WRKY transcription factors are a class of plant-specific proteins involved in the regulation of growth and development, secondary metabolism, senescence and response to biotic and abiotic stresses.The present study firstly cloned the cDNA sequence of WRKY72 (GenBank No.KC246585) from oilseed rape (Brassica napus) by the method of reverse transcription PCR (RT-PCR).The encoded protein of BnaWRKY72 included a conserved WRKY domain and a C2H2-type zinc finger motif.Subcellular localization study using green fluorescence protein revealed that BnaWRKY72 transcription factor localized in the nucleus.Dual luciferase reporter assay detected that BnaWRKY72 had trans-activation activity towards firefly luciferase gene driven by tandem repeat of W-box element.The expression of BnaWRKY72 was examined by qRT-PCR under various hormones, biotic and abiotic stresses.It was found that BnaWRKY72 responded to cold stress and Sclerotinia sclerotiorum infection.Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays were used to screen and confirm the interacting proteins of WRKY72 which included homologous BnaWRKY31 and BnaWRKY42 in the same subclade and itself.It was also found that BnaWRKY72 could interact with small ubiquitin-like modifier (SUMO) 3, suggesting that SUMO modification might participate in regulating of BnaWRKY72 activity.The present study explored the molecular characteristics of BnaWRKY72 for the first time, which provides a reference for further study of its stress resistance function and related molecular mechanism.

The function of follicular theca cells and granulosa cells of chicken (Gallus gallus) is different from that of mammals.The most important process in chicken follicular development is the development of small yellow follicle (SYF).In order to study the expression level of circular RNA (circRNA) in chicken follicle and enrich follicular development theory in chickens, the present study used SYF granulosa cells (SYFG) and SYF theca cells (SYFT) from Jinghai Yellow chicken as samples.RNA-seq libraries with linear RNA removed were prepared and sequenced with Hi-Seq 4000 platform instrument, and differentially expressed circRNAs were preliminarily screened by bioinformatics analysis.Then Gene ontoloty (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for differentially expressed circRNAs were performed to find the putative functional annotation and enrichment of the host genes.Lastly, a total of 9 904 circRNAs in SYFG and SYFT were identified with 4 199 pervasively distributed in the two kinds of cells.The number of circRNAs in SYFT was greater than that in SYFG.When coming to the feature of circRNAs, it was found that the most length of circRNAs spanned from 100 to 400 nt with an average length of 296 nt, and they distributed on all chromosome 1~28 and sex chromosome with more distribution in longer chromosome.After differentially expressed analysis, 625 dysregulated circRNAs were found with 271 up-regulated in SYFG and 354 up-regulated in SYFT.The up-regulated circRNAs in SYFG might involve in cell proliferation, cell recognition, fatty acid biosynthesis, and metabolic process, and the candidate genes such as TOX high mobility group box family member 2 (TOX2), insulin like growth factor 1 receptor (IGF1R),and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit β (PIK3CB) potentially affect granulosa development as circRNAs isoform.The up-regulated circRNAs in SYFT might participate in nucleotide binding, focal adhesion, and several pathways such as gonadotropin releasing hormone (GnRH) signaling pathway, apoptosis, oocyte meiosis associated with reproduction, and candidate genes as monoamine oxidase A (MAOA), fibroblast growth factor receptor 2 (FGFR2) and mitogen-activated protein kinase 1 (MAPK1) were selected.In conclusion, the circRNAs screened in present study might play their roles mainly by competing with linear transcripts or as competitive endogenous RNA (ceRNA).The results of present study enrich the basic genetic theory of chicken follicular development in the perspective of circRNA.

Insulin-like growth factor-1 (IGF-1) is an important endocrine hormone which regulates cell growth and differentiation, and participates in the regulation of immune system.To investigate the mechanisms of IGF-1 in regulating fish immune response to pathogens and explore the regulation mechanism of IGF-1 on fish monocytes/macrophages (MO/MΦ), the present study got Plecoglossus altivelis PaIGF-1 cDNA sequence (GenBank No.MK005188) from transcriptome data.The sequence had 929 bp in length, contained an ORF which encoded a protein of 171 amino acids with the molecular weight of 18.86 kD.The amino acid sequence of PaIGF-1 was relatively conservative, sequence comparison showed that PaIGF-1 shared 77% amino acid sequence identity with Atlantic salmon (Salmo salar).Analysis of qRT-PCR showed that the expression of PaIGF-1 was in a high level in liver, trunk kidney, head kidney and intestine.After infected with Vibrio anguillarum, the expression of PaIGF-1 increased at 8 h in spleen, and increased at 24 h in head kidney, and there was no change in gill and liver.Furthermore, prokaryotic expression system was used to induce the expression and purification of PaIGF-1 recombinant protein (rPaIGF-1), and the mononuclear macrophages were incubated with rPaIGF-1.It was found that rPaIGF-1 increased the phagocytic activity of ayu MO/MΦ and had no effect on bacterial killing.Additionally, the qRT-PCR analysis showed that rPaIGF-1 increased the expression of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), suppressed the expression of IL-1β and tumor necrosis factor-α (TNF-α) in ayu MO/MФ infected with V.anguillarum.Finally, mononuclear macrophages PaCXCR (C-X-C chemokine receptor) 3.1⁺ and PaCXCR3.2⁺ were incubated with rPaIGF-1.It was found that rPaIGF-1 promoted the phosphorylation of PaSTAT3 in PaCXCR3.2⁺ but had no effect on the phosphorylation levels of PaSTAT1 and PaSTAT3 in PaCXCR3.1⁺.Inhibition of PaSTAT3 expression by specific siRNA could significantly down-regulate the expression of IL-10 and TGF-β in PaCXCR3.2⁺ treated with rPaIGF-1 which indicated that rPaIGF-1 might inhibit the inflammatory response of ayu MO/MΦ through activating PaSTAT3.The above research provides a theoretical basis to study PaIGF-1 function and related mechanism in regulating fish immune response to pathogens.

Streptococcus agalactiae infection may cause abnormal behavior of tilapia (Oreochromis niloticus) and seriously endangers the development of the tilapia industry.To investigate the impacts of S.agalactiae on behavior of GIFT tilapia and its mechanism, the qRT-PCR and immunofluorescence were used to examine the concentration of S.agalactiae in the brain and intestine, the behavior changes, as well as the expression of 5-hydroxytryptamine (5-HT) in the cerebellum, intestine and stomach of tilapia infected with by intraperitoneal injection.The results showed that the copies of S.agalactiae in the brain and intestinal tissues of tilapia increased sharply from the 6th and 0 h after pathogen infection respectively, and reached the maximum at 18th h (61.2 copies /mg in the brain and 1 800.5 copies/ mg in the intestine).Furthermore, fish showed abnormal behavior from the 12th h after pathogen challenge, and the abnormal behavior continued until the end of the experiment or the death of the fish.In addition, 5-HT positive reaction was found in the cerebellum, intestine and stomach of tilapia before and after S. agalactiae infection.While the positive reaction of 5-HT in cerebellum and intestine showed a weakening trend with the prolongation of experiment, and the fluorescence intensity of cerebellum and intestine were 9.1 and 6.9 at 18th h, respectively.But the 5-HT positive reaction in stomach increased first and then decreased with experiment went on, reaching a minimum of 3.1 at the end of the experiment.Therefore, it could be speculated that after infection of S.agalactia, tilapia induces abnormal behavior of tilapia by regulating 5-HT in the central nervous system and the peripheral digestive system.The current study researched the abnormal behavior of tilapia after S.agalactiae infection from the perspective of 5-HT, which could provide a new insight into the mechanism of impulsive behavior of fish infected with pathogen.

Hybrids are used in maize (Zea mays) production.Based on natural populations, recombination inbred line (RIL), doubled haploid (DH), F₂, backcrossing (BC) and other traditional loci or regions can only reflect the contemporary genetic effects of F₁ indirectly.The genetic effects of F₁ could be studied directly by locating the genetic loci or regions of hybrids that control the target traits.In this study, 100 maize inbred lines with rich genetic background were used as female parents, and four lines, Mo17, E28, Zheng58, and Chang7-2 were used as test parents.According to the NCⅡ experiment, 400 hybrids (100 females×4 males) were constructed.Statistical analysis and genome-wide association study (GWAS) of grain quality-related traits, protein (Pro) content, oil content , and lysine (Lys) content, in 4 test cross populations were performed.The kernel quality traits were significant differences in F₁, because of the influence of 4 different genetic background test parents.The results showed that the Pro content in the test cross populations with pollens from E28 (E28s) and Chang7-2 (Chang7-2s) was significantly higher than other populations (P<0.05); the oil content in the test cross population with pollens from E28 was significantly higher than other populations (P<0.05); Lys content in the test cross population with pollens from Mo17 (Mo17s) was significantly higher than other populations (P<0.05).For Pro, Oil, and Lys, 2, 5, and 17 significantly associated SNPs were detected in Mo17s, respectively.The phenotype variation range that can be explained by a single locus was 1.99%~21.83%, 1.15%~16.83%, 1.48%~29.91%.And in populations of E28s, Chang7-2s, and Zheng58s, the correspondingly detected SNP loci associated Pro/Oil/Lys were 11/7/54, 8/8/6, and 38/79/11, respectively.The phenotype variation range that can be explained by a single locus was 1.15%~38.60%/4.01%~26.35%/0.25%~46.10%, 7.32%~34.63%/1.25%~8.08%/2.53%~18.64% and 1.07%~61.14%/1.00%~31.04%/1.02%~21.15%, respectively.The total shared SNPs detected for Pro, Oil, and Lys were 7, 2 for Pro, 4 for Oil, and 1 for Lys.The phenotype variation range that could be explained by a single locus was 0.12%~2.51%, 3.18%~11.59%, 0.25%~14.63%.These 7 SNPs were located on the Chr1, Chr4, Chr5, Chr6, Chr9, Chr10 and donated 6 candidate genes.The candidate genes were GRMZM2G143817, GRMZM2G446313, GRMZM2G146346, GRMZM2G104920, GRMZM2G083886, GRMZM2G148400, respectively.These results provided informative references for loci mining associated with maize kernel quality-related traits via QTL mapping or GWAS pathways, and also provided practical references for kernel quality improvement in maize breeding procedures.

Plant heat shock transcription factor (Hsf) is important regulating factor of signal transduction pathway activated transcriptively by genes under heat stress and other stresses.Hsf can specifically bind to heat shock elements (HSE) in the upstream promoter region of heat shock protein genes to realize the regulation to these genes expression.Plant Hsfs belong to multi-genes family, the members are different among varieties.Hsfs are divided into 3 classes of A, B and C, each class including several subclasses.Many previous works were mainly focused on class A, especially subclass A1 and A2.There are more than 56 members in wheat (Triticum aestivum) Hsf family reported in previous research, containing multiple subclass members, showing diverse characteristics and functions.Recently, the Hsf family members were found to reach to 82 based on wheat genome sequence.In this paper, The TaHsfA2f was isolated from wheat young leaves treated by heat shock at 37 ℃ for 1.5 h using homologous cloning methods.Sequence analysis showed that the coding sequence (CDS) of TaHsfA2f (GenBank No.MK045331) was 1 062 bp encoding a protein of 353 amino acids.The amino acid sequence analysis demonstrated that TaHsfA2f contained a DNA-binding domain (DBD), a nuclear localization signal (NLS) of MRKELEDAMSNKRRRR peptide, a nuclear export signal (NES) of LKRDKGLLM peptide and an aromatic, large hydrophobic and acidic amino residues (AHA) of DDFWEDLLHE peptide.Through transient reporter assays with tobacco (Nicotiana tabacum) epidermal cells, it was found that the TaHsfA2f protein was subcellular localized in the nuclei under normal growth conditions.Homologous analysis showed that TaHsfA2f protein shared higher identities with the HsfA2e proteins from some other crops such as barley (Hordeum vulgare), Sargassum mongolicus, Arabidopsis thaliana and so on.qRT-PCR analysis showed that TaHsfA2f was expressed in the majority of tissues and organs of wheat with higher expression level in mature roots and lower expression level in young shoots (P<0.05).TaHsfA2f expression in leaf were up-regulated by heat shock at 37 ℃, salicylic acid (SA) and H₂O₂ (P<0.05), respectively, and the peak values appeared at 60 min and 120 min after treatments.Through genetic transformation in wide type A.thaliana and assays of thermotolerances of transgenic line 10_19, line 13_9 and line 20_14 of overexpressing TaHsfA2f, the results revealed that overexpression of TaHsfA2f could improve both the basal and acquired thermotolerances of transgenic A.thaliana, and relative Hsp genes expression were upregulated to different degrees at 8 h after 2 different heat regimes treatments.Compared to WT, under heat stress, the seedlings growth potential of 3 transgenic lines were better and chlorophyll contents of the rosette leaves were higher and relative electric conductivity (REC) were lower, providing physiological evidences for phenotypes.These results could provide theoretical evidence for further understanding of biological characteristics and functions of subclass HsfA2 members of wheat Hsf family.

With the extensive planting of poplar (Populus spp.) in China, the pest problem of poplar comes along.Nowadays, the application of chemical insecticides can not only fail to effectively reduce the harm of pests to poplar trees, but also seriously damage the ecological environment.Therefore, it is feasible to improve the insect resistance of poplar through grafting and propagation of transgenic poplar and non-transgenic poplar.In this study, in order to understand the ecological safety of grafted transgenic poplar, hybrid Populus tomentosa and Hb1 transgenic Populus × euramericana cv.'Neva' with Bt-Cry1Ac gene were used as scions and rootstocks to study whether the mRNA and protein of Cry1Ac gene were transported between scions and rootstocks, and whether the leaves of grafted seedlings were resistant to the insect pest.The results of reverse transcription-PCR (RT-PCR) and third-generation digital PCR showed that no Cry1Ac gene mRNA was detected in the shoots and leaves of hybrid P.tomentosa as scions or rootstocks, and the copy number of Cry1Ac gene was much lower than that of positive control, indicating that the exogenous Cry1Ac gene mRNA could not be transported between rootstocks and scions.Cry1Ac protein was detected in leaves, phloem and xylem of scions and rootstocks in 2 grafting combination by enzyme-linked immuno sorbent assay (ELISA), which proved that Cry1Ac protein could be transported between rootstocks and scions by grafting.The non-transgenic poplar grafted on the transgenic poplar could inhibit the growth and development of the larvae of the Hyphantria cunea, and slow down the development of the larvae, showing a certain degree of insect resistance.This study clarified the expression, transmission and insect resistance of Cry1Ac gene expression products between transgenic and non-transgenic tissues, and could provide theoretical basis and scientific guidance for the rational use of grafted poplar in insect resistance practice.

Chimonanthus salicifolius belongs to Chimonanthus of Calycanthaceae family, the leaves of Ch.salicifolius are shattered with a very fragrant smell, while its flowers smell rather lighter.In order to investigate the key regulation gene and molecular regulation pathway related aroma components in leaves and flowers of Ch.salicifolius, the transcriptome sequencing, sequencing data assembly, and functional annotation and classification of the sequences were conducted by using the leaves and flowers of Ch. salicifolius as materials in this study, and the aroma synthesis related differentially expressed genes were analyzed by qRT-PCR.By using transcriptome sequencing and bioinformatics analysis, 153 059 Unigenes were obtained by De novo assembly in leaves and flowers.Among them, 56 964, 40 746, 22 173, 47 858, 46 196 and 37 029 unigenes were assigned into NR (Non-Redundant Protein Sequence Database), Swiss-Prot (Manually Annotated and Reviewed Protein Sequence Database), KEGG (Kyoto Encyclopedia of Genes and Genomes), Pfam (Protein Family), KOG (euKaryotic Ortholog Groups) and GO (Gene Ontology), respectively.Though the differentially expressed analysis of 2 materials, 5 517 differentially expressed genes were annotated, of which 2 104 genes up-regulated and 3 413 genes down-regulated.After GO and KEGG enrichment analysis, 314 GO annotations and 33 significantly enrichment metabolic pathways were obtained, including 7 aroma synthesis related significant enrichment of metabolic pathways involved 33 different enzymes, seven different enzyme genes expression in leaves were higher than that in flowers, while the remaining 26 enzyme genes expression were lower than that in flowers.Differentially expressed genes and regulation pathways of leaves and flowers of Ch.salicifolius were screened by transcriptional analysis, which showed that the genes AOC (allene oxide cyclase), TAT (tyrosine aminotransferase), CHLP (geranylgeranyl reductase), DXS(1-deoxy-D-xylulose-5-phosphate synthase), TES ((-)-α-terpineol synthase), LMS ((R)-limonene synthase) and GA3OX (gibberellin 3-beta-dioxygenase) high expression in leaves of Ch.salicifolius, and they may have a greater contribution to the formation of aroma components in leaves, promoting the synthetic biomass of aroma components.This study results will provide the valuable genome data sources in molecular biology of Ch.salicifolius.

As a lipid droplet protein, butyrophilin subfamily 1 member A1 (BTN1A1) is associated with the formation and secretion of milk fat globules.In order to investigate the effect of over-expression bovine (Bos taurus) BTN1A1 on lipid droplets in mammary epithelial cells, the coding region of BTN1A1 gene was amplified by PCR, and Lenti-BTN1A1 lentivirus recombinant vector was constructed.The virus was packaged into recombinant vector and infected bovine mammary epithelial cells.BTN1A1 over-expression cell lines were selected by applying puromycin in cell culture medium.DNA was extracted from the over-expressed cell lines and genotypes were identified.Expression of BTN1A1 gene and protein was detected by qRT-PCR and Western blot.The effects of BTN1A1 over-expression on cell lipid droplets were analyzed by fluorescent labeling with Nile red.BTN1A1-pEGFP-N1 vector was constructed to observe the co-localization of BTN1A1 protein and lipid droplet.The results showed that the CDS sequence of the cloned BTN1A1 gene was about 1 580 bp.The lentiviral vector recombinant with BTN1A1 was constructed.BTN1A1 cell line was selecteded by 5 μg/mL purinomycin.Genotypic identification showed that exogenous BTN1A1 gene was successfully inserted into the cell genome.Compared with control group, the expression level of BTN1A1 gene mRNA was 489 times greater (P<0.001) ,while protein expression was also increased in over-expression BTN1A1 cell line.Fluorescence values of lipid droplets showed that over-expression BTN1A1 significantly increased lipid droplet content with the extension of cell culture time (P<0.01).BTN1A1 protein was found to co-localized with lipid droplets in cells.In this study, over-expression BTN1A1 promoted lipid droplet synthesis in mammary epithelial cells.This study provides basic data to elucidate the mechanism of lipid droplet synthesis in mammary epithalial cells of dairy cows.

The number of thoracic vertebrae is an important economic trait in production, but few candidate genes have been identified in sheep (Ovis aries). The aim of this study is to explore the association between polymorphism of VRTN (vertnin) and NR6A1 (nuclear receptor subfamily 6 group A member 1) genes and thoracic vertebral number, and tissue expression levels of 2 genes were also detected. The association analysis of VRTN and NR6A1 polymorphisms with thoracic vertebral number were performed by PCR, and expression levels of VRTN and NR6A1 genes in Sunite sheep with different thoracic vertebral numbers (14 thoracic vertebral number, T14; 13 thoracic vertebral number, T13) were tested by qRT-PCR. The results showed that there were no significant association between g.82533904C>T, g.11206763T>C and g.11210075T>C and the thoracic vertebral number in Sunite sheep (P＞0.05), while there was a highly significant association between g.82533661C>A and the thoracic vertebral number in Sunite sheep (P＜0.01). NR6A1 expressed in heart, liver, spleen, lung, kidney, muscle and fat of Sunite sheep. There were no significant difference of expression levels between sheep with T14 and T13 in heart, lung, kidney, muscle and fat (P＞0.05), while there were significant difference in spleen (P＜0.05) and extremely significant difference in liver (P＜0.01) of Sunite sheep with different thoracic vertebral number. The expression levels of VRTN were all higher in Sunite sheep with T14 than those of T13 in heart, liver, spleen, lung and kidney, the difference of muscle was significant (P＜0.05), the difference of the other tissues were not significant (P＞0.05). There was no uniform expression pattern of Sunite sheep with different thoracic vertebral number. In summary, g.82533661C>A had potential application value for the selection of the Sunite sheep population with more thoracic vertebral number. At the same time, there was no significant association between NR6A1 and the thoracic vertebral trait of the Sunite sheep, and VRTN may be one of the candidate genes affecting the thoracic vertebral numbers in Sunite sheep. Determining candidate genes for thoracic vertebrae number trait in sheep is conducive to the selection of multiple thoracic vertebrae sheep population, which can effectively improve the economic benefits of the sheep industry.

Fat mass and obesity associated gene (FTO) is an obesity susceptibility gene discovered by genome-wide scanning technology.FTO could regulate fatty acid transport and fat metabolism-related genes by transcription factors and transporters, participating in the development, maintenance and deposition of adipose tissue.Qianbei Ma goat is one of the three local varieties in Guizhou province.It is delicious and has good meat production.In present study, Qianbei Ma sheep (Capra hircus) was taken as the investigated subject, and the association between FTO gene exon polymorphism and serum physiological and biochemical indexes related to lipid metabolism was studied.SNPs were firstly screened by hybrid DNA pool combined with PCR direct sequencing technology.The genotypes of the mutation sites were directly sequenced by single sample.The different genotypes of FTO gene mutations and serum leptin, hormone sensitive lipase (HSL), adiponectin, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, alanine aminotransferase (ALT), aspartate aminotransferase (AST) were determined by SPSS 17.0.A total of 9 SNPs were found in the tested goat population: g.C10943T, g.G109518T, g.T125300A, g.C125357T, g.G125405T, gT125417C, g.T426229C, g.C426244G, g.G426347A, where g.G109518T and g.G426347A site mutations were located in the second and ninth intron regions, respectively.Chi-square results showed that each mutation site belonged to moderate polymorphism (0.5<PIC<0.2) and did not deviate from the Hardy-Weinberg equilibrium state.There were 3 genotypes in each mutation site.Correlation analysis showed that the serum AST and ALT levels in g.T125300A and g.G426347A sites were significantly different among different genotypes (P<0.01); There were significant differences in serum AST and ALT levels between g.G125405T and g.T125417C sites (P<0.05); AST differences were significant at g.T426229C sites (P<0.05), while ALT levels were significantly different (P<0.01).Serum HSL levels were significantly different between genotypes at g.T426229C and g.G426347A (P<0.05).It is concluded that the FTO gene g.T125300A, g.G125405T, g.T125417C, g.T426229C and g.G426347A sites have a certain correlation with the lipid metabolism of Qianbei Ma goat which provide a theoretical reference for screening auxiliary selection markers of fat deposition and meat quality regulation in goats.

miRNAs (microRNAs) play a role in a variety of physiological and pathological processes.miRNAs play biological functions through the corresponding target genes.By combining bioinformatics prediction and experimental verification, miRNA target genes can be rapidly and efficiently screened.In our early study, high-throughput sequencing revealed that ssc-miR-339-3p was differentially expressed between control group and treatment group on piglets (Sus scrofa) infected with Clostridium perfringens type C, suggesting that it might have played an important role in the infection process.This study aimed to bioinformatics prediction and analysis of the target genes of ssc-miR-339-3p, and to verify the ssc-miR-339-3p and partial target genes, to explore its possibility influence and regulating mechanism of piglets diarrhea.In this study, bioinformatics databases such as miRBase, Ensemble, NCBI, miRTarBase, and bioinformatics software such as PITA, RNAhybrid, miRanda, Promoter Scan, Alibaba2.1, DAVID, Cytoscape, were used to perform the prediction of transcription factor binding site and conservative analysis among different species of its target genes, involved Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.qRT-PCR was used to verify the expressions of ssc-miR-339-3p and some target genes.The results showed that miR-339-3p was highly conserved among species, and multiple transcription factor binding sites such as Sp1, AP-1, C/EBPα, NF- κB, SRF and USF, were found in the ssc-miR-339-3p promoter region.The obtained 160 target genes are significantly enriched in positive regulation of MAP kinase activity, positive regulation of apoptosis, negative regulation of RNA polymerase Ⅱ promoter transcription, apoptosis signaling pathway and other biological processes and participated in NF-κB, PI3K-Akt, FoxO and Toll-like receptors signal pathways.qRT-PCR results showed that the expression level of ssc-miR-339-3p was significantly lower in the resistance groups (IR) and sensitive groups (IS) than in the control groups (IC) (P<0.05), this result was consistent with the high-throughput sequencing results; the expression levels of the target genes nuclear factor kappa-B 1 (NFKB1) and B-cell surface antigen CD40 (CD40) were significantly higher between IR and IS groups than the IC group (P<0.01); the expression of TNF receptor-associated factor 3 (TRAF3) and interleukin 1 receptor associated kinase 1 (IRAK1) were not significantly different among the 3 groups (P>0.05).Therefore, we assumed that ssc-miR-339-3p is regulated by various transcription factors such as Sp1, NF-κB and USF, which may activate NF-κB, PI3K-Akt, FoxO and Toll-like receptors signal pathways to regulate the piglets diarrhea through the target genes NFKB1, TRAF3, IRAK1, CD40.This study predicts and preliminarily validates the obtained target genes, which may provide experimental and theoretical basis for the function and regulating mechanism of miR-339-3p in piglet's resistance to Clostridium perfringens infection, thereby it can provide a theoretical basis for searching for the effective molecular genetic markers of resistance to Clostridium perfringens type C in piglets.

Potato common scab by Streptomyces spp.is a serious disease occurring within the western growing region of Inner Mongolia and is a major obstacle to potato (Solanum tuberosum) production.In order to clarify the pathogens and biological characteristics of potato common scab in Wuchuan county, Inner Mongolia, the pathogens were isolated by dilution separation method, and one isolate PS1 was obtained.The pathogenicity was determined by potato chip method and potted inoculation, and the pathogenic genes were detected by specific primers.In results, The strain PS1 could colonize in potato chips and produce brown necrosis, and produce typical scab symptoms in potted potato tubers.The pathogenic genes txtAB, tomA and nec1 (necrosis-inducing protein) were detected.The morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence were analyzed to identify the pathogens, and the isolate was identified as Streptomyces galilaeus.The results of biological characteristics indicated that the oat liquid medium for mycelium growth was optimum.When cultivated 10 d the mycelium dry weight was maximum.The optimum pH was 7, and the optimum cultivation temperature was 32 ℃.The optimum single carbon and nitrogen source were glucose and L-methionine.In this study, the pathogen species and biological characteristics in potato common scab were clarified, which laid the foundation for further study of epidemic patterns an control measures.

Plant diseases caused by pathogenic fungi are one of the main reasons for the decline in crop quality and yield in countries around the world, which result in 20% reduction on the yields of global grain and other crops.Therefore, biological control has become one of the hotspots in plant fungal disease controls.In previous study, a strain Bacillus subtilis 8-32 was screened, which was tested to have strong antagonism to Fusarium oxysporum causing soybean root rot, and 8-32 had significant growth-promoting and biocontrol effect in greenhouse experiments.In this study, the growth-promoting and antagonistic mechanism of B.subtilis 8-32 was investigated for the further development and application of this strain.In detail, the antifungal spectrum of B.subtilis 8-32 was studied by plate confrontation method.The inhibition of B.subtilis 8-32 on spore germination, germ tube elongation and mycelium growth of main plant pathogenic fungi such as Fusarium oxysporum was testified in this study.Besides, molecular level study was carried out to reveal related antagonistic genes according to the reported literature, these genes were directly amplified by PCR using the main antagonistic gene primers of Bacillus such as iturin, surfactin, fengycin.Meanwhile, the lipopeptide antagonists were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS).Plant growth-promoting substances (including indole acetic acid and siderophores) secreted by B.subtilis 8-32 were detected.The results showed that the antifungal spectrum of B.subtilis 8-32 strain was extensive.It could antagonize 7 pathogenic fungi which caused different crop diseases such as soybean root rot and wheat wilt, among which the effects were stronger for Rhizoctonia solani and Pomacea graminis, and the inhibition zone radius was 1.2 cm and 1.1 cm, respectively.The results showed that the strain 8-32 could inhibit spore germination and germ tube elongation of pathogenic fungi, and the maximum inhibition ratio reached to 100% and 95.40%, respectively.And the strain 8-32 also inhibited the growth of pathogenic fungi such as Fusarium oxysporum, Fursarium solani, Colletotrichum gloeosporivides, Alternaria brassicea through mycelial breakage.The results of PCR amplification of the antagonistic genes showed that 4 distinct single bands were amplified from 7 pairs of primers.After sequencing and NCBI Blast, the similarities between sequences and lipopeptide antibiotic genes subtilosin A (sboA), subtilisin gene (qk), fengycin gene (FenB) and hypothetical protein yndj gene (yndj) were 100%, respectively.Finally, the antagonistic substances secreted from B.subtilis 8-32 identified by HPLC-MS, including a family of lipopeptides, which were identified as homologues of iturin, that had antifungal effect.In addition, the quantitative detection on plant growth-promoting substances showed that B.subtilis 8-32 had a weak ability to produce siderophores, but strong on IAA production, which reached to 45.29 mg/L after 72 h fermentation.All the above results will lay a foundation for further study on the biological control of B.subtilis 8-32 at the molecular level, and will provide a theoretical basis for the commercial application of B.subtilis 8-32.

Plants terpenoids, such as triterpenes, which have efficacy on anti-inflammatory, anti-oxidation, tumor cell proliferation-resistant and so on, were widely used in medicine, cosmetics and processed food.However, the molecular mechanisms through which the compounds were produced were still unclear.This article reviewed the identifications of key critical transcription factors activate/repress the terpene biosynthesis.At the end, prospects were drawn on measures which would promote the construction of gene transcription network in plant terpene biosynthesis.

Hepatocyte nuclear factor 4α (HNF4α) is a member of the nuclear receptor superfamily and extensively controls the basal expression of many genes involved in glucose, cholesterol and fatty acid metabolism pathways.In order to obtain chicken (Gallus gallus) HNF4α specific antibody, this study firstly optimized and synthesized chicken HNF4α gene according to the codon preference of Escherichia coli, and constructed the recombinant vector pET-30a(+)-HNF4α for prokaryotic expression of HNF4α.Next, the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) to induce expression of HNF4α protein.After optimizing the induction temperature and isopropyl-β-dthiogalactoside (IPTG) concentration, the expression product was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE.The HNF4α protein was purified by Ni-NTA agarose columand and then immunized the New Zealand rabbit (Oryctolagus cuniculus) to prepare the polyclonal antibody against chicken HNF4α.The antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA), and the expression of HNF4α in chicken tissues was further analyzed by the obtained antibody.The results showed that the codon-optimized pET-30a(+)-HNF4α was highly expressed in Escherichia coli BL21 (DE3), and the soluble protein was obtained under induction of 0.5 mmol/L IPTG for 4 h at 37 ℃ .The expressed product was purified by Ni-NTA agarose column , and a fusion protein having a size of about 51.6 kD and a purity of more than 90% was obtained by SDS-PAGE and Western blot.The polyclonal antibody titer was 1∶512 000 by ELISA.The antibody was used for detection of HNF4α in different organs of chicken, and the results showed that the HNF4α was highly expressed in the liver and kidney, and expressed in small amounts in the small intestine and testis, but hardly expressed in other tissues.In summary, this study successfully prepared chicken HNF4α antibody with high titer and specificity, which provided basic data for further investigation of chicken HNF4α gene function.

Mycoplasma bovis (Mb) is one of the most important pathogens causing diseases in beef cattle (Bos taurus) and cow, which cause great economic loss to cattle industry.Enolase (eno) is the key rate-limiting enzyme in glycolysis pathway and catalyzes the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP).In order to study the enzymatic activity of Mb eno, the specific primers were designed according to the eno gene sequence of Mb PG45 strain in GenBank (NC_014760.1) and the eno gene of Mb Wuwei strain was amplified by PCR.Based on the sequence analysis and the gene optimization, the prokaryotic expression vector pET-eno was constructed and transformed into Escherichia coli Transetta (DE3).Then the recombinant protein was expressed with induction by isopropyl β-D-1-thiogalactopyr-Anoside (IPTG).Subsequently, the expression product was purified and the assay of its enzymatic activity was completed.The results showed that the optimized eno gene of Mb Wuwei strain was successfully expressed in DE3 in soluble form and the relative molecular weight of the recombinant protein His-eno was approximately 53 kD.The results of enzymatic activity analysis indicated that the catalytic activity of His-eno was slightly higher than that of rabbit muscle enolase, the optimum temperature was 42 ℃ and the optimal pH was 8.0.The michaelis constant (K_m) and maximum reaction velocity (V_max) were 4.1×10^-3 mol/L and 3.4 μmol/(L·min), respectively.The results of this study could provide basic data for further study on the biological function of Mb eno.

Mycoplasma ovipneumoniae (Mo) and M.mycoides subsp.capri (Mmc) are the main pathogens of M.pneumonia of sheep and goats (MPGS).In order to rapidly identify the main pathogens of MPGS, a duplex TaqMan probe Real-time fluorescence quantitative PCR (qRT-PCR) detection method was established for simultaneous detection of Mo and Mmc.Using Beacon Designer 7.9 combined with NCBI Blast software analysis, the specific primers and probes were designed based on the p113 sequence of Mo and the MLC_1770 (hypothetical protein gene) sequence of Mmc.The duplex TaqMan probe qRT-PCR method was established by optimizing the reaction conditions such as primer concentration, probe concentration and annealing temperature, and the specificity, sensitivity and repeatability of the method were validated.The result showed that the correlation coefficient (R²) of Mo and Mmc were 0.998 and 0.999, respectively, and the amplification efficiency of Mo and Mmc were 94.8% and 97%, respectively.The assay showed a good specificity without cross reaction from other common pathogens of sheep and goats.The lowest detectable limit (LDL) of the method for Mo and Mmc was the same, which was 100 copies/μL, and its sensitivity was 100 times more than conventional PCR.The inter- and intra-variation coefficients of the assay were less than 2%.Applying the assay to detect 187 cases of clinical samples which were collected from different areas of Fujian, the result showed that the positive rates of Mo and Mmc were 47.6% (89/187) and 11.8% (22/187), respectively, and the duplex positive rate of infection was 10.2% (19/187).The above results indicated that the assay could be used for the accurate and rapid detection of Mo and Mmc in clinic.The study provided technical support for rapid detection and epidemiology of Mo and Mmc.