The occurrence of viral diseases of rice (Oryza sativa) in agricultural production is mostly caused by multiple viruses coinfection. When rice is infected with different viruses, different disease characteristics and complex symptoms or disease-like symptoms caused by multiple viruses are often difficult to diagnose only by virological symptoms. Therefore, according to the current agricultural production of China's 6 common rice viruses, namely, Rice stripe virus (RSV), Rice grassy stunt virus (RGSV), Rice dwarf virus (RDV), Rice ragged stunt virus (RRSV), Rice black-streaked dwarf virus (RBSDV), Rice ragged stunt virus (RRSV) and Southern rice black-streaked dwarf virus (SRBSDV), specific primers were designed according to the coat protein (CP) gene sequence of each virus, respectively. A rapid and simultaneous one-step multiplex reverse transcription PCR (RT-PCR) method was developed to detect the 6 viruses on rice. The results showed that the primer (10 μmol/L) final volume, RSV, RDV, RGSV, RRSV, RBSDV and SRBSDV were 0.6, 0.4, 0.3, 0.4 0.6 and 0.2 μL, respectively. The dosage of OneStep RT/Taq Mix, 5×reaction buffer and the sample RNA of rice virus were determined to be 0.75 μL, 4 μL and 1 μg, respectively. Finally, add DEPC H₂O to 20 μL. Multiple RT-PCR parameters were set as RNA reverse transcription at 50 ℃ for 30 min; Pre-denaturation step at 94 ℃ for 2 min; 35 cycle denaturation step at 94 ℃, 30 s, annealed at 58 ℃ for 30 s, extended at 72 ℃ for 1 min; With a final extension at 72 ℃ for 10 min. The research results indicated that the system could be efficient and accurate in distinguishing the 6 rice viruses, greatly improved the efficiency of detection. This method can be widely used in laboratory-based accurate detection, field-based rice virus disease diagnosis and vector detection, and also provides technical support and reference basis for the research and development of related products.

Climate changes, feature as the increased temperature and concentration of CO₂ in the atmosphere, substantially affect the agricultural production. Soybean (Glycine max) is an important oil crop, but little is known about the gene regulation of soybean leaves response to elevated CO₂ and warming, which were important to gain insights of soybean adaption and feedback to climate change. In this study, an OTC (open top chamber) experiment was performed to simulate the conditions of elevated CO₂ and warming. Soybean leaves at the full pod stage (R5) were collected for transcriptome analysis. The results showed that 5 646 differentially expressed genes (DEGs) can be annotated as 44 functional terms by Gene Ontology (GO) analysis, among which, cell processes, cell and binding had higher enrichment degree. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the expression of 14 differentially expressed genes which were mainly related with the regulation of photosynthesis pathway increased under elevated carbon dioxide condition. However, the expression of 16 out of 17 differentially expressed genes which were mainly related with fatty acid metabolism pathway increased under elevated CO₂ and warming conditions. Warming did not induce any significant changes of differentially expressed genes. The results would provide theoretical basis for further exploring the molecular mechanism of soybean response to future climate changes.

Phosphatidylinositol signaling system plays an important role in plant development. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is one of the most crucial enzyme that catalyze PIP2 synthesis in this signaling system. The present study identified 19 CaPIP5K family genes in pepper (Capsicum annuum) genome database, among which 14 CaPIP5Ks were dispersedly distributed on 8 out of the 12 chromosomes and 5 CaPIP5Ks located on chromosome 0. Except 1 PIP5K domain was confirmed in every CaPIP5K proteins, 5~8 repeated MORN (membrane occupation and recognition nexus) domains were also detected in 8 CaPIP5Ks. All CaPIP5K proteins were predicted to locate in the cytoplasm. Moreover, cis-acting elements in CaPIP5K promoter region mainly responded to hormones, defense and stress, and low temperature. In addition, the expressional analysis indicated that CaPIP5K4-1 not only specifically expressed in flower instead of other tissues, but also gradually improved with the development of flower buds in fertile accessions as compared to sterile accession. This study provides a reference basis for further exploring the mechanism of CaPIP5K gene family in the development of anther and pollen in pepper.

Seed-related traits are important agronomic traits of crops, which are of great significance to melon (Cucumis melo) genetics and breeding and related molecular biology research.In this study, the thin-skinned melon small seed line 'P5' was used as the female parent, and the thick-skinned melon big seed line 'P10' was used as the male parent, F₁ and F₂ populations were obtained, 450 F₂ plants were used to carry out genetic analysis of seed related traits. The results showed that the segregation ratio of seed shape and seed coat color was consistent with 3∶1 (χ²=0.26, 0.07). Both of them were quality traits controlled by a pair of alleles. The results of F₂ population distribution showed that seed length, seed width and 100-seed weight showed unimodal normal distribution, which were quantitative traits. The 127 F₂ individual plants were used to carry out specific-locus amplified fragment sequencing (SLAF-seq) technique and construct a genetic map of high saturation melon, and preliminary QTL analysis of seed-related characters was carried out. The results showed that a melon genetic map containing 12 linkage groups was constructed, and 3 716 markers were obtained, the total map distance was 1 356.49 cM, and the average genetic distance between markers was 0.37 cM. The locus SS3.1 controlling melon seed shape (SS) was located between Marker555072 and Marker555263 on melon chromosome 3, and the distance from the linkage markers on both sides was 83.91 and 84.34 cM, respectively, with a contribution rate of 26.45%. The locus SC12.1 controlling melon seed color (SC) was located between Marker1467315 and Marker1479935 on melon chromosome 12, and the distance from the linkage markers on both sides was 62.60 and 63.44 cM, respectively, and the contribution rate was 14.07%. Two QTL loci (HSW6.1 and HSW7.1) were detected to control the 100-seed weight of muskmelon seeds. HSW6.1 is located between Marker795158 and Marker795692 on chromosome 6, and the distances from the linkage markers on both sides are 17.67 and 21.03 cM, respectively, with a contribution rate of 13.97%. HSW7.1 is located between Marker854935 and Marker857435 on chromosome 7, and the genetic distances to the linkage markers on both sides are 3.17 and 5.45 cM, respectively, with a contribution rate of 9.76%. A QTL locus (SL3.1) controlling the seed length of muskmelon was detected, which was located between Marker554875 and Marker554952 of melon chromosome 3, The genetic distances between the locus and the linked markers on both sides are 82.65 and 83.49 cM, respectively. The contribution rate is 27.43%. Two QTL loci (SW3.1 and SW12.1) were detected to control the seed width of melon. SW3.1 is located between Marker554875 and Marker554924 on chromosome 3. The genetic distances to the linkage markers on both sides are 82.65 and 83.07 cM, respectively, with a contribution rate of 16.03%. SW12.1 is located between Marker1411425 and Marker1411798 on chromosome 12, and the genetic distances to the linkage markers on both sides are 29.83 and 29.83 cM, respectively, with a contribution rate of 12.88%. The results provide a theoretical basis for further cloning gene mining and functional analysis of melon seed related traits.

Fat mass and obesity associated gene (FTO) belongs to the non-heme plus dioxygenase superfamily and plays an important role in mammalian fat development. This study takes Qinghai Datong yak (Bos grunniens) as the research object, the CDS region of FTO gene was amplified by RT-PCR, and its function and structure were predicted by bioinformatics online softwares. At the same time, the expression of FTO gene in different stages (18 and 30 months old), different tissues (heart, liver, spleen, lung, kidney, longissimus dorsi muscle, subcutaneous adipose) and different stages of yak preadipocyte differentiation (0, 4, 8 and 12 d) were analyzed by qPCR. The results showed that the full length of FTO (GenBank No. OM640141) coding region was 1 518 bp, encoding 505 amino acids. The FTO protein mainly consisted of α-helix (44.36%), irregularly coiled (38.42%), extended chain (10.89%) and β-turned (6.34%), without transmembrane structure and signal peptide, belonging to an unstable hydrophilic protein. The results of phylogenetic tree showed that the amino acid sequence of FTO was 99.80% homologous to cattle (Bos taurus) with the closest genetic distance, and was closely related to zebu (B. indicus) and wild yak (B. mutus), and most distantly related to horse (Equus przewalskii). The results of qPCR showed that FTO expression was highest in yak heart tissue and subcutaneous adipose tissue, and lowest in spleen. The expression of FTO was significantly higher at 30 months than that at 18 months age (P<0.05). The FTO expression was found to be significantly decreased at 4, 8 and 12 d (P<0.05) during yak preadipocyte differentiation, and the FTO protein expression detected by Western blot had the same changing trend. This study can provide basic material for further research on the biological function of FTO gene in yak fat development.

Apoptosis is an important way to maintain the homeostasis and function of immune organs, which is beneficial to maintain homeostasis in goats (Capra hircus) under transport stress. The study was to investigate the effects of transport stress on apoptosis and the expression of B-cell lymphocytoma-2 (Bcl-2) and Bcl-2 related X protein (Bax) in goats immune organs. Twelve healthy male Ganxi goats were randomly divided into 3 groups: Control group (non transported), transport 2 h group and transport 6 h group. The immune organs (spleen, mesenteric lymph nodes) of goats were collected after transportation. Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptosis in immune organs. Immunohistochemistry, Western blot, qPCR methods were used to locate and detect at the protein and gene level. TUNEL analysis showed that the apoptosis rate of spleen and lymph nodes in transport 2 and 6 h were significantly higher than the control group (P<0.01); Immunohistochemical results showed that Bax and Bcl-2 were positively expressed in goats splenic nodules and marginal area of spleen, and lymphatic nodule and surrounding diffuse lymphatic tissue of lymph nodes before and after transportation, but the expression intensity varied among the 3 groups. Western blot results showed that the proteins expression levels of Bax, Bcl-2 and Bax/Bcl-2 ratio were not statistically different among the 3 groups in spleen and lymph nodes (P>0.05). qPCR results showed that, compared with the control group, the mRNA expression levels of Bax and Bcl-2 in transport 2 and 6 h groups and Bax/Bcl-2 ratio in transport 6 h group were significantly increased in spleen. In the lymph nodes, the gene expression levels of Bax and Bcl-2 in transport 2 h group were significantly decreased (P<0.01), and the gene expression of Bcl-2 in 6 h group was significantly increased (P<0.01), Bax/Bcl-2 ratio in transport 2 and 6 h groups was significantly decreased (P<0.01). The results showed that transport stress could lead to increase apoptosis and significant change of the expression of Bax and Bcl-2 in immune organs. The present study provides a theoretical basis for preventing and treating the effects of transport stress in goats.

Oviduct epithelial cells (OECs) can provide an ideal physiological and biochemical environment to support embryonic development, but their proliferation ability in vitro is weak, which can not meet the needs of experimental research and application. In this study, exogenous telomerase reverse transcriptase gene (TERT) was transferred into Mongolian sheep (Ovis aries) oviduct epithelial cells (sheep OCEs, SOECs), and the proliferation ability of Mongolian sheep oviduct epithelial cells modified by telomerase gene was observed in vitro. Firstly, the SOECs system was established by enzyme digestion, and the recombinant plasmid pCI-neo-TERT was transfected into the primary SOECs by liposome method. The results showed that the expression of TERT gene was detected by qPCR; The growth curve and activity of cytokeratin18 (CK18), a specific marker of epithelial cells, were detected by immunofluorescence. The results showed that TERT gene was successfully introduced into SOECs, and PCR showed that 276 bp TERT gene fragment was expressed in the cells, and the expression of TERT gene was significantly higher than that of the uninfected and transfected pCI-neo empty vector (P<0.001). The transfected cells still retained the biological characteristics of SOECs. The doubling time of P₅ TERT-SOECs was 46.61 h, while that of P₅ SOECs was 65.03 h. It was obvious that the proliferation rate of TERT-SOECs was significantly higher than that of SOECs. In conclusion, the study successfully established the TERT-SOECs system with strong in vitro proliferation ability, which laid an important foundation for the seed cell supply of germplasm cell bank.

Nicotinamide mononucleotide (NMN) has a variety of biological functions, which are useful for heart and brain diseases, senile degenerative diseases, neurodegenerative diseases, and anti-aging. The skeletal muscle treated with NMN has significantly preventive effect on the transcriptional changes related to aging, but the effect of NMN on the proliferation and differentiation of skeletal muscle is still unclear. In order to understand the effect of NMN on cell myoblast differentiation and enrich the mechanism of muscle growth and development, this study used mouse ((Mus musculus) myoblasts (C2C12) as a model, and different concentrations of NMN (0, 0.1, 1 and 2 μg/mL) was applied to 2% horse serum (HS) to induce differentiation of C2C12. The Cell Counting Kit-8 method (CCK-8 method) was used to detect the viability of C2C12 cells after NMN treatment; The cells were stained by 2 fluorescent probes, 2',7'-dichlorodi hydrofluorescein diacetate (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide (JC-1), respectively, the effects of NMN on mitochondrial membrane potential and reactive oxygen species (ROS) of C2C12 cells were detected; Western blot (WB) and immunofluorescence technique were used to detect the expression of myogenin (MyoG), myogenic differentiation 1 (MyoD1) and myosin heavy chain (MYH). The results showed that 0, 0.1, 1 and 2 μg/mL NMN had no toxic effect on C2C12 cells, and a certain concentration of NMN could promote cell viability, increased mitochondrial membrane potential, and up-regulated the expression of myogenic differentiation related proteins MyoG, MyoD1 and MYH within a certain period of time. The above research results indicated that a certain concentration of NMN could promote the myogenic differentiation of C2C12 cells within a certain period of time. This research enriches the physiological role of NMN by exploring the role of NMN in cell myogenic differentiation, and provides a new reference basis for the subsequent application of NMN in the animal husbandry industry.

miR-458b-5p mediates the Wnt/β-catenin signaling pathway to regulates the development of chicken (Gallus gallus) ovarian follicles, thus affecting the level of laying in poultry. The SNP of pri-miR-458b affects the processing of miR-458b. To explore the association of pri-miR-458b gene polymorphisms with laying trait, in this study, SNPs in pri-miR-458b gene were detected with the direct sequencing and then their correlations with the laying traits were analyzed in Jining Bairi chickens. The results showed that a total of 30 SNPs were detected in the pri-miR-458b gene, and 10 loci of them were related to laying traits. Among them, rs316393825, rs734005133, rs737338503, rs733344248, rs740622221 and rs733777709 were linkage imbalance. Their combined genotype significantly affected egg number from 42 to 49 week of age and egg number until 49 week of age. The dominant haplotype of AGCGCG could increase egg number from 42 to 49 week of age by 3.29 and egg number until 49 week of age by 8.08. Compared with the free energy of AGCGCG type, the free energy of GGCGCG type decreased by 3.3 kcal/mol, while that of GTTAAA type increased by 5.3 kcal/mol. The secondary structure stability of GGCGCG type was the highest, AGCGCG type was the second, and GTTAAA type was the lowest. This study provides valuable molecular genetic markers for auxiliary selection of laying traits in Jining Bairi chickens.

Chicken occupies the first place in meat consumption, and excessive lipid deposition will reduce meat quality. Studies have shown that autophagy participates in liver lipid metabolism, improves lipid deposition and regulates lipid metabolism in poultry. This study aims to investigate the effect of autophagy related gene 5 (Atg5) on lipid metabolism related genes in chicken fibroblast cell line (DF-1). According to the amino acid sequence of chicken ATG5 protein in NCBI database, the physical and chemical properties of chicken ATG5 protein were analyzed by ExPASy database. The expression of Atg5 in chicken tissues was detected by qPCR. The overexpressed recombinant plasmid pcDNA3.1-Atg5 was constructed, and the expression of Atg5 was interfered by RNAi technology. The expressions of lipid metabolism-related genes: Acetyl CoA carboxylase (ApoB), fatty acid synthase (FASN), acetyl CoA carboxylase (ACACA), peroxisome proliferator-activated receptor-α (PPARα), forkhead box protein-1 (Foxo1), fatty acid-binding protein 7 (FABP7) and sortilin 1 (Sort1) were detected after interference and overexpression of Atg5. The results showed that chicken ATG5 protein was an unstable protein with relative molecular weight of 32.478 kD. Then Atg5 was expressed in all tissues, and the highest mRNA expression was found in breast muscle tissue. In addition, the overexpression vector pcDNA3.1-Atg5 was successfully constructed, and optimal interfering siRNA was siRNA-330. Overexpression or interference of Atg5 gene affected the expression of other lipid metabolism-related genes. Among them, after overexpression of Atg5, the expression level of ApoB decreased significantly, and the expression level of Sort1 increased significantly, the change of FABP7 was not significant. After interference with Atg5, the expression levels of FABP7 and ApoB were significantly increased, but the change of Sort1 was not significant, and it was speculated that the expression levels of Sort1, FABP7 and ApoB might be potentially related to Atg5. This study provides a reference basis for further study on the function of Atg5 gene in chicken.

The black Muscovy duck (Cairna moschata) is excellent lean meat ducks that is drought-tolerant and rough-fed, which has important value and special status in the poultry industry. However, the black Muscovy duck has strong broodiness and low fecundity, which restricts rapid development of the industry. The ovarian tissues of 3 black Muscovy ducks at the laying and broody stages were collected, respectively. The TRIzol method was used to extract total RNA from the ovarian tissues, the library was constructed and high-throughput sequencing technology was used to analyze the differential expression of miRNA between the 2 groups, then the known, novel miRNAs were predicted for target genes, and the functions of the target genes were enriched and analyzed by GO and KEGG, at last the high-throughput sequencing results were verified by qPCR. The results showed that the raw reads produced by sequencing exceeded 11 099 443 pieces in both groups, and the proportion of clean reads after filtering was all higher than 96.15%, which suggested the data could be used for subsequent analysis. A total of 344 miRNAs were identified, including 275 known miRNAs and 69 newly discovered miRNAs. Sixteen significantly differently expressed miRNAs were screened, including 5 down-regulated and 11 down-regulated in broody ducks with 440 predicted target genes, in which growth hormone secretagogue receptor (GHSR), follistatin (FST) and other target genes were related to reproduction and ovarian development. Thus, it was speculated that the corresponding gga-mir-16c-5p, gga-mir-146a-3p and novel_ 247、novel_ 325 and other miRNAs might be related to broodiness of black Muscovy duck. GO enrichment analysis showed that chromosome organization, nuclear chromosome, spindle microtubule, DNA binding, and nucleotide kinase activity were related to reproduction and development. KEGG pathway annotation placed 234 target genes into 101 signaling pathways, including Wnt and insulin signaling pathways which might be related to germ cell differentiation and development. qPCR confirmed that relative expression levels of up-regulated and down-regulated miRNAs were consistent with the high-throughput sequencing results. This study screened key miRNAs related to broodiness in Muscovy ducks, providing basic material for analyzing broodiness mechanism of Muscovy duck from transcriptional or post transcriptional regulatory level and accelerating the breeding of new high-yield strains.

The Ichthyophthirius multifiliis has high incidence and high mortality, and lacks effective treatment. It poses a great threat to the aquaculture breeding industry. To observe the infection of Ichthyophthirius multifiliis in rainbow trout (Oncorhynchus mykiss), the gill and skin of rainbow trout (treatment group and control group) were observed by tissue section and scanning electron microscope, and the dynamic expression of Toll-like recepotor (TLR) signaling pathway TLR2, TLR3, TLR4 genes and cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ) genes in gill, skin, liver, head kidney, spleen and intestine were detected by qPCR method. The results showed that the structure of skin and gill were seriously damaged by I. multifiliis. There were a large number of irregular secretions on the skin surface. There were gaps between epidermis and dermis, and the trophozoites of different sizes were parasitic in the gaps. The gill filament was damaged and shed, the gill lamellae were deformed and twisted, and the gill filament bone was swollen and deformed. Compared with the control group, TLR2 gene expression in the treatment group was significantly higher in gill and skin by 17.19 and 30.57 times (P<0.05), and significantly lower in spleen by 0.65 times than the control group, with no significant difference in other tissues. The expression of TLR3 gene in gill, skin, spleen and intestine was significantly increased (P<0.05) and were 3.8, 14.09, 1.66 and 1.89 times higher than those in the control group, respectively, and there was no significant difference in liver and head kidney (P>0.05); TLR4 gene expression was significantly higher in the skin by 2.24 times and lower in the liver and gill by 0.36 and 0.22 times than in the control group (P<0.05). There was no significant difference in other tissues (P>0.05). Compared with the control group, the treatment group expression of TNF-α in gill, skin and head kidney increased significantly, IL-1β and IFN-γ were significantly increased in all tissues in this study. The expression of TNF-α in skin was the highest, it was 8.22 times higher than the control group. The expression of IL-1β in head kidney was the highest, it was 14.6 times higher than the control group. The expression of IFN-γ in intestine was the highest, it was 5.1 times higher than the control group. TLR signaling pathway was involved in the defense mechanism of rainbow trout after being infected with I. multifiliis, and the gene expression in this pathway was tissue specific. After rainbow trout were infected by I. multifiliis, the organism established a systemic immune mechanism and produced multiple immune responses. This study revealed the pathological changes and the role of TLR signaling pathway after rainbow trout infection, and provides a theoretical reference for the prevention and treatment of rainbow trout infestation with I. multifiliis.

Light intensity is an important environmental factor that can affect the physiological behavior and metabolism of cultured varieties in aquaculture. In this study, 3 light intensities were used to stress the golden pompano (Trachinotus ovatus), including low light intensity (10 lx), medium light intensity (about 250 lx), and high light intensity (1250 lx). The metabolome analysis was performed to illustrate the effect of extreme lights on metabolism of T. ovatus. The liquid chromatography-mass spectrometry (LC/MS) was employed to detect the metabolites. The principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used to screen differential metabolites in the liver, and the MetaboAnalyst database was used to analyze metabolism-related pathways. The results showed that compared with the control group, 102 differential metabolites in 10 lx group were found and enriched in 39 metabolic pathways, including β-alanine metabolism, tyrosine metabolism, L-glutamate metabolism and biosynthesis of unsaturated fatty acids. 55 differential metabolites in 1 250 lx group were found and enriched in 27 metabolic pathways, including glucose metabolism, biotin metabolism, thiamine metabolism, pyruvate metabolism and taurine and hypotaurine metabolism. Pathway analysis showed that low light stress inhibited amino acid metabolism and fatty acid synthesis in T. ovatus, and the fish might try to adapt low light environment by reducing movement, enhancing immune function and antioxidant capacity. The high light stress promoted glucose production, inhibited pyruvate metabolism and taurine synthesis, and might affect metabolism and immune function of T. ovatus. This study provides a valuable reference for setting culture parameters in T. ovatus aquaculture.

The bZIP (basic leucine zipper, bZIP) transcription factors are widely present in phytopathogenic fungi, and play an important regulatory role in the morphogenesis and infection of pathogens. Systematic studies of the bZIP transcription factor family in Botrytis cinerea have been rarely reported. In this study, the bZIP family genes in Botrytis cinerea were genome-wide identified, the protein physicochemical properties, phylogenetic evolution, conserved domain, and the expression pattern in pathogen conidia during development and infection period were analyzed. Meanwhile, qPCR was used to detect the expression of bZIP family genes after NaCl and H₂O₂ treatment. The results showed that there were 16 bZIP family genes identified from Botrytis cinerea genome, which were divided into 4 subfamilies by phylogenetic analysis. All bZIP family genes of Botrytis cinerea contained the typical BRLZ domain of bZIP family. Some genes showed high expression levels at different development of conidia and infection stages of Botrytis cinerea. All bZIP family genes of Botrytis cinerea were significantly down-regulated after NaCl and H₂O₂ treatment. These results indicated that bZIP family genes of Botrytis cinerea played an important role in the growth and development, infection process and response to salt stress and oxidative stress. This study provides a theoretical basis for further revealing the function and molecular mechanism of bZIP family genes of Botrytis cinerea.

Pepper blight is a devastating soil-borne disease caused by Phytophthora capsici, which occurs generally in pepper (Capsicum annuum) growing areas worldwide, and causes huge economic loss. The antagonistic activity of Bacillus subtilis BS193 was assessed by dual culture test on V8 plates using 6 plant pathogenic oomycetes. The results showed that BS193 greatly inhibited the targeted pathogens with more than 50% inhibition, and showed the best inhibitory effect on Ph. capsici with the rate of 66.7%. When the BS193 cultural filtrates and Ph. capsici were inoculated simultaneously on pepper leaves, 63.50% disease suppression was observed. In pepper seedling inoculation test, the BS193 strain with concentration of 1×10⁸ cfu/mL at 3 d post inoculation of Ph. capsici showed 89.52% pepper blight disease reduction. In addition, BS193 displayed the better potential in promoting plant growth in greenhouse environment. The height, root length, fresh weight and dry weight of pepper seedlings increased by 50.04%, 48.16%, 73.27% and 48.83%, respectively, after 30 d of root irrigation treatment with 20 mL (1×10⁸ cfu/mL) fermentation broth of BS193 strain. The fermentation filtrate of BS193 could inhibit the mycelium growth and lead to a significant increase in mycelial branching, deformity and shrinkage. Meanwhile, 40% fermentation filtrate could significantly inhibit sporangia production, zoospore release and cyst germination with inhibition rate of more than 90%. Further, BS193 were evaluated for biocontrol traits by solid medium and transparent circle detection, and found that BS193 could form the biofilm and exhibited production of protease and cellulase. Overall, the results indicated that B. subtilis BS193 has significant inhibitory activity against Phytophthora pathogens, and the excellent effect of promoting plant growth, which can be considered as a candidate with great development potential for biological control of pepper blight.

Surface immunogenic protein (Sip) is a surface immunogenic protein of Streptococcus agalactiae, which can be used as a target for the potential vaccine antigen against S. agalactiae in tilapia (Oreochromis niloticus). In order to improve the expression level of recombinant Sip protein, the prokaryotic expression and immunogenicity analysis of codon optimized Sip protein from S. agalactiae was present in this study. The codon of Sip gene was optimized according to the Escherichia coli codon preference, and then the optimized gene was inserted into the expression vector pCzn1. Then, the newly constructed plasmid pCzn1-Sip was transfected into E. coli BL21 (Plys) cells for expression. Based on ultrasonication and purification by Ni column, the recombinant protein of Sip was obtained. The expression level and specificity of the recombinant codon optimized Sip were detected by SDS-PAGE and Western blot. The results showed that the 1 299 bp fragment of the optimized Sip gene was obtained by PCR amplification. The constructed recombinant plasmid pCzn1-Sip included 2 fragments about 4 400 bp and 1 299 bp after double digestion, which was consistent with the predicted value. The sequencing results showed that the optimized Sip gene was successfully transferred into the expression vector and the encoded amino acid sequence of Sip protein remained the same. SDS-PAGE and Western blot analysis showed that the recombinant Sip protein was obtained, which with a relative molecular weight of about 53 kD, and the recombinant optimized protein had S. agalactiae antigenicity. After purification by Ni column, it was found that the expression level of optimized soluble Sip was about 4 times compared with that of the non-optimized. Moreover, after immunization, the optimized Sip recombinant protein could supply about 69.23%~74.35% relative percent survivals against S. agalactiae challenge in tilapia. The results showed that the codon optimization of S. agalactiae surface protein Sip could obviously improve its expression level in E. coli. Moreover, the optimized Sip protein still has shown immunogenic. This study provides a scientific basis for the research and development of genetic engineering vaccines for tilapia streptococcus disease.

Gene editing technology is one of the modern breeding technologies for stable and accurate modification of target genes. CRISPR/Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most widely used gene editing technology available today.It recognizes target sites of target genes to break the double-stranded DNA and activates DNA damage repair pathway, so as to achieve accurate gene editing. Now CRISPR/Cas9 has been widely used in rice (Oryza sativa), corn (Zea mays),peanut (Arachis hypogaea) and other important crops. In addition, it can be applied to polyploid crops such as wheat (Triticum aestivum), cotton (Gossypium hirsutum) and soybean (Glycine max) which are thought difficult to be edited. In this paper, the principle of CRISPR/Cas9 mediated gene editing system, its application in polyploid crops and existing problems are discussed on the basis of summarizing some achievements. Meanwhile, suggestions for improving gene editing efficiency are also put forward, hoping to provide reference for future research.

Identification of species origin of animal fibres is important in textile inspection work. In this study, a universal primer set for yak (Bos grunniens), camel (Camelus dromedarius and Camelus bactrianus), sheep (Ovis aries), goat (Capra hircus), cattle (Bos taurus) and pig (Sus scrofa) were designed according to their mitochondrial genomes. Moreover, species-specific primer sets for yak, camel, sheep and goat as well as a universal primer set for goat and sheep were also generated. After that, several DNA extraction methods were compared to find out the most optimal method for animal wool shaft fiber. Then the preservation status of samples were determined rapidly and examined whether it was mixed accurately by sequencing the amplicon of the universal primer set. By combining the species-specific primers amplification and electrophoresis detection, we are able to identify the animal species sources of wool shaft fibers with limit time and cost. In this study, an accurate, rapid and simple molecular identification system was established to distinguish the animal species sources of wool shaft fibers, which provides a technical reference for the quality control of textile products.

Hepatocyte nuclear factor 3β (HNF 3β) is an important transcription factor involved in the foregut and liver development in mammalian and energy metabolism in adult liver. However, the function of HNF 3β in chicken (Gallus gallus) is still unknown. In order to provide effective antibodies for studying the roles of HNF 3β in glucose and lipid metabolism in chicken liver, the encoding region segment of chicken Hnf 3β was optimized according to the codon preference in Escherichia coli and synthesized. Then this segment was cloned into pET 30a and the HNF 3β recombinant protein was expressed in E. coli. The recombinant protein was purified from the denatured inclusion body with 6 mol/L urea using Ni²⁺ affinity chromatography. The denatured protein was diluted into the refolding buffer at ration of 1∶20 for protein renaturation. The purified recombinant protein emulsified with Freund's adjuvant was used to immunize rabbits (Oryctolagus cuniculus) to prepare chicken HNF 3β antiserum. The antiserum titers were determined using the enzyme linked immunosorbent assay (ELISA) method, and the antibody was purified by the Protein A method. The effects of the antibody were verified by the detection of HNF 3β protein expressed in the hepatocellular carcinoma cell line, leghorn male hepatoma (LMH). The results showed that the recombinant protein HNF 3β was effectively express in E. coli. after the sequence of the chicken Hnf 3β was optimized. The recombinant protein HNF 3β was expressed as the inclusion bodies in E. coli. The recombinant protein was purified by Ni²⁺ affinity chromatography from the inclusion bodies denatured with 6 mol/L urea. The soluble recombinant protein HNF 3β was obtained with a purity of more than 95% after a denaturing and renaturing procedure. The titer of the antiserum from rabbits was over 1∶64 000 after immunization with the recombinant protein for 4 times by ELISA detection. The polyclonal antibodies of HNF 3β were then purified with the protein A affinity chromatography. The western blot results showed that the prepared antibody could specifically detect the expression of HNF 3β protein in LMH cells. The expression of HNF 3β was more than 5 times in LMH cells transfected with the pcDNA3.1(+)-ggaHnf 3β for 48 h in comparison with the control. In conclusion, the high specificity and affinity polyclonal antibodies against HNF 3β were obtained from the rabbits immunized with the purified recombinant protein HNF 3β, which contribute to detect the chicken HNF 3β and provide a tool to investigate its roles in chicken metabolism regulation.

Duck astrovirus-3 (DAstV-3) is a newly emerging infectious disease pathogen in ducks (Anas platyrhynchos domestica) in recent years. In this study, specific primers were designed based on the RNA-dependent RNA polymerase gene (RDRP) sequences of DAstV-3 strains isolated and published in GenBank, and a SYBR Green Ⅰ real-time quantitative PCR assay for detecting DAstV-3 was developed. The results demonstrated that a good linear relationship with the standard curve cycle threshold (Ct) and the template concentrations in the range of 1.15×10¹~1.15×10⁷ copies/μL, the correlation coefficient R² and amplification efficiency were 0.999 and 2.08, respectively. Besides, the assay had good specificity for DAstV-3 and had no cross-reaction with other common infectious disease pathogens (H9 subtype Avian influenza virus, Avian tambusu virus, Duck reovirus, Muscovy parvovirus) in ducks. High sensitivity, minimum detection limit in each rection of the assay was 23 copies for DAstV-3. Good reproducibility, the coefficient of variations (CV) in intra- and inter-assays were 0.14%~0.65% and 0.53%~0.94%, , respectively, both less than 1%, indicating that the method had a good repeatability. 209 suspected DAstV-3 samples during June 2018 and March 2021 were detected by the established assay, and the DAstV-3 detection rate was 56.5% (118/209). SYBR GreenⅠ real-time PCR assay established had good specificity, sensitivity and repeatability, which could provide an effective technical support for detection and epidemiological investigation of DAstV-3.