Barley (Hordeum vulgare) is an important crop and industrial raw material, and spot blotch is one of the diseases that seriously endanger barley production. The disease mainly damages leaves and leaf sheaths, and its occurrence and prevalence seriously affect the yield and quality of barley. In order to explore the molecular markers related to barley spot blotch resistance, 142 barley materials were identified by artificial inoculation of spot blotch pathogen at seedling stage, and SSR molecular markers combined with phenotypic identification results of barley resistance to spot blotch were used for correlation analysis, so as to find effective resistance sites. The results showed that 504 alleles were detected from 70 SSR markers, with an average of 7.2 alleles per marker. The variation range of major allele frequency was 0.196 3 to 0.503 5, with an average of 0.344 2, and the variation range of PIC was 0.521 2 to 0.847 0. The genetic diversity index ranged from 0.600 7 to 0.862 4, and the population was divided into 5 groups by cluster analysis and population genetic structure analysis. A total of 6 SSR markers were detected, which were located on the 3H, 4H and 5H chromosomes of barley, respectively, and a QTL locus that confered resistance to barley spot blotch was finally determined on the 4H chromosome. The results of this study will be helpful for the breeding of barley variety, and the associated loci of barley spot blotch resistance obtained can provide reference for molecular marker-assisted breeding of barley.

TCP (teosintebranchⅠ, cycloidea, proliferating cell factors 1 and 2) is a class of plant-specific transcription factors and play an important regulatory role in plant growth and development. Dormancy and sprouting of potato (Solanum tuberosum) tuber is extremely important for potato breeding, tuber production and processing. Our previous study found that the expression of the StTCP23 (GenBank No. NW _ 006239107.1) in potato was continuously up-regulated in the process of tuber dormancy release. To clarify the biological function of the StTCP23, this study carried out cloning, subcellular localization, and bioinformatics analysis, and used qPCR technology to analyze its tissue specificity and expression during tuber dormancy release. The results showed that StTCP23 was mainly located in the nucleus. StTCP23 contained a typical TCP conserved domain, belonging to non-transmembrane proteins, and the total phosphorylation site accounted for 22.44%. The expression of StTCP23 gene was the highest in potato leaves and there was a variety difference. The expression level of StTCP23 gene showed a continuous upward trend from dormancy to sprouting of tubers, and the expression difference of StTCP23 gene was extremely significant at dormancy and dormancy release stages (P<0.01). The results provide a basis information for elucidating the function of StTCP23 gene in potato.

Abscisic acid (ABA) and its receptor pyrabactin resistance like (PYL) play an important role in plant resistance to drought stress. In order to explore the drought resistance function of the MePYL4a (GenBank No. XM_021766535) gene of cassava (Manihot esculenta), the MePYL4a gene was cloned from the cassava variety 'SC8', and its bioinformatics analysis was carried out. The subcellular localization was carried out using the Agrobacterium tumefaciens mediated transient transformation method; The expression of MePYL4a gene under drought stress was detected by qPCR; MePYL4a overexpression vector was constructed and transformed into Arabidopsis thaliana for drought resistance analysis of A. thaliana transgenic plants. The results showed that MePYL4a was an unstable protein, and the subcellular localization characteristics of MePYL4a protein showed that it was located in the nucleus and cytoplasm; qPCR analysis showed that MePYL4a gene expression level was significantly induced by drought stress, reached the highest level in 1 h, and then slowly decreased. After drought treatment, the phenotype observation showed that MePYL4a could improve the drought tolerance of A. thaliana seedlings under drought stress. The average biomass of over expressed plants was 1.5 times that of wide type (WT). The expression of key rate limiting enzyme gene 9-cis-epoxycarotenoid dioxygenase (NCED3), and drought stress response index gene responsive to dehydration (RD29A) for ABA synthesis was significantly higher than that of WT and Atpyl4 mutant plant. And the activity of antioxidant enzymes was about twice higher than that of WT, indicating that the ability of over expressed plants of MePYL4a to remove active oxygen was stronger than that of WT and Atpyl4 plant. Under drought stress, it could accumulate more biomass and enhance the drought resistance of A. thaliana. In conclusion, MePYL4a overexpression reduced the accumulation of reactive oxygen species in A. thaliana under drought stress and induced higher expression of stress resistance genes, which enhanced drought resistance of A. thaliana. This study provides a reference for MePYL4a to participate in cassava stress resistance.

Molybdenum cofactor sulfurase (MCSU or LOS5) of plants plays an important role in resisting non-biological stress resistance. At present, few studies reported on the regulation of abscisic acid (ABA) synthesis by VvMCSU gene in response to light stress in grape (Vitis vinfera). VvMCSU gene was cloned by PCR with a full length of coding region, then predicted the gene function from the encoded amino acid sequence by bioinformatics. Subsequently, the expression profiles of VvMCSU in response to light shading stress in leaves were analyzed by qPCR in combination with their leaf photosynthetic characteristics. The results were shown below: Under heavy shading, the maximum photosynthetic quantum yield (Fv/Fm) and non-photochemical quenching parameter (qN) increased by 4.32 % and 28.13 % respectively, and the leaf width and the content of ABA significantly increased by 10.16% and by 1.50 times compared with the control group. The coding region of VvMCSU gene was 2 478 bp, which encodes 825 amino acids, and its promoter region had several cis elements in response to abiotic stress and hormone. VvMCSU was a hydrophilic protein with MCSU structure, which interacted with proteins related to ABA synthesis, and belongs to the same evolutionary branch as camellia (Camelia sinensis). The expression of VvMCSU was up-regulated under 60 % light shading, which was 2.77 times higher than that of the control group 0 % shading or under natural light; and 4.01 times higher than that of 80% severe shading. The expression of VvMCSU gene was positively correlated with ABA content and ratios Fv/Fm (P<0.05). In this study, chlorophyll fluorescence and ABA content were used to analyze the expression of VvMCSU under different light conditions. The results suggested that VvMCSU was an important regulatory gene involved in ABA metabolism in response to light shading stress of grape. This study provides a theoretical basis for viniculture studies to improve grape shading tolerance.

As the direct receptor of abscisic acid (ABA), Pyrabactin resistance 1-like (PYL) protein plays an important role in plant growth, development and stress. In order to explore the function of PYL gene family in strawberry, this study identified PYL gene family members in woodland strawberry (Fragaria vesca) and pineapple strawberry (F. ananassa) using bioinformatic methods, and analyzed the expression of PYL gene under different simulated stress conditions by qPCR. The results showed that there were 16 FvPYL genes distributed on 7 chromosomes in woodland strawberry and 40 FaPYL genes distributed on 18 chromosomes in pineapple strawberry. The gene size span of FaPYL gene family was larger than that of FvPYL. Their protein structure was similar and mainly contained α-helix and random coil. In addition, except FvPYL15 and FaPYL40, the other proteins were hydrophilic and mainly expressed in chloroplast, cytoplasm and nucleus. Phylogenetic analysis showed that FaPYL and FvPYL genes were distributed in 5 subfamilies, and the gene structure and motif position of the same species in the same subtribe were basically the same. There were only 3 pairs of segmental duplications in FvPYL genes, but a large number of segmental duplications and some tandem repeats in FaPYL genes. Selection pressure analysis of genes found that strawberry PYL genes were biased toward purification selection. Cis-acting elements analysis found that PYL family genes mainly contained elements related to light, hormone, stress, growth and development. As detected by qPCR, the relative expression levels of strawberry PYL genes were significantly different under different treatments. Under ABA treatment, FaPYL and FvPYL were up-regulated except FvPYL8. Under NaCl treatment, most FaPYL genes were up-regulated to varying degrees, among which FaPYL21 and FaPYL40 were strongly induced by 200 μmol/L NaCl. FvPYL genes expression were overall upregulated, with the relative expression of FvPYL11 was 60 times higher than that of the control. Under PEG treatment, FvPYL genes were positively regulated, FvPYL1 and FvPYL7 were significantly up-regulated. Compared with woodland strawberry, the upregulation range of FaPYL genes were significantly different, among which the relative up-regulation levels of FaPYL2 and FaPYL7 were extremely significant. This study provides a theoretical basis for functional mining and application research of PYL gene family in strawberry.

Sunite sheep (Ovis aries) is high-quality mutton sheep resources in China and one of the main sources of mutton in northern China. Long non-coding RNA (LncRNA) plays an important role in muscle development in O. aries. To predict the regulatory network of Lnc_000588 in skeletal muscle development of Sunite sheep, in this study, the real-time quantitative PCR analysis was performed on its tissue expression in different parts, and the prediction of bioinformatics interaction of targeted miRNA-mRNA was carried out. Meanwhile, the expression of Lnc_000588 was detected in mouse (Mus musculus) C2C12 cells. Lnc_000588 was expressed in all tissues, and the expression level in the longissimus dorsi muscle was significantly higher than that in other tissues (P<0.01); there was no significant difference in Lnc_000588 during cell proliferation, and the expression level in C2C12 cells increased with the prolongation of differentiation time and the increase of myotubes, the expression after 48 h was extremely significantly higher than that in the earlier period (P<0.01). From Lnc_000588 involved in the regulation of the muscle growth and development through miRNA, a total of 32 miRNAs were predicted, of which 3 miRNAs were found to be related with muscle development. The target genes of these 3 miRNAs are enriched in the Ras signaling pathway (oas04014: Ras 66 pathways including), Rap1 signaling pathway (oas04015: Rap1 signaling pathway) and AMPK signaling pathway (oas04152: AMPK signaling pathway). In conclusion, the expression level of Lnc_000588 in Sunite sheep muscle tissue was significantly higher than that in other tissues, which may be involved in the differentiation of C2C12 cells. This study provides basic data for exploring the regulatory function of LncRNA involved in the muscle tissue of Sunite sheep.

Reproductive performance and litter size are important economic traits for sheep (Ovis aries), and luteinizing hormone/choriogonadotropin receptor (LHCGR) has an important impact on sheep reproductive performance and litter size. To verify the effect of LHCGR gene on litter size in sheep, this study used Dorper sheep, Small Tail Han sheep and Tan sheep and their hybrid offspring (D, TH, F₁, F₂ and H₁) 5 sheep populations as research subjects. Flight mass spectrometry (Sequenom MassARRAY^® SNP) technology was used to genotype the 4 mutation sites of LHCGR gene: g.75709226 G>T, g.75733123 G>A, g.75747421 A>C and g.75748150 A>G. The association analysis of litter size was performed. The results showed that, the g.75709226 G>T and g.75733123 G>A loci were low polymorphism (polymorphic information content, PIC<0.25) in 5 sheep populations, and the g.75747421 A>C and g.75748150 A>G loci were moderate polymorphism (0.25≤PIC<0.5) in 5 sheep populations. In the g.75747421 A>C site, the number of litter size in wild-type AA individuals in H₁ sheep population was significantly higher than that in heterozygous AC and mutant CC individuals (P<0.05); there was no significant difference in litter size among the genotypes of g. 75709226 G>T, g.75733123 G>A and g.75748150 A>G sites (P>0.05). There was complete linkage disequilibrium between g.75747421 A>C and g.75748150 A>G loci, resulting in 4 haplotypes and 6 diplotypes; the litter size of the diplotype H3H3 individuals was significantly higher than that of the diplotype combination H1H1, H1H2, H1H3 and H2H2 individuals (P<0.05), and there was no significant difference with the diplotype combination H2H3 individuals (P>0.05). The results of this study showed that, the polymorphism of LHCGR gene g.75747421 A>C locus was associated with the lambing number of H₁ sheep population, and this locus could be used as a molecular genetic marker to improve lambing traits of H₁ sheep population; the AAAA diplotypes in LHCGR gene SNP sites g.75747421 A>C and g.75748150 A>G could be used as potential molecular markers for screening lambing traits in sheep. This study provides an important reference for LHCGR gene selection in sheep lambing number traits, and provides a theoretical basis for sheep breeding work.

As a major degradation/recycling pathway, autophagy plays an important role in maintaining intracellular homeostasis and reproductive development. In order to explore the potential role of autophagy in the development of epididymis in boars (Sus scrofa), the expression and distribution of autophagy-related molecules in the epididymis of Congjiang Xiang pig were analyzed by Western blot, immunohistochemistry, and qRT-PCR methods at 15 d (pre-puberty), 30 d (in-puberty), 60 d (post-puberty) and 180 d (sexual maturity) after birth. The results of Western blot showed that the expression of Beclin1 was the highest at 60 d and the lowest at 180 d, the expression of mammalian target of rapamycin (mTOR) was the highest at 30 d and the lowest at 180 d, and the expression of microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3)-Ⅱ/LC3-Ⅰ was the highest at 180 d and the lowest at 30 d. Immunohistochemical staining results showed that Beclin1 protein was mainly distributed in the main cells and microvilli of epididymal epithelium at 15, 30, and 180 d, while it was strongly positive in the muscle-like cells around the epididymal epithelium at 60 d. LC3 distributed mainly in the main cells and microvilli in the epididymis at 15, 30, and 60 d, but was specifically expressed in the epithelial narrow cells at 180 d. The results of qRT-PCR showed that the expression of autophagy-related gene Beclin1 was the highest at 15 d, the lowest at 180 d, and there was no significant difference between 15 and 30 d; The expression of MAP1LC3B gene was the highest at 180 d and the lowest at 60 d, and there was a significant difference among different age groups (P<0.05). The expression of autophagy related protein 12 (Atg12) and Unc-51-like kinase 1 (ULK1) genes showed the same trend, with the highest at 180 d and the lowest at 60 d, with significant differences among different ages (P<0.05); The expression of mTOR gene was the highest at 30 d and the lowest at 15 d, and there was a significant difference among different age groups (P<0.05).Above results indicate that there might be an autophagy regulation pathway through mTOR in the epididymis of Congjiang Xiang pigs at different ages. This study provides basic data for exploring the regulatory mechanism of autophagy in epididymis development.

Messenger RNA (mRNA) plays an important role in the complex dynamic network regulating skeletal muscle development. The role of New Zealand rabbit (Oryctolagus cuniculus) mRNA from fetus to adult skeletal muscle remains unclear. This study aims to screen for pathways and candidate genes associated with different stages of skeletal muscle development in New Zealand rabbits using transcriptome sequencing technology. In this study, the leg muscles of 2-week-old fetus, 6-week-old child rabbits and 6-month-old adult New Zealand rabbits were selected for transcriptome sequencing, and differential mRNAs and their potential effects were analyzed. The results showed that 12 845 mRNAs were identified, of which 1 891 mRNAs were differentially expressed in 3 stages. There were 929 differentially expressed genes (393 up-regulated, 536 down-regulated) between fetus group and child group, fetus group and adult group have 938 differentially expressed genes (up-regulated 449, down-regulated 489); and 24 differentially expressed genes (9 up-regulated and 15 down-regulated) between the child and adult groups. GO enrichment analysis showed that the differential transcripts between fetus and child groups were associated with transverse muscle contraction and skeletal muscle contraction. KEGG analysis showed that differential transcripts in the fetus and child groups were mainly enriched in PI3K/AKT signaling pathway, FoxO signaling pathway and AMPK signaling pathway. Differential transcripts in the child and adult groups were enriched in the cGMP-PKG signaling pathway and HIF-1 signaling pathway, which control muscle growth and development. The expression of 10 randomly selected differential genes verified by qPCR were consistent with the analysis of RNA-seq. Genes related to muscle development such as ARG2, ATPTA2, TPM1, PRKAG3, PRKAA2 and FoxO3 were obtained in the fetus and child groups, and genes related to muscle growth such as GOLGA4 and PFKFB3 were obtained in the child and adult groups. This study screened the genes and related pathways associated with skeletal muscle development in New Zealand rabbits, which can provide some theoretical basis for the subsequent study of skeletal muscle development in New Zealand rabbits.

A massive number of genome wide association studies have found that ADP-ribosylation factor-like 15 (ARL15) variation is significantly associated with energy metabolism-related diseases. To investigate the biological function of this gene, CRISPR/Cas9 technique was used to construct ARL15 knockout mice (Mus musculus) model for the first time, and its phenotype was analyzed. First-generation sequencing results showed that 1 841 bp of ARL15 was knocked out, including exon 2. Compared with wild-type mice, the mRNA and protein expression levels of ARL15 were significantly decreased in ARL15^-/- knockout mice (P<0.05). The embryo survival ratio of ARL15^+/+, ARL15^+/- and ARL15^-/- mice on embryo 16.5 d was present at Mendelian Genetics; ARL15^-/- mice had lower body weight on postnatal 0.5 d (P<0.05), and died shortly. qPCR showed that deletion of ARL15 inhibited adiponectin, C1Q and collagen domain containing (Adipoq) and diacylglycerol O-acyltransferase 2 (DGAT2) mRNA expression levels in fat and intestinal tissues of mice, respectively (P<0.05). In conclusion, ARL15 plays an important role in the early postnatal survival and lipid metabolism in mice. The ARL15 knockout mouse model has been successfully established, which can be used to further study the function of ARL15.

The Wenshang Barred chicken (Gallus gallus) is the only barred-feather breed in China as an indigenous breeds of chicken. This study aimed to investigate the genetic diversity and genetic structure, detect the genomic selection signals and explore the important germplasm characteristic genes of Wenshang Barred chicken population from the genomic level. According to multiple factors such as geographical distribution and genetic background, the Wenshang Barred chicken and 23 other indigenous chicken breeds as well as 3 introduced chicken breeds were selected as the research objects. The genomic SNPs were identified using restriction-site associated DNA sequencing (RAD-seq), which was used to compare the genetic differences between Wenshang Luhua chicken and other chicken populations, and annotate the selected genes in the process of evolution. The results showed that 300 543 SNP markers were detected in Wenshang Barred chicken population, and 14 breed-specific SNPs as well as 5 genes were identified. In terms of genetic structure, Wenshang Barred chicken and Yuanbao chicken, Tianjin monkey chicken, Langya chicken and Langshan Chicken as separate branches on the evolutionary tree. Functional analysis showed that the selected genes were mainly involved in physiological functions such as hormone regulation, nervous system development, amino acid metabolism and germ cell differentiation. This study can provide crucial reference information and theoretical basis for germplasm conservation and genetic diversity evaluation of Wenshang Barred chicken population.

Setosphaeria turcica melanin regulation factor 1 (StMR1) is a melanin transcriptional regulatory factor with a double zinc finger domain in Setosphaeria turcica, which affects the growth, metabolism and pathogenicity by regulating gene expression. Therefore, it is very important to elucidate the regulation mode of this transcription factor for analyzing the pathogenic mechanism of S. turcica. The previous study of our group found that after the StMR1 gene was knocked out, the synthesis of melanin in the mutant was blocked, the colony changed from black to gray-white, and the pathogenicity was significantly reduced. In this study, the RNA sequence (RNA-seq) data of wild-type strains of S. turcica and StMR1 gene deletion mutants were used to screen differentially expressed genes (GEGs). Gene Ontology (GO) and KEGG function annotation analysis were conducted for DEGs. The screening obtained 1 383 up-regulated genes and 1 710 down-regulated genes. The down-regulated genes were related to molecular functions, cellular components and biological processes, involving 25 metabolic pathways in 6 categories. Six down-regulated genes, SETTUDRAFT_172122, SETTUDRAFT_168776, SETTUDRAFT_166944, SETTUDRAFT_162601, SETTUDRAFT_131269 and SETTUDRAFT_163249, were analyzed of the expression level by qRT-PCR. The qRT-PCR results showed that the expression levels of the 6 genes were down-regulated in the mutant, suggested that StMR1 might have a positive regulation effect on the genes. Bioinformatics analysis showed that gene SETTUDRAFT_162601 had a typical Catalase domain, and the expression level of SETTUDRAFT_162601 was significantly increased under H₂O₂ by qRT-PCR (P<0.05), suggested that this gene was related to antioxidant stress. These results provide reference for further study on the mechanism of transcription factor StMR1 regulating pathogenicity, and also provide a reference for analyzing the function of zinc finger transcription factors.

Fusarium verticillioides and F. graminearum are the main pathogens that cause stalk rot, ear rot and other diseases in maize (Zea mays), which can reduce the yield and quality of maize, and affect mechanized harvesting. At the same time, mycotoxins produced by pathogens can seriously threaten the health of human and livestock. Heat shock proteins (HSP) can be expressed largely when the organism is under environmental stress to prevent protein denatured and improve the organism's resistance to various adversities. Currently, Hsp70 is the most conservative and widely studied type. In order to investigate whether the growth of F. verticillioides and F. graminearum were regulated by temperature, F. verticillioides and F. graminearum were inoculated into the culture medium with the same growth period, and place them in the incubator at 20, 25 and 30 ℃ respectively. It was found that F. verticillioides grew rapidly at 30 ℃, with dense hyphae, while F. graminearum grew better at 20 ℃. In order to further clarify whether HSP70 gene family is involved in regulating the growth and development of F. verticillioides and F. graminearum under adverse temperature, nine HSP70 genes were identified in F. verticillioides genome and 6 HSP70 genes were identified in F. graminearum genome through genome-wide search. The relative expression levels of Hsp70 gene family in F. verticillioides and F. graminearum cultured at 20, 25 and 30 ℃ were detected by qPCR. The results showed that the expression levels of FvHsp70-2, FvHsp70-4, FvHsp70-5 and FvHsp70-6 in the F. verticillioides Hsp70 gene family were significantly increased at 20 ℃ (P<0.05), especially the gene FvHsp70-2, which was about 4.5 times higher at 20 ℃ than at 30 ℃. In F. graminearum, while the expression levels of FgHsp70-6 closely related to FvHsp70-6, FgHsp70-2 closely related to FvHsp70-2, FgHsp70-4 closely related to FvHsp70-4, and FgHsp70-3 closely related to FvHsp70-5 were significantly increased at 30 ℃ (P<0.05). These results indicated that Hsp70 genes are responsive to adverse temperature in F. verticillioides and F. graminearum, and the expression patterns of Hsp70 family genes established at different temperatures can provide a theoretical basis for analyzing the molecular mechanism of Hsp70 influencing temperature change to regulate the growth of F. verticillioides and F. graminearum.

Bacterial angular spot of cucumber is one of the common bacterial diseases in the greenhouse cucumber (Cucumis sativus) cultivation. Using biocontrol bacteria is an important method to control the disease. In order to improve the antagonistic activity of Bacillus velezensis against bacterial angular spot of cucumber, the methods of ultraviolet (UV) mutagenesis and the atmospheric room temperature plasma (ARTP) mutagenesis were used to mutagenize the Bacillus velezensis strain ZF145. Taking Pseudomonas amygdali pv. Lachrymans as the indicator bacteria, screening was carried out by plate confrontation and in vivo experiment, and the mutation efficiency of the 2 mutagenesis methods was compared. The results showed that the control effect of positive mutant strain A561 on cucumber bacterial angular spot was 63.61%, which was much higher than that of ZF145 and the 5% Zhongshengmycin Wettable Powder. The expressions of the surfactin synthetase gene clusters (srf) genes srfAA, srfAB and srfAC associated with the synthesis of surfactin were significantly up-regulated (P<0.05). The positive mutation rate of ARTP mutation was 10.86%, much higher than that of UV mutation, which was more suitable for mutation breeding of Bacillus. This study provides a theoretical support for the mutation breeding of Bacillus velezensis, and is of great significance for the biological control of bacterial angular spot of cucumber.

The yeast two-hybrid (Y2H) system is one of the most effective molecular biology methods to identify protein-protein interactions. Y2H system has played an important role in proteomics, functional genomics, pathology and other research fields. Due to the success of Y2H, many derivative technologies based onY2H have been developed in recent years. The principle of Y2H, one-hybrid system, three-hybrid system, reverse two-hybrid system, cytosolic yeast two-hybrid system and other derivative systems were reviewed in this paper. The application of Y2H in high-throughput screening, quantitative analysis of protein interaction, protein interactome, pathogenic mechanism, search for new drug targets, and agriculture were introduced as well. The prospect of Y2H was also brought out in this article. This review will help researchers comprehend the principles and development process of Y2H. It can also help researchers understand the application scope of Y2H. This review would provide references for those who need to use Y2H to study protein-protein interactions.

Plant diseases are caused by pathogenic microorganisms, such as bacteria, fungi and viruses, which seriously limit crop productivity and increase economic losses. With the development of sustainable agriculture, the accuracy, sensitivity, operability and portability of pathogen rapid detection technologies are the urgent need for agricultural producers. With the multidisciplinary fusion of biotechnology, material science and other cutting-edge technologies, new biosensors are developing towards miniaturization, high sensitivity, real-time detection and gradually plays an important role in the study of plant pathogens. In this review, the recent advancement in the development of advantageous biosensing systems for plant pathogen detection based on both nucleic acid and protein identification is reviewed. The current development status of nucleic acid based biosensors using nano materials, glassy carbon electrode, and nano chips, and protein based biosensors using enzymes, antibodies and aptamers are summarized. The practical application of real-time detection of plant pathogens using these sensors are prospected. This article provides reference and promotion for further research of biomolecular sensing techniques in the field of agricultural plant protection.

Herbicide-tolerant soybean (Glycine) 'DBN9004' is a new genetically modified soybean event with tolerance to both glyphosate and glufosinate. It has obtained safety certificate and has important industrial application prospect in China. In order to meet the technical requirements of safety supervision for genetically modified crops, it is urgent to develop quantitative detection methods for 'DBN9004'. In this study, the 3' flanking sequence of herbicide-tolerant soybean 'DBN9004' was used as the target, primers and probes were designed, and a qPCR assay was established. The specificity, accuracy, precision, detection limit, quantitative limit and other indicators were tested. The results showed that the method was specific and reproducible, with a quantitative limit of 0.1% (40 copies) and a detection limit of 0.05% (20 copies) , the relative standard deviation (RSD) was 6.00%~10.95%, and the quantitative results of qPCR and droplet digital PCR method were consistent. This method provides a new technical means for the accurate quantitative detection of new herbicide-tolerant soybean event 'DBN9004', and provides technical support for the regulation of agricultural transgene crops.

For the detection of enzootic nasal tumor (ENT) caused by Enzootic nasal tumor virus 2 (ENTV-2), the RT-PCR followed by restriction enzyme digestion method, RT-PCR, and qPCR had been established. However, the existence of endogenous retroviruses (ERVs) can interfere with the accuracy of the results. In this study, ENTV-2 was compared with ERVs and Jaagsiekte sheep retrovirus (JSRV) by bioinformatics methods, the conserved sequence of envelope protein (env) gene of ENTV-2 was found, and a pair of specific primers were designed. By optimizing the reaction conditions, a high resolution melting curve (HRM) detection method was established. The optimization results showed that the best reaction system was 0.4 μmol/L upstream and downstream primers, 1 μL template, 1 μL styo9 (2.5 μmol/L), 2×Premix Taq 10 μL, add ddH₂O to a final volume of 20 μL. The reaction condition was 94 ℃ for 2 min; 94 ℃ for 15 s; 59 ℃ for 15 s, 72 ℃ for 15 s, 40 cycles. The env gene recombinant plasmid was constructed as standard positive plasmid and used to determine the specificity, sensitivity, and reproducibility of the HRM method. The results showed that the established HRM method could only specifically detected ENTV-2 but not ERVs which was highly homologous with ENTV-2. The sensitivity of the HRM was 82.7 copies/μL for the positive plasmid. The repeatablity of the method was good, the coefficient variations (CV) of intra-batch and inter-batch were < 1%.The positive detection rate of 92 clinical samples was 18.7%, and the coincidence rate with TaqMan detection method was 100%. This study provides technical support for the early and rapid detection of the diseases related to ENT.

Porcine epidemic diarrhea (PED) is a highly contagious, highly lethal disease caused by Porcine epidemic diarrhea virus (PEDV). It causes severe diarrhea in 3~10 day-old piglets (Sus scrofa). The immune protection of PEDV depends on the neutralizing activity of colostrum and serum neutralizing antibodies against PEDV S1 protein. The establishment of rapid and high-throughput detection method of neutralizing antibodies is conducive to the prevention and control of PED. In order to evaluate PEDV neutralizing antibodies, an indirect ELISA method based on the S neutralizing peptide of PEDV genotype Ⅱ strain were established. In this study, the optimal conditions were determined that coating concentration of antigen was 0.2 μg/well by the equation titration. The dilution of serum was 1∶100, the reaction time of serum was 30 min, the optimal concentration of secondary antibody was 1∶6 000, the reaction time of secondary antibody was 60 min, and the reaction time of substrate was 10 min. The sensitivity, specificity of indirect ELISA were determined to compare with the neutralization test. The sensitivity and specificity of the indirect ELISA employed in the clinical sera samples were 94.74%, 91.67%, respectively and the Kappa was 0.93. The coefficient of variation of each serum sample after 3 repetitions was less than 10% in which 3 PEDV negative sera and 3 positive sera were tested repeatedly in inter group and intra group by the indirect ELISA. The sera against other pathogens (Swine fever virus, Porcine pseudorabies virus, Porcine reproductive and respiratory syndrome virus) showed no cross-reactivity with neutralizing peptide of PEDV S1 by the indirect ELISA. The correlation between the indirect ELISA and the virus neutralization test were performed by statistical analysis from 29 positive sera and 18 negative sera, thus the formula Y=0.370 8X+1.377 2 (X: The S/P value; Y: lg (1/neutralizing titer-32), R²=0.963) was obtained by correlation analysis. Since the neutralizing antibodies were indication about the evaluation of PEDV vaccine efficacy, this indirect ELISA provides a better tool for the large scale detection of anti-PEDV neutralizing antibodies, compared with the neutralization test.