The N⁶-methyladenosine (m⁶A) modification is the most abundant RNA modification in animals, and is dynamically regulated by methyltransferase, demethylase and m⁶A binding protein. In mRNA, m⁶A modification can regulate most RNA metabolism processes and play an important physiological role in animals. In this review, the research progress of distribution and expression, detection methods, enzymes related to methylation metabolism and their physiological functions of m⁶A modification in animals are well introduced. Besides, the existing problems or challenges in the current m⁶A modification research are prospected in the present study, which will provide reference for further study on m⁶A modification.

The N⁶-methyladenosine (m⁶A) modification is the most abundant RNA modification in animals, and is dynamically regulated by methyltransferase, demethylase and m⁶A binding protein. In mRNA, m⁶A modification can regulate most RNA metabolism processes and play an important physiological role in animals. In this review, the research progress of distribution and expression, detection methods, enzymes related to methylation metabolism and their physiological functions of m⁶A modification in animals are well introduced. Besides, the existing problems or challenges in the current m⁶A modification research are prospected in the present study, which will provide reference for further study on m⁶A modification.

In recent years, the research on the function of gut virome has attracted increasing attention. As a main component of the gut virome, intestinal phage has become a research hotspot. Numerous studies have shown that the diversity and abundance of gut phage not only directly affect the gut microbiota, but also have close correlation with host function, playing an important role in modulation of host health. In this review, we summarize current research progress in the interaction between gut phage and bacteria, the interaction between phage and animal host, and the relationship between gut phage and diseases such as inflammatory bowel disease and metabolic disease. This review will provide a theoretical basis and new perspective for further study on the effect of gut phage on animal health.

In recent years, the research on the function of gut virome has attracted increasing attention. As a main component of the gut virome, intestinal phage has become a research hotspot. Numerous studies have shown that the diversity and abundance of gut phage not only directly affect the gut microbiota, but also have close correlation with host function, playing an important role in modulation of host health. In this review, we summarize current research progress in the interaction between gut phage and bacteria, the interaction between phage and animal host, and the relationship between gut phage and diseases such as inflammatory bowel disease and metabolic disease. This review will provide a theoretical basis and new perspective for further study on the effect of gut phage on animal health.

Small non-coding RNA (sRNA) is a type of RNA molecule that is transcribed but not translated into protein in the genome and controls gene expression at the post-transcriptional level. Unlike protein-mediated regulation system, sRNA-mediated regulation can respond to environmental changes quickly when bacteria confront adverse growth environment. RyhB-1 and RyhB-2 are two highly homologous sRNAs in Salmonella, which regulate target genes expression singly or together with the help of regulating factors by base pairing. RyhB-1 and RyhB-2 are major components of Salmonella iron homeostasis regulation at post-transcriptional level by increasing the iron uptake, limiting non-essential iron-utilizing proteins synthesis and accelerating the storage of iron-sulfur protein when iron is scarce. In addition, when Salmonella encounters various environmental stresses such as oxidative stress, hypoxia or acidic environment, RyhB can also control the production of reactive oxygen species, balance the stability of inorganic substances such as nitrate, and regulate bacterial motility and Salmonella virulence to cope with environmental changes. This paper is concerned with the physiological characteristics, regulatory mechanisms and functions of Salmonella RyhB to provide guidance for the subsequent study of RyhB.

Small non-coding RNA (sRNA) is a type of RNA molecule that is transcribed but not translated into protein in the genome and controls gene expression at the post-transcriptional level. Unlike protein-mediated regulation system, sRNA-mediated regulation can respond to environmental changes quickly when bacteria confront adverse growth environment. RyhB-1 and RyhB-2 are two highly homologous sRNAs in Salmonella, which regulate target genes expression singly or together with the help of regulating factors by base pairing. RyhB-1 and RyhB-2 are major components of Salmonella iron homeostasis regulation at post-transcriptional level by increasing the iron uptake, limiting non-essential iron-utilizing proteins synthesis and accelerating the storage of iron-sulfur protein when iron is scarce. In addition, when Salmonella encounters various environmental stresses such as oxidative stress, hypoxia or acidic environment, RyhB can also control the production of reactive oxygen species, balance the stability of inorganic substances such as nitrate, and regulate bacterial motility and Salmonella virulence to cope with environmental changes. This paper is concerned with the physiological characteristics, regulatory mechanisms and functions of Salmonella RyhB to provide guidance for the subsequent study of RyhB.

Quorum sensing (QS) was first discovered in bacteria. It has also been found in fungi in recent years. The key signal molecule of QS are called Quorum sensing molecules (QSM), which excreting, sensing, responsing and acting on itself, and having no toxic action on itself when at the concentration that just cause the reaction. Farnesol is the QSM of Candida albicans, which influence multitudes factors of C. albicans including drug resistance, hypha formation, adhesive power and apoptosis; and influence the C. albicans host's immunity and host cell apoptosis. Furthermore, C. albicans has higher tolerance to farnesol than other fungi, but the mechanism is unclear. At present, because of the little understanding on the mechanism of the QSM farnesol, the interaction between the QSM farnesol and C. albicans is reviewed in this paper.

Quorum sensing (QS) was first discovered in bacteria. It has also been found in fungi in recent years. The key signal molecule of QS are called Quorum sensing molecules (QSM), which excreting, sensing, responsing and acting on itself, and having no toxic action on itself when at the concentration that just cause the reaction. Farnesol is the QSM of Candida albicans, which influence multitudes factors of C. albicans including drug resistance, hypha formation, adhesive power and apoptosis; and influence the C. albicans host's immunity and host cell apoptosis. Furthermore, C. albicans has higher tolerance to farnesol than other fungi, but the mechanism is unclear. At present, because of the little understanding on the mechanism of the QSM farnesol, the interaction between the QSM farnesol and C. albicans is reviewed in this paper.

The objective of this study was to investigate the expression of porcine PLAC1 gene,and preliminarily identify its gene structure, biological function and inheritance patterns. The placenta tissue of pigs on 26, 50 and 95 days of pregnancy were collected, the full-length of PLAC1 mRNA and its splicing variants were cloned by using the RACE-PCR(rapid-amplification of cDNA ends) and RT-qPCR(real-time quantitative PCR). Moreover, the expression patterns of PLAC1 gene in placenta of pigs was verified by in situ hybridization. In addition, the sequence variation of PLAC1 gene was detected. The results showed that there were 3 different transcripts of the PLAC1 gene in pigs, and the CDS regions of the 3 transcripts were identical, with a length of 525 bp, encoding 174 amino acids; The results of the expression profile and hybridization in situ of PLAC1 gene indicated that the PLAC1 gene was specifically expressed in pig placental chorionic epithelium, and the expression of PLAC1 mRNA was different during different stages of pregnancy. The expression of PLAC1 mRNA was significantly higher on 50 and 95 days of gestation in placenta of pigs than 26 days of gestation. The genotype of SNP on PLAC1 gene confirmed that it was a hemizygous gene, and followed the inheritance patterns of hemizygous gene. The experiment data provide a basis for exploring the biological function of porcine PLAC1 gene in placenta development.

The objective of this study was to investigate the expression of porcine PLAC1 gene,and preliminarily identify its gene structure, biological function and inheritance patterns. The placenta tissue of pigs on 26, 50 and 95 days of pregnancy were collected, the full-length of PLAC1 mRNA and its splicing variants were cloned by using the RACE-PCR(rapid-amplification of cDNA ends) and RT-qPCR(real-time quantitative PCR). Moreover, the expression patterns of PLAC1 gene in placenta of pigs was verified by in situ hybridization. In addition, the sequence variation of PLAC1 gene was detected. The results showed that there were 3 different transcripts of the PLAC1 gene in pigs, and the CDS regions of the 3 transcripts were identical, with a length of 525 bp, encoding 174 amino acids; The results of the expression profile and hybridization in situ of PLAC1 gene indicated that the PLAC1 gene was specifically expressed in pig placental chorionic epithelium, and the expression of PLAC1 mRNA was different during different stages of pregnancy. The expression of PLAC1 mRNA was significantly higher on 50 and 95 days of gestation in placenta of pigs than 26 days of gestation. The genotype of SNP on PLAC1 gene confirmed that it was a hemizygous gene, and followed the inheritance patterns of hemizygous gene. The experiment data provide a basis for exploring the biological function of porcine PLAC1 gene in placenta development.

The objective of this study was to analyze the expression profile and imprinting status of a novel long noncoding RNA (LINC24065) located in the DLK1-DIO3 imprinted domain in natural reproduction(NR) and somatic cell nuclear transfer (SCNT) calves, which would provide a theoretical basis for further understanding the role of DLK1-DIO3 imprinted domain in the reprogramming of SCNT. The brain of NR calves was used as experimental material, a novel long intergenic non-coding RNA was identified by RACE and RT-PCR methods and named LINC24065. The expression and imprinting status of LINC24065 in NR and SCNT calves were analyzed by SNP-based direct sequencing of RT-PCR product. Sequence analysis revealed that LINC24065 contained 6 splicing variants which were expressed in 7 detected tissues of NR calves. But in SCNT calves, LINC24065-V3 was not detected and the other 5 isoforms showed tissue-specific expression. Imprinting status analysis indicated that LINC24065 exhibited monoallelic expression in 7 tissues of NR and SCNT calves; but in brain of SCNT calves, the monoallelic expression of LINC24065 had different parent origin from that in other tissues. The aberrant expression patterns and imprinting status of LINC24065 occurred in SCNT calves. The expression of splicing variants also changed in tissues. These results suggest that LINC24065 gene is associated with abnormal development of SCNT cattle.

The objective of this study was to analyze the expression profile and imprinting status of a novel long noncoding RNA (LINC24065) located in the DLK1-DIO3 imprinted domain in natural reproduction(NR) and somatic cell nuclear transfer (SCNT) calves, which would provide a theoretical basis for further understanding the role of DLK1-DIO3 imprinted domain in the reprogramming of SCNT. The brain of NR calves was used as experimental material, a novel long intergenic non-coding RNA was identified by RACE and RT-PCR methods and named LINC24065. The expression and imprinting status of LINC24065 in NR and SCNT calves were analyzed by SNP-based direct sequencing of RT-PCR product. Sequence analysis revealed that LINC24065 contained 6 splicing variants which were expressed in 7 detected tissues of NR calves. But in SCNT calves, LINC24065-V3 was not detected and the other 5 isoforms showed tissue-specific expression. Imprinting status analysis indicated that LINC24065 exhibited monoallelic expression in 7 tissues of NR and SCNT calves; but in brain of SCNT calves, the monoallelic expression of LINC24065 had different parent origin from that in other tissues. The aberrant expression patterns and imprinting status of LINC24065 occurred in SCNT calves. The expression of splicing variants also changed in tissues. These results suggest that LINC24065 gene is associated with abnormal development of SCNT cattle.

To explore the regulation mechanism of duck LXRα gene on fat deposition,in this experiment,the pEGFP-N3 was used as the vector to construct the LXRα gene eukaryotic over-expression vector carried HIS-tag that was transfected into the duck primary hepatocytes and PC3 cells, respectively. The over expression level of LXRα gene in duck hepatocytes and PC3 cells were detected by the duck HIS-tag kit,and the correlation between the over expression of LXRα gene in the two kinds of cells and the lipid index contents was analyzed,and the regulation mechanism of LXRα gene on lipid metabolism in duck hepatocytes and PC3 cells was explored. The results showed that the over expression of LXRα gene in both cells were significantly positively correlated with the contents of triglyceride (TG) and high-density lipoprotein (HDL) (P<0.01),and significantly negatively correlated with the contents of total cholesterol (TC) and low density lipoprotein (LDL) (P<0.01). The results reveal that over expression of LXRα gene has a positive regulatory effect on the anabolism of TG and HDL,and has a negative regulatory effect on TC and LDL,and the regulatory mechanisms in the two types of cells are similar. The results of this study will lay a theoretical foundation for the further study of the role of LXRα gene in the lipid metabolism network pathway and the solve of excessive fat deposition in duck body.

To explore the regulation mechanism of duck LXRα gene on fat deposition,in this experiment,the pEGFP-N3 was used as the vector to construct the LXRα gene eukaryotic over-expression vector carried HIS-tag that was transfected into the duck primary hepatocytes and PC3 cells, respectively. The over expression level of LXRα gene in duck hepatocytes and PC3 cells were detected by the duck HIS-tag kit,and the correlation between the over expression of LXRα gene in the two kinds of cells and the lipid index contents was analyzed,and the regulation mechanism of LXRα gene on lipid metabolism in duck hepatocytes and PC3 cells was explored. The results showed that the over expression of LXRα gene in both cells were significantly positively correlated with the contents of triglyceride (TG) and high-density lipoprotein (HDL) (P<0.01),and significantly negatively correlated with the contents of total cholesterol (TC) and low density lipoprotein (LDL) (P<0.01). The results reveal that over expression of LXRα gene has a positive regulatory effect on the anabolism of TG and HDL,and has a negative regulatory effect on TC and LDL,and the regulatory mechanisms in the two types of cells are similar. The results of this study will lay a theoretical foundation for the further study of the role of LXRα gene in the lipid metabolism network pathway and the solve of excessive fat deposition in duck body.

To clone and characterize short stature homeobox 2 (Shox2) gene in New Zealand rabbit, predict its structure and function, and to investigate its expression pattern, the full-length CDS sequence of Shox2 was cloned, and the biological characteristics of Shox2 protein were analyzed in this study, such as homology, hydrophilicity, secondary and tertiary structure characteristics and phylogenetic tree. Western blot was employed to verify fusion protein TrxA-Shox2. Quantitative real-time RT-PCR (RT-qPCR) was employed to detect the expression patterns of Shox2 in kidney, aorta, heart, dermis, lung, brain, liver, spleen, gallbladder and muscle tissues at 4 different developmental phases(E20.5 d, E28.5 d, 10 d and adult) of 3 animals for each phase. Shox2 gene was successfully cloned in present study (GenBank accession number:KP726285), CDS of Shox2 was composed of 666 bp encoding 221 amino acids. According to the results of bioinformatics analysis, Shox2 belonged to the unstable alkaline protein, 61.99% of which amino acids was alpha helix, 33.48% for random coil, 4.52% for extended strand. The result of Western blot verified the expression of fusion protein TrxA-Shox2. The analysis of homology and phylogenetic tree showed that the amino acid sequence of Oryctolagus cuniculus Shox2 protein had high homology and close genetic distance with tarsier, gorilla, rattus norvegicus and human, which meant that Shox2 gene was conservative during evolutionary process. According to the result of RT-qPCR, Shox2 gene universally expressed in most tissues detected in this study. A relatively higher Shox2 mRNA expression level was detected in kidney, gallbladder and muscle tissues in embryo for E20.5 d, in heart and spleen in embryo for E28.5 d, in dermis and liver for 10 d, and in derminds and spleen for adult. In this study, Shox2 gene was successfully cloned and characterized. Shox2 gene universally expressed in all tissues detected, its expression level was related to the function of Shox2 in different tissues and at different developmental stages, which was strictly regulated.

To clone and characterize short stature homeobox 2 (Shox2) gene in New Zealand rabbit, predict its structure and function, and to investigate its expression pattern, the full-length CDS sequence of Shox2 was cloned, and the biological characteristics of Shox2 protein were analyzed in this study, such as homology, hydrophilicity, secondary and tertiary structure characteristics and phylogenetic tree. Western blot was employed to verify fusion protein TrxA-Shox2. Quantitative real-time RT-PCR (RT-qPCR) was employed to detect the expression patterns of Shox2 in kidney, aorta, heart, dermis, lung, brain, liver, spleen, gallbladder and muscle tissues at 4 different developmental phases(E20.5 d, E28.5 d, 10 d and adult) of 3 animals for each phase. Shox2 gene was successfully cloned in present study (GenBank accession number:KP726285), CDS of Shox2 was composed of 666 bp encoding 221 amino acids. According to the results of bioinformatics analysis, Shox2 belonged to the unstable alkaline protein, 61.99% of which amino acids was alpha helix, 33.48% for random coil, 4.52% for extended strand. The result of Western blot verified the expression of fusion protein TrxA-Shox2. The analysis of homology and phylogenetic tree showed that the amino acid sequence of Oryctolagus cuniculus Shox2 protein had high homology and close genetic distance with tarsier, gorilla, rattus norvegicus and human, which meant that Shox2 gene was conservative during evolutionary process. According to the result of RT-qPCR, Shox2 gene universally expressed in most tissues detected in this study. A relatively higher Shox2 mRNA expression level was detected in kidney, gallbladder and muscle tissues in embryo for E20.5 d, in heart and spleen in embryo for E28.5 d, in dermis and liver for 10 d, and in derminds and spleen for adult. In this study, Shox2 gene was successfully cloned and characterized. Shox2 gene universally expressed in all tissues detected, its expression level was related to the function of Shox2 in different tissues and at different developmental stages, which was strictly regulated.

The aim of this study was to investigate the effect of PNPLA5 gene on male rats' fertility. We used male PNPLA5 knockout rats(KO) as the experimental group and wild type rats(WT) as the control group, feeding them with regular diet. The epididymis spermatozoa and testis tissue were collected. We detected the pregnancy rate and average litter size by breeding assay, the sperm motility by Computer Aided Sperm Analysis(CASA), the expression level of mitochondria function related proteins (cytochrome C,Cytc and cytochrome C Oxidase subunit IV) in sperms were detected by Western blot, and histochemical morphology of testis tissue by HE staining.The results showed that PNPLA5 knockout significantly reduced the pregnancy rate of the mated female rats (P<0.01), significantly decreased the curvilinear velocity of sperm(P<0.05) and percentage of sperm with progressive movement (P<0.01) compared to the wild type male rats.We also found that the PNPLA5 knockout downregulated the expression level of Cytc and upregulated expression level of COX IV in sperms compared with those in wild type rats(WT), but no significant difference were observed between 2 groups (P>0.05). In addition, the histological structure of testis of PNPLA5 knockout rats (KO) appeared loose, unarranged, and epithelial cells detached to the lumen. In conclusion,this study shows that PNPLA5 knockout reduces the fertility of male rats and it may provide an important evidence for understanding the animal reproductive performance.

The aim of this study was to investigate the effect of PNPLA5 gene on male rats' fertility. We used male PNPLA5 knockout rats(KO) as the experimental group and wild type rats(WT) as the control group, feeding them with regular diet. The epididymis spermatozoa and testis tissue were collected. We detected the pregnancy rate and average litter size by breeding assay, the sperm motility by Computer Aided Sperm Analysis(CASA), the expression level of mitochondria function related proteins (cytochrome C,Cytc and cytochrome C Oxidase subunit IV) in sperms were detected by Western blot, and histochemical morphology of testis tissue by HE staining.The results showed that PNPLA5 knockout significantly reduced the pregnancy rate of the mated female rats (P<0.01), significantly decreased the curvilinear velocity of sperm(P<0.05) and percentage of sperm with progressive movement (P<0.01) compared to the wild type male rats.We also found that the PNPLA5 knockout downregulated the expression level of Cytc and upregulated expression level of COX IV in sperms compared with those in wild type rats(WT), but no significant difference were observed between 2 groups (P>0.05). In addition, the histological structure of testis of PNPLA5 knockout rats (KO) appeared loose, unarranged, and epithelial cells detached to the lumen. In conclusion,this study shows that PNPLA5 knockout reduces the fertility of male rats and it may provide an important evidence for understanding the animal reproductive performance.

The objectives of the present study were to clone the Lin28A cDNA of sheep, investigate its expression patterns and regulation properties during the onset of puberty in sheep. In this study, 5 Duolang sheep were slaughtered in the prepuberty (1 week before the first estrus), puberty and postpuberty (1 week after the first estrus),respectively,hypothalamus, pituitary and ovarian tissues were collected. The Lin28A cDNA of these tissues was cloned, the homology of the amino acids of Lin28A between Duolang sheep and other species were compared by DNAman 6.0. The expression patterns of Lin28A, let-7a and let-7b in hypothalamus, pituitary and ovary during the puberal transition in Duolang sheep were detected by real-time PCR.The results showed that the fragment of Lin28A cDNA was 3 581 bp which contained 2 963 bp of 3' UTR and 618 bp of the coding region.The deduced protein sequence of the Lin28A coding region shared 87.56%, 88.52%, 92.68%, 89.95%,76.10% and 61.84% to that of human, house mouse, cattle, sheep, original chicken and zebrafish. The expression of Lin28A was significantly decreased in the hypothalamus and ovary from prepuberty to puberty (P<0.05), the expression levels of let-7a and let-7b did not change significantly (P>0.05).The results provided a foundation for revealing the function of Lin28A gene and its role in the onset of puberty in sheep.

The objectives of the present study were to clone the Lin28A cDNA of sheep, investigate its expression patterns and regulation properties during the onset of puberty in sheep. In this study, 5 Duolang sheep were slaughtered in the prepuberty (1 week before the first estrus), puberty and postpuberty (1 week after the first estrus),respectively,hypothalamus, pituitary and ovarian tissues were collected. The Lin28A cDNA of these tissues was cloned, the homology of the amino acids of Lin28A between Duolang sheep and other species were compared by DNAman 6.0. The expression patterns of Lin28A, let-7a and let-7b in hypothalamus, pituitary and ovary during the puberal transition in Duolang sheep were detected by real-time PCR.The results showed that the fragment of Lin28A cDNA was 3 581 bp which contained 2 963 bp of 3' UTR and 618 bp of the coding region.The deduced protein sequence of the Lin28A coding region shared 87.56%, 88.52%, 92.68%, 89.95%,76.10% and 61.84% to that of human, house mouse, cattle, sheep, original chicken and zebrafish. The expression of Lin28A was significantly decreased in the hypothalamus and ovary from prepuberty to puberty (P<0.05), the expression levels of let-7a and let-7b did not change significantly (P>0.05).The results provided a foundation for revealing the function of Lin28A gene and its role in the onset of puberty in sheep.

The aim of this study was to investigate the expression pattern of KDM2B in mouse oocytes and preimplantation embryos, which provided important foundation for the mechanism study of KDM2B in meiosis and embryonic development. Twenty mice aged 6-8 weeks were selected as experimental material, GV and MⅡoocytes, 2-cell, 4-cell, 8-cell, blastocyst were collected. The primer was designed based on Mus musculus KDM2B sequence in GenBank, and the expression pattern of KDM2B was detected by RT-qPCR in different stages of preimplantation embryos. Then immunofluorescence staining was used to locate the distribution of KDM2B protein in embryos of each period. The result showed that the mRNA expression level of KDM2B in GV oocyte was extremely significant higher than that in MⅡ(P<0.01). During the early embryonic development, the KDM2B was expressed in 2-cell to blastocyst, and extremely significant higher in blastocyst than that in other stages (P<0.01). In preimplantation embryos, KDM2B was observed in the nucleolar at GV oocyte stage, and fluorescence signatures in the nucleus clearly weakened at the MⅡ stage (P<0.01), however KDM2B was not detected in 2-to 8-cell, and became detectable at blastocyst. This study established the spatial and temporal expression profiles of KDM2B, and the specific mechanism of KDM2B in regulating meiosis and embryonic development requires further investigation.

The aim of this study was to investigate the expression pattern of KDM2B in mouse oocytes and preimplantation embryos, which provided important foundation for the mechanism study of KDM2B in meiosis and embryonic development. Twenty mice aged 6-8 weeks were selected as experimental material, GV and MⅡoocytes, 2-cell, 4-cell, 8-cell, blastocyst were collected. The primer was designed based on Mus musculus KDM2B sequence in GenBank, and the expression pattern of KDM2B was detected by RT-qPCR in different stages of preimplantation embryos. Then immunofluorescence staining was used to locate the distribution of KDM2B protein in embryos of each period. The result showed that the mRNA expression level of KDM2B in GV oocyte was extremely significant higher than that in MⅡ(P<0.01). During the early embryonic development, the KDM2B was expressed in 2-cell to blastocyst, and extremely significant higher in blastocyst than that in other stages (P<0.01). In preimplantation embryos, KDM2B was observed in the nucleolar at GV oocyte stage, and fluorescence signatures in the nucleus clearly weakened at the MⅡ stage (P<0.01), however KDM2B was not detected in 2-to 8-cell, and became detectable at blastocyst. This study established the spatial and temporal expression profiles of KDM2B, and the specific mechanism of KDM2B in regulating meiosis and embryonic development requires further investigation.

This study aimed to investigate the effects of weaning age and dietary nutrition levels on the digestion and metabolism of major nutrients of Shaanbei White Cashmere goats aged from 4 to 6 months old. In the experiment, fifty-four female Shaanbei White Cashmere goats aged 54 days old (average body weight was (10.73±1.03)kg) were selected and randomly assigned to 6 groups, with 3 replicates per group and 3 for each replicate. Goats in group I, Ⅱ and Ⅲ were fed standard diets, and the weaning age was 90, 75 and 60 d, respectively. Goats in group IV, V and VI were all weaned at 60 d, and the energy and protein levels of their diet were 85%, 115% and 130% of the standard diet, respectively. The experiment lasted for 180 days. Three digestion and metabolism trails were conducted at the age of 114-120, 144-150, 174-180 d, respectively. One goat (a total of 18 goats) was randomly selected from each replicate for a 3-phase digestion and metabolism test. Samples of feed, feces and urine were collected for measuring the apparent digestibility of main nutrients and the utilization rate of energy and nitrogen. The results showed that:1)The average daily gain of weaned lambs in the group I, Ⅱ and Ⅲ were 95.97, 104.58 and 108.75 g·d^-1, respectively, and the value of group Ⅲ was significantly higher than that of group I (P<0.05). The dry matter intake of goats in the group Ⅱ and Ⅲ was significantly higher than that of group I (P<0.01). 2) The intake energy, digestive energy, metabolic energy, digestible nitrogen, retention nitrogen and the apparent retention rate of nitrogen of 4-6 months old weaned lambs were significantly increased in the group V (P<0.05). 3) The dry matter intake and the average daily gain of 4-6 months old weaned lambs increased firstly and then decreased with the increase of dietary nutrient levels (P<0.05). 4) The dietary nutrition levels significantly affected the digestion and metabolism of DM, EE, NDF, ADF, Ca, P, N and energy (P<0.05). 5) Except for DM, the digestive and metabolic rates of EE, NDF, ADF, Ca, P, N and energy increased with the increasing age. In this study, lambs of Shaanbei White Cashmere goats weaning at 60 d had the best growth performance. As for the overall nutritional balance and the digestion and metabolism levels of 4-6 months old weaned lambs, the optimal dietary energy and protein level is recommended to be 1.15 times higher than that of the current standard diet.

This study aimed to investigate the effects of weaning age and dietary nutrition levels on the digestion and metabolism of major nutrients of Shaanbei White Cashmere goats aged from 4 to 6 months old. In the experiment, fifty-four female Shaanbei White Cashmere goats aged 54 days old (average body weight was (10.73±1.03)kg) were selected and randomly assigned to 6 groups, with 3 replicates per group and 3 for each replicate. Goats in group I, Ⅱ and Ⅲ were fed standard diets, and the weaning age was 90, 75 and 60 d, respectively. Goats in group IV, V and VI were all weaned at 60 d, and the energy and protein levels of their diet were 85%, 115% and 130% of the standard diet, respectively. The experiment lasted for 180 days. Three digestion and metabolism trails were conducted at the age of 114-120, 144-150, 174-180 d, respectively. One goat (a total of 18 goats) was randomly selected from each replicate for a 3-phase digestion and metabolism test. Samples of feed, feces and urine were collected for measuring the apparent digestibility of main nutrients and the utilization rate of energy and nitrogen. The results showed that:1)The average daily gain of weaned lambs in the group I, Ⅱ and Ⅲ were 95.97, 104.58 and 108.75 g·d^-1, respectively, and the value of group Ⅲ was significantly higher than that of group I (P<0.05). The dry matter intake of goats in the group Ⅱ and Ⅲ was significantly higher than that of group I (P<0.01). 2) The intake energy, digestive energy, metabolic energy, digestible nitrogen, retention nitrogen and the apparent retention rate of nitrogen of 4-6 months old weaned lambs were significantly increased in the group V (P<0.05). 3) The dry matter intake and the average daily gain of 4-6 months old weaned lambs increased firstly and then decreased with the increase of dietary nutrient levels (P<0.05). 4) The dietary nutrition levels significantly affected the digestion and metabolism of DM, EE, NDF, ADF, Ca, P, N and energy (P<0.05). 5) Except for DM, the digestive and metabolic rates of EE, NDF, ADF, Ca, P, N and energy increased with the increasing age. In this study, lambs of Shaanbei White Cashmere goats weaning at 60 d had the best growth performance. As for the overall nutritional balance and the digestion and metabolism levels of 4-6 months old weaned lambs, the optimal dietary energy and protein level is recommended to be 1.15 times higher than that of the current standard diet.

The aim of this research was to assess the effects of dietary protein and energy levels on the growth, digestion performance and serum biochemical indexes of 21-60 days old Hu sheep lambs. In a 2×2 factorial design arrangement, sixty-four healthy Hu sheep lambs with similar body weight and birth date were assigned to 4 groups, including high energy-high protein (HE-HP), high energy-low protein (HE-LP), low energy-high protein (LE-HP) and low energy-low protein (LE-LP) groups. Each group had 4 replicates with 4 lambs in each replicate. After a three-day adaptation period, all lambs were weaned and fed with milk replacer and starter. The trial lasted for 40 days. The feed intake of lambs were recorded every day. The body weight was measured and blood samples were collected before morning feeding every 20 days. The digestibility tests were carried out from 31 to 40 and 51 to 60 days of age. The results showed that:1) There was no significant interaction between energy and protein levels on growth performance of lambs (P>0.05), but the ADG and feed conversion efficiency significantly decreased with the decrease of dietary protein or energy levels (P<0.05). 2) There was an significant interaction between energy and protein levels on the digestibility of DM and total energy in lambs from 31 to 40 days of age(P<0.05). Specifically, fed with the high energy diet, the digestibility of DM and total energy of lambs significantly decreased with the decrease of protein levels (P<0.05). 3) The serum glucose concentration significantly decreased with the decrease of dietary energy level for 40 days old lambs (P<0.05). At 60 day, decreased the protein levels significantly decreased the serum urea nitrogen content(P<0.05) and significantly increased the serum GH content (P<0.05), decreased the energy levels significantly increased the serum urea nitrogen content (P<0.05) and triglyceride content(P<0.05). In summary, dietary energy and protein levels can affect the growth performance of lambs from 21 to 60 days old, appropriate dietary energy and protein levels should be provided to guarantee the digestion and growth of lambs.

The aim of this research was to assess the effects of dietary protein and energy levels on the growth, digestion performance and serum biochemical indexes of 21-60 days old Hu sheep lambs. In a 2×2 factorial design arrangement, sixty-four healthy Hu sheep lambs with similar body weight and birth date were assigned to 4 groups, including high energy-high protein (HE-HP), high energy-low protein (HE-LP), low energy-high protein (LE-HP) and low energy-low protein (LE-LP) groups. Each group had 4 replicates with 4 lambs in each replicate. After a three-day adaptation period, all lambs were weaned and fed with milk replacer and starter. The trial lasted for 40 days. The feed intake of lambs were recorded every day. The body weight was measured and blood samples were collected before morning feeding every 20 days. The digestibility tests were carried out from 31 to 40 and 51 to 60 days of age. The results showed that:1) There was no significant interaction between energy and protein levels on growth performance of lambs (P>0.05), but the ADG and feed conversion efficiency significantly decreased with the decrease of dietary protein or energy levels (P<0.05). 2) There was an significant interaction between energy and protein levels on the digestibility of DM and total energy in lambs from 31 to 40 days of age(P<0.05). Specifically, fed with the high energy diet, the digestibility of DM and total energy of lambs significantly decreased with the decrease of protein levels (P<0.05). 3) The serum glucose concentration significantly decreased with the decrease of dietary energy level for 40 days old lambs (P<0.05). At 60 day, decreased the protein levels significantly decreased the serum urea nitrogen content(P<0.05) and significantly increased the serum GH content (P<0.05), decreased the energy levels significantly increased the serum urea nitrogen content (P<0.05) and triglyceride content(P<0.05). In summary, dietary energy and protein levels can affect the growth performance of lambs from 21 to 60 days old, appropriate dietary energy and protein levels should be provided to guarantee the digestion and growth of lambs.

The purpose of this study was to elucidate changes in miRNA and mRNA expression profiles of PCV2 infected PK-15 cells, and to illuminate the interaction between differentially expressed miRNAs and mRNAs. Stem-loop quantitative PCR was used to detect and identify differential expression levels of multiple hot spot miRNAs in PCV2 infected and uninfected groups. The results of proteomic study of PCV2 infected cells at the 12th hour post infection (hpi) were analyzed (GEO Accession No.:GSE71945) to obtain differentially expressed mRNA and to analyze the interaction between differentially expressed miRNA and mRNA. It was found that miR-10b, miR-128, miR-155-5p, miR-21, miR-29b, miR-361-3p were differentially expressed at 12 hpi. By using miRecords to predict their target genes, 7 105, 9 741, 7 451, 7 183, 7 971 and 8 793 target genes were found, respectively. Totally there were 158 differentially expressed mRNAs that belonged to various cell components and participated in many important biological pathways, and there was a large number of common target genes between the differentially expressed miRNAs candidate target genes and differentially expressed mRNAs. In summary, PCV2 infection can lead to disruption of multiple miRNA expression in cells, resulting in disorganization of miRNA target genes, which in turn influences the normal life events of cells.

The purpose of this study was to elucidate changes in miRNA and mRNA expression profiles of PCV2 infected PK-15 cells, and to illuminate the interaction between differentially expressed miRNAs and mRNAs. Stem-loop quantitative PCR was used to detect and identify differential expression levels of multiple hot spot miRNAs in PCV2 infected and uninfected groups. The results of proteomic study of PCV2 infected cells at the 12th hour post infection (hpi) were analyzed (GEO Accession No.:GSE71945) to obtain differentially expressed mRNA and to analyze the interaction between differentially expressed miRNA and mRNA. It was found that miR-10b, miR-128, miR-155-5p, miR-21, miR-29b, miR-361-3p were differentially expressed at 12 hpi. By using miRecords to predict their target genes, 7 105, 9 741, 7 451, 7 183, 7 971 and 8 793 target genes were found, respectively. Totally there were 158 differentially expressed mRNAs that belonged to various cell components and participated in many important biological pathways, and there was a large number of common target genes between the differentially expressed miRNAs candidate target genes and differentially expressed mRNAs. In summary, PCV2 infection can lead to disruption of multiple miRNA expression in cells, resulting in disorganization of miRNA target genes, which in turn influences the normal life events of cells.

The purpose of this study was to verify the in vitro interaction between Newcastle disease virus (NDV) matrix (M) protein and chicken importin β1 protein. The products of NDV M gene and chicken importin β1 gene were obtained by PCR amplification, and then subcloned into the prokaryotic expression vectors pGEX-6p-1 and pET-32a(+) to construct plasmids pGEX-6p-M and pET-32a-importin β1, respectively. The recombinant proteins were expressed by transforming the plasmids into BL21(DE3) under the condition of IPTG and then analyzed by SDS-PAGE. The obtained inclusion body weight histones were dealt with through denaturation and renaturation to recover activity. Then the interaction between GST-M (bait protein) and His-importin β1 (prey protein) was examined by GST pull-down assay. The results showed that the recombinant prokaryotic expression vectors pGEX-6p-M and pET-32a-importin β1 were successfully constructed, and the recombinant proteins were correctly expressed in BL21 (DE3). However, GST-M existed in the form of inclusion bodies, but His-importin β1 existed in the form of both solubility and inclusion bodies. The activated GST-M recombinant protein was recovered by protein refolding kit and could capture the His-importin β1 protein when used as bait protein, while GST alone could not. The in vitro interaction between NDV M protein and chicken importin β1 protein was verified by GST pull-down assay, which will provide foundation for further studying the role of importin β1 in the nuclear localization of NDV M protein and the replication and pathogenicity of NDV.

The purpose of this study was to verify the in vitro interaction between Newcastle disease virus (NDV) matrix (M) protein and chicken importin β1 protein. The products of NDV M gene and chicken importin β1 gene were obtained by PCR amplification, and then subcloned into the prokaryotic expression vectors pGEX-6p-1 and pET-32a(+) to construct plasmids pGEX-6p-M and pET-32a-importin β1, respectively. The recombinant proteins were expressed by transforming the plasmids into BL21(DE3) under the condition of IPTG and then analyzed by SDS-PAGE. The obtained inclusion body weight histones were dealt with through denaturation and renaturation to recover activity. Then the interaction between GST-M (bait protein) and His-importin β1 (prey protein) was examined by GST pull-down assay. The results showed that the recombinant prokaryotic expression vectors pGEX-6p-M and pET-32a-importin β1 were successfully constructed, and the recombinant proteins were correctly expressed in BL21 (DE3). However, GST-M existed in the form of inclusion bodies, but His-importin β1 existed in the form of both solubility and inclusion bodies. The activated GST-M recombinant protein was recovered by protein refolding kit and could capture the His-importin β1 protein when used as bait protein, while GST alone could not. The in vitro interaction between NDV M protein and chicken importin β1 protein was verified by GST pull-down assay, which will provide foundation for further studying the role of importin β1 in the nuclear localization of NDV M protein and the replication and pathogenicity of NDV.

The purpose of the present study was to establish an indirect enzyme linked immunosorbent assay (ELISA) detection method via obtaining prokaryotic expression products of elongation factor Tu (EF-Tu) referring Mycoplasma bovis (M. bovis) and analyze its subcellular localization in M. bovis, which could provide theoretical basis for studying the biological function of EF-Tu in M. bovis and lays the essential foundation for establishing effective serological diagnosis method and construction of subunit vaccine against M. bovis. In this study, specific primers were designed referred to gene sequence of EF-Tu from M. bovis PG45 reported in Uniprot. And Overlap PCR-amplified products of EF-Tu of M. bovis was inserted into the pMD19-T vector. With right sequences, prokaryotic expression vector of pET32-EF-Tu was constructed and converted into Escherichia coli (BL21) competent cells. Then, the expression of target protein was induced with 1 mmol·L^-1 of IPTG. After that, the recombinant protein of EF-Tu was purified via Ni-NTA affinity chromatography, with which New Zealand rabbits were immunized to prepare polyclonal antibodies, and the titer of antibody was measured by ELISA. Subsequently, subcellular localization of EF-Tu was analyzed using Western blot and ELISA. The indirect ELISA detection method was established by recombinant protein of EF-Tu. Results were as follows:Recombinant protein of EF-Tu showed an expected molecular mass of 66 kD and was expressed in soluble form. The antibody titer of rabbit serum was 1:6 400. The results of ELISA and Western blot showed that the recombinant protein of EF-Tu showed good immunogenicity, and elongation factor was detected in both cytoplasm and cell membrane of M. bovis with similar expression amounts. The indirect ELISA method for the detection of M. bovis established with EF-Tu showed good specificity and sensitivity. The recombinant protein of EF-Tu was successfully obtained, and elongation factor was expressed in both cytoplasm and cell membrane of M. bovis showing similar expression amounts. The indirect ELISA method showed great potential for serological diagnosis of M. bovis.

The purpose of the present study was to establish an indirect enzyme linked immunosorbent assay (ELISA) detection method via obtaining prokaryotic expression products of elongation factor Tu (EF-Tu) referring Mycoplasma bovis (M. bovis) and analyze its subcellular localization in M. bovis, which could provide theoretical basis for studying the biological function of EF-Tu in M. bovis and lays the essential foundation for establishing effective serological diagnosis method and construction of subunit vaccine against M. bovis. In this study, specific primers were designed referred to gene sequence of EF-Tu from M. bovis PG45 reported in Uniprot. And Overlap PCR-amplified products of EF-Tu of M. bovis was inserted into the pMD19-T vector. With right sequences, prokaryotic expression vector of pET32-EF-Tu was constructed and converted into Escherichia coli (BL21) competent cells. Then, the expression of target protein was induced with 1 mmol·L^-1 of IPTG. After that, the recombinant protein of EF-Tu was purified via Ni-NTA affinity chromatography, with which New Zealand rabbits were immunized to prepare polyclonal antibodies, and the titer of antibody was measured by ELISA. Subsequently, subcellular localization of EF-Tu was analyzed using Western blot and ELISA. The indirect ELISA detection method was established by recombinant protein of EF-Tu. Results were as follows:Recombinant protein of EF-Tu showed an expected molecular mass of 66 kD and was expressed in soluble form. The antibody titer of rabbit serum was 1:6 400. The results of ELISA and Western blot showed that the recombinant protein of EF-Tu showed good immunogenicity, and elongation factor was detected in both cytoplasm and cell membrane of M. bovis with similar expression amounts. The indirect ELISA method for the detection of M. bovis established with EF-Tu showed good specificity and sensitivity. The recombinant protein of EF-Tu was successfully obtained, and elongation factor was expressed in both cytoplasm and cell membrane of M. bovis showing similar expression amounts. The indirect ELISA method showed great potential for serological diagnosis of M. bovis.

The purpose of this study was to isolate and identify pathogenic bacteria from brain tissues and other organs of piglets suffering from meningitis. The species and serotype of the isolated strain were identified by PCR and serum agglutination test. The ultrastructure of the isolated strain was observed by transmission electron microscope, and the sequence type (ST) was determined by multilocus sequence typing tests (MLST). Minimal inhibitory concentration (MIC) was determined to analyze the antibiotic resistance of the isolated strain, while the virulence was detected by mice infection experiments. Results were as follows:a strain of Streptococcus suis was isolated from the brain tissue of piglets with meningitis which belongs to serotype 9 and named WH1609. The result of electron microscope showed that WH1609 had capsular structure. The result of MLST showed that WH1609 belonged to a new ST. WH1609 was a multi-drug resistant strain but sensitive to vancomycin, penicillin and ampicillin. The LD₅₀ of BALB/c mice infected with WH1609 was 1.89×10² CFU·mL^-1, and histopathological observation showed that the infected mice had obvious lesion in brain tissue. All the results suggest that a virulent strain of Streptococcus suis 9 isolated from the brain tissue of piglets suffering from meningitis exhibited a 10⁵ times higher LD₅₀ than that of other Streptococcus suis virulent strains, which is of great value to the study of the molecular mechanism meningitis caused by Streptococcus suis.

The purpose of this study was to isolate and identify pathogenic bacteria from brain tissues and other organs of piglets suffering from meningitis. The species and serotype of the isolated strain were identified by PCR and serum agglutination test. The ultrastructure of the isolated strain was observed by transmission electron microscope, and the sequence type (ST) was determined by multilocus sequence typing tests (MLST). Minimal inhibitory concentration (MIC) was determined to analyze the antibiotic resistance of the isolated strain, while the virulence was detected by mice infection experiments. Results were as follows:a strain of Streptococcus suis was isolated from the brain tissue of piglets with meningitis which belongs to serotype 9 and named WH1609. The result of electron microscope showed that WH1609 had capsular structure. The result of MLST showed that WH1609 belonged to a new ST. WH1609 was a multi-drug resistant strain but sensitive to vancomycin, penicillin and ampicillin. The LD₅₀ of BALB/c mice infected with WH1609 was 1.89×10² CFU·mL^-1, and histopathological observation showed that the infected mice had obvious lesion in brain tissue. All the results suggest that a virulent strain of Streptococcus suis 9 isolated from the brain tissue of piglets suffering from meningitis exhibited a 10⁵ times higher LD₅₀ than that of other Streptococcus suis virulent strains, which is of great value to the study of the molecular mechanism meningitis caused by Streptococcus suis.

This experiment was conducted to explore the characterization and the antibacterial activity of an antibacterial factor in Baylisascaris schroederi. The gene BsABF was cloned and expressed with prokaryotic expression system. Western blot and immunofluorescence histochemical staining were used to determine the immunological characteristics and the location in the parasite. The antibacterial activity was evaluated by the preliminary antibacterial experiments. Results were as follows:Open reading frame of BsABF gene (276 bp) encoded a 10.01 kDa protein with an N-terminal signal peptide while sharing 94% identity with Ascaris suum. The protein is mainly distributed in hypodermis, intestinal and eggs. The prokaryotic expression of rBsABF had a good immunoreactivity. Meanwhile the antibacterial assessment revealed that the recombinant protein has the antibacterial activity towards Enterococcus faecalis and Escherichia coli in giant pandas. In this study, we have first identified the BsABF gene, and rBsABF does have the antibacterial activity, and it lays the foundation for further study on the functional properties of BsABF in the future.

This experiment was conducted to explore the characterization and the antibacterial activity of an antibacterial factor in Baylisascaris schroederi. The gene BsABF was cloned and expressed with prokaryotic expression system. Western blot and immunofluorescence histochemical staining were used to determine the immunological characteristics and the location in the parasite. The antibacterial activity was evaluated by the preliminary antibacterial experiments. Results were as follows:Open reading frame of BsABF gene (276 bp) encoded a 10.01 kDa protein with an N-terminal signal peptide while sharing 94% identity with Ascaris suum. The protein is mainly distributed in hypodermis, intestinal and eggs. The prokaryotic expression of rBsABF had a good immunoreactivity. Meanwhile the antibacterial assessment revealed that the recombinant protein has the antibacterial activity towards Enterococcus faecalis and Escherichia coli in giant pandas. In this study, we have first identified the BsABF gene, and rBsABF does have the antibacterial activity, and it lays the foundation for further study on the functional properties of BsABF in the future.

The study was conducted to investigate whether estrogen can affect the regulation of primary sensory neurons, and to explore the role of Ca²⁺ in this mechanism. The distribution of estrogen receptor (ER) in primary culture dorsal root ganglion (DRG) from newborn rabbits was observed by Immunocytofluorescent (ICF), and the effects of 17β-E₂ (1, 10, 100, 1 000 nmol·L^-1) on cytosolic free Ca²⁺ fluorescence in DRG neurons cultured for 72 h were monitored by Laser Scanning Confocal Microscopy (LSCM). It was found that, in primary cultured DRG neurons, ERα, ERβ and GPR30 were 45.61%, 32.39%, 39.82% positive or strongly positive neurons respectively, and the neurons proportions of different sizes in different ER positive or strongly positive neurons were different. ERα and ERβ were strongly positive in DRG neuron nuclei, medium positive in cytoplasm, and weakly positive in a little neurite. GPR30 were weakly positive or negative in DRG neuron nuclei, medium positive or strongly positive in cytoplasm, and weakly positive in neurite. A certain degree of "heterogeneity" of three types of ERs (ERα, ERβ, and GPR30) was observed in the number, size type, degree of response and distribution in positive neurons. According to the variation of Ca²⁺ fluorescence intensity induced by 17β-E₂, DRG neurons were divided into 3 types, excitatory (19.28±0.70)%, inhibitory (3.54±0.02)% and insensitive neurons (77.17±1.48)%, respectively. Excitatory neurons were divided into strongly excitatory neurons (0.195±0.118) and weakly excitatory neurons (0.032±0.003) depending on the intensity of Ca²⁺ fluorescence changes. Interestingly, with the increase of 17β-E₂, the proportion of strongly excitatory neurons decreased, but no difference was detected in the proportion of total excitatory neurons at each group. It was indicated that with the increasing of 17β-E₂, the increasement of[Ca²⁺]_i was inhibited in strongly excitatory neurons, but not completely inhibited. These results suggest that estrogen may have distinct effects on various sensory neurons; Ca²⁺ pathway is one of the mechanisms in the effects of estrogen on the activity of some primary sensory neurons, and estrogen inhibits the effect of Ca²⁺ in DRG neurons in two ways:a direct inhibition of the effect of Ca²⁺ signaling pathway in several neurons, and a certain inhibitory impact on the effect of Ca²⁺ increment in strongly excitatory neurons with the increasing estrogen.

The study was conducted to investigate whether estrogen can affect the regulation of primary sensory neurons, and to explore the role of Ca²⁺ in this mechanism. The distribution of estrogen receptor (ER) in primary culture dorsal root ganglion (DRG) from newborn rabbits was observed by Immunocytofluorescent (ICF), and the effects of 17β-E₂ (1, 10, 100, 1 000 nmol·L^-1) on cytosolic free Ca²⁺ fluorescence in DRG neurons cultured for 72 h were monitored by Laser Scanning Confocal Microscopy (LSCM). It was found that, in primary cultured DRG neurons, ERα, ERβ and GPR30 were 45.61%, 32.39%, 39.82% positive or strongly positive neurons respectively, and the neurons proportions of different sizes in different ER positive or strongly positive neurons were different. ERα and ERβ were strongly positive in DRG neuron nuclei, medium positive in cytoplasm, and weakly positive in a little neurite. GPR30 were weakly positive or negative in DRG neuron nuclei, medium positive or strongly positive in cytoplasm, and weakly positive in neurite. A certain degree of "heterogeneity" of three types of ERs (ERα, ERβ, and GPR30) was observed in the number, size type, degree of response and distribution in positive neurons. According to the variation of Ca²⁺ fluorescence intensity induced by 17β-E₂, DRG neurons were divided into 3 types, excitatory (19.28±0.70)%, inhibitory (3.54±0.02)% and insensitive neurons (77.17±1.48)%, respectively. Excitatory neurons were divided into strongly excitatory neurons (0.195±0.118) and weakly excitatory neurons (0.032±0.003) depending on the intensity of Ca²⁺ fluorescence changes. Interestingly, with the increase of 17β-E₂, the proportion of strongly excitatory neurons decreased, but no difference was detected in the proportion of total excitatory neurons at each group. It was indicated that with the increasing of 17β-E₂, the increasement of[Ca²⁺]_i was inhibited in strongly excitatory neurons, but not completely inhibited. These results suggest that estrogen may have distinct effects on various sensory neurons; Ca²⁺ pathway is one of the mechanisms in the effects of estrogen on the activity of some primary sensory neurons, and estrogen inhibits the effect of Ca²⁺ in DRG neurons in two ways:a direct inhibition of the effect of Ca²⁺ signaling pathway in several neurons, and a certain inhibitory impact on the effect of Ca²⁺ increment in strongly excitatory neurons with the increasing estrogen.

The elucidation of the producing swainsonine biosynthesis pathway of Alternaria Section Undifilum oxytropis, an endophytic fungi isolated from locoweed, which can provide theoretical basis and technical support for the biological fermentation to obtain a large number of SW for anti-tumor applications and detoxification breeding of locoweed. Used different doses of nitrosoguanidine, reacted with spore suspension of Alternaria Section Undifilum oxytropis at 120 r·min^-1 for 5, 10, 15, 20, 25, and 30 min at 28℃, respectively, and selected good growth strains were inoculated with PDA medium. The culture was continued for 24 days, and the α-mannosidase inhibition method was used to detect SW content in the mycelia and the fermentation broth to evaluate the genetic stability of growth. The mycelium was pretreated with glass beads and liquid nitrogen disruption method. The genomic DNA extraction kit was used to extract the genomic DNA of the original strain and the mutant strain D4. The purified and endonuclease-strained DNA library was constructed and detected by fluorescent quantitative PCR. Evaluate library construction quality and use the Ion torrent PGM^TM sequencing platform to complete high-throughput whole-genome sequencing of mutant and original strains. The results showed that a mutant D4 was successfully obtained by chemical mutagenesis. The high-throughput sequencing technology was spliced and assembled using MIRA software, and 19 and 21 related genomic fragments of mutant D4 and original strain were obtained respectively. The experimental results were analyzed by BLAST and GO software, and annotation to the key synthesis genes of SW in Alternaria Section Undifilum oxytropis, such as SwnK, SwnH2, SwnH1, SwnR putative amino acid transporter, etc., and the key synthesis enzymes, such as pyrroline-5-carboxylate reductase, formate/glycerate dehydrogenase catalytic, saccharopine reductase-like protein, combined with KEGG database, the possible biosynthetic pathway of SW is predicted in Alternaria Section Undifilum oxytropis. The above results will contribute to clarify the SW biosynthesis mechanism of Alternaria Section Undifilum oxytropis.

The elucidation of the producing swainsonine biosynthesis pathway of Alternaria Section Undifilum oxytropis, an endophytic fungi isolated from locoweed, which can provide theoretical basis and technical support for the biological fermentation to obtain a large number of SW for anti-tumor applications and detoxification breeding of locoweed. Used different doses of nitrosoguanidine, reacted with spore suspension of Alternaria Section Undifilum oxytropis at 120 r·min^-1 for 5, 10, 15, 20, 25, and 30 min at 28℃, respectively, and selected good growth strains were inoculated with PDA medium. The culture was continued for 24 days, and the α-mannosidase inhibition method was used to detect SW content in the mycelia and the fermentation broth to evaluate the genetic stability of growth. The mycelium was pretreated with glass beads and liquid nitrogen disruption method. The genomic DNA extraction kit was used to extract the genomic DNA of the original strain and the mutant strain D4. The purified and endonuclease-strained DNA library was constructed and detected by fluorescent quantitative PCR. Evaluate library construction quality and use the Ion torrent PGM^TM sequencing platform to complete high-throughput whole-genome sequencing of mutant and original strains. The results showed that a mutant D4 was successfully obtained by chemical mutagenesis. The high-throughput sequencing technology was spliced and assembled using MIRA software, and 19 and 21 related genomic fragments of mutant D4 and original strain were obtained respectively. The experimental results were analyzed by BLAST and GO software, and annotation to the key synthesis genes of SW in Alternaria Section Undifilum oxytropis, such as SwnK, SwnH2, SwnH1, SwnR putative amino acid transporter, etc., and the key synthesis enzymes, such as pyrroline-5-carboxylate reductase, formate/glycerate dehydrogenase catalytic, saccharopine reductase-like protein, combined with KEGG database, the possible biosynthetic pathway of SW is predicted in Alternaria Section Undifilum oxytropis. The above results will contribute to clarify the SW biosynthesis mechanism of Alternaria Section Undifilum oxytropis.

To investigate the role of GSTO1 in the fluoride-induced autophagy and apoptosis in the osteoblasts, RNA interference (RNAi) technology was used to inhibit the expression of GSTO1 gene in mouse osteoblasts, and the expressions of autophagy and apoptosis related genes were detected. Three pairs of 21 nucleotide sequence siRNA (siRNA1, siRNA2, siRNA3) were designed and synthesized according to the mouse GSTO1 mRNA sequence, and the best effective fragment was screened. Then, the mRNA expression levels of GSTO1 under different concentrations of sodium fluoride (NaF) (10⁴, 10³, 10², 10, and 1 mg·L^-1) were evaluated to determine the optimal fluoride concentration. This experiment included control group, negative control group (NC-siRNA), NaF group (1 mg·L^-1), silencing target gene group (siRNA-GSTO1) and NaF+ silencing target gene group (NaF/siRNA-GSTO1). RT-PCR was used to detect the transcription of autophagy-related genes (LC3, p62, Beclin1, Atg3, Atg5) and apoptosis-related genes (BCL2, Caspase-3) in osteoblasts. The protein expression of LC3, p62, Beclin1, Atg3 and Atg5 were detected by Western blot. Results were as follows:Osteoblasts were identified by HE, Giemsa, alkaline phosphatase, and alizarin red staining. In the experimental model, 50 nmol ·L^-1 was found to be the optimal transfection concentration of siRNA, and siRNA2 was the best silent transfection fragment, at 1 mg·L^-1 NaF optimal concentration. The NaF treatment group and siRNA-GSTO1 group inhibited the transcription of LC3, Beclin1, Atg5 and BCL2, and decreased the transcription of p62 and Caspase-3. However, compared with the NaF group, the transcription of LC3, Beclin1 and Atg5 mRNA were significantly decreased (P<0.05), while the transcription of p62 and Caspase-3 were significantly increased in the NaF/siRNA-GSTO1 group (P<0.05). The expression of autophagy-related proteins LC3, Beclin1, Atg5 and p62 in osteoblasts were completely consistent with the gene transcription. In summary, both low-dose fluoride and silencing of GSTO1 expression increased autophagy and decreased apoptosis in the osteoblasts. GSTO1 had synergistic effect on low-dose fluoride-induced autophagy enhancement and apoptosis inhibition in the osteoblasts.

To investigate the role of GSTO1 in the fluoride-induced autophagy and apoptosis in the osteoblasts, RNA interference (RNAi) technology was used to inhibit the expression of GSTO1 gene in mouse osteoblasts, and the expressions of autophagy and apoptosis related genes were detected. Three pairs of 21 nucleotide sequence siRNA (siRNA1, siRNA2, siRNA3) were designed and synthesized according to the mouse GSTO1 mRNA sequence, and the best effective fragment was screened. Then, the mRNA expression levels of GSTO1 under different concentrations of sodium fluoride (NaF) (10⁴, 10³, 10², 10, and 1 mg·L^-1) were evaluated to determine the optimal fluoride concentration. This experiment included control group, negative control group (NC-siRNA), NaF group (1 mg·L^-1), silencing target gene group (siRNA-GSTO1) and NaF+ silencing target gene group (NaF/siRNA-GSTO1). RT-PCR was used to detect the transcription of autophagy-related genes (LC3, p62, Beclin1, Atg3, Atg5) and apoptosis-related genes (BCL2, Caspase-3) in osteoblasts. The protein expression of LC3, p62, Beclin1, Atg3 and Atg5 were detected by Western blot. Results were as follows:Osteoblasts were identified by HE, Giemsa, alkaline phosphatase, and alizarin red staining. In the experimental model, 50 nmol ·L^-1 was found to be the optimal transfection concentration of siRNA, and siRNA2 was the best silent transfection fragment, at 1 mg·L^-1 NaF optimal concentration. The NaF treatment group and siRNA-GSTO1 group inhibited the transcription of LC3, Beclin1, Atg5 and BCL2, and decreased the transcription of p62 and Caspase-3. However, compared with the NaF group, the transcription of LC3, Beclin1 and Atg5 mRNA were significantly decreased (P<0.05), while the transcription of p62 and Caspase-3 were significantly increased in the NaF/siRNA-GSTO1 group (P<0.05). The expression of autophagy-related proteins LC3, Beclin1, Atg5 and p62 in osteoblasts were completely consistent with the gene transcription. In summary, both low-dose fluoride and silencing of GSTO1 expression increased autophagy and decreased apoptosis in the osteoblasts. GSTO1 had synergistic effect on low-dose fluoride-induced autophagy enhancement and apoptosis inhibition in the osteoblasts.

This study was conducted to investigate the effect of Arula-7 powder on mechanical barrier, immune barrier and chemical barrier in rats infected with Escherichia coli O₁. A total of 60 SD rats, 6-8 weeks old, were equally divided into the control group, untreated group and ciprofloxacin group, Arula-7 powder high (1.08 g·kg^-1), medium (0.54 g·kg^-1) and low (0.27 g·kg^-1) dosage groups (n=10). The rate of protection was calculated. We collected the small intestine and the intestinal morphology indexes were measured by HE staining. Meanwhile, the serum and ileal tissue were also collected and the levels of D-lactic acid, DAO, sIgA and intestinal trefoil factor were detected using ELISA kit. The results showed that:(1) Arula-7 powder with medium dose had the highest protection rate of 50%. Furthermore, the medium dose of Arula-7 powder had positive effects on the improvement or recovery of the morphology of intestinal mucosa in rats infected with E. coli O₁ and its effect was similar with that of ciprofloxacin. (2) There was no significant difference between the control group and the ciprofloxacin group in the level of D-lactic acid (P>0.05). The level of DAO had no significant difference between the control group and the Arula-7 powder medium/high dosage groups (P>0.05). (3)Except for Arula-7 powder low dosage group,the significant level of sIgA was observed in ileal mucosa of other four groups compared with the untreated group (P<0.05). The level of ITF in ileal mucosa of untreated group was significantly lower than other medicine treatment groups (P<0.05). In conclusion, Arula-7 powder has the best effect for repairing the intestinal mucosa injury in rats infected with E. coli O₁ at medium dose (0.54 g·kg^-1), and its effect is similar as that of the antibiotic ciprofloxacin.

This study was conducted to investigate the effect of Arula-7 powder on mechanical barrier, immune barrier and chemical barrier in rats infected with Escherichia coli O₁. A total of 60 SD rats, 6-8 weeks old, were equally divided into the control group, untreated group and ciprofloxacin group, Arula-7 powder high (1.08 g·kg^-1), medium (0.54 g·kg^-1) and low (0.27 g·kg^-1) dosage groups (n=10). The rate of protection was calculated. We collected the small intestine and the intestinal morphology indexes were measured by HE staining. Meanwhile, the serum and ileal tissue were also collected and the levels of D-lactic acid, DAO, sIgA and intestinal trefoil factor were detected using ELISA kit. The results showed that:(1) Arula-7 powder with medium dose had the highest protection rate of 50%. Furthermore, the medium dose of Arula-7 powder had positive effects on the improvement or recovery of the morphology of intestinal mucosa in rats infected with E. coli O₁ and its effect was similar with that of ciprofloxacin. (2) There was no significant difference between the control group and the ciprofloxacin group in the level of D-lactic acid (P>0.05). The level of DAO had no significant difference between the control group and the Arula-7 powder medium/high dosage groups (P>0.05). (3)Except for Arula-7 powder low dosage group,the significant level of sIgA was observed in ileal mucosa of other four groups compared with the untreated group (P<0.05). The level of ITF in ileal mucosa of untreated group was significantly lower than other medicine treatment groups (P<0.05). In conclusion, Arula-7 powder has the best effect for repairing the intestinal mucosa injury in rats infected with E. coli O₁ at medium dose (0.54 g·kg^-1), and its effect is similar as that of the antibiotic ciprofloxacin.

This experiment was conducted to study the effects of dietary supplementation of palmitic acid on lactation performance, blood biochemical indexes and hormone concentration in early lactation Holstein dairy cows. Thirty healthy Chinese Holstein dairy cows with similar body weight ((598.49±30.98) kg), parity (2-4 fetuses), lactation stage(15-37 days of calving) and lactation production ((20.76±3.32) kg) were randomly divided into 3 groups, each group with 10 cows and one cow in each replicate. Cows in the control group were fed the basal diet, and cows in the experimental groups were supplemented with 2.0% and 4.0% palmitic acid in the basal diet, respectively. The trial lasted for 60 days. Milk samples and blood samples were taken at the beginning and end of the test to determine the milk compositions and the blood biochemical indexes and hormone concentrations. The results showed that:1) The daily dry matter intake, initial milk yield, feed efficiency, initial milk fat rate, milk protein rate, milk non-fat solid (NFS) rate, and the the contents of initial lactose, free fatty acids, glucose, acetoacetic acid, β-hydroxybutyrate, insulin-like growth factor 1 (IGF-1), insulin, glucagon and leptin were not significantly different among the 3 treatment groups (P>0.05); 2) Compared with the control group, 2.0% and 4.0% palmitic acid groups significantly increased milk yield, standard milk (FCM) yield and the post-treatment milk fat rate, glucose, insulin and IGF-1 concentrations (P<0.05), and significantly decreased the post-treatment acetoacetic acid, β-hydroxybutyrate, free fatty acid, glucagon and leptin concentrations (P<0.05); 3) There was no significant difference in milk yield, FCM yield, milk protein rate, milk NFS rate and lactose, glucose, acetoacetic acid, β-hydroxybutyrate, free fatty acid, IGF-1, insulin, glucagon and leptin concentrations between the 2.0% and 4.0% palmitic acid groups (P>0.05). These results indicated that dietary supplementation of 2.0% or 4.0% palmitic acid could reduce the ketone body production, improve milk production and quality by regulating hormone secretion in early lactation dairy cows. Under the conditions of this test, the best dose for the effect is to supplement the diet with 2.0% palmitic acid.

This experiment was conducted to study the effects of dietary supplementation of palmitic acid on lactation performance, blood biochemical indexes and hormone concentration in early lactation Holstein dairy cows. Thirty healthy Chinese Holstein dairy cows with similar body weight ((598.49±30.98) kg), parity (2-4 fetuses), lactation stage(15-37 days of calving) and lactation production ((20.76±3.32) kg) were randomly divided into 3 groups, each group with 10 cows and one cow in each replicate. Cows in the control group were fed the basal diet, and cows in the experimental groups were supplemented with 2.0% and 4.0% palmitic acid in the basal diet, respectively. The trial lasted for 60 days. Milk samples and blood samples were taken at the beginning and end of the test to determine the milk compositions and the blood biochemical indexes and hormone concentrations. The results showed that:1) The daily dry matter intake, initial milk yield, feed efficiency, initial milk fat rate, milk protein rate, milk non-fat solid (NFS) rate, and the the contents of initial lactose, free fatty acids, glucose, acetoacetic acid, β-hydroxybutyrate, insulin-like growth factor 1 (IGF-1), insulin, glucagon and leptin were not significantly different among the 3 treatment groups (P>0.05); 2) Compared with the control group, 2.0% and 4.0% palmitic acid groups significantly increased milk yield, standard milk (FCM) yield and the post-treatment milk fat rate, glucose, insulin and IGF-1 concentrations (P<0.05), and significantly decreased the post-treatment acetoacetic acid, β-hydroxybutyrate, free fatty acid, glucagon and leptin concentrations (P<0.05); 3) There was no significant difference in milk yield, FCM yield, milk protein rate, milk NFS rate and lactose, glucose, acetoacetic acid, β-hydroxybutyrate, free fatty acid, IGF-1, insulin, glucagon and leptin concentrations between the 2.0% and 4.0% palmitic acid groups (P>0.05). These results indicated that dietary supplementation of 2.0% or 4.0% palmitic acid could reduce the ketone body production, improve milk production and quality by regulating hormone secretion in early lactation dairy cows. Under the conditions of this test, the best dose for the effect is to supplement the diet with 2.0% palmitic acid.

In order to reveal the distribution and expression of estrogen receptor α (ERα) and estrogen receptor β (ERβ) in mammary gland of yak during mid-lactating and non-lactating periods. The mammary tissues from each of 5 healthy yaks in the mid-lactating (3-4 months after parturition) and non-lactating periods (1 month after weaning) were collected, the localization, distribution and protein content of ERα and ERβ were tested by using the SP immunohistochemical staining method and the Western blot method, respectively. The results showed that predominant cytoplasmic staining for ERα were found in gland bubble epithelial cells and weak nuclear staining for ERα were found in fat cells in interlobular connective tissue and vascular endothelial cells in mammary gland during mid-lactating period. But during non-lactating period, distribution of ERα-positive material was contrary to that in mid-lactating period. Predominant cytoplasmic staining for ERβ were found in the gland bubble epithelial cells during mid-lactating period and weak nucleus staining for ERβ were found in vascular endothelial cells and fat cells. During non-lactating period, ERβ-positive material were found in cytoplasm of duct epithelial cells, in the nucleus of stroma cells in interlobular connective tissue and vascular endothelial cells, and in cytoplasm of acinar epithelial cells, but the number of positive cells was less than that during mid-lactating period. The relative expression of ERα protein in yak mammary gland tissue in non-lactating period was significantly higher than that in mid-lactating period(P<0.05), while there was no significant difference in the relative expression of ERβ protein between mid-lactating and non-lactating periods (P>0.05). The different expression patterns of estrogen receptor subtypes in mammary gland of yak in different periods suggested that ERα and ERβ in mammary gland of yak might perform different functions during the process of mammary development and lactation.

In order to reveal the distribution and expression of estrogen receptor α (ERα) and estrogen receptor β (ERβ) in mammary gland of yak during mid-lactating and non-lactating periods. The mammary tissues from each of 5 healthy yaks in the mid-lactating (3-4 months after parturition) and non-lactating periods (1 month after weaning) were collected, the localization, distribution and protein content of ERα and ERβ were tested by using the SP immunohistochemical staining method and the Western blot method, respectively. The results showed that predominant cytoplasmic staining for ERα were found in gland bubble epithelial cells and weak nuclear staining for ERα were found in fat cells in interlobular connective tissue and vascular endothelial cells in mammary gland during mid-lactating period. But during non-lactating period, distribution of ERα-positive material was contrary to that in mid-lactating period. Predominant cytoplasmic staining for ERβ were found in the gland bubble epithelial cells during mid-lactating period and weak nucleus staining for ERβ were found in vascular endothelial cells and fat cells. During non-lactating period, ERβ-positive material were found in cytoplasm of duct epithelial cells, in the nucleus of stroma cells in interlobular connective tissue and vascular endothelial cells, and in cytoplasm of acinar epithelial cells, but the number of positive cells was less than that during mid-lactating period. The relative expression of ERα protein in yak mammary gland tissue in non-lactating period was significantly higher than that in mid-lactating period(P<0.05), while there was no significant difference in the relative expression of ERβ protein between mid-lactating and non-lactating periods (P>0.05). The different expression patterns of estrogen receptor subtypes in mammary gland of yak in different periods suggested that ERα and ERβ in mammary gland of yak might perform different functions during the process of mammary development and lactation.

By the Gateway technology and the lentiviral expression system, goat signaling lymphocyte activation molecule (SLAM,also known as CD150) was stably integrated into the genome chromosome of Vero cell,to establish a Vero/g-SLAM positive cell clones, with which the ability of replication of peste des petits ruminants virus (PPRV) can be improved. On the other hand, to verify the activity, genetic stability and proliferation of PPRV expressing SLAM of Vero/g-SLAM. Peripheral blood lymphocytes were isolated from goat, of which genomic total RNA was extracted, SLAM gene with complete ORF were obtained by one step RT-PCR. Using of the BP and LR loci gene recombination technology to construct the entry clone vector pDONR/SLAM and expression skeleton of pDEST/SLAM, with packaging plasmids pLP1, pLP2 and VSV-G to co-transfect 293-FT cells to produce a lentiviral stock, use the lentiviral stock to infect Vero cell. The Vero cells with SLAM were named Vero/SLAM, and were resistant to the blasticidin (3.5 μg·mL^-1). By the target gene amplification, Confocal laser scanning microscope, indirect immunofluorescence, Western blot and cytopathic effect, the SLAM genome integration, transcription, expression and reaction activity of SLAM protein, and PPRV proliferation effect in Vero/SLAM were verified respectively. Results were as follows:SLAM was stably integrated in the genome of Vero cells; The receptor protein was expressed in the periplasmic part of the cell; After continuous subpassages of the cells, SLAM are not lost; Vero/SLAM were inoculated virus The cytopathic time was shortened from 4-7 d to 3.5 d, and TCID₅₀·0.1 mL^-1 increased from 4.25 to 5.67. Compared with the normal Vero cells, Vero cells that integrate SLAM have significantly enhanced their susceptibility to PPRV because of expression of PPRV specific cell receptors. In this study, a new cell line, Vero/SLAM cells was established successfully. The SLAM expressed by this cell has the obvious function of enhancing PPRV replication, the critical target genes that enhance PPRV replication can be elucidated at the cellular model level. It also provides information for the study of host cell changes caused by PPRV infection and the pathogenesis of the organism.

By the Gateway technology and the lentiviral expression system, goat signaling lymphocyte activation molecule (SLAM,also known as CD150) was stably integrated into the genome chromosome of Vero cell,to establish a Vero/g-SLAM positive cell clones, with which the ability of replication of peste des petits ruminants virus (PPRV) can be improved. On the other hand, to verify the activity, genetic stability and proliferation of PPRV expressing SLAM of Vero/g-SLAM. Peripheral blood lymphocytes were isolated from goat, of which genomic total RNA was extracted, SLAM gene with complete ORF were obtained by one step RT-PCR. Using of the BP and LR loci gene recombination technology to construct the entry clone vector pDONR/SLAM and expression skeleton of pDEST/SLAM, with packaging plasmids pLP1, pLP2 and VSV-G to co-transfect 293-FT cells to produce a lentiviral stock, use the lentiviral stock to infect Vero cell. The Vero cells with SLAM were named Vero/SLAM, and were resistant to the blasticidin (3.5 μg·mL^-1). By the target gene amplification, Confocal laser scanning microscope, indirect immunofluorescence, Western blot and cytopathic effect, the SLAM genome integration, transcription, expression and reaction activity of SLAM protein, and PPRV proliferation effect in Vero/SLAM were verified respectively. Results were as follows:SLAM was stably integrated in the genome of Vero cells; The receptor protein was expressed in the periplasmic part of the cell; After continuous subpassages of the cells, SLAM are not lost; Vero/SLAM were inoculated virus The cytopathic time was shortened from 4-7 d to 3.5 d, and TCID₅₀·0.1 mL^-1 increased from 4.25 to 5.67. Compared with the normal Vero cells, Vero cells that integrate SLAM have significantly enhanced their susceptibility to PPRV because of expression of PPRV specific cell receptors. In this study, a new cell line, Vero/SLAM cells was established successfully. The SLAM expressed by this cell has the obvious function of enhancing PPRV replication, the critical target genes that enhance PPRV replication can be elucidated at the cellular model level. It also provides information for the study of host cell changes caused by PPRV infection and the pathogenesis of the organism.

To investigate the formation reasons of caseous nodules in the liver of a yak, bacteria were isolated and cultured from the liver, and the 16S rRNA and rpoB gene sequences of isolated bacteria were amplified and sequenced. The biochemical phenotypes were identified by Vitek 2 automatic microbiology analyzer and drug susceptibility test was performed by K-B method. Results were as follows:A Gram-positive, rod-shaped and suspected pathogen was isolated. The amplification lengths of 16S rRNA and rpoB were 1 483 and 434 bp, while the accession number were MH000696 and MH006608, respectively. The similarity of 16S rRNA of clinical isolate to Corynebacterium xerosis was as high as 99% and phylogenetic tree showed it lied in the same branch with Corynebacterim xerosis. The results of Vitek 2 fully automatic Microbial Identification System showed that it was identified as Kocuria varians, with the probability of 93%. The bacteria was highly sensitive to tobramycin, aztreonam, cefoperazone, minocycline and other medicines. In summary, the isolated bacteria is Corynebacterium xerosis. But the gene sequence, such as rpoB, and biochemical property have a certain degree of mutations. This study provides reference for the isolation and identification in the veterinary clinic, and is the foundation of pathogenesis research of this bacteria.

To investigate the formation reasons of caseous nodules in the liver of a yak, bacteria were isolated and cultured from the liver, and the 16S rRNA and rpoB gene sequences of isolated bacteria were amplified and sequenced. The biochemical phenotypes were identified by Vitek 2 automatic microbiology analyzer and drug susceptibility test was performed by K-B method. Results were as follows:A Gram-positive, rod-shaped and suspected pathogen was isolated. The amplification lengths of 16S rRNA and rpoB were 1 483 and 434 bp, while the accession number were MH000696 and MH006608, respectively. The similarity of 16S rRNA of clinical isolate to Corynebacterium xerosis was as high as 99% and phylogenetic tree showed it lied in the same branch with Corynebacterim xerosis. The results of Vitek 2 fully automatic Microbial Identification System showed that it was identified as Kocuria varians, with the probability of 93%. The bacteria was highly sensitive to tobramycin, aztreonam, cefoperazone, minocycline and other medicines. In summary, the isolated bacteria is Corynebacterium xerosis. But the gene sequence, such as rpoB, and biochemical property have a certain degree of mutations. This study provides reference for the isolation and identification in the veterinary clinic, and is the foundation of pathogenesis research of this bacteria.