In order to study the regulation of Turpan black sheep (Ovis aries) in breeding and cultivate the high fecundity breeds of sheep, ovis aries microRNA-150(oar-mir-150), which was significantly differently expressed in follicular and luteal phase of Turpan black sheep, was studied as the research object in this study. The target genes of oar-mir-150 were predicted by bioinformatics, and the expression of oar-mir-150 and steroidogenic acute regulatory protein gene(STAR) in follicular and luteal phase of Turpan black sheep ovaries were detected by qRT-PCR. In addition, the base sequence of wild, mutant and deletant-type STAR gene were synthesized and ligated with the PGL3-control vector after annealing for constructing the luciferase reporter gene vector. Then, the oar-mir-150 mimic or negative control mimic with the recombinant plasmid were co-transfected into 293T cells, and the luciferase activity was detected by the double luciferase activity assays. The results indicated that oar-mir-150 had a total number of 540 target genes, and 8 target genes were differently expressed between follicular and luteal phase. STAR gene as a target gene was up-regulated when the follicular phase compare to luteal phase that had the target gene binding site (UUGGGAG) of the oar-mir-150 which located in the 3'UTR. The relative quantitative results illustrated that the relative expression of oar-mir-150 was 0.73 which is the significant decrease (P＜0.05) and STAR mRNA is 1.90 which is the significantly increase(P＜0.05) in the Turpan black sheep ovaries when the follicular phase compare to the luteal phase. The wild, mutant and deletant-type luciferase reporter gene vectors were all successfully constructed. The transfection ratio of transfection reagent and green fluorescent protein vector was 1∶1 which was determined as the best transfection efficiency and the transfection ratio of PGL3-control and PRL-TK was 200:1 which was determined as the optimal transfection ratio. The double luciferase activity assays demonstrated that oar-mir-150 effectively negatively regulated the expression of STAR. Studies have shown that there was a definite relationship of target regulation between oar-mir-150 and STAR, and oar-mir-150 regulated the expression of STAR gene negatively, which would provide a theoretical basis for the study of microRNA in regulating the reproduction process of Turpan black sheep at the different stages of estrus cycle.

Brucella is a Gram-negative facultative intracellular bacterium that is the causative agent of various animal and human brucellosis. The manB gene of Brucella, which encodes mannose phosphate mutase, is an important component of Brucella virulence factor. In order to analyze the biological function and immunogenicity of ManB protein of lipopolysaccharide, this study constructed the prokaryotic expression vector of Mannose phosphate mutase manb gene and predicted the bioinformatics of ManB protein and identified its immunogenicity. The structure and function of ManB protein was analyzed by bioinformatic tools. The manB gene of Brucella was amplified by PCR and ligated with pET-28a expression vector and then transformed into Escherichia coli BL21 (DE3). The expression of target protein was detected by SDS-PAGE and its reaction was detected by Western blot. Then the immunogenicity was analyzed by mouse immunoassay. Bioinformatics analysis showed the protein had no transmembrane domain and had no signal peptide. Its secondary structure was mainly α-helix. The three-dimensional structure of the protein was constructed by Phyre2 Server. SDS-PAGE analysis showed the fusion protein was about 56.3 kD. Western blot showed the protein could react with sheep serum against Brucella, and indicated it possessed reactogenicity. Indirect ELISA results showed that the mice (Mus musculus) in the experimental group produced antibodies in 7th, 14th and 21st d after immunization. The antibody level increased with time and reached the peak in 21st d. The ManB fusion protein had good reactogenicity, immunized mice were able to induce high level of antibody, and had good immunogenicity, which could provide new materials for the development of new vaccine of Brucella in the future.

To pinpoint genetic model of cucumber (Cucumis sativus) internode length and function mode of gene, and also to evaluate the genetic effect and heritability of major genes and minor genes, two culcmber parents with significantly different internode length, namely 'SJ57-h' (short internode, P1' hereinafter) and 'SJ11-1' (long internode, 'P2' hereinafter), were used to generate F1、B1、B2 and F2 populations for joint analysis. Mixed major-gene plus polygenes inheritance model were used to genetic law of internode length in both spring and autumn seasons. The results showed that the best fitted genetic model for cucumber internode size in both autumn and spring was D-0 model which internode length was controlled by one pair of additive-dominant-major genes plus additive-dominant-epitasis polygenes. Moreover, progeny internode length was closer to long-internode parent P2, namely, the progenies has long-internode parent heterosis. In spring, the major gene heritability of B1, B2 and F2 populations were estimated to be 68.5%, 36.2% and 56.3%, respectively, while the heritability of polygene was 0.6%, 17.1% and 0.6%, respectively. The variance of environment accounted for an average of 40.2% of the phenotypic variation among three generations. In autumn, heritability of the major gene among B1, B2 and F2 generations was estimated as 68.5%, 40.5%, 65.1%, while the heritability of polygene was 1.2%, 40.6% and 5.4%, respectively. Environment variance accounted for about 26.3% of the phenotypic variation. These results indicated that cucumber internode length was mainly controlled by a pair of major genes while the internode length was also obviously affected by environment factors in the B2 generation. Selection efficiency of major genes was relatively high in early generation of F2 and B1, whereas for selection efficiency of polygene was higher in B2 generation. Therefore, the trait of cucumber internode length was suitable for selection in early generation. The finding in the study will lay a theoretical foundation for breeding new cucumber vareties with improved plant architecture.

Loquat (Eriobotrya japonica) is an important plant species resources of traditional Chinese medicine. It has been reported that the main activity components in E. japonica were pentacyclic triterpenoids, which have been extensively known for its important and wide biological activity. Comparing with original plant plants, the content of tpentacyclic triterpenoids in E. japonica obtained by suspension culture is much higher. In this study, the genetic basis of these active components enrichment after suspension culture was analyzed to improve the synthesis mechanism of tricyclic triterpenoids in plant and provide reference for the artificial regulation of terpenoids in large-scale loquat cell suspension culture. The loquat leaves (sampleⅠ), the loquat callus on solid media (the necessary stage of suspension culture, sampleⅡ), the loquat suspension cell lines (two regulations rich in pentacyclic triterpenoids, sample Ⅲ and sample Ⅳ) were sequenced by Illumina Hiseq4000. The results are as follows: De novo assembly , protein function annotations, Gene Ontology (GO) annotations and Metabolic pathway analysis (the Kyoto Encyclopedia of Genes and Genomes) of Unigenes were obtained using bioinformatics. A total of 123 685 Unigenes were obtained with an average length of 804.44 bp. The genes related to triterpene synthesis expression profiles of samples Ⅰ and three other cultured cells (samples Ⅱ, Ⅲ, Ⅳ) were significantly different, but the difference was not significant among the three cultured cells. From expression profile of the differentially expressed genes in the skeleton formation and modification metabolic pathways of triterpenoid, 10 potential-selection genes were selected by the intersection of the different expressed genes between the samples and the importance of the genes reported. Those genes are two acetyl-CoA acetyltransferase genes (AACTs), squalene synthase gene (SQS), two squalene monooxygenase-like genes (SQEs), two amyrin synthase genes (ASs), cytochrome P450 gene (CYP450) and two UDP-glycosyltransferase–like genes (UGTs). And more from the overall analysis of those genes indicated that the expression level of those genes and the content of the target products had some relevance. Those genes will contribute significantly to further research and analysis for the pathway of triterpenes in this species. At the same time, the synthesis route of the pentacyclic triterpenoid in the loquat callus stage (sampleⅡ)had been opened extensively, which is an important intermediate stage of the suspension culture of the loquat cells. This study will help to improve the synthesis mechanism and the applied research of plant pentacyclic triterpenoids.

14-3-3 proteins play important regulated roles in plant growth, development, stress physiology and starch metabolism. Prerious studies have found a differentially expressed 14-3-3 protein in development anther of wolfberry (Lycium barbarum). In order to investigate the regulation of 14-3-3 protein on the starch metabolism during the anther development of L. barbarum, A differentially expressed 14-3-3 protein gene was isolated from the anther of wolfberry cultivar 'Ning qi No. 1', named as Lb14-3-3a (GenBank accession No.MG189703) and the protein structure was predicted. Furthermore, The plant overexpression vectors of the gene were constructed, and the functions of Lb14-3-3a in potato (Solanum tuberosum) were studied. The full-length cDNA of Lb14-3-3a was determined by RT-PCR technique. The phylogenetic tree of 14-3-3 proteins was constructed using a neighber-joining method with the MEGA5.1 program (http://www.megasoftware.net/). The image of phylogenetic tree of 14-3-3 proteins which was drawn in MEGA5.1 Homology researches of the NCBI (National Center of Biotechnology Information) databases were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) with default parameters. The plant overexpression vector pMDC83-Lb14-3-3a was constructed using gateway technology. The pMDC83-Lb14-3-3a was transformed into the potato cultivar 'Zihuabai' by Agrobacterium mediated method. The positive seedling was selected by PCR and the starch content in leaves at different growth stages was determined by spectrophotometric method. Biological information analysis indicated that the gene belonged to the 14-3-3 protein gene family. The opened reading frame (ORF) of Lb14-3-3a gene was 768 bp long and its encoded protein contained 256 amino acid residues, the molecular weight was 61.9 kD and the isoelectric point was 4.99, which had a conserved domain of 14-3-3 protein family. Lb14-3-3a was associated with tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) on the same branch of the phylogenetic tree. Positive transgenic plant was obtained by resistance screening and PCR identification. Under the strong promoter of CaMV35s (35S promoter of cauliflower mosaic virus), the exogenous gene Lb14-3-3a stably expressed in potato. Determination of starch content showed that the starch content of wild type plants had little difference with genetically modified plant, but starch content of tuber stage and mature stage of the transgenic plant leaf were higher than that of wild type. It is speculated that the Lb14-3-3a gene may affect the synthesis of potato starch. The result provides a reference for further studies on gene function of Lb14-3-3a on the accumulation and hydrolysis of starch during anther development of wolfberry.

Myocyte enhancer factor 2D (MEF2D) is a member of the myocyte enhancer factor 2 (MEF2), a supergene family, and thought to be related to skeletal muscle growth and development. To investigate the effects of polymorphism of MEF2D gene on slaughter traits of Xingyi ducks (Anas platyrhynchos domestica), this study used PCR and DNA sequencing methods to find SNP loci and analyzed their correlation with slaughter traits. Meanwhile, the expression profile of MEF2D gene was investigated by qRT-PCR of different stages Xingyi ducks. Finally, the results showed 6 SNPs in exons, respectively, C3894T, G3957A, T16236C, A16305C, G16359C and A21071G. All of the six sites were synonymous mutation, and those did not cause a change of amino acid encoding. By population genetics analysis, the results showed that 6 loci were at moderately polymorphic status (0.25＜PIC＜0.5). The χ2 tests showed that the loci were all in Hardy-Weinberg equilibrium status (P＞0.05). Analysis of matching chain disequilibrium and haplotype analysis show that there was a strong chain of balance between the six loci. Analysis of association of polymorphism with slaughter traits at all mutation loci showed that T16236C locus and A16305C locus were significant effects on live weight, carcass weight, semi-eviscerated weight, eviscerated weight, and breast muscle weight (P＜0.05 or P＜0.01); G16359C locus was significant effects on semi-eviscerated weight and eviscerated weight (P＜0.05); A21071G locus was significant effects on carcass weight, eviscerated weight, leg muscle weight, dressing percentage and eviscerated weight rate (P＜0.05 or P＜0.01). Seven haplotype combinations were found in 52 Xingyi ducks, the CGCCCG haplotype have significant effects on semi-eviscerated weight and semi-eviscerated weight rate (P＜0.05 or P＜0.01); TACCCA (TATAGA) haplotype have significant effects on leg muscle weight (eviscerated weight rate) (P ＜ 0.05). The results of qRT-PCR showed that MEF2D was expressed in all 10 involved tissues, which illustrated that the expression of this gene was of broad spectrum, and exists significant difference between ducks and drakes. In all of the duck tissues, the highest expression of leg muscle was the highest, the expression level of heart, liver, lung, kidney, brain, muscle duodenum and glandular stomach were relatively higher. And in all of the drake issues, the expression level of cardiac muscle and liver were the highest, duodenum, brain, muscle, leg muscle and glands in the stomach were relatively high. The results suggested that the variation trend of mRNA expression level of MEF2D in most issues of ducks at different age stages was first increased and then decreased and then increased; drakes showed the trend of decrease- increase- decrease. The results suggested that four SNPs and three haplotype combinations might have potential effects on slaughter traits in the above mentioned duck populations and could be used for marker-assisted selection. This study could provide a theoretical basis for the follow-up analysis of structure and function of MEF2D gene in ducks, further enrich the research results of MEF2D.

TMEM9B (transmembrane protein 9 domain family, member B), found in recent years, is a multifunctional transmembrane glycoprotein. Several studies have shown that TMEM9B protein plays key roles in multiple signaling pathways. However, the studies on the expression and functional properties of Tmem9B gene are seldom reported. In this study, Hy-Line brown chicken (Gallus gallus), Arbor acres plus broiler (AA+ broiler) and Sanhuang broiler were selected. The full-length coding sequences of Tmem9B gene were cloned from different chicken breeds and analyzed using bioinformatics tools. The expressional changes of Tmem9B gene in three breeds of normal chicken (AA+ broiler, Sanhuang broiler and Hy-Line brown chicken) and Escherichia coli lipopolysaccharide(LPS)-induced chicken inflammation model (AA+ broiler and Hy-Line brown chicken) were determined respectively to study the expressional activities and functional characteristics using quantitative real time polymerase chain reaction (qRT-PCR) technique. The results showed that there was only one SNP (single nucleotide polymorphism) site in the coding sequence of Sanhuang broiler Tmem9B gene, which meant that the gene was highly conserved among different chicken breeds. Tissue expression profile analysis showed that Tmem9B gene was all expressed in various tissues of AA+ broiler, Sanhuang broiler and Hy-Line brown chicken, but there were a little differences between different chicken breeds. Interestingly, the Tmem9B gene had high transcriptional activity in brain of all the three breeds. The expressions of Tmem9B gene of immune-related tissues were significantly up-regulated in the inflammation model chicken induced by LPS (lipopolysaccharide). This study showed hat Tmem9B may be a multifunctional gene that played important roles in many chicken biological processes, such as growth, development and inflammatory response. This study may provide theoretical basis for further explanation of the function and characteristics of Tmem9B gene.

Screening immune-related genes is important for pig (Sus scrofa) disease resistant breeding. In our previous genome-wide association study (GWAS) of T lymphocyte subpopulation traits in piglets (Sus scrofa)，an immune regulation region of 1.05 Mb was identified, which contained 6 immune-related genes, that is, cluster of differentiation 4 (CD4), lymphocyte activation gene-3 (LAG3), cluster of differentiation (CD27), lymphotoxin beta receptor (LTBR), tumor necrosis factor receptor superfamily member 1a (TNFRSF1A) and cluster of differentiation (CD9). In the present study, to unravel the 6 genes expression differences between breeds with different disease resistance, one typical Chinese indigenous breed (Dapulian) with higher disease resistance in North China and one commercial breed (Landrace) with lower disease resistance were used. Blood samples of clinically healthy Dapulian (n=104) and Landrace (n=171) piglets of 35 d old were collected, and their total RNA was extracted and reverse-transcribed into cDNA. Firstly, the mRNA expressions of these 6 genes and the reference gene beta-2-microglobulin (B2M) were determined by quantitative real-time PCR (qRT-PCR). Then, after normalization with reference gene B2M, Six immune-related genes expression characteristics and breed effects were performed analyses in the 2 breeds. The piglets used in this study were sampled from 13 Dapulian sows and 28 Landrace sows, finally, litter effect on these genes expression was also analyzed. The results of this study showed that the expressions of these 6 immune-related genes in Dapulian and Landrace were lower than the reference gene (B2M), and individual difference in the population was small, with variation coefficients of 5.92~15.96 and 5.99~12.43 in Dapulian and Landrace respectively. Comparison between the 2 breeds, there were significant difference in the expression levels of CD4, CD27, TNFRSF1A and CD9. Specifically, the expressions of CD4, CD27 and TNFRSF1A in Dapulian piglets were significantly lower than those in Landrace at 0.01 or 0.05 level, and expression of CD9 in Dapulian piglets was significantly higher than that in Landrace at 0.01 level. The litter effect of CD4 and CD27 reached significance level (P＜0.01). Our results indicated that CD4, CD27, TNFRSF1A and CD9 were differently expressed between Dapulian and Landrace piglets, which may be considered as candidate genes affecting the high resistance of Dapulian, and used in the study of marked assisted selection.

Reproductive performance in dairy cows is highly correlated with energy metabolism. The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear transcription factor that plays an important role in regulating the processes of energy homeostasis, reproductive, and immune physiology. A potentially important candidate gene for reproductive traits of dairy cows is PPARγ, which is encoded by the PPARG. In the present study, a Q448H mutation of the PPARG gene exon7 in Chinese Holstein cattle was detected with polymerase chain reaction products-single strand conformational polymorphism (PCR-SSCP) and DNA sequencing, and assessed through reproductive traits. Results demonstrate the presence of two alleles and 3 genotypes in the locus of the PPARG gene exon7. The 2 alleles were named G and T. The allelic frequencies of the G/T alleles in the three groups were 0.65/0.35, 0.75/0.25, and 0.69/0.31 for the Low-Hybrids, Improved-Hybrids, and Pure Holstein populations, respectively; the GG genotype was preponderant. Except for the Improved-Hybrids (P＜0.01), Low-Hybrids and Pure Holsteins were in Hardy-Weinberg equilibrium (P＞ 0.05) at this locus. Polymorphism information content was moderately high for Low-Hybrids, Improved-Hybrids, and Pure Holsteins at 0.35, 0.31, and 0.34, respectively. Comparing this data with database sequence of Bos taurus (Accession #: NC_007320.6), the mutation of G to T occurred at the 64 947 bp of allele T, which resulted in the substitution of glutamine into histidine, allele G was the same sequence as that of NC_007320.6. Analysis by least squares methodology showed that days-to-first-breeding (DFB) with genotype GG were significantly lower than that with the genotype GT (P＜0.05) and TT (P＜0.01). The days open (DO) with the genotype GG were significantly lower than that with genotype GT (P＜0.05). The number of services-per-conception (NSC), with genotype GG were significantly lower than that with the genotype GT (P＜0.01) at first lactation, and with the genotypes GT (P＜0.05) and TT (P＜0.05) at second lactation. It can be explained by the PPARG gene Q448H mutation that phenotypic variances were 3.82%, 2.55%, 2.24%, and 3.46% for the DFB, DO, and NSC at first and second lactations, respectively. There were no differences in the ages-at-first-calving (AFC) between any genotypes (P＞0.05). Additive effects of the G allele were -6.97 d, -11.2 d, and -0.215 t for DFB, DO, and NSC at second lactation, respectively. DFB and DO were reduced by 8.8 days and 15.7 days, respectively, while NSC was reduced by a factor of 0.25 with the allelic substitution of T to G, thereby indicating that the G allele of PPARG gene could benefit from further investigation as its role appears to be associated with reproductive performance. We conclude that the PPARG gene Q448H mutation could be used as a genetic marker for studies seeking to improve reproductive traits in Chinese Holstein cattle. This study has provided the theoretical foundation for maker-assistant-selection (MAS) of reproductive performance in Chinese Holstein cattle.

Myo-inositol-1-phosphate synthase (MIPS) is one of the key enzyme in biological inositol metabolism. In order to explore the role of MIPS in the stress response of Larimichthys crocea under cold treatments, MIPS gene was cloned and then its sequence were analyzed and the expression changes were detected under chronic and acute cold stress. The full length of MIPS cDNA (GenBank accession No. MG760436) was 2 599 bp which contained a 1 659 bp open reading frame (ORF) and encoded 553 amino acids, the molecular weight of putative MIPS protein was 60.5 kD. Sequence alignment showed that the amino acid sequence of L. crocea MIPS shared high similarity (85.5%~88.4%) to other teleost and all of these MIPS had 4 highly conserved core regions. In the phylogenetic tree, L. crocea MIPS gathered with MIPS of other teleost and was most closely related to the MIPS of Fugu rubripes (Takifugu rubripes). During chronic cold stress (water temperature slowed decreased from 12 ℃ to 6 ℃), the MIPS expression in gill, skin and heart significantly increased at first and then significantly decreased (P＜0.05), and the maximum appeared at 8 ℃ which were respectively 13.71, 89.50 and 50.83 fold, respectively, compared to 12 ℃. The expression continuously elevated in brain and muscle during chronic cold stress and at 6 ℃ the expression were 99.89 and 110.17 fold higher than before cold stress. And there were no significant changes in intestine, kidney, liver and spleen (P＞0.05). During acute cold stress (from 12 ℃ to 8 ℃ immediately and keeping 8 ℃ for 4 h), the expression level of MIPS were significantly up-regulated (P＜0.05) in all the detected tissues except intestine. The 2 biggest expression changes were appeared in brain and muscle, which were 54.53 and 32.89 fold higher than before cold stress. The expression changes in both chronic and acute cold treatments indicated MIPS participates in the response to cold stress and has possible relationship to cold adaptability of L. crocea, brain and muscle are the 2 most important tissues in the response to cold stress. These results provide reference for researching the function of MIPS gene and the mechanism of response to cold stress, as well as breeding low-temperature tolerant cultivars of L. crocea.

Melon (Cucumis melo) was mainly cultivated in the temperate zone to the tropics of the world. In order to explor in the relationship between the occurrence of male sterility in melon and the metabolism of superoxide and the related oxidase activities from the transcriptional level, this study used the male sterile AB line of stamens of the fertile and sterile plants of 5 mm in melon to do RNA sequencing (RNA-seq), and screened differentially expressed genes which related to the oxidase. In addition, the study carried out the real time fluorescence quantitative analysis in 2 stages of microspore development to identify the expression quantities of differential genes, and determined enzymatic activities of the buds of male sterile and fertile plants. The results showed that there were total of 1 364 differentially expressed genes in transcriptome sequencing , including 834 up-regulated genes and 530 down regulated genes. Then, it did cluster analysis to the screened differentially expressed genes according to the same or similar expression patterns, it showed that these differentially expressed genes divided into 28 categories, and eighth of them have 18 differentially expressed genes, these genes had extremely significant correlation (P＞0.001) with pathway of superoxide metabolism. Gene Ontology (GO) function analysis of differentially expressed genes showed that a total of 1 118 genes were involved in the 3 branches including the biosynthesis, molecular function and cell component, 576 differentially expressed genes were involved in the catalytic process of catalase, of which 17 genes were differentially expressed significantly. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation results showed that there were 10 genes related to the oxidoreductase among them including 9 peroxidase homologous genes and a catalase homologous gene. In the biosynthetic pathway of phenylpropanoidand, compared with the male fertile plants, the gene expression of peroxidase (POD) related gene MELO3C012183 was significantly up regulated in male sterile plants, and the expression of gene MELO3C014656 was down regulated. Furthermore, in the pathway of tryptophan metabolism, compared with the male fertile plants, the gene expression of catalase (CAT) related gene MELO3C017024 was significantly up regulated in male sterile plants. Quantitative real time polymerase chain reaction (qRT-PCR) validation showed that fertile plants' expression quantities of peroxidase 2-like precursor gene and peroxidase 11 gene which related to POD were lower than sterile plants, on the contrary, fertile plants' expression quantities of catalase isozyme 1-like gene which related to CAT were higher than sterile plants, the enzymatic activities of POD and CAT in male sterile plants were higher than that of fertile plants. The results supposed that the differential performances of enzymatic activities of POD and CAT in the male sterile line were caused by the self protection and inducing of plants, the accumulations of oxygen free radicals caused the abnormal increase in the level of membrane lipid peroxidation in male sterile lines, this phenomenon was likely to cause damage to microspore development and pollen abortion. This study would provid a scientific basis for the internal cause and mechanism of the pollen abortion in melon.

Superoxide dismutase 2 (SOD2) gene is an important component of endogenous antioxidant defense barrier and plays important roles in the prevention of disease, anti-aging, anti-tumor and anti-inflammation. To study the expression and localization of SOD2 gene in breast tissue of normal and clinical mastitis sheep (Ovis aries), Breast tissue of 3 normal and 3 clinical mastitis sheep were selected in this study. The expression of SOD2 mRNA and protein in breast tissue of normal and clinical mastitis sheep were detected by qRT-PCR and Western blot. And the SOD2 protein was located in breast tissue of normal and clinical mastitis sheep by immunohistochemistry (IHC) method. The results showed that the expression of SOD2 mRNA and protein in the breast tissue of clinical mastitis sheep were extremely significantly higher than that of control group (P＜0.01). Immunohistochemical staining found that SOD2 positive expression was located in breast epithelial cells of normal and clinical breast tissue. SOD2 positive reaction was stronger in breast tissue of the clinical mastitis sheep. The results of this study show that the expression of SOD2 mRNA and protein in the breast tissue of clinical mastitis sheep are up-regulated, will provide basic data for the further study of the mechanism of the gene and the pathogenesis of sheep mastitis.

Bacillus licheniformis could secrete a kind of keratinase which can hydrolyze keratin such as feather, hair, nail and so on. In previous work, the results indicated that a thermophilic Bacillus licheniformis Y6 was able to live at elevated temperature, it also could degrade feather rapidly, and lived on feather as the only source of carbon and nitrogen. Based on these properties, this paper finally extracted a kind of thermostable keratinase with excellent characteristics was extracted. Hence, it possess the potential to manage poultry waste and can be exploited in some specific industries. aiming at cloning a keratinase gene from Bacillus licheniformis Y6, this paper designed a pair of primers, and finally obtained the gene (GenBank No. MF327392) which included a complete Open Reading Frame with the size of 1 140 bp. Aligned in NCBI, this keratianse remained to be conserved during evolution, and shared the same catalytic triad Ser-His-Asp with other serine proteases. Analyzed by some online software, this entire gene encoded 379 amino acids with pre-, pro- and mature protein regions, and its mature protein, with the molecular weight 27.27 kD and the theory isoelectric point 6.57, was classified as stable protein. After cloning the gene into vector pET-32a, The recombinant vector pET-32a kerY6 was transformed into Escherichia coli BL21(DE3) induced with 0.5 mmol/L isopropyl β-D-thiogalactoside(IPTG), then the precipitation and culture supernatant were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis which indicated that the recombinant keratinase was successfully induced with a band at 58 kD corresponding to expectation, but could not be detected in the broth. In order to detect whether it developed into inclusion body, precipitation and supernatant of bacteria lysate were subjected to SDS-PAGE, results showed no band in the supernatant lane. Subsequently, the recombinant keratinase was purified by Ni-chelating affinity chromatography. Followed by characterization, the recombinant keratinase displayed a wide range of reaction temperature 60~80 ℃, and retained 85% maximum activity for a long time which indicated an excellent thermost ability. As the time accumulation, deactivation of enzyme was observed by incubating the recombinant keratinase over 80 ℃. Incubated with a variety of pH values, the maximum activity was obtained at pH value 8.5. The results of pH values assay demonstrated that the recombinant keratinase was a typical alkaline protease. Ethylene diaminete traacetic acid (EDTA), sodium dodecyl sulfate (SDS), phenylmethanesulfonyl fluoride (PMSF), Mn2+ and Fe3+ could remarkly inhibited its activity by 76%, 49%, 59%, 34%, 75%, respectively; Ca2+ and Mg2+ prompted its activity to 118%, 125%, respectively. Organic solvent, for instance, isopropylalcohol, glycerin and dimethyl sulfoxide (DMSO) almost had no influence on its activity. In conclusion, the keratinase from thermophilic bacillus licheniformis Y6 has great potential to apply to industry.

Dermal papilla cells (DPCs) are specialized mesenchymal-originated fibroblasts with prominent ability to induce hair follicle morphogenesis and development in mammals. They are situated at the bottom of hair follicle and direct hair follicle remodeling by sending various growth factors to follicular keratinocytes. Isolation and culture of DPCs from hair follicle is laborious and time-consuming job, thus present study was designed to optimize the culture condition of goat DPCs in order to acquire enough DPCs for other studies. In this study, DP was separated and cultured from goat hair follicle under stereoscope by microdissection, and confirmed the identity of cultured cell by using antibodies specific to their marker genes α-smooth muscle actin and CD133/prominin-1, then cell counting, (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay, alkaline phosphatas analysis and other methods were used to compare the combination of serum concentration and medium types. The results indicated that (1) goat DPCs shared the expression of specific markers as previously reported, and they were successfully cultured in four tested culture medium supplemented with different proportions of serum respectively; (2) Growth of goat DPCs depends on the nutritional supplement: the higher proportion of serum added in culture medium resulted faster proliferation rate and stronger ability to migrate and induce hair growth; (3) Advanced medium does contain effective factors to maintain or strengthen goat DPCs proliferation and inducing ability, thus it is more suitable for propagating goat DPCs than conventional medium, otherwise when the serum addition proportion is 10%, the growth and proliferation of goat DPCs cultured advanced medium are significantly higher than that in conventional media (P＜0.05); (4) the effect of Advanced media added with 4%~6% serum for gaot DPCs propagation is as good as the normal medium added with 10% serum (P＞0.05). In conclusion, we successfully cultured goat DPCs, identified them and proved that Advanced medium are more suitable for propagation of them in vitro than conventional medium.

Bacillus subtilis FJ-3-16 is an efficient keratinase-producing bacterium strain and its extracellular keratinase has many prospective applications in livestock wastes degradation, animal furs dehairing, detergent additive and so on. In order to increase the industrial yield of the extracellular keratinase produced by the strain, its fermentative condition was optimized using one-variable-at-a-time method, Plackett-Burman (PB) design and central composite design (CCD). The optimized medium contained corn meal 1.41%, soybean flour 3.62%, casein 2%, CaCl2 0.16%, K2HPO4 0.3%, KH2PO4 0.1% (pH 8.0) and optimized condition was 48 h cultivation at 37 ℃ and a rotate shaking of 250 r/min. The total activity of the extracellular keratinase was increased from 73.82 to 166.08 U/mL. Then, the optimized cultivation condition was further amplified in 50 L fermenter. The total activity of the extracellular keratinase was further increased to 207.6 U/mL in the amplified 50 L fermenter, and meanwhile the fermentative time was reduced from 48 to 18 h. The dehairing capacity of the ferment toward the skins of the Bama pig's (Sus scrofa) hoof and head was also evaluated using enzymatic dehairing experiments. Pieces of skins obtained from the hoof and head of the Bama pig, and the whole hoof of the Bama pig were treated by the crude extracellular keratinase at 50 ℃ for 6 h, 10 h, respectively, and dehairing effects were obvious. This study would pave the way for the application of strain FJ-3-16 in pig dehairing.

Mycoplas macapricolum subsp. capripneumoniae (Mccp) is the causative agent of contagious capri pleuropneumonia (CCPP). A SYBR Green I real-time fluorescent quantitative PCR (SYBR Green I qRT-PCR) was established based on primers derived from Mccp strain F38 (No. LN515398.1) arcD (MCCPF38_00114) gene deposited in GenBank. The results showed that the assay could only detect Mccp, and had no cross-reactivity with Mycoplasma mycoides subsp. capri (Mmc), Myplasma ovipenumoniae (Mo), Orf virus (ORFV)，Staphylococcus aureus (SA), Pasteurella multocida (Pm), Escherichia coli (Ec) and Haemophilus parasuis (HPS), indicating a high specificity. The sensitivity of the assay was 279 copies/μL with 1000 times higher than that of the conventional PCR. In addition, the variations in intra- and inter-assays were both less than 1%, indicating a good repeatability. The results indicate that the SYBR Green I qRT-PCR assay could provide a early, rapid and more sensitive diagnostic method for CCPP and a quantitative analysis assay for Mccp.

Foot-and-mouth disease (FMD) is an acute, hot, and highly contagious animal disease caused by Foot-and-mouth disease virus (FMDV). This study is to establish a gene chip that distinguishes FMDV serotypes O, A and AsiaⅠ. Three specific primers were designed according to the VP1 gene sequences of FMDV, and three target fragments of 248, 206 and 239 bp were obtained by RT-PCR. Three kinds of probes were designed and labeled with primer markers. A genotyping microarray encoding O, A and AsiaⅠ was established and its sensitivity, specificity, reproducibility and shelf life were evaluated. The optimum reaction conditions were as follows: hydration temperature was 37 ℃, confluent concentration was 0.25% BH4Na, 25% ethanol, and hybridization temperature was 42 ℃. Sensitivity test showed that FMDV-O, FMDV-A and FMDV-AsiaⅠ were 10-5, 10-7, 10-5 after the band is not visible. The PCR detection limits were: FMDV-O 118 pg/mL, 4.3×106 copies/μL; FMDV-A 18.4 pg/mL, 8.1×104 copies/μL; FMDV-AsiaⅠ 129 pg/mL, 4.9×105 copies/μL. The FMDV-O dilution reaches 10-6; the FMDV-A dilution reaches 10-8; the FMDV-AsiaⅠ dilution reaches 10-6, the sensitivity of the method was 10~100 times higher than that of a conventional PCR. There was no cross-hybrid among the detecting surfaces O, A and A1; the chip was reproducible and the prepared chip could be stored for at least three months. In this study, the experimental conditions were optimized, and a fast and simple FMDV classification method was established to provide a new technique for the classification of FMDV.

Infectious hematopoietic necrosis virus (IHNV) is an economically important pathogen causing significant mortalities in a wide variety of salmonid species, including the main aquaculture species, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Aimed to disease control and aquatic biosecurity, In this study, a new electrochemical biosensing method for IHNV on-site detection was developed to identify the IHNV-carrying salmonid fries or eggs. Based on evolutionary conservation analysis of 357 non-redundant IHNV sequences from different origins, the matrix gene (M) was found most conserved and selected as target sequence. Six IHNV specific primers were designed for reverse transcription isothermal amplification, according to M gene sequence of the IHNV Chinese strain Ch20101008. Biotinylated dUTP was mixed into dNTPs in the amplification system, thus amplification products were able to be captured and anchored on avidin modified electrode surfaces. And the 5' ends of 2 primers were labeled with ferrocene, which made the amplification products electrochemically active and capable to be detected by cyclic voltammetry (CV). The amplification for nucleic acid was able to be finished in 15 min, and the current value of oxidation peak had obvious positive correlation with the copy numbers of IHNV in the template. With amplification temperature at 63 ℃ and signal to noise ratio at 3, the limit of detection (LoD) was calculated to be 6.2 copies/μL, which was capable to distinguish asymptomatic IHNV carriers. Detectable products could be produced across a wide amplification temperature range from 59 to 65 ℃, thus the IHNV detection could be performed without precise thermal control. Therefore, the electrochemical biosensing method has promising application prospects in IHNV detection in aquaculture industry, especially salmonid farms and hatcheries.

Bacteria and archaea have evolved an adaptive immune system, which was known as clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR -associated protein (Cas 9) system, and that uses short RNA to degrade the target sequences present in invading viral and phage DNAs. This system can identify the target sequence and cut the double-stranded DNA. So, it has been harnessed by thousands of laboratories for genome editing applications in a variety of experimental model systems since 2013. As a new type of genome editing techniques, the CRISPR/Cas9 system has advantages in simple in design, strong specificity and high efficiency. CRISPR/Cas9 system has brought a breakthrough in genome directional regulation and application, and rapidly applied in the basic theory, transgenic animal production, genetic diagnosis, clinical therapy fields. Whereas, every new technology has its limitations, CRISPR/Cas9 system also exists off-target effects and difficulty to insert genes. This limits its applications to some extent. Therefore, around the CRISPR system, many scientists improved a series of modification that can be used for the gene editing and gene expression of new tools in recent years. Here, we reviewed the research progress and application of new gene editing tools and gene expression modification based on the CRISPR system evolution and looking forward to the application prospect and development direction.