Deubiquitinating enzyme is a reverse modulating enzyme of ubiquitination pathway and is involved in the regulation of protein degradation. In order to explore the gene function of deubiquitinating enzyme family member WTG1 (wide and thick grain 1) in wheat (Triticum aestivum), the physical and chemical properties of TaWTG1 encoded protein as well as structure were analyzed by bioinformatics method in this study. The prokaryotic expression vector pET28a-TaWTG1 was constructed and transformed into Escherichia coli expression strain BL21(DE3). The induced-expression conditions, including culture temperature, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration and induction time, were optimized. The solubility of recombinant protein His-TaWTG1 was analyzed, and its deubiquitination activity was verified in vitro after purified by nickel column. The results showed that the TaWTG1 protein consists of 319 amino acid residues with a theoretical molecular weight of 35.38 kD and an isoelectric point of 4.62. The tertiary structure of the protein is mainly composed of α-helix, which contains the otubain conserved domain of the ovarian tumor-related proteases (OTU)-related protease family. The optimal induced expression conditions of the recombinant protein His-TaWTG1 were 0.4 mmol/L IPTG at 28 °C for 6 h. The recombinant protein His-TaWTG1 mainly existed in supernatant in compatible form. Western blot analysis identified the recombinant protein purified by nickel column as the target protein. In vitro activity assay showed that TaWTG1 had deubiquitination enzyme activity and could cleave lysine (K) 48 and K63-linked tetrameric ubiquitin chains. These results provide general data for further functional study of TaWTG1 gene in wheat.

As an important pathogenic factor, melanin directly affects the infection ability of plant pathogenic fungi, and the transcription factor MR (melanin regulation factor) plays an important role in melanin biosynthesis. In this study, the transcription factor gene Stmr1 (GenBank No. NW007359937) in Setosphaeria turcica was identified and cloned through genome BLAST. The gene was 3 282 bp in length and consisted of 3 introns and 4 exons, encoding 1 021 amino acids. The homology analysis of amino acid sequence indicated that the encoded protein StMR1 had the highest similarity to the MR transcription factors of Alternaria brassicicola and Cochliobolus heterostrophus. It contained 2 Cys2His2 zinc finger motifs and 1 Zn(Ⅱ)2Cys6 zinc cluster protein. Self-activation assay showed that StMR1 had high transcriptional activation activity and was located in the nucleus by fluorescence localization. Detected by qRT-PCR, the expression of Stmr1 gene was significantly different in the 5 stages from germination of conidia to penetration, and the expression was significantly up-regulated in the invasive nail stage, so it was speculated that it was involved in infection and expansion of the pathogen. The results of this study provide a theoretical basis for further exploration of the mechanism of regulation of melanin synthesis and StMR1 transcription factors in germ infection.

Reducing the content of seed glucosinolates (GSL) has a positive impact on the seed quality of Brassica napus. In this study, genome-wide association study (GWAS) of seed GSL content was performed by using the 60K Brassica infinium single nucleotide polymorphism (SNP) array in 203 oilseed rape accessions. A total of 20 SNPs were detected significant associated with GSL content and located on the A02, A03, A09, C03, C08, and C09 chromosomes. A linkage disequilibrium analysis was performed on flanking sequence of these significantly associated SNP loci, and a haplotype region (57830409~58283210 bp; r² = 0.96) was detected significantly associated with GSL content on the C03 chromosome. This haplotype region carrying an orthologues of Arabidopsis gene BCAT4 (branched-chain aminotransferase 4) was involved in GSL biosynthesis process. Based on the above results, regional association analysis revealed BnBCAT4-C03(BnaC03g68450D) gene region structural variation effected the accumulation of GSL in this haplotype region by genome-wide resequencing data of 50 accessions. Meanwhile,co-expression network analysis suggested BnBCAT4-C03 gene relationship with genes in the pathway of glucosinolates synthesis that formed molecular networks regulation in the synthesis and accumulation of GSL content. Our results would be benefit for the development of haplotype functional markers to further reduce GSL content in rapeseed.

In plant cell wall, the ferulic acid can form covalent cross-linking in lignin-ferulic acid-arabia xylan complexes with hemicellulose and lignin, which is an important molecular basis for the formation of anti-degradation barrier in lignocellulose. Ferulic acid esterase (FAE) can break the ester bond between hemicelluloses as well as hemicellulose and lignin. In this study, the codon of a thermophilic ferulic acid esterase gene from Clostridium thermocellum was optimized and then constructed into the plant binary expression vector containing different signal peptide coding sequence which the thermophilic FAE protein expressed in Arabidopsis thaliana targeting to the cytoplasm, apoplast, endoplasmic reticulum, chloroplast or mitochondria, respectively. The results indicated that the thermophilic FAE was successfully expressed in different transgenic lines, and with the highest value when expressed in the cytoplasm, its enzyme activity at 70 °C was much higher than that at 25 °C (P<0.05). The plant height, fresh weight and 100-seed weight between different transgenic lines and wild type were not significantly different, but an early flowering was observed in the lines with FAE proteins expressed in chloroplasts or mitochondria. This study can provide new ideas and methods for efficient resource utilization of energy plants, pastures and crop straws.

Heat shock transcription factors (Hsfs) are important components in response to stress signals. Carnation (Dianthus caryophyllus) variety 'Fancy' was used as experimental material to clone the complete coding sequence of DcHsfA4 (GenBank No. MN096327). The full length of the sequence was 1 173 bp and encoded 390 amino acid. Bioinformatics analysis showed that the molecular formula of DcHsfA4 protein was C₁₉₆₃H₃₀₅₁N₅₇₇O₆₂₅S₈, the molecular weight was 44.99 kD, the theoretical isoelectric point was 5.48, the fat coefficient was 71.95, and the instability coefficient was 49.72, which was predicted to be unstable protein. Hydrophobic/hydrophilic prediction indicated that the DcHsfA4 protein was a hydrophilic protein. Protein transmembrane analysis revealed that DcHsfA4 was a non-transmembrane protein. The secondary structure prediction showed that the amino acid composition of DcHsfA4 included α-helix (42.56%), extended chain (6.67%), β-sheet (3.08%), and irregular curl (47.69%), which belonged to an irregular structure. Amino acid sequence alignment revealed that DcHsfA4 contained a highly conserved DNA binding domain, in addition, two hydrophobic hepated repeat, a nuclear localization signal sequence and a C-terminal activation domain. Phylogenetic analysis indicated that DcHsfA4 had the highest homology with Arabidopsis thaliana AtHsfA4. The expression characteristics of DcHsfA4 under different abiotic stress treatments were analyzed by qRT-PCR. The results showed that the 42 ℃ stress, ABA or mannitol treatment significantly increased the expression level of DcHsfA4. Moreover, the expression pattern of DcHsfA4 was different at 4 ℃ stress. The transcripts of DcHsfA4 were up-regulated by drought treatment for 24 h and NaCl treatment for 12 h. The results provide a basis for further exploration of the characteristics of carnation heat shock transcription factors and the biological functions in response to stress.

Chinese fir (Cunninghamia lanceolata) has the characteristics of naked ovule, which provides favorable conditions for colchicine to enter the ovule quickly and improve induction efficiency. In order to study the induction effect of colchicine on ovule of Chinese fir, the gene expression changes were analyzed by transcriptome sequencing technique, and the expression characteristic of related genes were verified by qRT-PCR. The results showed that, for colchicine treatment group and control group, 6.39 and 6.96 Gb original data were obtained by transcriptome sequencing, respectively. A total of 60 790 unigenes were obtained after de novo assembly, and 1 430 differentially expressed genes were screened between the two groups, from which 4 spindle formation-related genes, 8 DNA replication-related genes, and 6 cell cycle-related genes were obtained and all showed significant down-regulated expression. qRT-PCR analysis showed that the gene expression trends of NEDD1 (neural precursor cell expressed, developmentally down-regulated gene 1), MCM3 (minichromosome maintenance complex component 3), MCM7, CDC6 (cell division cycle 6) and ATXR6 (trithorax-related protein 6) were consistent with those of transcriptome data. In this study, colchicine was directly acted on ovules and differentially expressed genes were screened, which provides basic information for further studying the molecular mechanism on polyploidy induction by colchicine.

As the demand for goat (Capra hircus) products increased, the long-distance transportation of goats increased, and the transport stress would cause weight and meat quality decline, immunity decline, disease and even death of goats, and the management of goats during the transport would affect the profitability and animal welfare. The effects of transport stress on the microstructure and ultrastructure as well as the expression of heat shock proteins (HSP) of liver and kidney of goats were investigated. 12 healthy male goats from West Jiangxi were randomly divided into control group, 2 h transport stress group and 6 h transport stress group. HE staining, transmission electron microscopy, immunohistochemistry and Western blot were used to analyze the degree of injury and the distribution and expression of HSP27, HSP70 and HSP90 of liver and kidney in goats at different transport time. The results of microscopic and ultramicroscopic observation showed that different degrees of injury of the liver and kidney in the transportation stress group occurred. In the liver, hepatocytes showed obvious granule and vesicular degeneration, and part of chromatin aggregated in cell nuclei. In the kidney, the structure of renal tubules and collecting tubules were collapsed, the glomeruli were hyperemic and swollen, and the renal capsule lumen was narrowed. Moreover, the mitochondria in the cells of liver and kidney were both swollen and deformed, and the cristae was fractured and disappeared. The number of mitochondria was reduced, and some mitochondria even underwent membrane structure rupture due to swelling and vacuolation. The lesions of 6 h transport stress group were more serious, some liver cells showed nuclear lysis, and the damage degree of lumen and glomerular cells was also more serious. Immunohistochemical results showed that 3 HSP were mainly expressed in the cytoplasm of hepatocytes near the central vein and portal area, while in the kidney, the expressions were mainly in the renal tubules and collecting tubules, and the expressions were stronger in the transport group. In addition, HSP27 also showed strong positive results in vascular endothelium and bile duct cells in the liver and renal corpuscles in the transport group. Western blot results showed that there were no significant differences in the expression of these 3 HSPs in the liver and kidney between 2 and 6 h transport stress group (P>0.05), and the expression of HSP90 in liver between transport group and control group was also no significant differences (P>0.05). The expressions of HSP27 and HSP70 in liver in 2 and 6 h transport stress groups were extremely significantly (P<0.01) and significantly (P<0.05) higher than the control group, respectively. In the kidney, the expressions of HSP27 and HSP90 in 2 and 6 h transportation stress groups were both extremely significantly (P<0.01) higher than the control group, and the expressions of HSP70 in 2 and 6 h transport stress groups were significantly (P<0.05) and extremely significantly (P<0.01) higher than the control group, respectively. In conclusion, transport stress caused severe pathological damages to the liver and kidney of goats, and the damages worsened with the extension of transport time. These 3 HSP were distributed differently in liver and kidney. The expression levels of HSP27 and HSP70 in goat liver and kidney were both significantly increased after transport stress, which improved the stress resistance of hepatorenal cells. The results provide theoretical reference for the further study of solutions of goat transport stress.

Rainbow trout (Oncorhynchus mykiss) is a typical cold-water fish, which has poor tolerance to higher water temperature. With the global warming, the impact of heat stress on rainbow trout is becoming more and more serious. To further understand the mechanism of heat shock proteins (Hsps) in rainbow trout during heat stress, in this study, 36 Hsp40 family genes were identified by bioinformatics analysis of rainbow trout genome database and RNA-seq data. The physiochemical properties, Phylogenetic, gene structure and gene expression profile of rainbow trout Hsp40 family gene were systematically analyzed at the whole gene level. The results showed that these genes were divided into three subtypesⅠ,ⅡandⅢ according to their structural similarities. The number of amino acid ranged from 190 to 760, and the theoretical pI distribution ranged from 5.01 to 9.09. Gene structure analysis showed that the Hsp40 family genes of rainbow trout had only 1 exon. Subcellular localization analysis showed that the Hsp40 gene was mainly expressed in cytoplasm, nucleus and mitochondria, and the secondary structure was mainly alpha-helix and irregular curl. The expression profile of Hsp40 in rainbow trout was analyzed by RNA-seq data. The results showed that 8 and 10 Hsp40 genes were up-regulated in liver and head kidney, respectively, and in total of 7 Hsp40 genes were up-regulated in both tissues. The significant expression regulation of Hsp40 family genes after heat stress indicates that Hsp40 genes are involved in the heat stress of rainbow trout, which provides theoretical basis and basic data for studying the regulation mechanism of Hsp40 family gene members against heat stress.

Secreted insecticidal protein (Sip) is a secretory insecticidal protein of Bacillus thuringiensis (Bt) insecticidal family, which is toxic to Coleoptera larvae. Colaphellus bowringi is a common pest of Cruciferae. Sip protein has high insecticidal activity against the Colaphellus bowringi. In this experiment, the isotope relative labeling and absolute quantification technique (iTRAQ) was used to analyze the differential expression protein of Colaphellus bowringi protein, and the differential proteins were screened and subjected to functional annotation and enrichment analysis. Quantitative analysis was performed by Proteinpilot^TM V4.5 software, and Gene Ontology (GO) function annotation, Cluster of Orthologous Groups of proteins (COG)function annotation and KEGG analysis were performed, and functional enrichment analysis (GO, KEGG enrichment) was performed on all significantly differential proteins. Compared with the control group, 47 proteins were identified in the experimental group with quantitative information, including 27 up-regulated proteins and 20 down-regulated proteins. Through GO function annotation, COG and KEGG analysis, it was found that these differential proteins mainly involved molecular functions such as binding and catalytic activity, among which the functions unique to up-regulated proteins were electron carrier activity, antioxidant activity and molecular converter activity. These proteins mainly performed biological processes such as cell processes and metabolic processes, in which only up-regulated proteins were involved in death, immune system processes, exercise, and viral replication. These proteins mainly consisted of cells, cell parts, organelles, etc. It was predicted that these differential proteins might have functions such as post-translational modification, protein turnover, chaperone, translation, ribosome structure and biosynthesis, translation, ribosome structure and biosynthesis, etc. In the biochemical metabolic pathway and signal transduction pathway, the up-regulated protein and the down-regulated protein shared a common pathway for the biosynthesis of secondary metabolites. The pathways that were important for up-regulating differential proteins were metabolic pathways, microbial metabolism in different environments, secondary metabolic biosynthesis, and pyruvate metabolism. The specific pathways for down-regulating proteins included the ribosomal pathway, systemic lupus erythematosus, and pancreatic secretory pathway. Through enrichment analysis, the main biological functions of these differential proteins were found to be nuclear nucleosomes, non-membrane bounded organelles, intracellular non-membrane bounded organelles, nucleosomes, ribosomes, COPI envelope vesicles, protein-DNA complex, secretory granules, nuclear chromatin, Golgi heap, etc. After treatment with Sip toxin protein, some receptors common to Bt toxin were found to be differentially expressed, among which alkaline phosphatase (Alp), G-protein, cadherin and aminopeptidase N (APN) were differentially expressed. The results provide a reference for the study of the mechanism and for the proteomics study of Sip toxin.

Since 2015, the number of cases of inclusion body hepatitis and pericardial effusion in domestic chickens (Gallus domesticus) with Fowl adenovirus serotype 4 (FAdV-4) infection has been increasing. In order to prepare a serological diagnostic antigen of FAdV-4 with good reactiongenicity, the FAdV-4 Fiber2 whole gene synthesis sequence after codon optimization was amplified and cloned into the pCold expression vector to construct the prokaryotic expression vector pCold-Fiber2. Then pCold-Fiber2 was transformed into PG-Tf2.The supernatant induced by IPTG (isopropyl β-D-thiogalac toside) was purified and immunized rabbits (Oryctolagus cuniculus). The polyclonal antibodies were successfully prepared. The results of Western blot and indirect immunofluorescence assay (IFA) showed that the purified polyclonal antibodies could react specifically with Fiber2 protein and FAdV-4. It was proved that Fiber2 protein demonstrated good immunogenicity. The indirect ELISA method for FAdV-4 antibody showed that the sensitivity and specificity of the method were 96% and 100%. Polyclonal antibodies with good Western blot and IFA reactivity were successfully prepared. An indirect ELISA method for detection of FAdV-4 was established with good reaction specificity, high sensitivity, simple operation and stable and reliable reaction results. This method would be suitable for monitoring, evaluating and detecting early latent infection of chicken group antibody level. It provides basic data for the development of serological diagnosis technology and prevention and control of the disease.

The livestock wastewater can be reused by biogas slurry irrigation in farmland to solve the problem of subsequent disposal to a certain extent. But the loss of nitrogen and phosphorus will lead to the risk of groundwater pollution after long-term biogas slurry irrigation. In this study, the adsorption and migration behaviors of ammonia nitrogen (NH₃-N) and total phosphorus (TP) were investigated by simulated biogas slurry irrigation soil column with different soil media. Illumina MiSeq high-throughput sequencing technology was applied to study the soil bacteria diversity variation before and after biogas slurry irrigation. The results showed that different soil media had different effects on the adsorption capacity of NH₃-N and TP. Purple soil 1 had the strongest adsorption capacity of NH₃-N and TP, while river sand had the weakest adsorption capacity. Two days after biogas slurry irrigation, purple soil 1 quickly adsorbed NH₃-N in the surface soil, while the adsorption of ammonia nitrogen decreases in the bottom soil with time. After biogas slurry irrigation, NH₃-N in biogas slurry migrated to -25 cm soil layer in purple soil 1, and the concentration of NH₃-N in leachate was 11.52 mg/L; NH₃-N migrated to -45 cm soil layer in river sand, and the concentration of NH₃-N was 211.80 mg/L, which had a trend of continuous downward migration. The concentration of TP in leachate of purple soil 1 and sandy loam soil at -35 cm layer was 0.23 mg/L and 0.63 mg/L, respectively, which could adsorbed and fixed total phosphorus and prevented phosphorus from migrating downward. Meanwhile, the irrigation of biogas slurry reduced the bacteria diversity and abundance, and changes the community structure in purple soil 1. Shannon index and Chao index of purple soil 1 after biogas slurry irrigation were 5.510 and 1122.408, respectively. TM7 (Saccharibacteria) was the most abundant and Verrucomicrobia was the least abundant. This study provides a certain theoretical basis for the adsorption and migration process of nitrogen and phosphorus elements in soil after biogas slurry irrigation. The impact of soil bacteria diversity is also revealed after biogas slurry irrigation. In addition, the subsequent impact on purple soil and surrounding environment after biogas slurry irrigation is in favor of further comprehension.

China is abundant in peas (Pisum sativum), which have important processing and utilization value in food, medicine and other fields. In recent years, researches on its functional components and bioactive substances have become popular gradually. In this study, pea meal as raw material for ultrasound-assisted extraction was used to get the crude soluble polysaccharide, and the yeild of polysaccharide, measured by the method of phenol-sulfate, was 7.26%. The ultrastructural morphology of the soluble polysaccharides of pea with different purification levels was analyzed by scanning electron microscopy (SEM). By using different level of purification methods, the crude soluble polysaccharides extracted from pea were classified into 4, as follows, crude polysaccharide (CPP), deproteined polysaccharide (TPP), DEAE-sepharose Fast Flow ion-exchange chromatography polysaccharide (W-DEPP (water elution -diethylaminoethyl fast flow- ion exchange column chromatography), N-DEPP (NaCl elution -diethylaminoethyl fast flow- ion exchange column chromatography)) and sephadex G-100 chromatography polysaccharide (W-DE-GPP-a, W-DE-GPP-b, N-DE-GPP1-a, N-DE-GPP1-b). As the results, ultrastructural morphology observations were analyzed and summarized according to the characteristics of ultrastructural morphology of these different polysaccharides by changing three definitions, which were 100 fold, 1 000 fold and 10 000 fold, respectively. In this study, the soluble polysaccharides of pea with different purification levels were investigated by SEM, from which a series of photos were obtained preliminarily. The results of this study provide a reliable reference for the morphological characteristics and further studies of pea polysaccharides.

Since the birth of the gene editing technology CRISPR/Cas9, it has been widely used in the fields of gene knockout, knock-in, and base repair. But its inefficiency and off-target cutting, low security, greatly limits the traditional CRISPR/Cas9 application in high-resolution single-base editing. The emergence of the base editor (BE) based on traditional CRISPR/Cas9 overcomes these drawbacks. The cytosine base editor (CBE) converts C•G to T•A, adenine base editor (ABE) enables the conversion of A•T to G•C, enabling efficient single base substitutions without introducing double-strand breaks, which avoids an uncontrollable insertion or deletion mutation (Indels) induced by the traditional CRISPR/Cas9 non-homologous end joining (NHEJ). Most of the genetic diseases in human are caused by base mutations, and the emergence of single-base editing tools can correct a certain proportion of pathogenic SNPs to some extent. Therefore it has broad application prospects in animal model construction, functional genomics research, molecular breeding, clinical medicine, and translational medicine. In this paper, the principle, development, application, opportunities and challenges of the just emerging ABEs with lower off-target efficiency are reviewed, in order to provide reference for the research and application of single-base editing technology.

Copy number variation (CNV) might be one main factor that affect the phenotypic diversity and evolutionary adaptation of animals. It uses a variety of mechanisms to regulate the body, such as changes in gene dose and transcription structure. Since the first generation CNV map of the human (Homo sapiens) genome was constructed, CNV has been increasingly studied in humans and animals. At present, studies have shown that CNV is related to the pathogenic mechanism of some human diseases, genetic variation of animals and important economic traits of domestic animals. In this paper, the definition, formation mechanism, detection method and research status of CNV for livestock and poultry were described based on relevant research reports at home and abroad. The problems faced by the research on CNV for livestock and poultry were summarized and the application prospect was prospected. This review provides reference materials for further research on CNV.

In recent years, with the development of immobilized microbial technology, its application has been extended to various fields. In view of the importance of the immobilization technology of agricultural microbial agents in the agricultural field, this paper summarizes the research progress of agricultural microbial preparation immobilization technology in recent years, reviews the characteristics of immobilized microbial technology, summarizes the research situation of immobilized carrier of agricultural microbial agents, introduces the application research progress of immobilization technology of agricultural microbial agents, and discussed existing problems and prospects for the future. This review provides references for the preparation technology, carrier selection and application of agricultural immobilized microbial agents.

Functional research experiments on frogs (Xenopus laevis) have confirmed that TraB domain containing 2A (Tiki1) plays a decisive role in the process of head induction, but the function of Tiki1 in the early development of mammals and even humans (Homo sapiens) is not yet clear. Pigs (Sus scrofa) that are very similar to humans in physiology and pathology have become ideal mammalian models for studying Tiki1 functions. In this study, a pair of transcription activator-like effector nuclease (TALEN) plasmids tageting exon 4 of the pig endogenous gene Tiki1 were designed by TALEN technology. TALEN plasmids were validated at the level of porcine parthenogenetic embryos. The targeting efficiency and the affect on embryo development of TALEN plasmids injected at different concentrations were compared. Then plasmids transfection and screening of cells were carried out. In this study, a total of 8 sows were transplanted. 3 sows were pregnant and delivered smoothly, and a total of 12 live pigs and 1 stillborn fetus were obtained. The results showed that 4 of them belonged to the Tiki1 gene targeting positive pig model that expected by enzyme digestion and sequencing. 3 live cloned piglets were obtained from the first sow, and one of the 3 pigs (33.3%) was identified by enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. 6 live pigs and 1 stillbirth were obtained from the second sow. 2 of the 7 piglets (28.6%) were identified by the enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. 3 live cloned piglets were obtained from the third sow. One of the 3 piglets (33.3%) was identified by enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. The Tiki1 gene targeting positive pig model and WT mating were used to obtain 5 live F₁ generation piglets. The sequencing analysis showed that the constructed Tiki1 gene targeting positive pig model was inherited by the germline, not chimera. In this experiment, TALEN technology was used to modify the endogenous gene Tiki1 of pig genome for the first time. It provides a powerful tool for efficient gene editing in large animals and a technical basis for the establishment of various biomedical models and agricultural genetically modified pigs in the future.

Cryptochrome 1 (CRY1) gene is a member of mammalian clock genes. Its expressed protein is both a photoreceptor and a part of the circadian clock, which could be delivered to the effector via the humoral and neural pathways to regulate mammalian physiology, biochemistry and behavior. In this study, the samples (the hypothalamus, pituitary, pineal and testicular tissues) of yak were collected, and the sequence of CRY1 coding region was cloned and the expression of CRY1 in adult male yak reproductive axis and yak testes of different ages (30 d, 2, 4 and 6 years old) was analyzed by qRT-PCR (real-time fluorescent quantitative PCR), Western blot and immunohistochemical staining. The results showed that the CRY1 gene CDS was 1 712 bp in length and the ORF was 1 257 bp (GenBank No. MN460656), encoding a total of 418 amino acid residues, without transmembrane domain, and the encoded protein was a soluble protein. Tissue expression and localization revealed that CRY1 mRNA and protein were expressed in the hypothalamus, pituitary, testis and pineal tissues of yak, and the expression level in pineal tissue was significantly higher than other tissues (P<0.01). Immunohistochemical staining showed positive expression in the hypothalamic paraventricular nucleus, pituitary cells, testicular spermatogenic cells, pineal cells and glial cells. The expression of CRY1 mRNA and protein in the testis of 30 d was the lowest in the testis tissues of different age groups, and the expression level gradually increased with age. The results showed that the CRY1 gene was relatively more conservative in animal evolution and was expressed in the male yak gland axis and the yak testes of different ages, suggesting that it might play an important role in the reproductive process of yak. The above results provides the basis for the biological function of the yak biorhythm gene CRY1 and its related research on the regulation of yak reproductive physiology.

The central nervous system is an important regulatory system for the movement of various organs of the body. And the brain as a major part plays an important role in the animal growth and adaptation to the hypoxic environment. In order to investigate the expression of hypoxia-inducible factor 1α (HIF1α) and homolog of yeast ATG6 Beclin1 and their relationship between the process of hypoxia adaptation of adult yak (Bos grunniens) brain tissues, the expression of HIF1α and Beclin1 mRNA and protein in adult yak brain tissues were detected by qRT-PCR, ELISA and immunohistochemistry (IHC). The results of qRT-PCR and ELISA showed that the mRNA and protein levels of HIF1α and Beclin1 were the highest in adult yak hippocampus, followed by cerebral cortex, corpora quadrigemina, medulla oblongata and cerebellum. Immunohistochemistry staining showed that HIF1α and Beclin1 positive products were mainly distributed in the cytoplasm. And in the cerebral cortex positive products were mostly concentrated in pyramidal cell-like neurons. In hippocampus, they were distributed in the molecular layer, the granular layer and the polymorphic cell layer. Additionally in the corpora quadrigemina and medulla oblongata positive products were concentrated in the neuronal. Moreover, the positive products of HIF1α and Beclin1 were distributed in the cerebellar Purkinje cells, and also presented in a small amount of cells in the granular layer and the molecular layer. The above results suggested that hippocampus and cerebral cortex might be susceptible to hypoxia, and HIF1α might promote Beclin1 expression and improve brain tissues adaptation to hypoxia. But the specific mechanism needs further studying. Therefore, this study provides theoretical basis for further exploration of brain tissues hypoxia adaptation mechanisms.

Ganjia Tibetan sheep (Ovis aries) grow at high altitude and has a low reproductive rate. The aim of this study was to elucidate the gene expression and tissue distribution of follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the uterus of the estrous cycle of Ganga Tibetan sheep, to explore the regulation of FSH and LH in Tibetan sheep breeding activities. In the experiment, 32 healthy and non-pregnant Ganjia Tibetan sheep were selected and the uteruses were collected at different stages of the estrous cycle. The detection results of qRT-PCR showed that FSHR and LHR were expressed in uterus tissue during the estrous cycle of Ganjia Tibetan sheep, and the expression level of FSHR was the highest in Estrus, which was significantly higher than that in Proestrus and Metaestrus (P<0.01), and the expression level of LHR was the highest in Diestrus, which was significantly higher than that in Proestrus and Estrus (P<0.01). The detection results of immunohistochemistry showed that FSHR and LHR distributed in the Cervix, Corpus uteri and Cornual uteri of Ganjia Tibetan sheep during estrus cycle, mainly distributed in stromal cells, glandular epithelial cells, vascular endothelial cells and myometrial smooth muscle cells, and FSHR had the most distribution in glandular epithelial cells, stromal cells and myometrial smooth muscle cells in Estrus, and the most distribution in vascular endothelial cells in Diestrus; LHR had the most distribution in glandular epithelial cells, stromal cells and myometrial smooth muscle cells in Diestrus, and the most distribution in vascular endothelial cells in the Metaestrus. Above results indicated that FSHR and LHR might be involved in the regulation of the physiological function of Tibetan uterus during the estrus cycle. This study provides reference data for studying the regulation mechanisms of reproductive activity in Tibetan sheep at the cellular and molecular levels.

Ganjia Tibetan sheep (Ovis aries) are endemic animal in Gansu, and the reproduction rate is low due to the particularity of their living environment. In order to study the expression and distribution of prolactin receptor (PRLR) in pituitary and ovarian tissues of Ganjia Tibetan sheep during estrous cycle and illuminate the regulation of gene expression in reproductive activity, 32 Ganjia Tibetan sheep were selected, and the expression and distribution of PRLR in pituitary and ovarian tissues during estrous cycle were detected by immunohistochemical staining and real-time fluorescent quantitative PCR. The results showed that PRLR and its gene expression were found in the pituitary and ovarian tissues of estrous cycle of Ganjia Tibetan sheep. In the pituitary, the gene expression of PRLR in oestrus was the highest, which was significantly higher than that in the metestrus (P<0.05); PRLR-positive cells were mainly distributed in the cytoplasm of the distal eosinophils of the pituitary gland, and PRLR had the most distribution in the proestrus and the least in the oestrus. In the ovarian, PRLR gene expression was highest in oestrus, significantly higher than that in metestrus, proestrus and diestrus (P<0.05); Positive PRLR cells were mainly distributed in follicular granule layer, and had the most distribution in proestrus and the least in estrus period, and the pre-estrus and inter-estrus period had more distribution than that in post-estrus period and estrus period (P<0.05).The distribution and gene expression of PRLR in pituitary and ovary tissues of Ganga Tibetan sheep during estrous cycle were different, suggesting that there might be differences in the regulation of PRLR on estrous cycle. The results of this study provide basic data for the regulation of reproductive performance of Tibetan sheep.