MicroRNAs (miRNAs) are 18~22 nt and highly conserved non-coding RNAs involved in plant response to various abiotic stresses. Some members of the miR169 family were found to respond to heat stress based on previous high-throughput small RNA sequencing. This study used bioinformatic tools to analyze and predict the chromosome distribution, sequence structural characteristics, phylogenetic relationships, and duplication of the miR169 family; qRT-PCR was used to detect the expression of miR169 family members and their target genes in rice (Oryza sativa) seedlings at multiple time points under high-temperature stress treatments. The results showed that the miR169 family had 2 major evolutionary branches, and the members expanded mainly through tandem duplication events and chromosome segmental duplication. MiR169h, miR169l, miR169i and miR169m locating in the same branch were active gene clusters in the evolution of rice. Among them, 3 members, miR169h, miR169l and miR169i, all responded actively to heat stress, and the predicted target gene, nuclear factor Y-A1 (NF-YA1), showed down-regulated expression trend under high temperature treatment for 16~24 h. In summary, the rice miR169 gene family miR169h, miR169i and miR169l responded to intense heat stress in rice and might play an important role in rice heat tolerance. This study provides basic data for in-depth exploration of the molecular regulatory mechanism of miRNA involved in heat stress in rice.

Rice (Oryza sativa) grain length is an important agronomic trait, which is closely related to rice yield and appearance quality. In order to further explore new molecular markers for improving rice grain length, this study developed a fluorescent molecular markers of GW7 (Gene ID: 10208) gene based on the single base mutation of GW7 gene in the functional region, combined with penta-primer amplification refractory mutation system (PARMS). Combined with grain length phenotype and PCR product sequencing, the detection results of the marker in 13 rice parent materials showed that the marker could accurately identify different GW7 genotypes. Then, 16 homozygous GW7 genotypes were selected from 94 rice materials by using this marker. The results of marker-assisted selection showed that excellent rice materials with increased grain length could be obtained in F₂ generation of 'R91' and 'zhuo20'. The marker this study developed can help to selecte individual plants with target traits at the seedling stage, and perform cross-breeding and backcrossing during the flowering period, without having to wait until maturity and harvest thereby accelerating the breeding process for rice grain quality traits.

Watermelon (Citrullus lanatus) is one of the most popular fruits. With people's continuous pursuit of quality of life, improving products quality has become a key factor in high sales of crops. Plant growth promoting rhizobacteria are a group of beneficial bacteria that promote plant uptake of nutrients from the soil and improve plant health. To investigate the growth promoting effect of Bacillus velezensis WB on watermelon plants. Strain WB and watermelon seeds were selected as materials, the growth promoting ability of strain WB was analyzed using functional culture medium, the growth promoting effect of strain WB was validated using seed germination experiments and pot experiments, and the growth promoting mechanism of strain WB was explored using RNA sequencing (RNA-seq). The results showed that B. velezensis WB had the ability to produce auxin (IAA) and cellulase, as well as the ability to solubilize phosphorus, potassium, and to fixate nitrogen. The results of seed germination and pot experiments indicated that strain WB had a promoting effect on the growth of watermelon plants. In addition, strain WB upregulated the expression of genes related to plant growth promoting signaling pathways, including sesquiterpenoid and triterpenoid biosynthesis,phenylpropanoid biosynthesis, and plant hormone signal transduction pathway. Transcription factors were expressed in promoting growth, such as myoptosis (MYB), NAC (NAM, ATAF and CUC), and Dof (DNA binding with one finger). In conclusion, this study found that B. velezensis WB could promote the growth of watermelon and elucidated its growth promoting mechanisms, and providing theoretical basis for its application in future.

It is a global challenge that the remediation of cadmium contaminated farmland. Kenaf (Hibiscus cannabinus) can be used to remediate Cd-contaminated farmland, as it has strong stress resistance. In order to explore the response patterns to Cd stress of HcWRKY gene family in kenaf, in this study, HMMER and BLAST software were used to identify HcWRKY family genes in genome-wide. The ExPasy website was used to obtain the molecular information of the HcWRKY members. The HcWRKY members were mapped to kenaf chromosomes using TBTOOLS v1.6 software. The results suggested that 33 HcWRKY family members were unequally scattered on 12 chromosomes. It was difference that the physical and chemical properties, such as the number of amino acids, molecular weight, and theoretical isoelectric point in each HcWRKY family member. Excepting the HcWRKY4-2 located in cytoplasm, the other HcWRKY family memebers were located in nucleus. Phylogenetic analysis showed that the 33 HcWRKY family members were divided into 4 groups. There was only HcWRKY member in Group Ⅱ and Group Ⅳ; while Groups Ⅰ and Ⅲ contained HcWRKY and AtWRKY proteins. qRT-PCR analysis indicated that 24 HcWRKY family members were induced by Cd stress. During the response to cadmium stress in kenaf, the expression levels of 22 HcWRKY family member genes were up-regulated, while the expression levels of 2 HcWRKY family member genes were down-regulated, which indicated that these HcWRKY genes improved greatly camdium tolerance of kenaf. These results provides basic information for further exploring the biological role of the HcWRKY gene family response to cadmium stress.

As plant specific transcription factors, WOX gene family plays an important role in regulating plant growth and development, tissue and organ genesis and formation. This study used bioinformatics methods to identify 15 members of WOX gene family in the whole genome of Lagerstroemia indica, named LfiWOX1~LfiWOX15, which were distributed on 14 chromosomes. There were large differences in molecular weight and isoelectric point between proteins. All subcellular predictions were located in the nucleus. Systemic evolution analysis showed that genes were classified into 3 categories, and 5 orthologous WOX members closely related to Arabidopsis thaliana and rice (Oryza sativa) WOX genes. Additionally, some WOX genes with specific motifs in L. indica were classified into the same branch on the evolutionary tree, and all family members contain motif 1, motif 2, and motif 5. The WOX family genes contain 9 cis-regulatory elements related to plant growth and development, hormone regulation, and stress, with different members containing different elements. qRT-PCR analysis showed that the expression levels of LfiWOX12, LfiWOX13 and LfiWOX15 in callus tissue increased by 1.5~1.9 times compared to leaves, while the expression levels of other genes in callus tissue decreased by 0.1~90% compared to leaves. It was speculated that LfiWOX12, LfiWOX13 and LfiWOX15 were related to callus formation. The results provide basic data for further studying the regulation of WOX gene on callus induction and theoretical reference for the establishment of efficient regeneration and genetic transformation system in L. indica.

Genomic imprinting is a mammalian epigenetic phenomenon in which genes show parental-specific monoallelic expression. Imprinted genes play an important role in embryonic development and placental nutrition transport. The Ras protein specific guanine nucleotide releasing factor 1 (RASGRF1) gene encodes 140 kD RAS specific guanine nucleotide releasing factor, and it is related to the growth and development traits of animals in infancy. Rasgrf1 gene is a paternal imprinted gene in mice (Mus musculus), but its imprinting status in cattle (Bos taurus) has not been studied. In order to study the imprinting status and regulatory mechanism of RASGRF1 gene in cattle, the expression of RASGRF1 gene in bovine 6 tissues (heart, liver, spleen, lung, kidney, brain) and placenta was analyzed by quantitative RT-PCR, and then the allele expression of RASGRF1 gene in bovine tissue and placenta was analyzed by direct sequencing of RT-PCR products based on SNP. Finally, the methylation status of the promoter region of RASGRF1 gene was studied by sulfite direct sequencing. The results showed that RASGRF1 gene showed tissue-specific expression in cattle, its transcript was not detected in heart and liver. RASGRF1 gene is monoallelically expressed in spleen, lung, kidney and brain, and paternally expressed in bovine placenta. There was no differential methylation region (DMRs) was found in the promoter and exon 1 region of bovine RASGRF1 gene. Hypomethylation was shown in heart, liver, spleen, lung, kidney, brain, placenta and sperm. These results indicated that DNA methylation in this region was not involved in the regulation of imprinting expression of RASGRF1 gene, the imprinting of bovine RASGRF1 gene might be regulated by other epigenetic modifications. This study provides a reference for the further study of the function and imprinting regulation mechanism of bovine RASGRF1 gene.

The processes of maternal pregnancy and delivery in mammals are also related to the processes of inflammatory occurrence and development. Premature inflammation or unbalance immunity could cause pregnant termination or abortion. Elastase, neutrophil expressed (ELANE) plays a crucial role in inflammatory occurrence and development. The current study aimed to explore the localization, expression patterns and potential functions of ELANE in yak (Bos grunniens) placenta in middle and late pregnant phases (n=3/group) using hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) staining, immunofluorescence (IF) staining, qRT-PCR and Western blot. Meanwhile, the potential biological function of ELANE was predicted based on data-independent acquisition (DIA) proteomics data. HE staining revealed that abundant trophoblast giant cells and uninucleate trophoblast cells in yak middle pregnant placenta, while late pregnant placenta of yak exhibited degenerative changes and a reduction in trophoblast cell numbers. IHC and IF showed that ELANE protein was localized primarily in the cytoplasm of yak placenta trophoblast giant cells and uninucleate trophoblast cells. Compared with middle pregnant placenta of yak, qRT-PCR and Western blot results manifested that the relative expression levels of ELANE gene and its protein levels were up-regulated extremely significantly in late pregnant placenta of yak (P<0.01). Bioinformatics analysis suggested that ELANE might regulate phagocytosis, extracellular space, serine-type endopeptidase activity, cytokine binding and other pathways involved in growth and development of yak placenta in middle and late pregnancy. In conclusion, ELANE may participate in placental dynamic development and pregnant maintenance through regulating the function of placental trophoblast cells in yaks. This study provides a reference for further exploration the function of ELANE in yak placental tissues.

Leptin plays an important role in the regulation of energy balance and fat deposition, but its role in energy metabolism in yaks (Bos grunniens) is not yet clear. This study collected perirenal and subcutaneous brown and white adipose tissues of 1- and 30-day-old healthy male yak calves, cloned the CDS of the leptin gene through RT-PCR, analyzed its sequence characteristics using bioinformatics methods, and detected its expression and distribution in yak perirenal and subcutaneous adipose tissues using qRT-PCR and immunohistochemistry (IHC) staining methods. The results showed that the CDS of yak leptin gene (GenBank No. PP385937) was 504 bp, encoding 167 amino acids, and the homology with wild yak (Bos mutus) reached 99.01%, indicating that the gene was relatively conserved during evolution. The gene expression analysis showed that the leptin expression in the perirenal and subcutaneous adipose tissues of 30-day-old yak were significantly higher than those of 1-day-old yak (P<0.05). The IHC showed that the leptin positive staining was observed on the cytomembrane of borwn and white adipocytes in perirenal and subcutaneous brown and white adipose tissues, and the leptin positive staining intensity of 30-day-old yak was significantly higher than that of 1-day-old yak (P<0.05). This study provides basic materials for further elucidating the mechanism of leptin in cold adaptation of yaks.

Global warming has made the issue of heat stress in cows (Bos taurus) increasingly prominent, causing huge economic losses to the dairy industry. Elucidating the molecular mechanisms underlying the occurrence and response of heat stress in cows can better address the issue of heat stress. Differentially expressed long non-coding RNA (lncRNA), microRNA (miRNA), and mRNA profiles between heat and non-heat stressed conditions were screened using high-throughput sequencing in our previous study. In this study, LOC112447378/miR-15a/PRLR was identified as a key competing endogenous RNA (ceRNA) network in heat stress response through bioinformatics analysis based on the high-throughput data. By constructing an in vitro heat stress model using mammary alveolar cells-large T antigen (MAC-T), the mRNA expression levels of LOC112447378, miR-15a, and prolactin receptor (PRLR) were determined using qRT-PCR. The protein levels of PRLR were determined using Western blot. These results were consistent with high-throughput sequencing results. The mRNA expression level of LOC112447378 was significantly downregulated when miR-15a was overexpressed; The mRNA and protein levels of PRLR were significantly downregulated when miR-15a was overexpressed. After knocking down miR-15a, the mRNA expression level of LOC112447378 was significantly upregulated; The mRNA and protein levels of PRLR were significantly upregulated. The methyl thiazolyl tetrazolium (MTT) test results showed that miR-15a inhibited the proliferation of MAC-T cells. The results of Annexin V-FITC/PI dual staining showed that miR-15a promotes apoptosis of MAC-T cells. MiR-15a could target and regulate the target genes LOC112447378 and PRLR, thereby achieving regulatory effects of inhibiting MAC-T cell proliferation and promoting apoptosis. These results indicated that the LOC112447378/miR-15a/PRLR signaling axis could affect the proliferation and apoptosis of MAC-T cells, thereby playing an important regulatory role in the heat stress response process of MAC-T cells. This study provides new ideas and theoretical basis for molecular breeding of heat-resistant and high-yield cows in the future.

CRISPR/Cas12i is a recently developed CRISPR gene editing system by Chinese scholars, which has independent intellectual property rights and has been proven to have targeting efficiency comparable to the CRISPR/Cas9 system. Homology-directed repair (HDR) is one of the main repair mechanisms for double-stranded DNA breaks (DSBs). Gene editing based on the HDR mechanism can be used to correct any form of mutation in the genome, but it is often limited by the generally lower HDR efficiency in mammalian cells. In this study, the activity of the CRISPR/Cas12i system at different target sites in Homo sapiens embryonic kidney cell line HEK293T, the optimal dosage of single-stranded oligonucleotides (ssODN) donor templates for mediating HDR editing, and the appropriate concentration of small molecule drugs were verified through single-strand annealing (SSA) reporter experiment, dose gradient and concentration gradient experiment. The effects of adding different small molecule drugs on the HDR editing efficiency mediated by the CRISPR/Cas12i system in HEK293T cells and sheep (Ovis aries) fetal fibroblasts were then evaluated using flow cytometry sorting, genomic PCR, Sanger sequencing, and online prediction tools. The results showed that the CRISPR/Cas12i system exhibited high activity at 18 different target sites in HEK293T cells, with efficiencies around 80%, except 2 slightly lower sites. Different dosages and lengths of ssODN had some influence on HDR efficiency, and the appropriate concentrations of small molecule drugs varied slightly in different species and cell types. The addition of histone deacetylase inhibitor (HDACi) and RS-1 (C₂₀H₁₆Br₂N₂O₃S) significantly enhanced the HDR editing efficiency mediated by the CRISPR/Cas12i system in both HEK293T cells and sheep fetal fibroblasts. Among them, RS-1 showed the least cytotoxicity and did not significantly reduce the insertion/deletion mutation (InDel) efficiency while improving HDR efficiency. Furthermore, Entinostat increased HDR editing efficiency by approximately 148 fold at the bone morphogenetic protein receptor 1B (BMPR1B) site in sheep fetal fibroblasts. In conclusion, the CRISPR/Cas12i system exhibited high activity and could mediate efficient and precise HDR editing using ssODN as a donor in model cells and primary sheep cells. The appropriate concentrations of HDACi and RS-1 could effectively improve HDR editing efficiency. This study provides reference and guidance for the application and popularization of the CRISPR/Cas12i gene editing system.

MIR206 is a member of the MIR-1 family, which specifically expressed in skeletal muscle of vertebrates. Skeletal muscle is an important part of the body, so MIR206 gene may play an important role in the regulation of animal growth and development. Insertion/Deletion (InDel) is a kind of important genetic variation that may affect gene function by regulating gene expression levels. Therefore, this study investigated the effects of MIR206 InDel variation on growth traits and MIR206 promoter activity of Anhui white goat (Capra hircus) in order to provide theoretical reference for molecular breeding of goat. The body weight, body height, body length, chest circumference, cannon circumference, and rump width data of 431 Anhui white goat were collected. The InDel mutations of MIR206 gene were identified by direct sequencing of PCR products. The genotypes of Anhui white goat were detected by PCR and agarose gel electrophoresis. The distribution of InDel variation in Anhui white goat population was analyzed by calculating genetic parameters. SPSS 25.0 was used to analyze the association between the genotypes and the growth traits of goats. The effect of the genetic variation in the promoter region of MIR206 on promoter activity was analyzed by dual luciferase reporting system. The results of PCR product sequencing and sequence alignment showed that there was a 6 bp InDel mutation (rs653221349) at the 389~394 locus in front of the transcriptional start site of MIR206. There were 3 genotypes of this variant in Anhui white goat population: II (wild genotype), ID (heterozygous genotype) and DD (homozygous deletion genotype), which belonged to moderate polymorphism (0.25<PIC<0.5) and was in Hardy-Weinberg imbalance (P<0.05). The results of association analysis showed that different genotypes of the 6 bp InDel were significantly correlated with body weight, body length and chest circumference of Anhui white goat (P<0.05). The body weight, body length and chest circumference of ID genotype individuals were significantly better than those of II and DD genotype individuals. Results of the dual luciferase reporting system showed that the 6 bp deletion significantly reduced gene promoter activity. In summary, this study found that there was a 6 bp InDel mutation in the promoter region of MIR206 gene of Anhui white goat, which was significantly associated with growth traits of Anhui white goat and reduced promoter activity, which could be used as a candidate DNA molecular marker for molecular marker-assisted selection of Anhui white goat.

Methionine enkephalin (Met-Enk) is an opioid neuropeptide with regulatory effects on cell proliferation and metabolism. In order to investigate the effect of Met-Enk on apoptosis, mitochondrial structure and function of porcine (Sus scrofa) precursor adipocytes, in this study, Met-Enk was used to treat porcine primary precursor adipocytes, and detected apoptosis by using cell counting kit 8 (CCK-8) and annexin V/propidium iodide (AV/PI) staining, and observed mitochondrial structural changes by means of transmission electron microscope (TEM) and atomic force microscope (AFM) and detected the changes of mitochondrial function by JC-1 fluorescent probe and other methods. The results showed that Met-Enk treatment resulted in decreased activity and increased apoptosis rate of porcine precursor adipocytes, causing changes in the expression levels of apoptosis markers Caspase 3 and Caspase 9, etc. The TEM results showed that the number of mitochondria in the precursor adipocytes was reduced, the volume was swollen, and mitochondrial cristae were reduced. The AFM results showed that the mitochondria were deformed, and the surface of the membrane appeared to be concave and the roughness was increased; Immunoblot analysis and qPCR revealed that revealed that Met-Enk treatment led to impaired mitochondrial function and promoted apoptotic factors such as Bax/Bak through opioid growth factor receptor (Ogfr), which prompted the release of cytochrome C from mitochondria and promoted apoptosis. This study deeply investigated the mechanism of Met-Enk inducing apoptosis in porcine precursor adipocytes by affecting mitochondrial structure and function, so as to provide a reference for the use of Met-Enk to improve the development of adipose tissues in livestock and poultry.

Cervical cancer (CC) is a gynecological malignancy, and most of the flavonoids have anti-cervical cancer activities. However, whether the total flavonoids of Allium polyrhizum also have anti-cervical cancer activity is rarely reported. Quantification of cell counting kit-8 (CCK-8) assay, scratch assay, flow cytometry, real-time quantitative PCR, immunoblotting, spot hybridization, N6-methyladenine (m⁶A), methylated RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) methods were used to detect the growth of cervical cancer cells treated with total flavonoids of A. polyrhizum, and found that total flavonoids of A. polyrhizum had a significant effect on HeLa, SiHa and CaSki cervical cancer cells, which could effectively inhibit their proliferation and migration, and promote the apoptosis of HeLa cells. After treatment of HeLa cells with A. polyrhizum total flavonoids, the expression of a pro-apoptotic protein of B-cell lymphoma-2 associated X protein (BAX) was significantly up-regulated, and the high level of global m⁶A modification was related to significant downregulation of m⁶A demethylase fat mass and obesity-associated protein (FTO) expression. In addition, the expression of the m⁶A-reading protein insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) was significantly increased. Bioinformatics prediction revealed that there was a high confidence m⁶A modification site on BAX mRNA, and IGF2BP2 also bound to this site. Similarly, both the m⁶A antibody and IGF2BP2 antibody could significantly enrich BAX mRNA. In conclusion, this study demonstrated that the total flavonoids of A. polyrhizum had good anti-cervical cancer activity and may played an anticancer role by promoting apoptosis of cervical cancer cells through the m⁶A-IGF2BP2 related molecular mechanism. Finally, the results of this study could provide a theoretical basis for the related research on the anti-tumor mechanism of flavonoids, as well as some theoretical reference for the development and application of novel drugs.

Peimine and Peiminine are representative alkaloid medicinal component in the lily family herb Fritillaria thunbergii. They play an important role in the anti-inflammatory effect of F. thunbergii. In order to further investigate the anti-inflammatory effects of these 2 types of alkaloids at the cellular level, this study used lipopolysaccharide (LPS) induced Raw264.7 cells as an inflammatory model, Griess, ELISA, Western blot and qRT-PCR were used to analyze the effects of peimine and peiminine on LPS-induced secretion of pro-inflammatory factors nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in Raw264.7 cells, the results showed that both peimine and peiminine could inhibit the differentiation of Raw264.7 cells and reduce the pseudopodia produced by LPS stimulation, NO, TNF-α, IL-6 and IL-1β in Raw264.7 cell supernatant of medium and high dose groups of peimine and peiminine significantly decreased (P<0.05, P<0.01). Peimine and peiminine inhibited the expression of iNOS and COX-2 protein in Raw264.7 cells, and the inhibition of peiminine on iNOS protein was higher than that of peiminine on COX-2 protein, the inhibitory effect of peiminine a was higher than that of peiminine. The expression of IL-6 and IL-1β in Raw264.7 cells was significantly decreased by both peimine and peiminine. In conclusion, both peimine and peiminine have good anti-inflammatory effect, which provides some data support for the treatment of inflammation by alkaloids.

Ten-eleven translocation 2 (TET2) regulates the innate immune response of mammals in 2 ways: Methylation-dependent and methylation-independent. However, the innate immune regulation of chicken (Gallus gallus) TET2 has not been elucidated. The aim of this study was to clone chicken TET2 gene and construct the eukaryotic expression vector of TET2 and its truncated form, and preliminarily explore the effect of TET2 and its functional domain on chicken innate immune response. According to the chicken TET2 genome (GenBank No. NM_001277794.1) information, primers were designed to clone the full-length CDS sequence. The full length of TET2 was divided into N-terminal N1126 and C-terminal CD 2 truncated fragments, and then the truncated gene fragment was amplified. It was connected to the pCAGGS-Myc eukaryotic expression vector by homologous recombination. The recombinant plasmid pCAGGS-Myc-TET2 and its truncated form were transfected into chicken embryo fibroblasts DF-1, respectively. The protein expression and cellular localization of chicken TET2 and its truncated form were detected by Western blot and immunofluorescence. The effects of overexpression of pCAGGS-Myc-TET2 and truncated vectors on the expression of genes related to the innate immune response induced by ploy (I:C) were detected by qRT-PCR. PCR results showed that the full-length sequence of chicken TET2 and its truncated N1126 and CD were successfully cloned. Bioinformatics analysis showed that Anser cygnoides and Anas platyrhynchos were most closely related to G. gallus TET2. The results of Western blot showed that the full-length and truncated TET2 fused with Myc tag could be expressed normally in DF-1 cells. The results of immunofluorescence showed that the TET2 were localized in the nucleus. The results of qRT-PCR showed that overexpression of full-length and truncated chicken TET2 could significantly promote the expression of melanoma differentiation associated gene 5 (MDA5) and tripartide motif containing 25 (TRIM25) induced by poly (I:C)(P<0.05). However, there was no significant effect on the expression of interferon regulatory factor 7 (IRF7)(P>0.05).Compared with overexpression of N1126 truncation, overexpression of chicken TET2 full-length and CD domain significantly promoted poly (I:C)-induced IFN-β expression (P<0.05). In this study, the effects of TET2 and its truncated mutants on chicken innate immune response were preliminarily explored, which provides a reference for the study of the molecular mechanism of chicken TET2 regulating innate immune response.

White king pigeon (Columba livia) is an important economic poultry in China. To explore the relationship between calpain 11 (CAPN11) gene polymorphism and slaughtering traits is of great significance to the genetic improvement of slaughtering traits and the improvement of economic traits. In this study, SNP of CAPN11 gene of white king pigeon was screened by PCR direct sequencing method, and the relationship between SNP locus and slaughtering traits was analyzed. Through the identification of the SNPs of the CAPN11 gene of the white king pigeon, it was found that g.3641644C>T and g.3641746C>T 2 synonymous mutations were found at positions 636 and 738 in the exon 6 region of CAPN11 gene of the white king pigeon, all of which had 3 genotypes, the dominant genotypes and alleles were CT and C, 0.25<PIC<0.5. χ² test showed that the genotypic distribution of the two SNPs loci were in accordance with Hardy-Weinberg equilibrium. Linkage disequilibrium analysis showed that D'>0.80 and r²>0.33 were not satisfied between the 2 SNPs, indicating that there was no strong linkage disequilibrium effect. Correlation analysis showed that g.3641644C>T locus had a significant effect on antemortem live weight (P<0.05). The effects of g.3641746C>T locus on pre-slaughter live weight, dressed weight, eviscerated weight, chest muscle weight and leg muscle weight of white king pigeons reached significant level (P<0.05). Diplotype analysis showed that 4 haplotypes and 10 diplotypes were detected at 2 SNPs loci in the experimental population. The antemortem live weight, dressed weight, eviscerated weight and leg muscle weight of individuals with haplotype H2H2 (CCTT) were significantly higher than those of the other 9 diplotypes, and the chest muscle weight of individuals with haplotype H2H2 (CCTT) was significantly higher than that of individuals with other diplotypes except H3H3 (TTCC) and H3H4 (TTCT). Through the analysis of the secondary structure of haplotype mRNA, it was found that haplotypes H1 (CC), H2 (CT) and H3 (TC) all caused changes in the mRNA secondary structure of CAPN11 gene, which might affect the efficiency of transcription and translation, and then affect the slaughtering performance. In summary, CAPN11 gene can be used as a potential candidate gene to improve molecular marker selection for slaughtering traits of white king pigeons. This study provides a theoretical basis for accelerating the breeding process of white king pigeons and reference for improving molecular marker breeding of slaughtering traits of white king pigeons.

Japanese encephalitis virus (JEV) is a serious zoonotic virus, and the envelope protein (E) plays a key role in the neurovirulence of JEV. Previous studies have found that the mutation of EI176R site of recombinant Japanese encephalitis virus (rJEV-EI176R) can weaken its neurovirulence. In order to identify the biological characteristics of rJEV-EI176R, the E gene sequence of rJEV-EI176R was cloned and analyzed, and the bioinformatics characteristics of E protein were analyzed. The growth curve of rJEV-EI176R, the adsorption difference of various cells and the inflammatory level of mouse microglia cell line (BV-2) induced by rJEV-EI176R were determined by qPCR, plaque assay and indirect immunofluorescence assay (IFA). The results showed that the positive charge residue of E protein of rJEV-EI176R increased by 1, the isoelectric point increased by 0.37, and the instability index increased by 0.39. The number and proportion of α-helix, β-sheet, extended strand and random coil of E protein changed, and the amino acid at E176 site had different connections with nearby amino acids. The growth curve of rJEV-EI176R on BV-2 cells did not change significantly and could infect nerve cells and renal cells from different sources. The adsorption capacity of rJEV-EI176R was significantly improved (P<0.05). After inoculation of BV-2 cells with rJEV-EI176R, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ-induced protein-10 (IP-10) were significantly lower than those of the parental strain at 12 and 24 h (P<0.05). This study enriched the biological characteristics of E protein of JEV strain, and provides basic data for further study of JEV attenuated mechanism and vaccine development.

Somatic cell nuclear transfer (SCNT) has been widely used in protecting endangered species, propagating better varieties and treating diseases. Histone acetylase is an important epigenetic mark regulating the development of early embryo in pig (Sus scrofa), and its correct obliteration and reconstruction are the basis of the embryonic development. With the development of low input next generation sequencing, many studies found that a variety of abnormal epigenetic modification have been found during the reprogramming of somatic cell nuclear transfer embryos. Histone acetylase modification is an important factor that leads to the development block of porcine somatic cell nuclear transfer embryos. In recent years, some small molecule compounds of histone deacetylase inhibitors have been gradually discovered for the improvement of abnormal modification level of histone acetylase during embryonic development. This paper reviews the studies of the effects and mechanisms of histone deacetylase inhibitors on the development of porcine somatic cell nuclear transfer embryos, which provides reference for improving the cloning efficiency in pig.

Listeria monocytogenes is one of the most serious foodborne pathogens. The aim of this study is to establish a rapid, sensitive, and accurate droplet digital PCR (ddPCR) method for detecting L. monocytogenes, and apply the method to develop the reference materials. In the study, the ddPCR method was established with the invasion associated protein gene (iap) as the target gene, the reaction conditions were optimized, the specificity, sensitivity, and repeatability of the method were tested, and the established method was used to detect clinical samples and the standard material was developed by using the method. The results showed that the ddPCR method had the best amplification effect when the amount of 10 μmol/L primer was 1.5 μL, the amount of 10 μmol/L probe was 0.45 μL and the annealing temperature was 60 ℃. Finally, the ddPCR method for L. monocytogenes was established. The established ddPCR method had good specificity and did not cross-react with other non-specific strains. Repeatability test results showed that the coefficient of variation between groups was 1.57%~4.32%, which proved that the method had good repeatability. The method had high sensitivity, and the lower limit of detection was 6.65 copies/μL. The results of clinical samples showed that the ddPCR method and fluorescence quantitative PCR method were 100% consistent with the results of 47 clinical positive samples stored in the laboratory. This method was used to measure the uniformity and stability of the reference materials, and 9 laboratories determined the reference materials. The fixed value was 5.79×10³ copies/μL, and the uncertainty was 0.47×10³ copies/μL. The ddPCR method established in this study can be used for the detection of L. monocytogenes in the laboratory and the development of reference materials, etc., and can be a technical means to monitor L. monocytogenes infection.

Lilies (Lilium spp.) are susceptible to complex viral infections. Virus detection and sequence analysis are helpful in identifying the types of infecting viruses and revealing viral variations. In this study, based on the conservative gene sequences of Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), and Plantago asiatica mosaic virus (PLAMV), 4 pairs of primers were used to establish a multiplex RT-PCR detection method for these 4 viruses by optimizing the PCR extension time. This method was used to sample virus infections in lilies grown in the fields in Beijing. Furthermore, the 4 viral products were isolated for sequencing and alignment to construct a systematic evolutionary tree. The results showed that in the quintuplex RT-PCR reaction, extending the extension reaction time to 90 s resulted in the amplification of all 4 viruses and the 18S rRNA (reference gene). Sampling results indicated that lilies grown in the fields were often co-infected with 2 or more viruses, with detection rates in the order of LSV (100%)>CMV (92.31%)>LMoV (30.77%)>PLAMV (15.38%). Sequence analysis revealed that the average similarity of the isolated viral products to their respective viral sequences was above 84.4%, confirming the identification of the 4 target viruses. Variability analysis of the 4 viruses showed that when CMV was hosted by lilies, it formed a distinct branch, indicating host specificity. LMoV could be divided into 2 branches in the evolutionary relationship, with the experimental isolates belonging to branch Ⅱ. No clear branching patterns were observed for LSV and PLAMV. This study provides technical support for the routine molecular diagnosis and research of lily viral diseases, offering a foundational reference for disease prevention and control.

To develop an indirect ELISA method for rapid detection of Duck tembusu virus (DTMUV) antibodies, in this study, the prokaryotic recombinant expression vector pET-28a-NS3 was constructed, and NS3 protein obtained through induced expression was used as the coated antigen. After optimizing a series of conditions, an indirect ELISA method for detecting DTMUV antibodies was established. Antibody analysis was performed on 30 serum samples for challenge test and 150 collected serum samples by the established ELISA method. The results showed that the intra-assay coefficient of variation and the coefficient of variation (CV) of the indirect ELISA method was 1.49%~4.31% and 1.22%~6.07%, respectively, which met the national standard. DTMUV antibody could be specifically detected by this method. And the indirect ELISA method had no cross-reaction with Avian influenza virus (AIV) H5 subtype, AIV H7 subtype, Newcastle disease virus (NDV), Duck Circovirus (DuCV) and Duck hepatitis A virus (DHAV-1) positive sera. The coincidence rate of positive and negative detection numbers of challenge test samples and commercial kit was 100%, and the coincidence rate of positive and negative detection numbers of farm samples and commercial kit was 97.33%. In summary, the DTMUV NS3 protein antibody indirect ELISA method established in this study has good specificity and repeatability, and provides a rapid and effective detection method for the investigation of DTMUV epidemiological situation.