 was highly conserved to SAMS proteins from different species,and had the highest similarity of 93.06% to Aegilops tauschii SAMS2. Subcellular localization revealed that HvSAMS2 was located in the nucleus and cell membrane with a higher level in the nucleus. The qRT-PCR analysis results in different tissues indicated that HvSAMS2 was preferentially accumulated in root, and then leaves, following by stem. Furthermore, HvSAMS2 expression level was significantly enhanced upon drought, low temperature, salt, and methyl jasmonate (MeJA) treatment. Complete gene of HvSAMS2 was inserted into expression vector pYES2 and transformed into Saccharomyces cerevisiae INVSc1 for heterologous expression, and the expressing HvSAMS2 improved the salt tolerance of the engineered strain under salt stress. In summary, HvSAMS2 might play an important role in responding to abiotic stresses, and this study could provide basic data for further study on the molecular mechanism of salt resistance of SAMS genes.

Carotenoids, as important members of photosynthetic system and antioxidant system in plants, play important roles in plant growth and responding to stress. In order to study the effects of lycopene β-cyclase (Lcyb) and brassinazole-resistant 1 (BZR1) genes on the regulation of carotenoid biosynthesis, the virus-induced gene silencing (VIGS) system of Lcyb and BZR1 gene mediated by Tobacco rattle virus (TRV) was constructed in pepper (Capsicum annuum 'Shan Zao hong'). The gene expression of Lcyb and BZR1 and seedlings of RNAi lines were obtained by vein injection. The uninfected pepper plants were used as the normal control, the infected plants with TRV2-PDS gene were used as the positive control, and the infected plants with empty TRV2 vector were used as the negative control. qRT-PCR was used to detect the Lcyb and BZR1 genes expression of silenced pepper, and the accumulation of carotenoids in silenced pepper leaves was determined. Results showed that TRV2-PDS leaves appeared bleaching phenomenon after 21 d, and Lcyb and BZR1 gene expression were significantly lower (P<0.05), and carotenoid content of 2 kinds of silenced pepper leaf was significantly lower than that of normal control plants. It is indicated that Lcyb and BZR1 genes were involved in synthesis of carotenoids in pepper leaf. The study of TRV-mediated gene-silencing could be used for subsequent functional gene identification of pepper.

Genomic imprinting is an epigenetic phenomenon that causes genes to be monoallelically expressed dependent on the parent origin. In mammalian, imprinted genes play an important role in placental and embryonic development, as well as in the growth of individuals. The zinc finger CCCH-type containing 12C (ZC3H12C) gene encodes a transcription factor containing a single CCCH-zinc finger domain and is involved in regulating a variety of human immune diseases. ZC3H12C gene was first identified imprinted in the human (Homo sapiens) placenta, but its imprinting status has not been established in cattle (Bos taurus). In this study, the structure of the bovine ZC3H12C gene was first analyzed. Then by the method of comparing genomic DNA and cDNA amplification products based on single nucleotide polymorphism (SNP), the allele expression of ZC3H12C gene was analyzed in bovine tissue. The results showed that the ZC3H12C gene exhibited monoallelic expression in all 6 tested tissues, including heart, liver, spleen, lung, kidney, and brain, but they showed biallelic expression in the bovine placenta. The methylation status of ZC3H12C gene were analyzed by sulfite sequencing method. A differentially methylated region was found in intron 1 of ZC3H12C X2 spliced transcript, but in the placenta this region showed hypormethylated. These results suggesting that the monoallele expression of the ZC3H12C gene in bovine tissues is regulated by DNA methylation modification. These results provide valuable information on the further study of the function of ZC3H12C gene.

Tree frog (Rhacophorus) antimicrobial peptide Cathelicidin is a kind of small molecular peptide with antibacterial activity, which has potential application value in medical treatment, animal husbandry and some other aspects, but its eukaryotic expression is rarely reported. In this study, tree frog antimicrobial peptide Cathelicidin gene was synthesized based on the preferential codon usage of Pichia pastoris, then ligated to the expression vector pPIC9K and transformed into P. pastoris GS115 by electroporation. The positive transformants containing multi-copy gene insertions were screened using high concentration G418 and confirmed by PCR and reverse transcription PCR (RT-PCR). The recombinant Cathelicidin was induced for 72 h with 1.0% methanol, and the supernatant of culture medium was collected for in vitro antibacterial activity detection and toxicity analysis. The results showed that Cathelicidin was expressed successfully in P. pastoris with antibacterial activity against gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherchia coli), the minimal inhibitory concentration were (1.175±0.002) and (2.35±0.001) μg/mL respectively, and it was no hemolysis under the minimum inhibitory concentration. This study provides basic data support for industrial production of a new tree frog antimicrobial peptide.

Finding suitable cellulose degrading bacteria is the key to the effective application of cellulose in Inner Mongolia and other northern regions. In order to obtain some efficient cellulose-degrading bacteria, traditional isolation and enrichment culture technologies were used to isolate cellulose-degrading bacteria from the soil in western region of Inner Mongolia, and the degradation characteristics of cellulose-degradation bacteria were analyzed. A preliminary identification was based on analyzing the similarities of 16S rDNA gene sequences of the isolates. Cellulase activity was determined by DNS (3,5-dinitrosalicylic acid) method. And the enzyme-producing conditions of the highest enzyme activity cellulase-producing strain was further optimized. The results showed that a total of 59 cellulolytic bacteria were isolated, including 36 cold-adapted cellulose-degrading bacteria isolated at 10 ℃ and 23 cellulose-degrading bacteria isolated at 28 ℃. The 16S rDNA gene sequences indicated that these strains belonged to Alpha-Proteobacteria, Gamma-Proteobacteria, beta-Proteobacteria, Bacteroidetes, and Actinobacteria, respectively. Alpha-Proteobacteria was the first dominant group of room temperature cellulose-degrading bacteria, beta-Proteobacteria and Actinobacteria were the second dominant group. Gamma-Proteobacteria was the first dominant group of cold-adapted cellulose-degrading bacteria, Bacteroidetes was the second dominant group. At the genus level, Pseudochrobactrum was the first dominant genus of room temperature cellulose-degrading, while Pseudomonas was the second dominant genus, and Pseudomonas was the first dominant genus of cold-adapted cellulose degrading bacteria at low temperature, while Sphingobacterium was the second dominant genus. Among these strains, strain CB04 (belonging to Cellulomonas) had the highest carboxymethyl cellulose (CMC) activity. After optimization, the optimal conditions for enzyme production were as follows: the yeast extract was nitrogen source, the culture time was 4 d, the pH value was 7, and the CMC enzyme activity was up to 114.6 U/mL. The strain CB04 was a cellulase producing bacterium with great development potential. This study preliminarily revealed the composition and ability of cellulose-degradation bacteria in western region of Inner Mongolia, which provides a theoretical and practical basis for understanding and using cellulose-degradation bacteria for other applications.

Porcine epidemic diarrhea virus (PEDV) is a common pathogen that causes diarrhea in piglets (Sus scrofa). The intestinal mucosal barrier is destroyed after PEDV-infected, which leads to diarrhea in piglets. Adherens junction is an important part of the intestinal mucosal barrier. In order to investigate the effects of PEDV on the intestinal mucosal barrier and expression of adherens junction protein genes in piglets, the effect of PEDV on the intestinal mucosa of piglets were observed by electron microscopy in paraffin sections in this study. The expression of E-cadherin, Integrin and Nectin-1 genes in the intestines of PEDV-infected and normal piglets was examined by qRT-PCR, as well as in PEDV infected intestinal porcine epithelial cell line J2 (IPEC-J2). And their relationship with intestinal barrier function was further analyzed. The results showed that the intestinal villi of PEDV-infected piglets were damaged and shed, the lamina propria was exposed and the intestinal glands were atrophied, while the intestinal mucosal barrier structure of normal piglets were intact and distinct. The results of qRT-PCR indicated that the expression of E-cadherin gene in duodenum of PEDV-infected piglets was significantly lower than that of normal piglets (P<0.05), and the expression of Nectin-1 gene in jejunum was significantly lower than that of normal piglets (P<0.01). There was no significant difference in other groups. In vitro cell infection experiments also showed that the expression of E-cadherin and Nectin-1 genes was significantly reduced after IPED-J2 cells infected by PEDV. These results revealed that the intestinal mucosal barrier of epidemic diarrhea piglets was damaged and the adherens junction was destroyed. The down-regulation of E-cadherin and Nectin-1 was not conducive to maintain the integrity of intestinal mucosal barrier, and the damage of intestinal mucosal barrier might further promote the occurrence of diarrhea in piglets. This study initially revealed the effects of PEDV infection on intestinal mucosal barrier and adherens junction of piglets, and provides a new insights into the pathogenic mechanism of PEDV from the perspective of the intestinal barrier.

It is vital to obtain the viral genome sequence for taxonomy, diagnostic reagent development and epidemiology studies of Reoviridae viruses. In this study, the whole genome sequences of Reoviridae viruses belonging to 8 different species were acquired through full-length cDAN amplification (FLAC) and next generation sequencing (NGS) techniques, including Bluetongue virus (BTV), Epizootic haemorrhagic disease virus (EHDV), Palyam virus (PALV), Guangxi orbivirus (GXOV), Tibet orbivirus (TIBOV), Mammalian orthoreovirus (MRV), Banna virus (BAV) and Mangshi virus (MSV). The FLAC technique established by this study showed highly sensitive and could amplify the complete genomic DNA of different species of Reoviridae viruses through one RT-PCR reaction, without any sequence information or primer designing for target viruses. The genome size of the viruses sequenced in this study ranged from 18 266 to 23 605 bp with 10 or 12 genomic segments, which were obtained in a week through NGS sequencing and de novo assembling. The amount of data generated by NGS for different Reoviridae virus strains ranged from 1.6 to 1.9 GB, with the number of reads (Q>30) used for assembling virus genome ranged from 293 016 800 to 423 210 600 and the sequencing depth for each base site in the viral genome ranged from 6 000 to 20 000. The establishment of the FLAC and NGS techniques could provide technical guarantee for rapid and accurate acquisition of the whole genome sequence of Reoviridae,which would promote the studies on evolution, diagnostic reagents development and epidemiology of Reoviridae viruses.

WRKY named for its special heptapeptide conserved sequence WRKYGOK, is one of the largest families of plant-specific transcription factors (TFs). It is widely involved in the response of plants to biotic, abiotic and hormonal stresses. WRKY TFs mainly regulate the expression of target genes by specifically binding cis-acting elements (T)TGAC(C/T) (W-box) of the promoters of target genes, participate in plant signaling pathways in response to different biotic, abiotic and hormonal stresses, and interact with multiple proteins to achieve their functions in different signal transduction pathways. In this review, the structure, classification, origin and evolution of WRKY TFs and its function and regulatory mechanisms in response to biotic, abiotic and hormonal signal were described, which aims to systematically summarize research progress of WRKY TFs in plants. This review provides relevant researchers with an in-depth understanding of WRKY TFs and the molecular mechanism how it mediates to improve plant resistance, and also provides a useful reference for crop genetic improvement.

Retrotransposons are a type of functional element that can complete self-replication in the genome, use RNA as an intermediary, and integrate into other sites of the genome under the action of reverse transcriptase. The increasing copy number of retrotransposons in the biological genome is a major reason for the enlargement of the genome, and it is also the main source of genetic variation and the driving force for genome evolution. The transposition of retrotransposons breaks, rearranges, and forms chromosomes. Structural mutations such as insertion mutations also participate in the apparent regulation of the genome and the formation of heterochromatin structures, and are closely linked to changes in the structure and function of genes; the effects of retrotransposons on genomes, transcriptomes, and functional genes have become postgenomes research hotspots of the times. This article reviews the structural characteristics of animal transposon, its function in gene expression, the role of transposon in animal genome evolution in recent years, and its application as a molecular marker in animal production. This review provids reference for the functional role of retrotransposons in animal genomes and transcriptomes, as well as in animal production, and animal genetics and breeding.

Transgenic technology is the core of modern biotechnologies, and is also one of the most controversial issues in public in recent years. To better understand the current cognition of genetically modified organisms (GMO) among university students, a questionnaire survey on 1 085 students from 174 universities in 31 provinces of China was conducted. The results showed that university students acquired relevant information about GMO from different sources in the following orders: The computer (or mobile phone) internet>television>books and periodicals>daily communication>courses or seminars. The ratio of "support" vs. "oppose" GMO among university students (3.22/1) was 11 times higher than that of the public opinion (0.29/1). The support was highly correlated with their educational levels, specialities, and knowledge of GMO. When shopping, nearly 55% of students did not care whether it was GM food. About 36.6% were willing or on demand to purchase GM food, 50.6% would comprehensively consider relevant factors such as "cost performance" and then decided whether to buy it, and the remaining 12.8% refused to purchase GM food. Approximately 25% students were optimistic about the industrialization of GM crops in China, whereas nearly 10% were pessimistic about it. Based on comprehensive analyses of this survey, a few suggestions on the science popularization of GMO and general course setting and reformation in universities were proposed. After analyses of the recent progresses of developing GM plants in China, some countermeasures were also summarized, which will be helpful to promote the healthy development of GM industrialization in future.

Along with the increased commercialization and planting of a range of genetically modified (GM) crops globally, the biosafety of GM crops has become a hot topic and major public concern. To elucidate the optimal molar ratio for co-transformation and develop a safe, high-efficiency wheat (Triticum aestivum) transformation system, in this study, a combination of β-glucuronidase (gus) and a herbicide resistance gene (bar) was used to select stable transformants. Six independent co-transformation experiments were performed using either the minimal cassette Ubi-bar or the whole of pAHC20, with the Ubi-gus minimal cassette at different molar ratios. The whole pAHC25 plasmid was used as a reference, which was transferred into immature embryos of the wheat variety 'Kenong 199'. The results showed that the transient expression rate of gus gene increased significantly with the increase of Ubi-gus molar concentration, but there was no correlation between transient expression and stable expression. The transformation frequency differed significantly among combinations, in which the highest was obtained by combining Ubi-bar or pAHC20∶Ubi-gus at a molar ratio of 1∶2. The transformation frequency was significantly lower using the whole plasmid than the minimal constructs with co-transformation at a molar ratio of 1∶2, but significantly higher than that with 1∶1 and 1∶3. The stable integration, expression, and inheritance of the gus were confirmed by GUS assays and Southern blotting. The positive transgenic lines exhibited normal morphology except for poor seed set. This system may facilitate future transformation studies.

China imports a large number of transformed soybeans (Glycine max) from abroad every year, mainly cp4 epsps transformed soybean. It is necessary to establish a rapid-effective ELISA method for identification of cp4 epsps transformed soybean. This study, CP4-EPSPS protein was expressed in segments by synthetic peptides method, the antigenic epitopes recognized by 5 strains of CP4-EPSPS monoclonal antibodies were mapped. The monoclonal antibodies that recognized different epitope were paired by matrix approach, the working antibody and working concentration of antibody were determined by the maximum value of P/N. The optimal detection conditions were determined by controlling variable method, and the cp4 epsps transformed soybean rapid double antibody sandwich ELISA detection method was established. The performance of the established detection method was evaluated by specific test, repeatability test and positive determination value test, at the same time, 130 samples were detected by the established detection method and commercial kits, and compared their coincidence rates. The monoclonal antibodies 1D10 and 2D3 were paired and had the best matching detection effect by matrix approach. The monoclonal antibody 2D3 and 1D10 were determined as capture antibody and detection antibody, working concentration of 2D3 was 20 μg/mL, 1D10 was 10 μg/mL. The capture antibody coating condition was 37 ℃ 2 h, 4 ℃ overnight. The sample and detection antibody were added in ELISA plate at the same time, then co-incubated at 37 ℃ for 10 min. The sensitivity of this method was 160 (g/mL) times dilution for leaf and seed, the optimal dilution rate for soybean leaves and seeds were 10~80 and 10~40 (g/mL), respectively. The intra-plate and inter-plate variation coefficient was less than 25%, and the positive determination value was 1.41. 70 soybean leaves, 40 soybean seeds and 20 soybean milk samples were detected, the coincidence rate was 100%, and no cross react with other proteins. This detection method has good accuracy, repeatability and specificity. This testing program can be completed within 30 min, and is suitable for rapid qualitative detection for plant, seed and soybean milk.

As a new type of CRISPR-derived tool, the base editors have the advantages of accuracy, efficiency, and low off-target, and have been used in many organisms. Single base editors are mainly divided into 2 types-adenine base editor (ABEs) and cytosine base editor (CBE), which can realize single base mutation of A:G and C:T, respectively. The base editor has been continuously modified and has been developed with multiple versions. The Booroola fecundity (FecB) gene is the major gene of sheep (Ovis aries) prolificacy. FecB gene is derived from A746G site mutation of bone morphogenetic protein receptor 1B (BMPR1B) of Booroola sheep, resulting in 249 amino acids encoded being converted from glutamine (Q) to arginine (R), further increasing ovulation and lambing. The fibroblast growth factor 5 (FGF5) gene can regulate the hair follicle cycle, and C-T base mutation on FGF5 gene can significantly affect the length of cashmere fiber, which is crucial for improving cashmere yield of cashmere goat (Capra hircus). In order to explore the editing efficiency of different base editors in sheep and goat, the Booroola fecundity (FecB) of Tan sheep and the fibroblast growth factor 5 (FGF5) of Shaanbei white cashmere goat were selected. By constructing sgRNA-U6 vector, cell culture, transfection, drug screening, Four single base editors, xCas9-ABE (adenine base editor), ABEmax4, xCas9-BE4 and BE4max, were used to perform single base fixed-point editing on Tan sheep and Shaanbei white cashmere goat fetal fibroblasts. The editing efficiency of xCas9-ABE, ABEmax4, xCas9 -BE4 and BE4max in Tan sheep and Shaanbei white cashmere goat were determined. The results showed that in sheep fibroblasts, the editing efficiency of ABEmax4 was 46.15%, and the editing efficiency of xCas9-ABE was 38.46%, which was 27.4% and 19.71% higher than that of ordinary editor ABE7.10. On goat fibroblasts, the editing efficiency of BE4max It was 92.86%, which was 56.5% higher than the ordinary editor BE3, but no editing is generated by xCas9-BE4. In summary, the base editing application of sheep fetal fibroblasts, the optimal base editors of ABEmax4 and BE4max were obtained. The study screened the optimal base editor in the application of base editing in sheep fetal fibroblasts, which proved the feasibility of efficient site-specific editing of sheep genome, and also provides technical support for the application of base editor in gene editing of large mammals.

Bacillus, an important biocontrol resource, widely exists in nature. The establishment of a simple and efficient genetic manipulation technique for Bacillus is helpful to the study of related molecular mechanism. In this study, the recombinant plasmid pBD1 was constructed by molecular biological methods, which can be used for gene knockdown in Bacillus. In order to verify the gene knockdown efficiency of pBD1 in Bacillus, the small guide RNA (sgRNA) of serine protease gene bace16 in B. nematocida B16 was designed and inserted into pBD1 to construct bace16 low-level expression vector, which then electrotransfected into B16 to compare the expression difference of bace16 in the mutant and wild type. The results showed that reversible Bacillus knockdown vector pBD1 was successfully created based on dCas9, and bace16 low-expression mutant strain was obtained by qRT-PCR and enzyme activity tests. The bace16 expression could be complemented to the level of wild type when the inducer ITPG (isopropyl β-D-thiogalactoside) was removed. The present study constructed a reversible gene knockdown recombinant plasmid suitable for Bacillus, and established the gene knockdown and complementary system of Bacillus, which could provide basic information for the study of gene function of Bacillus.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious viral diseases affecting pig (Sus scrofa) industry. In order to develop a high-throughput serological detection method for Porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV antibody detection was developed by multi analyze profiling solve for unknown x (xMAP) technology. Firstly, PRRSV nucleocapsid (N) protein was coupled with carboxylated fluorescent microspheres. The coupling efficiency was verified. Microspheres was then reacted with serum samples and biotinylated detection antibody thereafter, the fluorescent signal of microspheres was detected with Luminex 200 System. Next, in order to determine the detection threshold, 32 serum samples of special pathogen free (SPF) pigs was detected at 1∶100 dilution. The threshold was calculated based on the median fluorescence intensity (MFI) and standard deviation. The optimal amount of binding protein was investigated. The repeatability of the method was verified, and the sensitivity and consistency of the method were studied compared with commercial kits. To verify the specificity of the method, the positive sera of Vesicular stomatitis virus (VSV) and Pseudorabies virus (PRV) were used. Finally, 83 clinical pig serum samples were detected by enzyme linked immunosorbent assay (ELISA) kit and liquid chip analysis, respectively, to verify the consistency of the 2 methods. The results showed that the MFI detection threshold was 103.22, and the optimal concentration of binding protein was 8 μg/1.25×10⁶ beads; The coefficient of variation between and within batches was within 8%, suggesting a good repeatability. Compared with ELISA kit, the sensitivity of liquid chip detection method was higher. Neither VSV nor PRV positive sera showed positive by liquid chip analysis, indicated that the established liquid chip detection method had good specificity. When 83 clinical pig serum samples were detected by ELISA and liquid chip detection technology, results showed that the consistency between 2 methods were 91.57%. It showed that the high-throughput detection method of PRRSV using liquid chip detection technology had been successfully established. This study provides a new method for the serological detection of PRRSV and a reference for the detection of other pathogens.

Fluorescence microscopy image plays an important role in the research of life sciences. A large number of image data can be processed effectively using computer and machine learning, so that we could obtain the conclusion with statistical significance. In this study, a cell segmentation model was proposed based on U-Net for low signal-to-noise ratio (SNR) fluorescence images, and a cell image dataset for training and validation was constructed. In this method, convolution kernels were adopted to extract features, residual modules to deepen network, and weighted loss function to make machine learning process pay more attention to cell edges. Compared to several other methods, this algorithm shows better performance in cell segmentation for low signal-to-noise ratio fluorescence images with 87.6% pixel accuracy and intersection-over-union (IOU) 72.0%. This study provides technical support for cell morphology research and image-based high-throughput cell screening.