Special Issue

Animal husbandry
This special topic selects papers related to animal husbandry published in Acta Agriculurae Boreali-Sinica , involving genetics and breeding, biotechnology, physiology and biochemistry, livestock and poultry diseases of cattle, sheep, pigs, chickens, etc.Click on the relevant paper to open the web page and download the full text. In order to quote and share for readers, each article contains a complete citation format in Chinese and English (including international DOI number) and a proprietary  QR code. Long press the  QR code of the article to open the web page of the article and realize mobile sharing at the same time. Thank you for downloading, quoting, forwarding and sharing.
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  • YANG Yawen, GAO Hexuan, LIU Lili, BAO Yingying, HE Yuqin, YANG Zhijie, CHEN Weigang, GE Wenbo
    Abstract (41) PDF (26) RichHTML (3)

    The Pineal-hypothalamus-pituitary-thyroid axis(PHPTA)could significantly regulate reproductive activities of animals.In order to investigate the expression of Kiss1/GPR54 system on PHPT axis and its regulation on reproductive activities of Tibetan sheep,24 healthy and non-pregnant Tibetan sheep in estrus cycle were selected as experimental group and 6 non-breeding season Tibetan sheep as control group.The dynamic changes of plasma Kisspeptin were detected by ELISA,and the mRNA and protein expression of Kiss1 and GPR54 in optic nerve,pineal body,hypothalamus,pituitary body and thyroid gland were detected by Real-time quantitative PCR,Western Blot and immunohistochemistry.The results showed that the plasma Kisspeptin secretion in late estrus was significantly higher than that in other periods,and that in each period of breeding season was significantly higher than that in non-breeding season.The mRNA and protein of Kiss1 and GPR54 were expressed in optic nerve and PHPT axis.The relative expression of mRNA and protein of Kiss1 and GPR54 in optic nerve were significantly higher in early estrus than in other periods(P<0.05).The mRNA and its protein of Kiss1 of the pineal body reached its maximum value in estrus,significantly higher than that in reproductive cycle.The mRNA relative expression of Kiss1 and GPR54 of hypothalamus and thyroid gland were significantly increased in late estrus(P<0.05).The mRNA relative expression levels of the two in pituitary body were significantly higher in estrus than in other periods.Immunohistochemical results showed that Kisspeptin and GPR54 were mainly distributed in the glial nucleus and cytoplasm,respectively.Both were strongly positive in pineal cell cytoplasm.Kisspeptin was expressed in neuroendocrine small cells and glial cells in hypothalamus,GPR54 was expressed in cytoplasm of neuroendocrine small cells.Kisspeptin and GPR54 were mainly expressed in the cytoplasm of basophils in pituitary body.In thyroid gland,they were mainly expressed in the cytoplasm of follicular cells.The dynamic changes of plasma Kisspeptin and the differential expressions of Kiss1 and GPR54 in different tissues during estrus cycle of Ganjia Tibetan sheep indicated that Kiss1/GPR54 system was involved in regulating reproductive physiological activities of Ganjia Tibetan sheep.

  • SHI Yumei, CHEN Shaokang, XING Kai, ZHAO Yanhui, YUAN Jiani, SHENG Xihui, QI Xiaolong, NI Hemin, GUO Yong, WANG Chuduan
    Abstract (44) PDF (42) RichHTML (4)

    This study aims to use high-throughput sequencing technology to perform mRNA sequencing and differential analysis of longissimus dorsi muscle tissue samples from Songliao black pigs and Landrace pigs,and screen out key genes that affect pig muscle growth,meat quality and fat deposition,so as to provide pork quality research provides new reference information.The longissimus dorsi muscle tissue samples of 6 Songliao black pigs and 6 Landrace pigs were collected,their RNA was extracted,and the mRNA was sequenced by Illumina HiSeq 2500 high-throughput sequencing technology,and the obtained reads were compared,annotated and differentiated.For expression analysis,NOISeq was used to screen out differentially expressed genes and perform enrichment analysis of related biological functions.The results showed that 664 differentially expressed genes were screened from the two pig species,of which 364 genes were highly expressed in Songliao black pigs and 300 genes were highly expressed in Landrace pigs.Through the biological function analysis of differentially expressed genes,LPIN1,FADS1,FADS2,PLIN2,PPARGC1A,PRKAG2 and ACSL1 were screened to participate in the regulation of lipid metabolism and muscle development.The related pathways were fatty acid metabolism,PPAR signaling pathway,AMPK signaling pathway,insulin signaling pathway and adipocytokine signaling pathway and so on.

  • LIANG Peng, ZHANG Wen, FENG Dengzhen, QIANG Hao, RONG Xuan, MENG Ke
    Abstract (68) PDF (66) RichHTML (22)

    The aim of this study was to analyze the variation in meat quality and expression of intramuscular genes between Tan sheep,Dorper sheep and Small-tailed han sheep,and to preliminarily reveal the molecular mechanisms underlying the differences in their meat quality traits and also provide a theoretical basis for the selection and improvement of other sheep breeds. The meat quality of 8-month-old Dorper sheep,Tan sheep and Small-tailed han sheep was analyzed and compared,and the Illumina HiSeqTM 4000 platform was used to sequence and analyze the transcriptome of the longest dorsal muscle tissue to explore the regulatory genes associated with the differences in their meat quality traits. The results showed that a total of 820 differentially expressed genes were screened,including 99 between Dorper sheep and Tan sheep,436 between Dorper sheep and Small-tailed han sheep and 552 between Tan sheep and Small-tailed sheep. The results of GO functional enrichment analysis of the differential genes showed that each comparison group was significantly enriched in 224,517 and 657 GO entries;and the KEGG pathway enrichment analysis showed that DEGs were enriched in cAMP,MAPK,AMPK,purine metabolism,glycerophospholipid metabolism,amino acid and fatty acid metabolism and glycolysis/gluconeogenesis signalling pathways,further screening for candidate genes related to meat quality regulation and flavor substance metabolism such as FOS,PLA2G4E,LPIN1,AMPD1,AMPD3, NT5C1A,GPI,PFKM and PKM. Real-time fluorescence quantitative PCR validation of several randomly selected differential genes showed consistent expression trends with the transcriptome sequencing results,indicating the reliability of the sequencing results. These differential genes obtained in this study can be used as basic information for the breeding of new breeds(lines)of Ningxia high-quality meat sheep.

  • FU Yanfeng, ZHAO Weimin, LI Hui, LI Bixia, CHENG Jinhua, XU Xiaobo, REN Shouwen
    Abstract (52) PDF (13) RichHTML (23)

    The objective of the study was to screen the candidate genes and SNP markers for the total number born(TNB)and number born alive(NBA)in pigs,so as to prepare for the next step of improving the reproduction traits,and breeding new high-proliferation of Canadian line of large white pigs. Illumina porcine 50K chip was used to do a genome-wide SNP scan on 186 Canadian line of large white sows in one pig farm,followed by quality control,beagle fill and SNP genotyping. Combined with the phenotypic traits and population structure analysis results of these experimental pigs,genome-wide association study(GWAS)was carried out. The results of population structure analysis showed that there was no population stratification in the pig samples used in the experiment. A total of 36 867 effective SNP markers were obtained on 18 pairs(36)of autosomes for GWAS analysis of experimental pigs. The results of GWAS showed that NBA in all parities of these Canadian large white pigs was significantly associated with two SNPs:seq-rs323899658 and seq-rs329781338,respectively. The SNP of seq-rs329781338 was annotated with three genes:SSBP1,WEE2 and KIAA1147,respectively. NBA was significantly associated with 7 SNPs,namely:seq-rs81238474,seq-rs321377412,seq-rs340736313,seq-rs80782154,seq-rs81244816,seq-rs81467772 and seq-rs329781338. Five out of seven SNPs had gene annotation,and a total of 22 genes were annotated:EphA1, TAS2R60, FAM131B, CLCN1, CASP2, TMEM139, TRBV21OR9-2, PRSS2, TRBV19, TRBV24-1, U6, SSBP1, WEE2, KIAA1147, NKX2-8, ALKBH3, HSD17B12, U6, HOMER2, WHAMM, FSD2 and SCARNA15. The results of GO function and KEGG pathway analysis showed that the most significant biological process of GO function of these genes was the regulation of multi biological process,the molecular function was non transmembrane/transmembrane protein tyrosine kinase activity,and the KEGG pathway was mismatch repair.Combined with GWAS and gene function annotation results,WEE2 and EphA1 genes could be used as key candidate genes to increase the TNB and NBA of Canadian large white pigs,and provide some reference for the precise cultivation of high-yield pigs in the future.

  • AN Qingming, WANG Xing, WU Zhenyang, MENG Jinzhu, ZHAO Yuanyuan, SONG Xingchao
    Abstract (42) PDF (14) RichHTML (15)

    Carcass muscle growth and development is an important evaluation index that affects the breeding efficiency of goats.The aim of this study was to analyze key candidate genes for muscle growth and development of Guizhou white goats with different genders by RNA-Seq,and provide new reference information for the research on the muscle growth and development of Guizhou white goat.We collected the longissimus dorsi muscle to extract RNA and determined the slaughter performance of 6 Guizhou white goats with different genders,which were two years old and were feeded in same level.Then screened differentially expressed genes,analyzed the signal pathway of related genes.Meanwhile,RT-qPCR was used to verify the screened differentially expressed genes.The results showed that the raw reads which obtained by sequencing were filtered,a total of 78.99 Gb Clean Data were obtained in six samples.Each single sample was obtained Clean reads between 83 030 104 and 95 739 024,and the comparison efficiency with the reference genome was between 93.75%-94.79%,a total of 25 089 transcripts were obtained,of which 1 077 were significant differentially expressed genes,and 194 were new transcripts.Among them,563 were up-regulated and 514 were down-regulated in longissimus doris tissue of male sheep.GO functional enrichment analysis was performed on 1 077 differentially expressed mRNAs,which 587 were significantly enriched(Q-value≥0.05) and concentrated in 35 groups of three major categories.KEGG signaling pathway analysis revealed that annotated differential genes participated in 243 signaling pathways,among which the PI3K/AKT signaling pathway were the most enriched,including 32 genes,among them,17 genes were significantly up-regulated and 1 gene was down-regulated,meanwhile,the coexpression score of COL4A1 and COL4A2 genes were the highest in this pathway,the ITGAV protein interaction with other proteins was the most abundant.6 genes that may be closely related to muscle growth and development in white goat were screened out,among which FHL3,WFIKKN2 and SOX6 genes were up-regulated in male longissimus doris tissue,QSOX2,MYH2 and LAP genes were down-regulated.RT-qPCR showed that the expression trend of the 6 candidate genes were consistent with high-throughput sequencing results,and the expression levels differences of these genes were significant,which showed the sequencing results were reliable.Totally,1 077 differentially expressed mRNAs,which 194 were new transcripts were obtained of longissimus doris tissue in Guizhou white goat with different genders by RNA-Seq technology,the screened differentially expressed genes and PI3K/AKT signaling pathway were related to the growth and development of longissimus dorsi muscle of Guizhou white goats.

  • RAN Li, LÜ Jinshi, ZHANG Hao, WANG Yong, ZHU Jiangjiang, LI Yanyan, MENG Qingyong, LIN Yaqiu
    Abstract (23) PDF (17) RichHTML (13)

    In order to clarify the role of APOC3 gene in the differentiation of intramuscular adipocytes of goats,the APOC3 gene sequence was cloned by RT-PCR,and the biological information was analyzed by online software.Quantitative Real-time PCR(qPCR)was used to detect the expression of APOC3 gene in intramuscular adipocytes of goats at different tissues and differentiation stages.APOC3 overexpression vector was constructed by double-enzyme digestion method,and the effects of APOC3 gene overexpression on lipid droplet accumulation were determined by oil red O staining.At the same time,the mRNA relative expression level of marker gene of adipogenic differentiation was detected by qPCR.The results showed that the ORF region of APOC3 was 294 bp in length and encoded 97 amino acids with a functional domain of 23-88 aa. APOC3 was expressed in 14 tissues including heart,liver and spleen,and so on,which the highest level of APOC3 was found in liver.APOC3 expression was the highest at 48 h and extremely significantly higher than that before differentiation.After APOC3 was overexpressed,lipid drop accumulation increased,and the relative expression levels of marker genes SREBP1 and CEBPβ were extremely significantly up-regulated,PPARγ was significantly up-regulated,and Pref-1 was significantly down-regulated.APOC3 might promote the differentiation of intramuscular adipocytes by up-regulating SREBP1,CEBPβ,PPARγ and down-regulating Pref-1.

  • TANG Long, ZHAO Yuwei
    Abstract (430) PDF (158) RichHTML (57)

    Application of some hormonal signaling compounds,as brassinolides and their derivatives,could significantly improve the salt stress resistance in plants.The purpose of present work was to test whether the over-expressing of Methylsterol monooxygenase gene(SMO),a key gene coding a bio-synthesizing enzyme of sterol in plants,could promote the salt-stress tolerance of target plants.PnSMO1.1,gene encoding of methylsterol monooxygenase in the plant species of Pharbitis nil was firstly cloned and then used as target gene for following genetic transformation process,while wild-type Pharbitis nil seedlings were used as the receptor plants for constructing of the transgenic lines which over-expressed PnSMO1.1 genes.In this work,the PnSMO1.1 gene transformed Pharbitis nil lines were constructed via an ovary injection transformation method.Plantlets from individual PCR identified transgenic plant lines were used as materials to detect vegetative growth figures and some pivotal physiological indicators,for instance,contents of malondialdehyde (MDA),relative conductivity,as well as castasterone and 6-deoxo-castasterone contents in cells under stresses with gradient NaCl conditions varied from 0—200 mmol/L.The results showed that the over-expression PnSMO1.1 significantly improved the relative growth of roots and hypocotyls in transformants than in wild-type (WT) or vacant plasmid transformed control (BL) plants under 100—250 mmol/L NaCl stresses.Compared to WT and BL seedlings,significantly higher accumulation of 6-deoxo-castasterone,but lower relative conductivity (rEC) values,castasterone accumulation or MDA contents were found in transgenic lines under various NaCl stresses.Stresses such as salinity,drought and freezing temperatures,had severely suppressed the vegetative growth of plants,as well as their yields.These results highlighted that over-expression of the PnSMO1.1 gene could significantly improve the salinity-stress resistance of transgenic plants by delicately adjusting the dynamic homeostasis of brassinolides in cells,and protecting the structural integrity of plasma membrane.

  • WANG Yong, MENG Qingfeng
    Abstract (267) PDF (26) RichHTML (28)

    In order to explore the effects of cattle manure on soil salinity and sodicity on the sodic soil in long-term experiments,the experiments were performed in a randomized complete block design with four treatments;soils that received manure applications for 8,12,18 years were used as the experimental treatments,and soil that did not receive cattle manure application was used as the control treatment(CK).The results showed that the application of cattle manure to saline-sodic soil resulted in a reduction in the bicarbonate ion(HCO3-)contents,the elimination of carbonate ions(CO32-),the decrease in soil bulk density(ρb),the increases in soil porosity(ft)and soil organic matter(SOM),the decreases in the exchangeable and soluble sodium ion (Na+)contents associated with increases in the exchangeable calcium ion(Ca2+),soluble potassium ion(K+),and magnesium ion(Mg2+)contents compared to those in untreated soil.The soil exchangeable sodium percentage(ESP)and pH were both significantly and positively correlated with the exchangeable Na+ and HCO3-,and CO32- contents,and soil pH was significantly and negatively correlated with SOM.Regression analysis showed that the dominant factors affecting the sodium absorption ratio(SAR)were the soluble Mg2+ and Na+ contents in the soil.Pearson correlation analysis showed that there were significantly negative correlation between the accumulated amount of cattle manure among the indicators of soil salinization degree,such as pH,EC,ESP and SAR.It was concluded that long-term manure application significantly decreased the soil pH,ESP,electrical conductivity(EC)and SAR due to the replacement of soil colloidal Na+ with Ca2+,the leaching of soil soluble salts from the topsoil and changes in the soil soluble salt ion composition.These outcomes were likely due to the decrease of ρb associated with increase of ft and Ca2+ and Mg2+ contents caused by annual manure application.

  • LI Jun, YAN Zhihao, JIA Wanli, XU Mengmeng, ZHANG Jingfeng, XU Qiuliang, HAN Haoyuan, QUAN Kai
    Abstract (110) PDF (39) RichHTML (25)

    Adipose triglyceride lipase(ATGL)is the main rate limiting enzyme in the process of fat hydrolysis.In order to analyze the structure and transcriptional regulation mechanism of ATGL promoter in dairy goat,the 5' flanking sequence of ATGL was amplified by PCR and analyzed by bioinformatics.Luciferase reporter gene vectors of five deletion fragments with different lengths of promoter were constructed and transfected into mammary epithelial cells of dairy goat.The cells were treated with rosiglitazone and T0901317 respectively to detect the effects of PPARG and SREBP1 on ATGL promoter activity.The results showed that the 5' flanking sequence of ATGL gene of dairy goat was 2 721 bp,including 2 024 bp upstream from the transcription start site.The ATGL promoter contained eukaryotic promoter elements TATA-box and CpG island.It was also found that there were transcription factors-binding sites of FOXO,SREBF1,PPARG,C/EBPα and E2F1 by the online software prediction.The core region of ATGL promoter was located at -256—+1,and there were negative regulatory elements at -527—-256.When cells were treated with rosiglitazone and T0901317,it was found that both of them could significantly up-regulate ATGL promoter activity.The response regions of rosiglitazone and T0901317 were -527—-256 and -882—-527,respectively,indicating that PPARG and SREBP1 could regulate ATGL promoter activity.

  • MA Xiaojun, WANG Jiamian, LI Shiying, YAN Lijiao, MA Qiangsheng, ZHANG Xiaoli
    Abstract (111) PDF (28) RichHTML (18)

    In order to study the damage of mammary fibroblasts in sheep E.coli mastitis and the mechanism of TLR4,primary sheep mammary fibroblasts was obtained through in vitro culture,and E.coli artificially infected cells to establish E.coli mastitis cell model.Analyze the effect of E.coli on cell injury and the role of TLR4 in E.coli mastitis.Enzymatic digestion combined with differential digestion to obtain primary sheep mammary fibroblasts;MTT method to detect cell viability and cytotoxicity of the TLR4 blocker CLI-095;LDH method detects the damage of E.coli to sheep mammary fibroblasts and determines the optimal MOI;qRT-PCR to detect the relative expression of caspase-3 and TLR4 mRNA and the blocking effect of CLI-095;colorimetric method to detect the enzymatic activity of pyroptosis protein caspase-4,5;WB to detect the relative expression of TLR4 protein;the plate viable bacteria count method was used to detect the effect of CLI-095 on the adhesion of E.coli to sheep mammary fibroblasts.Sheep mammary fibroblasts was isolated,and the results of MTT assay showed good cell viability.After sheep mammary fibroblasts were infected with E.coli MOI=4:1,the release of LDH was significantly increased in each time period;the relative expression of caspase-3 mRNA increased significantly within 1-3 h of infection,and decreased significantly after 3 h;caspase-4,5 enzyme activity increased within 1-3 h of infection,and decreased after 3 h.The mRNA and protein expression of TLR4 were significantly up-regulated,and the CLI-095 could inhibit the adhesion of E.coli to cells.After E.coli infects fibroblasts,the release of LDH was increased,the expression of caspase-3 gene was up-regulated,and the activity of caspase-4,5 was increased,which promotes the injury,apoptosis and pyroptosis of sheep mammary fibroblasts.E.coli infection promotes the expression of TLR4 mRNA and protein in sheep mammary fibroblasts.The TLR4 blocker CLI-095 may inhibit the adhesion of E.coli to cells by inhibiting the expression of TLR4.

  • CHEN Wenxun, ZHOU Ting, YAN Qiongxian, TIAN Lina, CAO Manhu, TAN Zhiliang, ZOU Aihua
    Abstract (102) PDF (29) RichHTML (5)

    The aim of this study was to investigate the effects of low protein diet on meat quality,amino acid and fatty acid composition of skeletal muscle and blood,and mRNA expression of myogenic regulatary factor genes of black goat.A total of 24 female Xiangdong black goats were randomly divided into control group(CON,10.77% CP,n=12)and low protein group(LP,5.52% CP,n=12).The pre and post-trial lasted for 25 d and 45 d,respectively.The results were as follows:Low protein diet increased L* 24 h of meat color,but had no difference on pH value and water loss rate.The percentage of type Ⅰ muscle fiber and type Ⅱ muscle fiber had no difference between two groups,only the average fiber cross sectional areas of semitendinosus had a tendency to reduce.Valine,leucine,lysine,phenylalanine,glutamate and total essential amino acids of vastus lateralis significantly reduced,isoleucine,methionine,aspartate,arginine and total amino acid tended to decrease.Glutamate of semitendinosus in the LP group significantly reduced,threonine and serine tended to decrease.Compared with the control group,C20:2 and C22:1n9 of semitendinosus in the LP group significantly decreased.Total PUFA,C18:1n9c,and C20:4n6 of vastus lateralis in the LP group significantly decreased;C18:1n9c of longissimus dorsi in the LP group significantly decreased.Compared with the control group,the mRNA expression of MYOD in semitendinosus were significantly down-regulated in the CON group.In conclusion,low protein diet didn't affect type conversion and cross sectional area of muscle fiber.Dietary protein deficiency reduced the deposition of amino acid in muscle,while reduced the deposition of polyunsaturated fatty acids in muscle,which affected on muscle flavor of goat.

  • LI Qi, YANG Changheng, WANG Yong, LIN Yaqiu, XIANG Hua, ZHU Jiangjiang
    Abstract (90) PDF (23) RichHTML (2)

    The aim of this study was to obtain the CDS of CIDEB and CIDEC gene,detect the expression of CIDEB and CIDEC in different tissues of goats,predict their interaction proteins.It would provide basial data for revealing the regulatory role of CIDEs in lipid metabolism.The CDS of CIDEB and CIDEC gene were cloned by RT-PCR,and the biological characteristics and interaction proteins of CIDE proteins were analyzed by online tools.Real-time quantitative PCR(RT-qPCR)was used to detect the expression of CIDEs and the genes,encoding the interaction proteins,in different tissues of goats,following the correlation analyses by a SPSS software.The CDS of CIDEB was 660 bp,encoding 219 amino acids,while the CDS of CIDEC was 714 bp,encoding 237 amino acids.Phylogenetic tree analysis showed that goat CIDEA,CIDEB and CIDEC were closely related to corresponding genes from sheep.RT-qPCR detection showed that CIDEA,CIDEB and CIDEC were highly expressed in the rumen,liver and small intestine of goat,respectively.The expression of CIDEA was significantly correlated with the expression of DFFB (P<0.01),ELOVL3 (P<0.01),DIO2 (P<0.05),TMEM26 (P<0.05),PRDM16 (P<0.05)and PLIN1 (P<0.05)in rumen.The expression of CIDEB was significantly correlated with that of DFFA and SDR39U1 in liver(P<0.05).The expression of CIDEC was obviously correlated with that of PLIN2,GPLD1 and ADIPOQ in small intestine(P<0.05).Through the analysis,we found genes related to the expression level of CIDEs,which could provide a theoretical basis for further revealing the role of CIDE gene in lipid metabolism and its regulatory network.Moreover,the proteins of CIDEs were all lipid-droplet related proteins,and could also provide a reference for further studying the precise mechanism of lipid droplet formation.

  • WANG Xianjun, XIANG Hua, ZHANG Xumei, REN Yupeng, ZHU Jiangjiang
    Abstract (65) PDF (11) RichHTML (2)

    This aim of study was to explore the role of ORFV118 in regulating cell cycle,apoptosis and the immune-related cytokines(IL-1β,IL-6,IFN-γ and TNF-α)in goat testicular sertoli cells.We cloned the whole sequence of ORFV118 and constructed an expression vector of pEGFP-ORFV118.The expression of ORFV118 protein was then identified by using Western Blot method after the transfection of goat testicular sertoli cells with pEGFP-ORFV118.Following which,a flow cytometer equipment was used to measure the effect of ORFV118 expression on cell cycle and apoptosis of goat testicular sertoli cells,and also on regulating the expression of genes related to cell cycle,including CDK2 and P21,and genes relative to apottosis,including Bax,Bcl-2,Caspase3,Caspase7,P53 and BCL2L11.The expression of immune-related cytokines,including IL-1β,IL-6,IFN-γ and TNF-α,was detected by ELISA method and RT-qPCR after ORFV118 expression in goat testicular sertoli cells.The results showed that a total length of 309 bp ORFV118 gene sequence was cloned successfully.The cell's DNA synthesis phase(S phase)was inhibited and the late stage DNA synthesis(G2/M phase)was promoted by the expression of ORFV118,and the expression of CDK2 mRNA was decreased.The cell apoptosis blocked after ORFV118 expression,and the expression of Caspase3,Caspase7 and SOCS2 mRNA decreased.The immune-related cytokines,including IL-1β and IFN-γ,was inhibited in goat testis cells,while the expression of IL-6 and TNF-α were promoted.

  • SONG Xiangjun, SONG Zichao, SHEN Xiao, CHEN Zhe, JIANG Huyan, HOU Manman, SHAO Ying, TU Jian, QI Kezong
    Abstract (63) PDF (10) RichHTML (9)

    In order to study the effect of the effector protein Hcp2b on the transcriptomics of the spleen during the infection of Avian pathogenic E.coli(APEC)in chicks.The hcp2b-deletion strain Δhcp2b and the reverted strain Chcp2b(with AE17 strain as a positive control)constructed and preserved in the Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control were used to inject 7-day-old chicks intramuscularly.After 24 h,the spleen of depressed chicks was collected to detect the tissue load and make pathological sections.The deletion strain Δhcp2b and the wild strain AE17 were selected to infect spleen tissue for transcriptomics sequencing.Also,Real-time PCR was used to identify the sequencing results.Moreover,GO analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes.The results illustrated that the wild strain AE17,the deleted strain Δhcp2b and the reverted strain Chcp2b had no significant difference in the tissue load of the spleen.Observation of tissue sections that the spleens of wild strain AE17 and deletion strain Δhcp2b both suggested enlarged interstitial spaces and hyperemia.Transcriptomics analysis revealed that the deletion strain Δhcp2b induced the differential expression of spleen gene mRNA in chicks.515 differentially expressed genes were screened(185 genes were up-regulated and 330 genes were down-regulated).Real-time PCR confirmed that the expression of IL22,TNFRSF6B,and TNFRSF8 genes in the spleen was down-regulated after the deletion strain Δhcp2b infected chicks,which was consistent with the trend of the sequencing results.At 24 h,GO analysis found that the chick spleen differential genes were significantly enriched in items such as membrane,membrane composition,and extracellular space.KEGG analysis found that differential genes in the spleen were significantly enriched in the pathway of endoplasmic reticulum protein processing.The hcp2b gene had no effect on APEC colonization of spleen,the hcp2b participates in the pathogenic process through the signal pathway that affects the endoplasmic reticulum protein processing of the spleen in the body's immune organs.

  • FENG Lei, WEI Weiqun, ZHONG Xueqing, FU Mingjia, XIAO Shiping, YE Dexiao, HUANG Ziyan
    In order to improve the antigenicity of the expression product of Subtilomycin precursor gene of Bacillus subtilis SX3411, a three-times repeated tandem 3×subA gene was synthesized by chemical synthesis method. The prokaryotic expression vector of 3×subA was constructed and transferred into Escherichia coli.The results showed that the 3×subA gene was expressed in E. coli. SDS-PAGE was used to purify the E. coli expressed SubA. After immunizing rabbits, the antibody anti-3×SubA was prepared and had a high titer. The intracellular expression of Subtilomycin precursor polypeptide subA was detected by Western Blotting. The results showed that B. subtilis SX3411 was cultured at 30℃ for different time, and there were more subA expressions in the cells of B. subtilis 3411 at about the 10th and 12th hour. In conclusion, the fusion expression product 3×SubA obtained by tandem expression of the Subtilomycin precursor gene subA has good antigenicity, and the obtained antibody anti-3×SubA can be used for the detection and analysis of Subtilomycin precursors in B subtilis SX3411.
  • CHEN Mei, CHAI Zhixin, WU Zhijuan, WANG Jikun, ZHONG Jincheng, XIN Jinwei
    In order to obtain the CDS region sequence of the yak CTGF gene and the structure and function of the encoded protein, and to study the mRNA expression level of this gene in tissues such as kidney, heart, lung, liver and gluteal muscle. The experiment took Leiwuqi yak as the research object. RT-PCR was used to obtain the CDS sequence of CTGF gene of Leiwuqi yak. Fluorescence quantitative PCR(qPCR)method was used to detect the mRNA expression level of this gene in five tissues. The results showed that the yak CTGF gene contained two CpG islands, the open reading frame length was 1 050 bp(accession number:MT968972), which could encode 349 amino acids, and the CTGF encoded protein had a signal peptide, which was a water-soluble unstable surface protein. There were 21 phosphorylation sites and 7 glycosylation sites, which were more distributed in the endoplasmic reticulum and microsomes(peroxisomes), with 3 complete conserved functional domains. Fluorescence quantitative results showed that CTGF gene was expressed in kidney, heart, lung, liver and gluteal muscle tissues of Leiwuqi yak, and the expression level was highest in liver. The aim was to further understand the role of CTGF gene in regulating yak muscle growth and provide reference data.
  • SHI Fengyun, ZHU Guangqin, LIAO Yunqiong, WANG Bing
    Insulin-like Growth Factor Binding Proteins (IGFBPs) are closely related to the growth and development of animals.In order to explore the possibility of IGFBP-3 gene as a candidate gene for sheep slaughter traits, this study used Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) combined with sequencing to detect variation characteristics of IGFBP-3 gene and to explore the correlation between nucleotide variation and slaughter traits in 592 Gansu alpine merino sheep.The results showed that one single nucleotide variant site (g.87 A>C) was detected in the fourth exon region of sheep IGFBP-3, and two alleles A and B (alle frequency:56.76% and 43.24%), showing three genotypes of AA, AB and BB (genotype frequency:29.73%, 54.05%, 16.22%), and the population studied was in the Hardy-Weinberg equilibrium state at this locus (P>0.05).The correlation results showed that the mutation at g.87 A>C of Gansu alpine merino sheep IGFBP-3 gene had a very significant effect on live weight and hot carcass weight before slaughter (P<0.01);it had a significant effect on eye muscle area, GR value and tenderness.Degree had a significant impact (P<0.05).It showed that the sheep population of allele B had a higher slaughter rate, and it could be used as a molecular marker for the selection of slaughter traits of Gansu alpine merino sheep in production practice.
  • LONG Xi, ZHAO Jiugang, CHAI Jie, PAN Hongmei, ZHANG Liang, ZHANG Tinghuan
    The aim of this study was to explore the innate immune candidate genes in Rongchang pigs and Landrace. Thymus of newborn Rongchang pigs and Landrace were used as the research objects. The RNA-seq technology was used to analyze the differentially expressed genes in the thymus of Rongchang pigs and Landrace. Then, the GO and KEGG enrichment analysis of differentially expressed genes were performed, and the qRT-PCR was used to verify the accuracy of RNA-seq. The results showed that a total of 421 differentially expressed genes were identified, including 231 up-regulated genes and 190 down-regulated genes of Rongchang pig compared to Landrace. GO enrichment analysis showed that these differentially expressed genes were mainly enriched in the biological processes such as scavenger receptor activity, G protein-coupled receptor binding, T cell activation, regulation of secretion by cell, exocytosis and SNARE complex assembly. KEGG pathway analysis showed that these differentially expressed genes were mainly involved in signal pathways such as cytokine-cytokine receptor interaction, cAMP signal, cysteine and methionine metabolism, phospholipase D signaling and some diseases(African cone disease, cancer).The RT-qPCR results of 6 differentially expressed genes showed that the results of RNA-seq were accurate and reliable. Based on the results of screening, GO and KEGG of differentially expressed genes, TMSB15A and STAB2, SAA1 and HP were candidate genes for thymus innate immunity in Rongchang pigs and Landrace, respectively.
  • SUN Bin, TANG Lin, ZHANG Junfang, SUN Jianfu, CUI Yan, WANG Ying, WANG Enze, LI Qiang, LI Xiangzi
    The aim was to clone the CDS region sequence of Yanbian cattle UCP1 (Uncoupling protein-1), use RT-PCR technology and gene cloning to obtain Yanbian cattle UCP1 gene, conduct homology comparison with other species and construct a phylogenetic tree, using bioinformatics methods to predict the physical and chemical properties of UCP1 encoded protein, potential phosphorylation sites and other properties, and use Quantitative Real-time PCR (qPCR) method to detect the expression abundance of UCP1 gene in different tissues of Yanbian cattle. The results showed that the CDS region of Yanbian cattle UCP1 gene was 749 bp in length, encoding 249 amino acids. The homology comparison revealed that the UCP1 gene of Yanbian cattle had 99.47% homology with Indian cattle. The phylogenetic tree showed that Yanbian cattle and Indian cattle had the closest genetic relationship and the farthest related to South American alpaca. The UCP1 protein lacked stability, the fat solubility coefficient was 91.53, the half-life of reticulocytes in vitro was 30 h, and the total average hydrophilicity was equal to 0.212, which was predicted to have a certain degree of hydrophilicity. The secondary structure was a mixed protein composed of α-helix (54.03%), extended chain (12.90%), random coil (27.42%) and β-turn (5.65%). The analysis of phosphorylation sites and glycosylation sites showed that UCP1 had a total of 21 phosphate sites, 4 potential O-glycosylation sites, and 3 N-glycosylation potential sites. The encoded product of UCP1 gene had 0 transmembrane helix (TMHs) structure, the predicted value of the amino acid residues of the transmembrane helix was 19.008 57, and the predicted value of the transmembrane helix of the first 60 amino acids of the protein was 10.980 09, which was the total probability of being located on the cytoplasmic side of the membrane 22.995% and was a non-secreted protein. Real-time fluorescent quantitative PCR (RT-PCR) showed that the expression level of UCP1 gene in Yanbian beef fat and small intestine was relatively high, while the expression level in muscle and heart was low. The experimental results could provide a reference for further research on the function of this gene.
  • CHEN Dingshuang, WANG Ruilong, LIN Yaqiu, WANG Yong, ZHU Jiangjiang, LI Xin, ZHANG Hao, LI Yanyan
    Hoxa5 is a member of the HOX family and plays an important role in the process of growth,development and tumorigenesis.However,there is less research on it in ruminants.In order to obtain the goat Hoxa5 gene sequence,to clarify its expression characteristics and know its expression level in different tissues of goats.The Hoxa5 gene was cloned from the subcutaneous fat tissue of Jianzhou big ear sheep using RT-PCR technology,and its biological characteristics were analyzed by bioinformatics methods.Real-time fluorescent quantitative PCR technology was used to detect the expression of Hoxa5 gene in the heart,liver,spleen,lung,kidney,back,thigh,arm,etc.tissues of Jianzhou big ear sheep.The results showed that the open reading frame of the goat Hoxa5 gene was 813 bp,encoding a total of 270 amino acids.the relative molecular mass of Hoxa5 was 29.283 ku,the isoelectric point was 9.42,and there was no signal peptide and transmembrane domain.This protein was a non-secreted protein;the secondary structure prediction of Hoxa5 protein found that the content of random coils in Hoxa5 protein was the highest,followed by α helix,and β turn accounts for a relatively small number of amino acids;amino acid homology comparison results showed that the Hoxa5 amino acid sequence similarity between Jianzhou big ear sheep and Ovis aries,Bos taurus, Equus caballus, Sus scrofa, Rattus rattus, Cavia porcellus, Mus musculus and Homo sapiens were 100.00%,99.63%,99.26%,99.26%,98.89%,98.89%,98.89% and 98.15%.The results of Real-time fluorescent quantitative PCR showed that Hoxa5 gene was differentially expressed in selected tissues,with the highest expression level in the kidney;Fluorescence localization of pEGFP-Hoxa5 fusion vector transfected into goat subcutaneous adipocyte showed that the gene was mainly located in the nucleus.This study showed that the goat Hoxa5 gene was up-regulated during subcutaneous adipocyte differentiation and the protein was localized in the nucleus. The results suggested that this gene might act as a transcription factor to regulate goat subcutaneous adipocyte differentiation.
  • YUE Yongqi, HUA Yonglin, JIA Yige, LI Jian, XIONG Yan, XIONG Xianrong
    Yak was used as the experimental object to clone the yak CCAAT/Enhancer binding protein alpha(CEBPα) gene, predicted its protein structure and function, isolated yak adipocyte, and detected the expression of CEBPα in different tissues of yak and yak fat expression during cell induction and differentiation. Collected the heart, liver, spleen, kidney, stomach, small intestine, subcutaneous fat, visceral fat and muscle tissue of an adult yak (4 years old), and used PCR technology to obtain the CDS sequence of the CEBPα gene;the method of bioinformatics predicted the sequence homology and protein structure of the gene;Real-time fluorescent quantitative PCR(qRT-PCR) was used to detect the expression of CEBPα in different tissues of yak;the yak preadipocytes were isolated by collagenase digestion, qRT-PCR was used to detect the expression pattern of CEBPα gene at 0, 3, 6, 9 d of induced differentiation;the results of the study showed that the CDS sequence of the yak CEBPα gene was 645 bp in length and encoded 214 amino acids. The yak CEBPα gene had relatively high homology with Bos taurus, with a nucleic acid homology of 99.73% and amino acid homology of 99.19%. The results of constructing a phylogenetic tree showed that the yak was more similar to ordinary cattle and sheep. CEBPα protein was an unstable hydrophilic protein with a transmembrane structure and phosphorylation/dephosphorylation. The secondary structure of the protein was dominated by α helix (27.10%) and random coil (67.29%). The results of qRT-PCR showed that the CEBPα gene was most highly expressed in the lower adipose tissue of the yak, and lowly expressed in the kidney, spleen and visceral adipose tissue. The expression of CEBPα gradually increased during the induction and differentiation of yak adipocytes. In this experiment, we cloned and obtained the CDS region sequence of CEBPα, determined its expression in various tissues of yak and clarified the expression pattern of this gene in the process of induced differentiation of yak adipocytes. Provide basic data to reveal the function of CEBPα gene in yak adipose tissue and adipocyte.
  • ZHAO Man, QI Zhi
    To explore to study the effect of exogenous calcium on the tolerance of Leymus chinensis, the seeds which collected from Horinger County, Hohhot City, Inner Mongolia were used as experimental materials, and the method of aseptic culture was used to study the effect of CaCl2 on the growth and physiological characteristics of Leymus chinensis under four abiotic stresses of salt, alkali, osmotic and cold stress. The results showed that the growth of Leymus chinensis seedlings was significantly inhibited under 150 mmol/L NaCl stress. After adding 1 mmol/L CaCl2 to the culture medium, the germination rate of seeds was significantly increased, and the root length, leaf length and fresh weight of seedlings were also increased. Under pH 8.5 or 300 mmol/L Mannitol stress, the growth of Leymus chinensis seedlings was both significantly inhibited. The effects of alkali stress and osmotic stress on the growth of Leymus chinensis seedlings could not be effectively alleviated by adding 1-20 mmol/L CaCl2 to the culture medium respectively. Under 4℃ stress, the growth of Leymus chinensis seedlings was significantly inhibited. After adding 20 mmol/L CaCl2 to the culture medium, the leaf length and fresh weight of seedlings increased significantly, the content of malondialdehyde(MDA) in leaves decreased, the activity of superoxide dismutase(SOD), peroxidase(POD), catalase(CAT) and glutathione reductase(GR) increased, and the content of reduced ascorbic acid(AsA) increased significantly. In conclusion, the addition of exogenous CaCl2 could improve the salt and cold resistance of Leymus chinensis seedlings, but the ability to resist alkali and osmotic stress has not been improved.
  • WU Jianfei, LIU Yu, LI Heng, JING Tian, LU Jianyuan, ZI Xiangdong
    The objective of this study was to investigate the sequence and tissue expression characteristics of DNA damage inducible transcript 3(DDIT3) in female yaks. The yak DDIT3 gene was cloned by RT-PCR. The structure and function of DDIT3 protein were analyzed by bioinformatics software. The expression levels of DDIT3 gene in different tissues, and different reproductive organs and oocytes at different physiological stage of female yaks were detected by Real-time quantitative PCR(RT-qPCR). The results showed that the full-length CDS region of yak DDIT3 gene was 507 bp, which encoded 168 amino acids. Nucleotide sequence alignment showed that yak had the highest homology with bison(99.71%) and more than 88% homology with other species, indicating that DDIT3 gene was highly conservative in the process of evolution. The expression of DDIT3 gene in ovary was extremely significantly higher than that in heart, liver, kidney, spleen, lung, uterus and fallopian tube(P <0.01). In the ovary, the expression of DDIT3 gene was significantly higher during pregnancy than that during follicular phase, luteal phase and fetus stage(P <0.05), and it was significantly higher during luteal phase than that in fetus stage(P <0.05). In the uterus, the expression of DDIT3 was significantly higher than that in follicular stage and fetus stage(P <0.05). In oviduct, its expression was not significantly different at different physiological stages. The expression of DDIT3 gene in M Ⅱ stage oocytes was significantly higher than that in GV stage and M Ⅰ stage(P <0.05). Its expression in M Ⅱ granulosa cells was also significantly higher than that in GV stage and M Ⅰ stage(P <0.05). There was no significant difference in DDIT3 expression between oocytes and granulosa cells in GV stage and M Ⅰ stage. In conclusion, yak DDIT3 gene possible plays an important role in regulating ovary function and pregnancy maintenance, follicle development and maturation.
  • YU Kun, LIU Ruili, LIU Xianxun, BAI Xuejin, DONG Yajuan
    The aims to explore the molecular mechanism of bta-miR-133a involved in the regulation of longissimus dorsi development in beef cattle. The longissimus dorsi muscle of Black cattle and Luxi cattle was collected for sRNA database construction, and the data was used to screen bta-miR-133a related to the development of beef cattle skeletal muscle. Use bioinformatics analysis software to identify its conservation and predict its target genes, perform GO enrichment analysis and KEGG pathway enrichment analysis for its target genes, and verify the predicted potential target genes through the dual luciferase report test. C2C12 cells were selected for functional verification, bta-miR-133a was overexpressed and knockouted. After 48 h, 2% horse serum was used to observe the myotube differentiation process, CCK-8 to detect cell proliferation rate, Real-time quantitative polymerase chain reaction to detect the expression of cell proliferation and differentiation marker genes PCNA, CCND1, Myh1, Myod1 and target genes. The results showed that the mature sequence of bta-miR-133a was highly conserved among various species. The target gene was predicted to be PAX7. The dual luciferase report test verifies that bta-miR-133a had a target relationship with PAX7.The result of GO enrichment and KEGG pathways analysis showed that the predicted target genes were significantly enriched in the regulation of actin cytoskeleton organization tissue, the negative regulation of the classical Wnt signaling pathway, actin filament binding and other GO components, and MAPK, Ras, FoxO and other signaling pathways related to muscle development in. Overexpression of bta-miR-133a promoted myotube differentiation(P <0.01), at 72 h, reduced cell proliferation rate(P <0.01), inhibited the expression of target gene PAX7 (P <0.01);knocked out bta-miR-133a inhibited myotube differentiation(P <0.01), increased cell proliferation rate at 72 h(P <0.05), and promoted the expression of target gene PAX7 (P <0.01). In terms of cell proliferation, overexpression of bta-miR-133a reduced the expression level of its marker genes PCNA and CCND1 (P <0.05), while the knockout group was the opposite. In terms of cell differentiation, overexpression of bta-miR-133a increased the expression level of the marker genes Myh1 and Myod1 (P <0.01), while the knockout group was the opposite. In summary, bta-miR-133a may participate in the development of longissimus dorsi by targeting and negatively regulating the expression of PAX7 to inhibit the proliferation of myoblasts and promote their differentiation.
  • ZHANG Kunli, LIN Benfu, XI Zhenjun, YANG Dongxia, BIAN Zhibiao, SONG Shuai, JIANG Zhiyong, CAI Rujian, LI Chunling
    This study was to develop an efficient and sensitive nano PCR for the detection of Streptococcus suis (SS). The specific primers were synthesized for the Glutamate dehydrogenase gene (gdh) of SS. Then, the annealing temperature and primer concentration reaction conditions were optimized. The results of specificity assay showed that the method could only amplify 687 bp specific sequence from SS samples, and there was no cross reaction with Actinobacillus pleuropneumoniae (APP), Haemophilus parasuis (H. parasuis), swine Escherichia coli (swine E. coli), Salmonella suis (S. suis) and (S. hyicus). Moreover, the method also could detected the main serotypes 1, 2, 7 and 9 type of SS, respectively. The results of sensitivity assay showed that the lowest SS concentration detected by the nano PCR method was 10 cfu/mL, and its sensitivity was 100 times higher than ordinary PCR of SS. The SS nucleic acid was detected three times, and the results were consistent. Finally, 44 suspected SS infection samples which collected from clinical cases were detected by nano PCR and routine PCR, respectively. The clinical sample testing showed that the positive rates were 72.7% (32/44), 54.5% (24/44), respectively. In conclusion, the nano PCR detection method of SS was established in this study can accurately and efficiently detect SS, which provides technical support for the clinical diagnosis and epidemiological investigation of S. suis disease.
  • LI Juan, ZHENG Yao, WANG Li
    The aim of this study was to identify the Aquaporin 7 gene and analyze the expression level and protein localization of AQP7 gene in different tissues of yak.The AQP7 gene of Jiulong yak was cloned by RT-PCR and analyzed by bioinformatics.RT-qPCR was used to detect the expression levels in 8 different tissues of yaks.Immunohistochemical staining method was used to analyze the expression and location of AQP7 protein in tissues.The results showed that the CDS sequence of AQP7 gene was 993 bp, which encoded 330 amino acids. Bioinformatics analysis found that AQP7 was a stable hydrophobic protein.The phylogenetic tree showed that AQP7 gene of Jiulong yak was closely related to cattle, and its homology was 99.7%. The expression of AQP7 gene was higher in Jiulong yak heart and muscle, which were significantly higher than that in kidney, liver, spleen, lung, intestine and rumen(P <0.01). Immunohistochemical results showed that AQP7 protein was mainly distributed in the muscle cells of the heart and muscles and the proximal convoluted tubules of the kidney, and its expression in the heart, muscles and kidney were higher than that in the liver, spleen, lung, intestine and rumen of Jiulong yak(P <0.05).This study may provide basic data for the physiological function and energy metabolism mechanism in yak high altitude adaptation.
  • HUO Wentao, ZHOU Ting, YU Yue, YANG Qinglan, CHANG Xindan, LI Jian, XIONG Xianrong, XIONG Yan
    The aim of this study was to clone the gene sequence of cell death-inducing DNA fragmentation factor 45-like effector A (CIDEA) in yak, analyze its biological characteristics, and detect the expression pattern of CIDEA in yak different tissues and during the differentiation of adipocytes. The CDS sequence of CIDEA was cloned by PCR, using Jinchuan yak subcutaneous adipose tissue cDNA as a template, and the biological characteristic was analyzed;specific primers were designed to detect the expression of CIDEA in various Jinchuan yak tissues, including lung, heart, kidney, fat and other tissue of Jinchuan yak. And primary adipocytes were isolated to analyze the expression trend of CIDEA during adipocyte differentiation. The results showed that the length of Jinchuan yak CIDEA was 696 bp, of which CDS was 684 bp, encoding 227 amino acids. CIDEA of Jinchuan yak had the highest homology to that of Bos taurus, representing that the nucleotide identity was 94.14% and the amino acid homology was 99.54%, and the lowest homology to that of Gallus gallus, representing that the nucleotide identity only was 63.38% and the amino acid homology only was 59.05%. Also, phylogenetic tree constructed by MEGA 5.0 software showed that similarity was highest between Jinchuan yak and cattle, and similarity was lowest between Jinchuan yak and chicken. Moreover, the physical and chemical properties of protein were predicted that CIDEA protein was an unstable basic hydrophilic protein without transmembrane structure and signal peptide. The secondary structures of CIDEA protein mainly consisted of 33.92% α-helix and 51.98% random coils. Real-time quantitative PCR results showed that the expression quantity of CIDEA was highest in Jinchuan yak adipose tissue and was lowest in lung. And the differentiation of adipocytes in Jinchuan yak showed an increasing trend. It is concluded that the expression of CIDEA might involve in adipose differentiation and adipose deposition in yaks.
  • WANG Liang, LI Zhongqiu, TANG Xiaodong, ZHANG Haifeng, LIU Di, ZHANG Dongjie
    The ATP6V0A4 gene encodes a subunit of V-ATPase, which is related to H+ transport and is the causative gene of hereditary distal renal tubular acidosis. In the previous study, our group found that the transcription level of the ATP6V0A4 gene in the back fat of Min pig was significantly increased after cold stress(P<0.05). In order to analyze the regulatory mechanism of the ATP6V0A4 gene at the transcriptional level, this study constructed a series of dual-luciferase reporter gene plasmids with different lengths containing the 5'-end promoter region of the gene, transfected PK15 cells and detected the relative dual-luciferase active. According to the test results, the overlap PCR technology was used to delete the target sequence to predict the possible transcription regulatory factors. The results showed that the-1 504﹣-1 bp fragment of the promoter region of the gene had transcriptional activity. After two rounds of promoter truncation tests, it was determined that there were positive regulatory elements in the-265﹣-1 bp region and negative regulatory elements in the-581﹣-265 bp. After targeted deletion of small fragments of-205﹣-190 bp and-120﹣-110 bp, it was determined that there were binding sites of positive regulatory elements that regulate the transcription of the gene in the region of-205﹣-190 bp. After prediction by online software, E2F3, SP2, EGR1, E2F6, CTCFL, E2F1, SP1 and ETF might be the positive regulatory transcription factors of this gene. The promoter region of the pig ATP6V0A4 gene was cloned for the first time and the transcription factor binding sites were screened and identified, which laid the foundation for the follow-up study of its transcriptional regulation under cold stress.
  • YIN Shi, WANG Bin, QUNI Lamu, YANG Liuqing, YUAN Yujie, LI Jian
    The aim of this research is to clone the HDAC2 gene in yak, predict the structure and property of HDAC2 protein, and identify the expression profile of HDAC2 mRNA in different tissues and testis of different stages of yak. Yaks were divided into juvenile(0.5-1 year), adult(4-5 years) and old groups(7-9 years).The samples of liver, kidney, lung, stomach, spleen, brain, heart and ovary from adult group and testis of three stages were collected. The yak HDAC2 gene was cloned by reverse transcription PCR (RT-PCR). The homology of HDAC2 gene sequence, as well as the amino acid sequence and structure of HDAC2 protein were predicated by bioinformatics. Real-time PCR was applied to identify the mRNA expression profile of HDAC2 in different tissues of yak. RT-PCR was applied to detect the mRNA expression of HDAC2 in yak testis of different stages. Chromogenic in situ hybridization (CISH) was applied to analysis the localization of HDAC2 mRNA in adult yak testis. Result showed that the open reading frame (ORF) of yak HDAC2 contained 1 467 bases and encoded 488 amino acids. The sequence of yak HDAC2 was highly conserved in mammals. Structure prediction indicated that HDAC2 was a hydrophilic liposoluble protein with a histone deacetylase domain. The mRNA of HDAC2 was highly expressed in ovary and testis. The expression of HDAC2 in juvenile stage was significantly higher than that in other stages. The mRNA of HDAC2 localized in multiple kinds of testicular cells except spermatid. The sequence of yak HDAC2 gene was cloned and the spatiotemporal expression pattern of this gene in testis was verified. This study provides some experimental data for further study on the role of HDAC2 in testicular development and spermatogenesis of yak.
  • CUI Baowei, LIU Quanyong, QIN Guangli, ZHU Junpeng
    The harmless treatment of breeding waste (sheep manure) is an important link in the development of modern green production. Different configurations of sheep dung, soil loosening and root promoters, and conventional chemical fertilizers treated with high-temperature harmless treatment,the effects of single and combined application of soil loosening and root promoting agent and fermented sheep manure (fertilizer (CK), fertilizer+soil loosening and root promoting agent (T1), fertilizer+fermented sheep manure (T2), fertilizer+fermented sheep manure+soil loosening and root promoting agent (T3)) on the physical and chemical properties of soil, agronomic properties, yield and quality of red cluster pepper in the wet area were explored. The results showed that, compared with fertilizer (CK), application of soil loosening and root promoting agent and fermented sheep manure could improve soil physical and chemical properties, promote the growth and development of pepper and improve the yield and quality. Among them, fertilizer+fermented sheep manure+soil loosening and root promoting agent (T3) treatment was the most significant. Compared with fertilizer (CK), soil organic matter, alkali hydrolyzed nitrogen, available phosphorus and available potassium were significantly increased by 24.08%,18.08%,14.80%,15.56%,the soil powder has increased significantly by 9.05%,the plant height, stem diameter and effective branch number of red cluster pepper increased by 25.71%,20.69%,28.81%,fruit number per plant, fresh quality and dry quality per fruit increased by 29.05%, 17.04% and 18.94% respectively, the yield of fresh and dry pepper increased by 37.75%,39.43%, respectively, the content of Vc and reducing sugar in the fruit of red cluster pepper increased by 19.91% and 20.77%, respectively, but it had no significant effect on nitrate and free amino acid content. Comprehensive consideration, combined application of soil loosening and root promoting agent and fermented sheep manure and fertilizer(T3) had the best effect on improving soil and increasing quality and yield effects of red cluster pepper.
  • YANG Liuqing, YIN Shi, QIN Wenchang, YUAN Yujie, LI Jian
    This research was aimed to clone the Sirtuin7 (SIRT7) gene in yak, predict the structure and function of SIRT7 protein, and detect its mRNA expression in different tissues and ovary during different phases of oestrus. The liver, breast, large intestine, kidney and ovaries in follicular phase, red body phase, and luteal phase from adult yaks(3-5 years) were collected. Total RNA of each tissue was extracted and the gene sequence of yak SIRT7 was amplified by reverse transcription PCR (RT-PCR). Different bioinformatics softwares were used to compare sequence homology of SIRT7 gene construct phylogenetic tree, and predict the structure and function of yak SIRT7 protein. The expressions of SIRT7 mRNA in different yak tissues and ovaries during different stages of oestrus were detected by quantitative real-time PCR (qRT-PCR). The result showed that the open reading frame (ORF) of yak SIRT7 was 1 203 bp, encoded 400 amino acids. The SIRT7 gene nucleotide sequence of yak was highly homologous to that of cattle, sheep, and goat. The SIRT7 protein was a hydrophilic protein. The secondary structure of SIRT7 protein was made of alpha helix(47.4%), beta fold(29.7%), beta turn(15.7%) and random coil(7.2%). Phosphorylation and glycosylation analysis showed that SIRT7 protein contained 38 phosphorylation sites and 24 O-glycosylation sites. Quantitative real-time PCR result showed that SIRT7 mRNA was highest expressed in the kidney of yak. In the yak ovary, the expression levels of SIRT7 mRNA increased gradually in follicular phase, red body phase and luteal phase.The highest expression level was found in luteal phase. This research provides some basic data for revealing the regulatory role of SIRT7 in the growth and development of yak, especially in the development of ovaries.
  • WANG Jianglin, WANG Yong, MENG Qingyong, ZHU Jiangjiang, LIN Yaqiu
    This study was performed to obtain the sequence of Kruppel-like factor 6(Kruppel-like factor 6, KLF6) CDS, clarify its expression pattern in tissues and subcutaneous adipocyte of goats. The KLF6 gene sequence of goat was cloned by RT-PCR, and the sequence of KLF6 gene was analyzed by biological software and online website. Quantitative real-time PCR (qPCR) was used to detect the expression abundance of KLF6 in various goat tissues and the expression level of subcutaneous adipocytes at different stages of lipogenesis differentiation. The results showed that the full-length sequence of goat KLF6 gene was 1 233 bp, including 957 bp of CDS region, which encoded 318 amino acid residues, and closest relative was the cattle and the sheep. KLF6 gene was widely expressed in various the investigated tissues of goats, the expression level of KLF6 was higher in adipose tissue, which was significantly higher than that in other tissues(P<0.01). The expression of KLF6 in goat subcutaneous adipocytes induced for 60 h was significantly higher than that in preadipocytes (P<0.01). The sequence of goat KLF6 gene was obtained and its molecular characteristics were identified. It was found that it was highly expressed in adipose tissue, and the expression level after differentiation was significantly higher than that before differentiation.It provides important data for revealing the regulation of KLF6 on the differentiation of goat adipocytes.
  • MA Hongcheng, XIONG Xianrong, WANG Han, HAI Zhuo, MIN Xingyu, LI Jian
    The aim of this study was to investigate the sequence characteristics of phosphatidylinositol-3 kinase catalytic subunit β(PIK3CB/P110β) gene and compare its expression profile in different developmental stages of yak follicles.The sequence of PIK3CB gene was cloned from yak ovary tissue by RT-qPCR and analyzed by bioinformatics. The expression level of PIK3CB gene in different tissues of yak was detected by RT-qPCR method. The collected yak follicles were divided into three groups according to their diameter:large(≥ 7 mm), medium(3.0-6.9 mm) and small(≤ 2.9 mm). The total RNA was extracted from the oocytes and parietal granulosa cells of each group. Then, the relative expression of PIK3CB gene mRNA was detected by RT-qPCR.The results showed that the full-length CDS region of yak PIK3CB gene was 3 213 bp, encoding 1 070 amino acids. Bioinformatics analysis showed that PIK3CB protein was a hydrophilic acidic protein with no transmembrane structure and signal peptide, and the secondary structure was mainly composed of α-helix and random coil. The nucleotide homology and genetic tree analysis showed that yak PIK3CB gene was closely related to that of Bos mutus and Bos taurus. PIK3CB gene was expressed in heart, lung, kidney, liver, small intestine, stomach and muscle of yak, especially in spleen, uterus and ovary, the expression level was significantly higher than that in other tissues(P<0.05). The results of RT-qPCR showed that PIK3CB gene was expressed during follicular development, and in different follicular wall granulosa cells, the expression of mRNA increased with the development of follicles, and the expression in large and intermediate follicles was significantly higher than that in small follicles (P<0.01), but there was no significant difference in mRNA expression in oocytes(P>0.05).The results suggested that PIK3CB gene was involved in the regulation of yak follicular development, and it was one of the indispensable catalytic subunits of PI3K signal in regulating the function of granulosa cells. This study provides basic data for further study on the mechanism of PI3K-AKT signaling pathway in ovarian development.
  • LI Juan, WANG Li, LUO Xiaolin, GUAN Jiuqiang
    In order to identify the AQP8 gene of yak, analyze its expression differences in different tissues and livers at different growth stages, and explore the differences of expression and distribution of AQP8 protein, the CDS of yak AQP8 gene was cloned by RT-PCR and then the sequence was analyzed using bioinformatics. The RT-qPCR was used to detect the mRNA expression level of AQP8 in 8 tissues and liver at different growth stages. Immunohistochemistry was used to study the expression and distribution of livers in different ages. The results showed that the ORF of AQP8 gene was 792 bp, encoding 263 amino acids. The phylogenetic tree analysis showed that the AQP8 gene of Maiwa yak had the closest associated with that of Bos mutus. The AQP8 protein has a stable MIP domain specific to AQPs. The mRNA expression level of AQP8 in liver of adult yak was significantly higher than heart, spleen, kidney and other tissues(P<0.05), and the expression level of AQP8 in liver at juvenile(15 months) was significantly higher than fetus(1 day) and adult(5 years) (P<0.05). Immunohistochemical results showed that AQP8 protein expression was the highest in liver of juvenile(15 months) yak(P<0.05). This study provided relevant basis for further exploring the mechanism of yak AQP8 in the growth process, and provided implications for study of mammals in the alpine and hypoxic environment.
  • HUANG Xiangyue, XIONG Xianrong, HAI Zhuo, MU Songyin, LI Jian
    The aim of this research was to clone the aquaporin 2 (AQP2) gene, identify its expression pattern in various tissues, and to analyze the expression in different growth periods of the male reproductive system in yak.This research may provide important foundation for exploration the role of AQP2 for reproductive development in yak.The samples of yak kidney, testis, epididymis, spleen, brain, lung, heart and liver were collected after slaughtering. The total RNA of different samples were extracted and the coding sequence of AQP2 gene was cloned by RT-PCR. Meanwhile the function and structure of AQP2 gene were analyzed by bioinformatics softwares.Then, the mRNA expression of AQP2 in different tissues and in different growth periods of the male reproductive system was detected by quantitative real-time PCR (qRT-PCR). The results showed that the CDS region of AQP2 gene was 816 bp, encoding 271 amino acids. It had high homology with cattle, buffalo and goat. The tissues expression analysis showed that AQP2 highly expressed in testis and kidney, which was significantly higher than other tissues (P<0.05). The results of immunohistochemistry indicated that AQP2 was expressed in round spermatids of seminiferous tubules, but no expression was found in spermatogonia, spermatocytes, elongated spermatids, leydig cells and sertoli cells. The results of qRT-PCR was found that the expression of AQP2 in vas deferens was the highest(P<0.05). The expression trend of AQP2 mRNA in testis and vas deferens was increased with age (P<0.05). While the expression level in prostate was slightly decreased with age, but the difference was not significant(P>0.05). The above results indicated that AQP2 was highly conserved during evolution, and it was highly expressed in testis and kidney. It was involved in sperm maturation and transportation, which might be completed by water reabsorption and liquid formation.
  • FENG Ziyan, WANG Xiangguo, YUAN Mengyi, FU Bofan, XIE Tongtong, CHANG Di, WU Chunyang, NI Hemin, XIAO Longfei
    To investigate whether p38 MAPK and PI3K/Akt signaling pathways are involved in the regulation of estradiol(E2) on PGF secretion in cow endometrial epithelial cells, endometrial epithelial cells of pregnant dairy cows were cultured in vitro, and different concentrations of E2 and p38 MAPK and PI3K/Akt pathway inhibitors SB 203580 and LY294002 were used to treat cows' endometrial epithelial cells respectively. The expression of COX-2 gene and protein and the level of PGF were detected by Western Blot, qRT-PCR and ELISA. The results showed that E2 promoted the expression of COX-2 and the secretion of PGF. In addition, E2 activated the p38 MAPK and PI3K/Akt signaling pathways. Moreover, the addition of SB203580 and LY294002 inhibit the promotion effect of E2 on COX-2 expression and PGF secretion. Therefore, it proved that E2 promoted the expression of COX-2 and the secretion of PGF by activating the p38 MAPK and PI3K/Akt signaling pathways.
  • ZHANG Kang, ZHANG Kai, WANG Lei, CUI Dongan, ZHANG Jingyan, YAN Zunxiang, MA Xueqing, XUE Huan, LI Jianxi
    To understand the prevalence and molecular characteristic of Bovine viral diarrhea virus (BVDV)in Gansu Province,stool samples of cattle with diarrhea symptoms in a cattle farm in Gansu Province were collected and detected for BVDV by RT-PCR.The stool samples identified as BVDV positive were treated and then inoculated into MDBK cells for virus isolation and culture.Subsequently,the isolated virus was identified by RT-PCR,immunofluorescence detection and viral gene sequencing,and genetic evolution analysis was then carried out.The result revealed that a BVDV strain was successfully isolated and named BVDV-GSLY.BVDV-GSLY was identified as a cytopathogenic(CP)biotype since BVDV-GSLY could produce cytopathic effect(CPE)on MDBK cells.The 5'-UTR and the Npro of BVDV-GSLY were amplified by RT-PCR and the PCR products were the expected sizes,respectively.Immunofluorescence assay showed that BVDV-GSLY could react with BVDV antibody to generate specific fluorescence.The virus titer was 107.8TCID50/0.1 mL when BVDV-GSLY passaged 12 times in MDBK cells.The genetic evolution analysis of the 5'-UTR and Npro sequences indicated that BVDV-GSLY was closely related to SD1 strain,and the homology with SD1 strain was 95.5% in 5'-UTR and 91.5% in Npro,respectively.The phylogenetic tree also showed that BVDV-GSLY was in the same branch with SD1 strain,indicating that BVDV-GSLY belonged to BVDB-1a subgenotype.A BVDV subgenotype 1a strain was successfully isolated,and the research enriched the molecular epidemiology of BVDV and provided the theoretical foundation for prevention and control of BVDV in Gansu Province and the development of BVDV vaccine.
  • ZHONG Mei, WANG Jikun, CHAI Zhixin, WU Zhijuan, ZHONG Jincheng
    Complement C1q(complement 1q)protein consists of three polypeptide chains,A,B and C,C1q plays an important role in oxidative stress,cell apoptosis,and maintaining the stability of the internal environment of the organism.To explore the expression patterns in various tissues at different ages,and will lay the foundation for the further understanding of the functions of C1q proteins of yak.Total RNA were extracted from different tissues,including heart,spleen,lung,brain and cerebella.The mRNA expression levels of C1QA, C1QB,C1QC genes were determined by Quantitative Real-time PCR(RT-qPCR).SPSS 22.0 software was used to analyze the significant difference between different tissues.RT-qPCR results indicated that C1QA, C1QB and C1QC genes were expressed in all tissues.The expression trend of different age groups was basically the same.The expression levels in lung of C1QA,C1QB and C1QC genes were significantly higher than spleen,heart,brain and cerebella(P<0.05);in spleen was significantly higher than heart,brain and cerebella(P<0.05);in heart was significantly higher than brain and cerebella(P<0.05),and there was no significant difference in the expression level between brain and cerebella(P >0.05).The results also showed that the expression levels of C1QA, C1QB and C1QC genes were the highest in 1.5-year-old yak lungs,which were significantly higher than those in spleen,heart,brain and cerebella(P<0.01).With the growth of the yak, C1QA gene expression level in most tissues was decreased significantly.In stark contrast,we found that gene expression level of C1QC was increased significantly in all tissues.While,the mRNA expression of C1QB gene was dynamic,with the level dramaticlly increasing and then significantly decreased with aging.In a word,in the high-altitude adaptationregulation of yak, C1QA,C1QB genes maybe involved through the down-regulation of tissue expression level,and C1QC maybe through the up-regulation.
  • LI Jiajun, LI Jianzhen, LI Wanqiang, WANG Zhenhua, WANG Junrui, XIAO Dan, PAN Kangcheng, YANG Miao
    To explore the molecular mechanism of the effect of Bacillus cereus PAS38 on the immunity in broilers. 60 7-day-old broilers were divided into two groups,the control group was fed with basal diet,and the treatment group was fed with basal diet containing Bacillus cereus PAS38 1×106 cfu/g. Six broilers were randomly selected from each group at the age of 42 days,spleens were taken and total RNA was extracted,then,and reversed transcription to cDNA. SSH and ACSSH techniques were used to construct splenic differential gene library and screen immune-related genes respectively. The results showed that 119 differentially expressed genes were screened by SSH,including 63 up-regulated genes and 56 down-regulated genes,and including 9 immunoregulatory genes,while five differentially expressed genes were screened by ACSSH,all of which were down-regulated genes,and no immunoregulatory genes were found. The results indicated that feeding Bacillus cereus PAS38 could affect the expression of immune genes such as JCHAIN, FTH1 and P2RX7 in the spleen of broilers,SSH library was superior to ACSSH library,and ACSSH technology was defective.
  • WANG Haiwei, WANG Zhen, LI Xing, LI Jing, XIE Huadong, WANG Qigui
    In order to study the effects of feeding methods and feeding time on the quality of hybrid high-quality meat chicken,two cross combinations of DZ and QX of Chongqing local chicken were selected to analyze the differences of slaughter performance and meat quality traits under different feeding methods(cage and free range)and different feeding time(120,150 days old),and the composition of free amino acids and flavor nucleotides was determined TAV and EUC were used to evaluate the taste characters.The results showed that feeding methods,feeding days and cross combinations could affect the growth performance,slaughter performance and meat quality of high-quality broilers.The results showed that the meat performance of DZ and QX hybrids in cage was higher than that in free range(P<0.01),and that at 150 days was higher than that at 120 days(P<0.05);the meat color in free range was better than that in cage,and that in 150 days was better than that in 120 days;there was no difference in muscle shear force between different feeding methods and feeding ages(P>0.05);the content of total delicious amino acids in free range was higher than that in cage(P<0.05),and that in 150 days old was higher than that in 20 days old(P<0.01).The content of IMP in free range was higher than that in cage culture(P<0.05),the contents of IMP,GMP and AMP in 150 day old were higher than 120 day old(P<0.01,P<0.05). The results showed that 150 days old was better than 120 days old,free range rearing was better than cage rearing,DZ combination was better than QX combination,and the highest EUC was 135.52 mg/g at 150 days of age.Feeding time was more significant for improving meat quality and taste of DZ and QX cross combinations,which provided scientific basis for the development and utilization of local chicken resources and the establishment of related chicken quality evaluation methods.
  • ZHANG Ruiguo, LI Shaobin, WANG Jiqing, LIU Xiu, LUO Yuzhu
    In order to study the polymorphism of FGF18 gene in goats and its effect on some cashmere traits, PCR-SSCP combined sequencing method were used to analyze three regions(Exon 3, Exon 4 and Exon 5) of FGF18 gene in three goat breeds(Longdong cashmere goat, Chaidamu cashmere goat, and Zhongwei goat), then the association between gene polymorphism and variation of Longdong cashmere goat traits was analyzed. The results showed that FGF18 gene was conserved among the three goat breeds, and only one synonymous mutation was found in Exon 5(c.498T>C). Three genotypes EE, EF and FF caused by c.498T>C locus were detected in all three goat breeds, and the distribution in the three goat breeds was at Hardy-Weinberg equilibrium with respect to this SNP. Allele E was the dominant allele in all three goat breeds; the dominant genotypes of Longdong cashmere goats, Chaidamu cashmere goats and Zhongwei goats were EF, EE and EF, respectively. The cashmere yield and height of Longdong cashmere goats with FF genotype goats were significantly higher than those goats with EE genotype and EF genotype(P<0.05). The results showed that the FGF18 gene was moderately polymorphic in the three goat populations and the c.498T>C site was significantly associated with the cashmere yield and the height of down layer of the Longdong cashmere goats. It was suggested that FGF18 gene can be used as a candidate gene for molecular improvement when these two traits are used as the breeding targets.
  • HAN Xinrong, WU Huiguang, WU Jianghong, WANG Guofu, GAO Shuxin
    To identify differentially expressed genes related to fat deposition in Angus and Chinese simmental cattle and analyze differentially expressed gene functions and pathways, the total RNA was extracted from the backfat, then sequenced by BGISEQ-500. The differentially expressed genes were screened by bioinformatics analysis, GO and KEGG enrichment analysis were used to study the function of differentially expressed genes and the candidate genes related to fat deposition were screened. The accuracy of RNA-seq results was verified by qRT-PCR. The results showed that there were 1 296 significant differentially expressed genes in Angus cattle compared with Chinese simental cattle, including 802 up genes and 494 down genes. Differential transcripts of GO function enrichment analysis revealed that molecular functions, cell components, and biological processes were enriched to 890 entries, 3 149 entries,and 2 205 entries. KEGG analysis showed that the differential transcripts were significantly enriched in 6 pathways. The cAMP pathway associated with fat deposition was analyzed, and the differentially expressed genes associated with fat deposition were screened out, the genes DKKL1, ACACB and PTGS2 were verified by qRT-PCR, and they were preliminarily identified as candidate genes for fat deposition. At the same time, a large number of differentially expressed genes were obtained by sequencing the backfat tissues of Chnese simmental cattle and Angus cattle, which laid a foundation for the subsequent screening of genes related to fat deposition.
  • HAI Zhuo, XIONG Xianrong, MA Hongcheng, HUANG Xiangyue, MIN Xingyu, LI Jian
    The objective of this research was to explore the expression pattern and molecular mechanism of histone demethylase 7A(KDM7A) in the reproductive development of yak.Yak was used as research object. The coding sequence of KDM7A gene was cloned by RT-PCR. The protein structure and function of KDM7A were analyzed by bioinformatics software, and the KDM7A gene was detected by Real-time quantitative PCR (RT-qPCR)in theovary, kidney,stomach,small intestine,muscle, heart, uterus, spleen, lung and liver tissue. The expression pattern of KDM7A mRNA was analyzed during the meiotic maturation of yak oocytes.The 2 704 bp KDM7A gene of the yak was cloned, and the CDS was 2 409 bp in length and encoded a total of 802 amino acids. Compared with other species, the base sequence of yak KDM7A was higher homology with cattle, bison and goat. Yak KDM7A gene was widely expressed in various tissues,in which the uterus KDM7A mRNA relative expression was the highest and significantly higher than other tissues except the stomach (P<0.05), while the relative expression of KDM7A in kidney and muscle tissue was significantly lower than that in other tissues (P<0.05).There was spatiotemporal dynamic expression feature of the KDM7A gene during the meiosis of oocytes. The expression level of KDM7A in meiosis metaphase Ⅱ (MⅡ) stage was significantly higher than that in meiosis metaphase Ⅰ(MⅠ) and germinal vesicle (GV) stages of yak oocytes (P<0.05).In summary, this study suggested that KDM7A gene was highly conserved in genetic evolution and involved in meiosis process of oocytes, which had a dynamic expression pattern during the reproductive development of yak. This study provided relevant basis for further exploring the action mechanism of yak KDM7A in the process of reproduction.
  • LIANG Yugang, CHEN Yisha, CHEN Lu, MENG Xiangjie, CHEN Can, HUANG Huang, YU Zhengjun
    This research studies the effect of the combination of rice ridge cultivation and rice-fish-chicken cultivation on the growth of the rice root system. On the basis of the researches carried out by other scholars on rice ridge cultivation, rice-fish cultivation, and rice-chicken cultivation, this research came up with a creative planting and breeding technique that features combining chicken and fish breeding with rice ridge cultivation. This research was based on a field comparison experiment that consisted of conventional rice ridge cultivation (CK), rice ridge cultivation with fish breeding (RF), rice ridge cultivation with chicken breeding (RC), and rice ridge cultivation with fish and chicken breeding (RFC). The growth trait and biological activity of the rice root system under rice-fish-chicken ridge cultivation was explored. The results showed that there were differences in the structure and biological property of the roots among different cultivation models during the two-year experiment. RFC and RC had no insignificant differences in root dry matter, root shoot ratio, root volume, root number, maximum root length, and root antioxidant activity and bleeding sap after full heading, which were slightly higher than CK (some indicators had significantly difference). Compared with CK, the root volume of RFC and RC was increased by 0.77%-14.05% and 0.10%-13.88% respectively, and the antioxidant activity was increased by 2.15%-13.48% and 0.64%-10.05% respectively. Compared to CK, RF showed a downward trend of all indexes related to the root structure, the antioxidant activity, and bleeding sap, in which the root dry matter, root volume, the antioxidant activity, and bleeding sap were reduced by 24.62%-50.70%, 7.80%-47.45%, 10.49%-21.68% and 24.39%-39.25% respectively. In conclusion, rice-fish-chicken ridge cultivation and rice-chicken ridge cultivation had a higher level in root morphological index compared with conventional rice ridge cultivation,such as root volume, root shoot ratio, root number and maximum root length. The biological activity of root system was improved after full heading stage and the aging of the root system at later stage was reduced, thus ensuring the growth of the overground parts of the rice and its yield.
  • ZHAO Liling, WANG Hui, CHAI Zhixin, WANG Jikun, WANG Jiabo, WU Zhijuan, XIN Jinwei, ZHONG Jincheng, JI Qiumei
    The aim of this study was to clone and to identify the yak lncFAM200B, which might lay the foundation for exploring its regulatory role in yak muscle and fat during development. The primers for lncFAM200B gene were designed and used for the full-length cloning with the gluteal cDNA as template. The protein coding potential of lncFAM200B was predicted using the online prediction software CPC and CPAT, and then identified by prokaryotic expression assays. The tissue expression profile was analyzed by Real-time quantitative PCR (RT-qPCR), the target genes of miRNAs, which were interactions with lncFAM200B, were predicted by TargetScan 7.1 and miRanda. GO enrichment and KEGG pathway analysis were conducted with DAVID online software. The results showed that the full-length of yak lncFAM200B was 531 bp, which was 59 bp longer than that in cattle.Bioinformatics predicts results showed that its coding potential was -1.287 4, and prokaryotic expression assay further confirmed that lncFAM200B had no protein coding ability, suggesting that yak lncFAM200B was a real lncRNA. Tissue expression profiling showed that the expression of lncFAM200B had a higher level in yak lung tissue. The lncFAM200B interaction miRNA targets analysis revealed that many genes, such as Sirt1, SCD5, KLF9, PTEN and MYPN, were related to muscle and fat development.Taken together, the present study laid the foundation for further research on the function and regulatory mechanism of yak lncFAM200B in muscle and fat development.
  • CHI Yongdong, WANG Yong, ZHAO Yue, ZHU Jiangjiang, LIN Yaqiu
    This experiment was conducted to study the sequence characteristics, physicochemical properties of goat C/EBPα, C/EBPβ and C/EBPδ genes and proteins, and the expression patterns in goat tissues and in the differentiation of goat intramuscular precursor adipocytes.The C/EBPα, C/EBPβ and C/EBPδ of goat were cloned by RT-PCR method and the biological characteristics were analyzed using the relevant biological software. qPCR was used to detect the expression levels of C/EBPα, C/EBPβ and C/EBPδ mRNA in different tissues and preadipocyte of goat. The results showed that the sequence of C/EBPα was 1 062 bp, C/EBPβ was 1 056 bp and C/EBPδ was 873 bp. The proteins encoded by C/EBPα, C/EBPβ and C/EBPδ were all unstable hydrophilic basic proteins, and had no signal peptide and transmembrane domain. The expression of three genes was detected in different tissues of goats. The expression level of C/EBPα was highest in subcutaneous fat, and the expression pattern of C/EBPβ and C/EBPδ was similar, and the expression level was highest in lung.The expression patterns of C/EBPα and C/EBPδ were similar in the differentiation of adipose precursor cells, and the expression level was the highest at 0 d after differentiation, and the expression level was decreased with the increase of differentiation time. C/EBPβ was found in intramuscular precursor fat. The expression of C/EBPβ in the cells increased with the number of days of induced differentiation, and the expression level was highest at 7 days of differentiation. Based on the above studies, it is speculated that the three genes play an important role in the process of goat adipose differentiation.
  • LI Xin, HE Xiaofang, ZHANG Hao, ZHENG Jianying, WANG Yuxue, LIN Yaqiu, WANG Yong, ZHU Jiangjiang
    The aim of this experiment was to obtain the sequence of goat PHKG1,to clarify its biological characteristics,and to elucidate the tissue expression level and cell expression profiles of subcutaneous and intramuscular adipocytes. Jianzhou Big-eared goats were selected as experimental materials,the heart,liver,spleen,kidney,lung,longissimus muscle,subcutaneous fat were harvested after slaughtering,and the total RNA was extracted from these tissues. The sequence of PHKG1 gene was cloned by Real-time PCR(RT-PCR),the biological characteristics were analyzed by online tools,and the tissue and cell temporal expression of PHKG1 was detected by Real-time quantitative PCR (qPCR). The results showed that the cloned PHKG1 gene sequence was 1 233 bp in length,of which the CDS region was 1 164 bp. The PHKG1 gene encoded 387 amino acids,forming an unstable hydrophilic acidic nontransmembrane protein with no signal peptide,and subcellular localization indicated that it was mainly present in the cytoplasm. The amino acid sequence similarity between goats and sheep,cattle,pigs,horses and humans was above 90%,indicating that PHKG1 was highly conserved in different species. Constructing phylogenetic tree showed that goats and sheep were in the same branch,in line with the evolutionary laws of species. The PHKG1 gene was widely expressed in goat-tissues and was the highest in the longissimus muscle(P <0.01)and the expression levels in intramuscular adipocytes at 60 h and subcutaneous adipocytes at 96 h of adipogenic differentiation were significantly(P<0.05)and extremely significantly(P<0.01)higher than those in the preadipocytes. This study laid the foundation for further study of the role of PHKG1 in the differentiation of goat adipocytes.
  • YIN Shi, QIN Wenchang, WANG Bin, YANG Liuqing, ZHOU Jingwen, LI Jian
    This research was conducted to clone the SIRT1 gene in yak, predicated the structure and function of SIRT1 protein, identified its expression pattern in different tissues and testis of different ages. Nine healthy male yaks were divided into in calve (0.5 to 1 year), juvenile (2 to 3 years) and adult (4 to 5 years) groups (three in each group). Three samples from the same age were considered as biological duplications. The samples of liver, heart, spleen, brain, muscle, small intestine from adult sage and testis from three stages were collected. Total RNAs in different testis were extracted respectively and the sequence of yak SIRT1 gene was cloned by reverse transcription PCR(RT-PCR). Different bioinformatics softwares were used to predicate the homology of SIRT1 gene sequence, amino acid sequence, structure and properties of SIRT1 protein. Real-time PCR was applied to identify the mRNA expression profile of SIRT1 in different tissues of yak and chromogenic in situ hybridization(CISH) was applied to analyse the localization of SIRT1 mRNA in yak testis. Western Blot was conducted to detect the expression pattern of SIRT1 protein in yak testis of different stages. The open reading frame (ORF) of yak SIRT1 was 1 866 bp and encoded 621 amino acids. Sequence alignment analysis showed yak SIRT1 had a high sequence similarity with other mammals. Structure prediction indicated that SIRT1 was a hydrophilic liposoluble protein with a conserved silent information regulator 2 (Sir2) catalytic core domain. The mRNA of SIRT1 was widely expressed in different tissues of yak and was highly expressed in testis, ovary and liver. The testicular SIRT1 protein was highly expressed in calves and the expression decreased significantly with the growing of age. SIRT1 mRNA localized in multiple kinds of cells except spermatid. This research provides a theoretical basis for further understanding the mechanism of SIRT1 in regulating yak testicular development and is of great importance in providing new insights in improving yak reproductive performance in the future.
  • ZHONG Mei, WANG Jikun, CHAI Zhixin, WANG Hui, WANG Jiabo, WU Zhijuan, XIN Jinwei, ZHANG Chengfu, JI Qiumei, ZHONG Jincheng
    Complement C1q(Complement 1q) protein consists of three polypeptide chains,A,B and C,which plays an important role in stabilization of homeostasis,oxidative stress,and glucose and lipid metabolism. The aim of this study was to clone the CDS of C1QA,C1QB and C1QC genes and detect their expression patterns in various tissues,and then explore the molecular mechanism of these genes in Yak high altitude adaptation. Total RNA were extracted from various tissues,including heart,liver,spleen,lung and kidney. The mRNA expression levels of C1QA,C1QB and C1QC genes were determined by Real-time PCR. The cDNA length of C1QA,C1QB and C1QC genes were 735,744 and 732 bp,which encoded 244,247 and 243 amino acids,respectively. Bioinformatics analysis found that C1QA,C1QB and C1QC were stable hydrophilic proteins,which dominated by glycine (Gly) and proline (pro). The C1QA,C1QB and C1QC contained C1Q domain,signal peptide,and without transmembrane domain,which distributed in extracellular region. There were 18,21 and 15 phosphorylation sites in the three protein sequence,respectively. For the secondary structure of the three proteins,they were mainly composed of irregular curls,and the proportions were 61.85%,63.97% and 66.67%,respectively. Furthermore,for the expression patterns of C1QA and C1QB,lung and spleen had higher expression levels than heart,liver and kidney (P <0.01),for C1QC, heart,liver,spleen and kidney had lower expression levels than lung(P <0.01). This study may provid basic data for the functional and molecular mechanism in Yak high altitude adaptation.
  • XIANG Hua, HUANG Ren, ZHU Jiangjiang, YUE Hua, TANG Cheng, ZHANG Huanrong
    This experiment was conducted to study the localization of ORFV129 ankyrin repeat proteins from Orf virus on transfected cells. According to the sequence of ORFV SY 17 complete gene (GenBank accession number:MG 712417.1), primers were designed to amplify the 129 gene of ORFV,and ORFV129 gene was cloned into pEGFP-N1 eukaryotic expression vector directionally. The recombinant expression plasmid was identified by restriction enzyme digestion analysis,sequencing and named as pEGFP-ORFV129. Preliminary bioinformatic analysis of ORFV129 gene was predicted and analyzed,including the amino acid sequence,protein transmembrane region and subcellular localization. The recombinant plasmid pEGFP-ORFV129 was transfected into the primary testis cells of newborn goat,and identification of ORFV129 protein expression by Western Blot. Subcellular localization of ORFV129 was examined with laser confocal microscopy after DAPI staining. The sequence of ORFV129 gene was amplified and cloned successfully,with a total length of 1 551 bp. Amino acid sequence analysis showed that the gene encoded a protein containing 516 amino acids,composing of 8 multiple copies of the ankyrin repeat motifs(ANKs). The transmembrane region analysis showed that ORFV129 had no transmembrane regions,and subcellular localization predicted that the protein was mainly located in the cytoplasm. The result of Western Blot showed that recombinant plasmid pEGFP-ORFV129 transfected into the primary testis cells of newborn goat was successfully expressed. Subcellular localization experiment results showed that the ORFV129 protein was located in the cytoplasm,which was identical with the predicted ones.The results might lay a foundation for further research the function of ORFV129.