Octamer-binding transcription factor 4 (OCT4) is a POU (Pit-Oct-Unc)-family transcription factor, which is specifically expressed in early embryos, embryonic stem cells and germ cells. Studying the expression and function of OCT4 in early porcine (Sus scrofa) embryonic development is significant for the analysis of early developmental mechanisms and pluripotency regulation. In this study, the expression pattern of OCT4 in porcine early stage embryos was studied by immunofluorescence and qPCR technology. The results showed that in parthenogenetic, in vitro fertilization and somatic cell nuclear transfer embryos, OCT4 protein widely expressed in the blastomeres from 2-cell to murola, and expressed simultaneously in the inner cell mass and trophectoderm of the blastocyst; OCT4 expression patterns in parthenogenetic and in vitro fertilization embryos were similar, the expression level declined at the 2-cell stage and increased at the 8-cell stage, and Oct4 mRNA is widely expressed in the trophoectoderm of the blastocysts. When OCT4 was specifically overexpressed in the trophoectoderm, the diameter and cell number increased significantly, trophoblast development-related genes E74-like factor 5 (ELF5) was down-regulated, EOMES was significantly up-regulated, fibroblast growth factor (FGF) signaling pathway related genes were significantly down-regulated and polarization-related genes PKCα (protein kinase Cα) and ERZIN were significantly down-regulated, methylation gene TET1 was significantly up-regulated, and DNA methyltransferases 3B (DNMT3b) was significantly down-regulated. The results indicate that OCT4 keeps expressing in the trophectoderm and might regulate proliferation after the first lineage differentiation. This study provides basic material for further study of porcine early embryonic development mechanisms and the establishment of porcine embryonic stem cells.

Fusarium head blight (FHB), a serious threat to wheat production, has become a worldwide problem affecting the sustainable development of wheat (Triticum aestivum). Since the middle of 1990s, the disease occurred from spots to large areas and become one of the main diseases of wheat in Hebei province, with an average annual occurrence area of 267 000 hm² or more. In order to provide the basis for scientific prevention and control FHB, MaxEnt model was used to predict the risk area of the disease in Hebei province in this study. Z-score method was used to standardize the occurrence area and area ratio of FHB in Hebei province from 2003 to 2018, and the features in time and space of FHB occurrence were analyzed. Based on the distribution characteristics of FHB in Hebei and the data of environmental variables, MaxEnt model was used to predict the potential risk area of FHB in Hebei. The area under the curve (AUC) of receiver operating characteristic (ROC) was used to evaluate the accuracy of the prediction model. The occurrence and prevalence of FHB in Hebei had a certain periodicity and spatial correlation, and the AUC value of MaxEnt model was 0.816, which indicated that the predicted distribution area of FHB had a good fit with the actual distribution area. The high and middle risk areas of FHB accounted for 14.98% and 10.19% of the total area of the whole province, respectively, mainly concentrated in the middle and south of Hebei. Among them, 66 counties were high risk areas, including the south of Baoding, the middle and east of Shijiazhuang, Hengshui, the middle and East of Xingtai, and the middle and east of Handan. The results of environmental variables analysis showed that the mean temperature of the warmest quarter (bio 10), the max temperature of the warmest month (bio 5), the mean temperature of the coldest quarter (bio 11) and the minimum temperature of the coldest month (bio 6) had a greater impact on the potential distribution of FHB. Among them, the relative contribution rate of bio10 was the important, accounting for 67.9%, and its importance accounted for 22.2%. The high and middle risk areas of FHB in Hebei accounted for 25.17% of the total area of the whole province, mainly concentrated in the central and southern part of Hebei. The occurrence of the disease has a high risk in Hebei. The establishment of the FHB prediction model will provide the basis for disease prediction and effective prevention and control.

Germin-like proteins (GLPs), a group of defense-related proteins that are widely existed in plants, play an important role in the process of plant responses to pathogenic microorganism infection and abiotic stress. However, the identification of this protein family in the maize (Zea mays) genome and whether it is involved in the resistance process during Setosphaeria turcica infects maize have not been reported. In this study, the GLP family genes (ZmGLPs) in maize were genome-wide identified, the sequences, gene localization, gene duplication, conservative motifs and systematic evolution were analyzed. Based on transcriptome data obtained in our previous research, the expression level change of ZmGLPs in response to S. turcica infection was analyzed. Fifty-seven GLP family genes were identified from maize genome. The length of their coding proteins varied greatly, ranged from 113 to 671 amino acids. The chromosomal locations and collinearity analysis revealed that 52 ZmGLPs showed unevenly distributed on 10 chromosomes of maize, and 5 other ZmGLPs were located in the extranuclear ctg150. The ZmGLPs family members could be divided into 6 clades by phylogenetic analysis, among which clade1 does not contain GLPs of other species. Based on transcriptome data, 43 ZmGLPs were detected when maize was infected by S. turcica. These genes were divided into 6 categories, according to their expression level in 3 different time points (0, 24 and 72 h), and among them, 27 genes were significantly expressed during the infection process of S. turcica.This study will not only identify the numbers and characteristics of ZmGLP genes in maize genome and the expression changes during the infection of S. turcica, but also provide a theoretical basis for further revealing the molecular mechanism of maize against the infection of pathogenic fungi.

Ascorbate peroxidase (APX) is a key enzyme for scavenging reactive oxygen species. It can catalyze the transformation of H₂O₂ to H₂O and plays an important role in plant stress. In the early stage, the research group screened APX gene with sequence variation between drought resistant and drought sensitive materials through two-dimensional electrophoresis of root proteome of Gossypium hirsutum. In order to explore the drought resistance function of cotton APX gene, 18 GhAPXs were identified from Gossypium hirsutum genome in this study, named GhAPX01 to GhAPX18. Phylogenetic tree analysis showed that members of the GhAPX family were clustered into four subgroups; Cis-acting elements analysis showed that there were abscisic acid, salicylic acid and gibberellin elements upstream of most of the APX family genes; Transcriptome thermography and qPCR results showed that GhAPX07 and GhAPX16 were induced to express under drought stress, and showed different expression patterns in drought resistant and drought sensitive cotton materials. It was speculated that these two genes might play an important role in the response of cotton to drought stress. These results provide a reference for further study on the drought resistance function and molecular mechanism of GhAPX gene.

Soybean (Glycine max) is main grain and oil crops in China, salinity and drought have seriously affected the yield and quality of soybeans. Calcineurin B-like protein (CBL) is a type of calcium receptor, which plays an important role in the process of plant stress response. The results of the expression pattern analysis of soybean CBL gene family in the early stage of this research group showed that soybean GmCBL7 could be induced by high salt and drought.In order to further study the relationship between GmCBL7 gene and salt tolerance and drought resistance. In this study, GmCBL7 (GenBank No. XM 006578843.3) was cloned from soybean by reverse transcription-PCR (RT-PCR) technology. The CDS sequence of the gene was 672 bp in length, encoding 223 amino acids, the molecular weight of the protein was 25.8 kD, and the theoretical isoelectric point was 4.81. It was an acidic protein with a typical EF-hand domain and no signal peptide. It was a non-secreted protein. Analysis of systematic evolution showed that GmCBL7 was in the same evolutionary branch as the homologous protein in legumes, and had the closest relationship with the homologous protein in wild soybean (Clycine soja). The plant expression vector pCPB-GmCBL7 was constructed to genetically transform tobacco (Nicotiana tabacum), and analyzed the physiological functions of the GmCBL7 transgenic tobacco for stress resistance, the results showed that after high salt (200 mmol/L NaCl) and drought (20% PEG6000) stress treatments for 6 and 12 h, the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) and content of proline were significantly higher than those of wild type, while the content of malondialdehyde (MDA) was significantly lower than that of wild type. It was suggested that overexpression of GmCBL7 gene could improve salt tolerance and drought resistance of transgenic tobacco.This study provides a basis for further exploring the function of GmCBL7 gene, and provides a new gene for soybean salt and drought resistance breeding.

Plant expansin protein is a kind of plant cell wall protein, which plays an important role in plant growth and development. This group conducted transcriptome studies on the development of sweet potato (Ipomoea batatas) root tubers, and identified an expansin protein gene IbEXPA4 (Ipomoea batatas expansin-A4-like, IbEXPA4) that was significantly up-regulated in the middle period of expansion of sweet potato root tuber. The expansin protein gene IbEXPA4 were cloned and analyzed using sweet potato varieties 'Guijingshu 8' and 'Guizishu 1' as materials in this study. The results showed that the complete open reading frame of this gene contained 771 bp, encoding 256 amino acids. The sequence information of this gene was uploaded to NCBI (GenBank No. MW361490.1). Bioinformatics analysis showed that relative molecular weight of IbEXPA4 protein was 27.53 kD, theoretical isoelectric point was 8.8, instability coefficient was 37.96, average hydrophobicity was -0.117, and fat coefficient was 65.62, which indicated that IbEXPA4 protein was a stable hydrophilic protein. The prediction of secondary and tertiary structure showed that the protein mainly contained random curls. The structure of IbEXPA4 protein was analyzed, it showed that IbEXPA4 protein had a signal peptide at the N terminal without transmembrane domain, and contained 2 conserved domains, DPBB_1 and Pollen_allerg_1. Subcellular localization predicted its location in the cell wall. The phylogenetic tree analysis showed that the IbEXPA4 had the highest homology with the expansin-A4-like gene of Ipomoea triloba, which was 99.22%. Quantitative fluorescence PCR showed that IbEXPA4 was expressed in the root tuber of 'Guijingshu 8' and 'Guizishu 1', which showed a trend of increase at first and then decrease. Moreover, the expression levels of IbEXPA4 gene reached the highest in root tubers after 60 d of transplantation, which were 3.09 times and 9.23 times higher than those of the control group (fibrous roots of 20 d), respectively. Comparison and analysis of the expression changes of IbEXPA4 in the root tubers of the two varieties showed that the change of IbEXPA4 was more significant in 'Guizishu 1'. Agricultural character analysis showed that root tuber length of 'Guijingshu 8' were significantly higher than those of 'Guizishu 1', while the root diameter of 'Guizishu 1' was significantly higher than that of 'Guijingshu 8' after 60 and 90 d of transplantation. Correlation analysis showed that the expression of IbEXPA4 in root tuber were significantly positively correlated with root tuber diameter and root tuber length. These results indicated that IbEXPA4 promoted the longitudinal growth and transverse growth of root tuber, and participated in the regulation of sweet potato root tuber growth. This study provides a theoretical basis for elucidating the molecular mechanism of sweet potato root tuber growth.

In the practice of genetic engineering, such as improving the nutritional quality of seeds and using seed-based bioreactor to produce proteins with industrial or medicinal value, it is necessary to use seed specific promoters to drive the transcription of exogenous genes. Seed storage proteins are the general name of a large number of storage proteins synthesized and stored in the process of seed ripening in higher plants, the promoters of the genes coding seed storage proteins serve as an important source for seed specific promoters. In order to obtain the seed specific promoter of Medicago truncatula, 2 075 bp promoter sequence (named PMt1g072600) of upstream of coding region of the major storage protein legumin gene Mt1g072600 was cloned, and the putative cis-acting elements of the promoter were predicted using workflow embed in PLACE and PlantCARE, the results showed that this sequence contained E-box, A/T rich element, P-box and other cis-acting elements with highly relevant features to seed specific promoter. The plant expression vector to drive the GUS gene using this promoter was constructed and transformed into Arabidopsis thaliana, the result of analysis of GUS expression level in transgenic A. thaliana seeds showed that the expression of GUS driven by PMt1g072600 was significantly higher than that drived by PCaMV35S. The results of histochemical staining of tissues/organs of transgenic A. thaliana showed that GUS driven by PMt1g072600 showed the specificity of seed expression.This study identified a seed specific promoter PMt1g072600 of M. truncatula, which can be used to drive the specific transcription of exogenous genes in transgenic plant seeds. This study also provides a reference for selection and utilization of seed specific promoters in plant genetic engineering.

GIGANTEA (GI) is a rhythm related gene, which plays an important role controlling of rhythm output and flowering regulation in plants.To explore the biological function of GIGANTEA (GI) gene in tall fescue (Festuca arundinacea), in this study, tall fescue was used as the experimental material, and the gene sequence and promoter sequence were amplified by using RACE (rapid amplification of cDNA ends) cloning and chromosome walking method, and analyzed by bioinformatics. Sequence analysis showed that the total length of FaGI gene (GenBank No. MZ540915) was 3 869 bp, and its ORF was 3 447 bp, encoding 1 149 amino acids. The promoter sequence was 2 371 bp and had multiple cis-acting elements such as CURECORECR, GT1CONSENSUS, LTRE1HVBLT49 and MYB recognition site.The phylogenetic tree showed that the protein encoded by the FaGI gene had a close evolutionary relationship with the homologous proteins of gramineous plants such as Festuca pratensis, Lolium perenne, Secale cereale, Triticum aestivum and other grasses.The subcellular location showed that it was located in the nucleus, indicated that the protein might play a role in the nucleus. Fluorescence quantitative analysis of expression patterns under different light treatments and different developmental stages showed that the expression of FaGI gene was regulated by photoperiod and circadian clock. The expression level of FaGI gene was different in different developmental stages. The expression level was the highest in the seedling stage, and the expression level decreases in reproductive growth stage. The p1300-FaGI overexpression vector was constructed to transform Arabidopsis thaliana, and it was found that the Arabidopsis overexpression FaGI could up-regulate the expression of AtCCA1 (CIRCADIAN CLOCK ASSOCIATED 1), AtTOC1 (TIMING OF CAB EXPRESSION 1), and down-regulate the expression of AtFT (FLOWERING LOCUS T) and AtCO (CONSTANS). This study provides a theoretical basis for the follow-up verification of the FaGI gene function of tall fescue.

Prostaglandin E synthase (PTGES) is the terminal rate-limiting enzyme for prostaglandin E2 (PGE2) synthesis, which catalyzes the conversion of prostaglandin H2 (PGH2) into PGE2. PGE2 can specifically bind to receptors and participate in various physiological processes, including animal reproduction. This study investigated the expression of PTGES in yak (Bos grunniens) under normal physiological conditions and its role in the reproductive process. There were 9 experimental samples: Female yaks' uterus, ovaries and fallopian tubes at different stages of the estrus cycle (follicular phase, luteal phase and pregnancy). The mRNA and protein expression of PTGES were analyzed by gene cloning, qPCR and immunohistochemistry (IHC). The results showed that the predicted proteins encoded by PTGES gene (GenBank No. MW030282) were hydrophilic non-transmembrane proteins through bioinformatics analysis. PTGES gene was expressed in yak fallopian tubes, ovaries and uterus, but there were some difference. The expression of PTGES in the ovary during the luteal phase was significantly higher than that of the follicular phase and pregnancy (P<0.05). The expression of PTGES in the fallopian tube during the follicular phase was significantly higher than that in the luteal phase and pregnancy (P<0.05). The expression of PTGES in the uterus during the follicular phase was significantly higher than that in the luteal phase and pregnancy (P<0.05). It could be known from the results of immunohistochemistry that PTGES was expressed in the luteum cells, oviduct mucosa epithelium, ovarian follicular membrane, ovarian granular layer, ovarian reproductive epithelium, endometrium, uterine glands and serosal membrane. The results of this study was speculated that PTGES might be involved in the regulation of yak reproductive physiological processes. This research provides a part of theoretical support for deeper exploration of PTGES involved in mammalian reproductive physiology regulation, which is helpful to the study of the breeding rules of mammals in the plateau.

The aim of this study is to explore the renin-angiotensin system's (RAS) expression and location in bovine mammary epithelial cells (bMECs), and its regulatory role in LPS-induced inflammatory injury. After treating bMECs with different concentrations of LPS, CCK-8 method was used to examine cell viability, and qPCR was used to detect IL-1β, IL-6, IL-8, TNF-α, ACE2, ACE, MasR和AT1R mRNA expression. Enzyme linked immunosorbent assay (ELISA) was used to test the content of AngⅡ and Ang 1-7 protein in the supernatant. Western blot and cellular immunofluorescence were used to verify the expression and location of ACE2 protein in bMECs. The results showed that ACE2 was expressed and localized in the cell membrane and cytoplasm of bMECs. Producing obvious inflammatory damage by LPS treatment of bMECs for 24 h, the expression of IL-1β, IL-6, IL-8, TNF-α, ACE, AT1R gene, and AngⅡ proein increased with the LPS concentration. The expression of ACE2 and MasR mRNA firstly increased and then decreased, while the expression of Ang 1-7 protein decreased along with the LPS concentration. This study indicates the presence of RAS members such as ACE2, ACE, MasR, AT1R, AngⅡ and Ang 1-7 in bMECs, and the ACE2/MasR/Ang 1-7 and ACE/Ang Ⅱ/AT1R pathways involved in the regulation of bMECs inflammatory injury induced by LPS. This experiment lays the foundation for studying the regulatory role of RAS in mastitis and discovering new targets for the action of anti-inflammatory drug.

Bovine viral diarrhea virus (BVDV) infection causes Bovine viral diarrhea/mucosal disease (BVD/MD), which can cause persistent infection and diarrheal mucosal disease in calves. The fatality rate of mucosal disease is as high as 100%, which is a serious threat to large-scale breeding and safety of biological products. In the previous study, the Turbo ID system was performed to capture the differentially expressed genes post-infection with BVDV, and the results showed differentially expressed genes such as UDP-glucose-glycoprotein-glucosyltransferase 1 (UGGT1). To explore the effect of UGGT1 gene on the replication of Bovine viral diarrhea virus, CRISPR/Cas9 was used to knock down UGGT1 gene in Madin-Darby bovine kidney (MDBK) cells in this study. Western blot was used to identify the knockdown of UGGT1 gene. Cell morphology changes were observed under a microscope and cell proliferation was counted. The effects of UGGT1 gene on BVDV replication were detected by immunofluorescence staining, qPCR, cytopathic effects (CPE) and virus titer in UGGT1 knockdown cells and Scramble cells infected with BVDV TC strain. The results showed that the UGGT1 gene of MDBK cells was successfully knocked down. There was no difference in the morphology and growth rate of UGGT1 knockdown cells, Scramble cells and wild-type MDBK cells. Compared with the negative control Scramble, BVDV infected UGGT1 knockdown cells 36 h after labeling double, the green fluorescence of double strand RNA (dsRNA) was significantly reduced; the level of 5'UTR RNA was significantly reduced after 24 h of infection (P<0.05), and the difference was extremely significant at 48 h (P<0.01); BVDV titer decreased significantly after 36 h of BVDV infection (P<0.05); After 36 h of infection, a large number of cells in Scramble fell off, and UGGT1 knockdown cells were significantly lower than those in Scramble control. This study showed that UGGT1 gene knockdown could inhibit the replication of BVDV, which provides an important basis for the establishment of new methods to prevent and control BVDV.

Due to its small size and saving breeding space, dwarf chickens (Gallus domesticus) are often used in the production of chicken supporting lines. The purpose of this study was to clarify the type of growth hormone receptor (GHR) gene mutations in dwarf size Plymouth Rock chicken and to explore the differences in growth and development between the normal size Plymouth Rock chicken and the dwarf size Plymouth Rock chicken. Primers were designed for the four common mutation sites of GHR gene in dwarf size Plymouth Rock chickens. PCR amplification was performed using gDNA of dwarf size Plymouth Rock chickens as templates to analyze the genetic variation types. At the same time, the developmental characteristics of body weight and tibia length of normal and short size Plymouth Rock chickens were compared and analyzed. Three mathematical models of Logistic, Gompertz and Von Bertalanffy were used to analyze the optimal model and fitting parameters. The results showed that compared with the normal size Plymouth Rock chicken, the 1 781 bp deletion of the GHR gene in the dwarf sized Plymouth Rock chicken was the reason for its dwarfing. Normal and dwarf size Plymouth Rock chickens have obvious differences in development during 1 to 13 weeks of age, especially in body weight and tibia length. Logistic was the best fit model for body weight of normal and dwarf size Plymouth Rock chickens (R²>0.990). In terms of tibia length, among male Plymouth Rock chickens, the best fit model for normal sized Plymouth Rock chickens was Logistic, and the best fit model for dwarf sized Plymouth Rock chicken was Von Bertalanffy. Among female Plymouth Rock chickens, the best fit model for normal and dwarf sized Plymouth Rock chickens was Von Bertalanffy. This study provides a reference basis for fine breeding management of Plymouth Rock chickens and molecular marker-assisted breeding of the dwarf size Plymouth Rock chickens.

At present, the expression of splice variants of myocyte enhancer factor 2C gene (MEF2C) in ducks (Anas platyrhynchos) has not been deeply studied. In order to understand the expression difference of MEF2C variants in tissues, RT-PCR was used to clone the full-length CDS of MEF2C gene in Xingyi duck. The results showed that 2 splice variants of MEF2C-a (GenBank No. MZ497418) and MEF2C-b (GenBank No. MZ497419) were successfully cloned, and the encoded amino acid sequences shared more than 95% homology with pigeon (Columba livia), quail (Coturnix japonica) and chicken (Gallus gallus), which indicated that MEF2C gene was highly conserved in the evolution process. The results of qPCR showed that MEF2C-a and MEF2C-b were expressed in both male and female ducks, and highly expressed in brain, myocardium, leg muscle and chest muscle. The expression patterns of the 2 splice variants in female and male ducks were different. The expression level of MEF2C-a in most tissues of male ducks was higher than that of MEF2C-b, the opposite in female ducks. The expression of 2 splice variants in leg muscles and chest muscles did not increase with increasing age. The present results provide basic materials for further study on the mechanism of MEF2C splice variants in duck muscle development.

Bacillus subtilis Z-5 has significant antagonistic effect against many plant pathogenic fungi. In this study, antagonistic effect of strain Z-5 on Fusarium oxysporum was detected by confrontation culture. The activities of protease, amylase, cellulase and siderophore in the sterile filtrate were detected by the method of transparent circle. The antifungal substances were extracted by acid precipitation, and the antagonistic effect of the extract against F. oxysporum was detected by inhibition zone method. The extract was separated by high performance liquid chromatography(HPLC) and the activities of the separated components were detected by inhibition zone method. The structures of antifungal substances were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) and mass spectrometry/mass spectrometry(MS/MS). The results showed that strain Z-5 presented significant antagonistic activity against F. oxysporum, and the sterile filtrate had significant protease, amylase and cellulase activities as well as the siderophore. The extracts of the sterile filtrate showed significant antagonistic effect against F. oxysporum and three antifungal components were obtained, which were identified as lipopeptide antibiotics C14 iturin A, C15 iturin A and C14-C18 fengycin A. The detection of antifungal substances from B. subtilis strain Z-5 laid the foundation for the study of its antifungal mechanism and the effective use of biocontrol agents.

Pseudorabies virus (PRV) infection causes Pseudorabies (PR), which is an acute contact contagious disease in pigs (Sus scrofa). Vaccines are broadly used to control PR. But immunity failures commonly happen due to the prevalence of PRV variant (PRV typeⅡ). In recent years, cases of PRV infection in humans (Homo sapiens) made PRV a global public health threat. However, the pathogenesis of PRV remains vague. To fully understand the molecular change of host cells caused by PRV infection, the PRV typeⅡstrain PRV-DX was used to infect pig kidney cells PK-15 to explore the differential transcriptome by high throughput sequencing. Results showed that 3 595 genes were up-regulated and 3 604 genes were down-regulated. Twelve differentially expressed genes were randomly selected for qPCR verification, and the changing trend was consistent with that of transcriptome data. GO annotation and KEGG signaling pathway enrichment indicated that the differentially expressed genes mainly focused on the pathways such as metabolism, gene expression and immunity. The present study provides important basic data and research direction for further understanding the pathogenic mechanism of PRV infection.

With the rapid development of isothermalamplification technology, the method to detect amplicons has become the key restricting its rapid popularization and application. Given the characteristics of Loop-mediated isothermal amplification(LAMP) reaction and the current amplicons detection methods, we summarized the progresses which have been made in recent years, including methods based on by-product generation analysis, methods aiming for multiplex detection, and the sequence-specific ones with visual readouts (e.g. CRISPR/Cas based ones). The possible opportunities and challenges are also been discussed. This review provides support for the nucleic acid analysis industry and applications based on nucleic acid amplification.

The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction. The MLPA procedure includes DNA denaturation, hybridization of probes and target DNA sequences, ligation of the probes, PCR amplification, capillary electrophoresis and data analysis. MLPA has a variety of applications including diagnosis and prognosis of tumors, detection of human genetic diseases, prenatal diagnosis. Due to the increasing occurrence of multiple infections and diseases, there is an urgent need to develop sensitive, specific, and high throughput tools to detect pathogens. MLPA is becoming a promising method in the area of high throughput detection of pathogens. This review gives an overview on the principles and procedures, applications in detection of pathogens and diagnosis of tumors and genetic diseases, advantages and limitations, and perspectives of this technique aiming to provide insights into the application and improvement of MLPA technique in animal pathogen detection and disease diagnosis.

Porcine reproductive and respiratory syndrome (PRRS), one of the leading outbreaks on a world scale in swine farms, is caused by Porcine reproductive and respiratory syndrome virus (PRRSV). This study aimed to construct reverse genetic system of highly pathogenic PRRSV (HP-PRRSV) strain HN07-1. The whole genome of HN07-1 was first obtained by a method in which the genome was segmently cloned and sequenced. Based on the reverse genetics technique, fragments of the whole genome were cloned into pcDNA3.1(+) vector and full-length infectious clone of HN07-1 strain, designated as pcDNA3.2-rHN07-1, were constructed. Besides, the recombinant plasmid pcDNA3.2-rHN07-1-EGFP was constructed by inserting EGFP between PRRSV ORF1b and ORF2a. The recombinant viruses were rescued by transfecting the constructed plasmids into Marc-145 cells and identified by immunofluorescence assay (IFA) and Western blot. The results showed that the full-length of HN07-1 genome was 15 345 nt. The typical cytopathic effect (CPE) was observed at 48 h of culture after one blind passage on Marc-145 cells. The results of IFA and Western blot indicated that PRRSV N protein expression of the rescued viruses and parent strain was confirmed. Moreover, the rescued viruses exhibit similar growth dynamic characteristics with its parent strain. Taken together, these results indicate that the infectious clones can be used for reverse genetic operation, which lays a foundation for the research of pathogenesis and vaccine development of PRRSV.

The outbreak of pseudorabies poses a huge challenge to its prevention and control. The establishment of a new diagnostic method with high specificity and sensitivity and the acquisition of antibodies with virus neutralizing effects are essential for the prevention, control and treatment of the disease. The purpose of this study was to express a soluble nano-antibody against the envelope glycoprotein E (gE) protein of porcine (Sus scrofa) Pseudorabies virus (PRV) in prokaryotic cells and to explore the application of this antibody in the diagnosis and treatment of porcine pseudorabies. In this study, the gene sequence of anti-porcine pseudorabies gE protein antibody was designed and synthesized, which was named PRVgENb, and PRVgENb-pET21b vector was constructed. The fusion protein was expressed in Escherichia coli. After purification on a Ni²⁺ affinity chromatography column, the single nano-antibody PRVgENb was obtained, which was labeled with HRP for direct ELISA and PRV virus neutralization test. The results showed that the fusion protein PRVgENb had high purity after prokaryotic expression and purification. Direct ELISA results showed that under a certain concentration of antigen, a low concentration of nano-antibody PRVgENb bound to the specific antigen with high activity. While a certain concentration of antibody could sensitively recognize low to high concentrations of specific antigen. The results of the virus neutralization test showed that the nano-antibody PRVgENb inhibited the replication of PRV and had a certain neutralization activity. This experiment established a method to stably obtain the nanobody PRVgENb, and initially explored its use in direct ELISA, and proved that it can inhibit the replication of PRV. Nanobody PRVgENb lays an experimental foundation for the diagnosis, treatment and follow-up research of PRV.