Rapeseed (Brassica napus) and Arabidopsis thaliana belonging to the same family Cruciferae have analogical mechanisms in regulating seed fatty acid (FA) accumulation. Rapeseed is a significant oil crop in China. Elevate its seed oil content is an important goal for rapeseed breeders since many decades. The seed oil content of Arabidopsis varies 20%~35%, and Arabidopsis can serve as an ideal model to study the mechanism of synthesis of FAs and other storage compound in seeds. Abscisic acid (ABA) is a plant endogenous hormone that regulates seed development, maturation and the acquisition of seed dormancy. However, little is known about the effect of ABA on accumulation of FAs and storage proteins in cruciferae oilseeds. In this study, ABA with different concentrations was applied to rapeseed as well as Arabidopsis plants which were at reproductive stage. In order to explore the effect of ABA on biosynthesis of FAs and other storage compounds such as protein in seeds, a series of culturing mediums containing various concentrations of ABA were used for the culture of fertilized rapeseed embryos 28 days after pollination (DAP). The results showed that ABA promoted the senescence of Arabidopsis leaves and rapeseed pods reflected by early yellowing. The spray of ABA (200 μmol/L) on Arabidopsis plants before bolting inhibited the development of siliques. Moreover, the spray of ABA repressed the expression of fatty acid elongation 1 (FAE1), inhibited the biosynthesis of very long chain fatty acids (VLCFA, C＞20), elevated the proportion of 18-carbon polyunsaturated fatty acid (PUFA), and decreased the proportion of storage protein, indirectly. On the other hand, the culturing of fertilized rapeseed embryos in vitro demonstrated that ABA repressed the expression of fatty acid desaturases (FADs), which further resulted in the reduction of PUFA the increases of VLCFAs and storage proteins. Taken together, the concentration of ABA is critical to seed FA accumulation, i.e., high (or low) ABA concentrations repressed (or promoted) the expression of genes on the FA biosynthesis pathway. Therefore, different concentrations of exogenous ABA could be applied to regulate seed FA accumulation positive or negatively. This research enriches knowledge about the function of ABA and the understanding about the molecular mechanism regulating the accumulation of fatty acids in oilseeds.

Profilin(PRF) is a low molecular weight actin binding protein which is linked to the signal transduction cascade via the phosphoinositide pathways in eukaryotes, regulating both actin polymerization and depolymerization. To date, PRF has been identified in many higher plants, such as Arabidopsis thaliana, Zea mays, Akebia trifoliata, Brassica campestris. In this study, a cDNA clone(designated TaPRF7 (GenBank No. KY940299) in accordance with the accession sequence of wheat (Triticum aestivum) PRF in GenBank) encoding PRF gene was isolated from stamen of wheat photoperiod-thermo sensitive genic male sterile(PTGMS) line BS366 using RT-PCR technique. The cDNA sequence contains a 396 bp ORF, coding for 131 amino acids, with an estimated molecular mass of 14.2 kD and a pI of 4.81. Homology comparison revealed that the protein contains conservative PROF domain and actin, PIP2 and poly-L-proline (PLP) binding sites. The amino acid sequence among TaPRF7 and other different species (T. aestivum, Z. mays, Oryza sativa, Glycine max, Hordeum vulgare, Sorghum bicolor, A. thaliana, Nicotiana tabacum, Solarmum tuberosum) compared by Blast, revealing that there was high homology (70%~97%) with profiling protein of other plants and the similarity to TaPRF1(ACE82291.1) protein was the highest with 97%. Phylogenetic analysis showed that TaPRF7 has close relationship with ZmPRF2, ZmPRF4, ZmPRF5, HvPRF1, SbPRF and OsPRF. Subcellular localization analysis indicated that TaPRF7 was targeted to the nucleus and cytoplasm by detecting the fluorescent signals of eukaryotic expression vector in Arabidopsis protoplasts. Expressions of TaPRF7 had been observed in various tissues (root, stem, leaf, stamen, pistal and glume) of wheat, but was highest in stamen, followed by root, suggesting that TaPRF7 was reproductive expression and mainly expressed in reproductive organs. To investigate the involvement of TaPRF7 in signal transduction, TaPRF7 gene expression of wheat seedling in the abiotic stresses was studied. The results showed that expression of TaPRF7 was inhibited significantly by exogenous methyl jasmonate (MeJA) and salicylic acid (SA) treatments, while up-regulated under cold (10 ℃), abscisic acid (ABA), drought (PEG 6 000), gibberellin (GA), NaCl, indoleaceticacid (IAA) treatments. TaPRF7 expression speaked at different time in different abiotic treatments. The results showed that peaks appeared about 8 h after cold and NaCl treatments, 4 h after PEG and GA treatments and 2 h after IAA and ABA treatments, respectively, suggesting that TaPRF7 responses to many abiotic stresses. Further, the TaPRF7 expressions were decreased after they peaked. It was likely to be due to negative regulation which caused by accumulation of the gene. Microfilament is one component of cytoskeleton, and it is mainly composed of actin. Recent studies showed that movement of microfilament was involved in plant fertility. PRF as a microfilament-associated protein, maybe play important roles in fertility regulation. To investigate the role of TaPRF7 in fertility regulation, analysis of TaPRF7 expression profile between PTGMS line BS366 and coventional wheat line Jing411 during fertility transition was performed. It was found that TaPRF7 showed low expression and no significant change during fertility transitionin Jing411. However, the expression of TaPRF7 increased with the anther development in both sterile environment and fertile environment with the development of anther. Besides, the expressions of TaPRF7 were higher during fertility transition in sterile environment comparing with expression infertile environment. In summary, it was speculated that TaPRF7 may be involved in the signaling transduction for anther dehiscence and cold-induced male sterility associated with wheat male sterility. This study laid a basis for investigation of TaPRF7 in molecular mechanisms of wheat PTGMS line.

Rainbow trout (Oncorhynchus mykiss), which is a cold-water species of salmonid, is cultivated as the main variety in China. The high temperature is an important threat to aquaculture of it in summer. In this study, the experimental fish belonged to full-sibling inbreeding families and the average weight of (400±10.5) g. In order to define heat stress response (HSR) and the changes of sensitive parameters, the study took slow heating process which was considered to simulate natural heating process in cage culture water, and 10 fish were sampled to investigate the changes of some main serum non-specific immunity parameters at 18, 21, 23, 24, 25 and 26 ℃, respectively. The results showed that in the process of heat stress, alanine transaminase (ALT) and aspartase aminotransferase (AST) activity at 25 and 26 ℃ were significantly higher than that at other temperature points (P＜0.05), respectively. The difference of total bilirubin (T-Bill) content was not significant in the heating process of 18~23 ℃ (P＞0.05), increased significantly at 24 ℃ (P＜0.05), and peaked at 26 ℃. Alkaline phosphatase (AKP) activity maintained the highest level at 18 ℃, decreased gradually with the water temperature rising and at 21 ℃ was significantly lower than that at 18 ℃ (P＜0.05).The difference of creatine kinase (CK) activity was not significant in the heating process of 21~25 ℃, but increased significantly at 26 ℃ (P＜0.05). Lysozyme (LZM) activity increased gradually in the heating process of 21~24 ℃, peaked at 24 ℃, and at 25 and 26 ℃ were significantly lower than that at other temperature points (P＜0.05), respectively. Complement 3 (C3) content increased gradually in the heating process. The differences of serum proteins content which included total protein (TP), albumin (ALB), globulin (GLO) were all not significant in the heating process (P＞0.05). From the above results, a conclusion can be drawn by analyzing the changes of sensitive parameters. Significant decrease of AKP activity at 21 ℃ indicated that heat stress response occurred and the body changed from normal state into adaptive regulation state at 21 ℃. The fact of significant decrease of LZM activity, significant increase of ALT and AST activity after 24 ℃, and the significant increase of T-Bill content at 24 ℃ indicated that the rainbow trout body's immune function decreased significantly and the body appeared severe tissues damage after 24 ℃. Therefore, 24 ℃ was a "key high-temperature point", the point was a critical point that the body changed from adaptive regulation state to "injury". The fact that AST, ALT, CK activity and T-Bill content peaked at 26 ℃, AKP and LZM activity reached the nadir at 26 ℃ indicated that tissues were damaged seriously and the body might show upper limit of thermal tolerance to temperatures at 26 ℃. The above results will contribute to the development of heat resistant breeding programs. Meanwhile, the key high-temperature point of 24 ℃ has important application value in the future study of heat stress, for example, when screening differentially expressed genes under heat stress, this study can offer a theoretical basis.

To gain a better understanding for the relationship between the genome structure and the reproduction mechanism of Xiang pig, the genome sequence of Xiang pig (Sus scrofa) was detected using next-generation sequencing method. Plenty of structural variations (SVs) were detected from chromosome X (chrX) of two Xiang pig groups with high (XH) or low (XL) litter size using several bioinformatics programs. There were 3 246 SVs being screened out from chrX of Xiang pig, in which 311 SVs were unique in XH group and 138 SVs in XL group with 449 SVs in total. It was notified that a SV hotspot locating at 61~109 Mb of chrX group was found out from samples of group XH. There were 138 SVs in the SV hotspot region, with 65 of them in deletion genotype. In addition, about 47 proteins might be disturbed by 80 of specific SVs in genes of Xiang pig. Out of 31 in the 47 genes contained QTL which was related with reproduction traits such as genes of NLGN4X, GPR143, ZDHHC15, ATRX, NXF2, AMOT, GRIA3, THOC2. These genes were significantly enriched in the adrenal gland development, regulation of intracellular steroid hormone receptor signaling pathway, RIG-I-like receptor signaling pathway and lipoprotein metabolic process base on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. 10/47 SVs in populations of Xiang pig were genotyped by PCR methods. The frequency of SV_GPR143 genotypes was significantly different between XH and XL groups (P＜0.05). And the SV_GPR143 locus showed a correlation ship with the litter sizes (ρ=0.217) of Xiang pig with 1.96 piglets in DD genotype lower than that in NN genotype (P＜0.05). It indicated that much more amount of SVs presented in group XH and might contribute to the high reproduction trait of Xiang pig.

Agouti gene is one of the important influencing animal coat color genes, although the expression of Agouti gene was found in a variety of animal, Agouti protein (Agouti signaling protein, ASIP) reports was rare on effects of Alpaca (Lama pacos) proliferation of melanocytes and genes related to coat color. In order to observe effect of ASIP-YY (a C-terminal fragment of ASIP) on melanocytes and the formation of Alpaca skin melanin，the different concentrations of ASIP-YY was used in extra-organismal melanocytes of Alpaca skin. Different concentrations of ASIP-YY (0.0, 0.1, 1.0, 10.0, 100.0 nmol/L) treated melanocytes of Alpaca Skin at 72 h. The activity of melanocytes were detected using methyl thiazolyl tetrazolium (MTT) method, the melanin content was tested with ultraviolet spectrophotometric method and qRT-PCR was used to detect the expressions of TYR, TRP-1, TRP-2, MC1R and MTIF mRNA. The results showed that the melanocytes activity was inhibited; the expression of TYR mRNA was significantly inhibited by 0.1,10.0, 100.0 nmol/L ASIP-YY(P＜0.01), the expression of TRP-1 mRNA was significantly inhibited by 10.0, 100.0 nmol/L ASIP-YY(P＜0.01), the expression of MC1R mRNA was inhibited by 10.0 nmol/L ASIP-YY (P＜0.05), the expression of MITF mRNA was significantly inhibited by 0.1, 1.0, 10.0 nmol/L ASIP-YY(P＜0.01), 0.1, 1.0 nmol/L ASIP-YY promoted the expression of TRP-1, TRP-2 mRNA(P＜0.05), 1.0, 100.0 nmol/L ASIP-YY significantly promoted the expression of MC1R mRNA(P＜0.01). ASIP-YY can make the size of melanocytes to be smaller and inhibit the expression of melanin related gene. ASIP-YY may be competitive with MC1R to combine, so as to inhibit the expression of melanin related genes. This study provides a theoretical basis for further study on the regulation of ASIP on Alpaca skin melanin formation.

Antler is a special osseous organs coverd with blood vessel, nerve inside. Antler development refers to the forming process of male fawn pedicle and first antler after puberty. It has confirmed that the piece of periosteum overlying the lateral crest of prepubertal deer frontal bone is a foundation of antler development, which is known as antlerogenic periosteum (AP). AP holds the patterning information for antler formation. Studies suggest that androgens play an important role in antler development. The aim of the study is to investigate the changes of proteomics before and after AP development, which will help to reveal the underlying molecular mechanisms of androgen induced antler development. The study used androgen stimulated female deer until antlerogenic periosteum developed and extracted total proteins from the treated and control groups AP and applied two-dimensional fluorescence difference gelelectrophoresis (2D-DIGE) to obtain differential expression information of proteins. After that, differentially expressed proteins were identified by MALDI-TOF/MS and analysed the proteins based on the database retrieval. The results showed that there were 318 differentially expressed proteins searched and identified by database selected. Among them, 163 proteins were up-regulated and 155 were down-regulated significantly. MALDI-TOF/MS was used to identify 93 of 318 proteins and there were 17 proteins identified successfully. The 17 identified proteins were classified into 5 categories with different structures and functions and these proteins were associated with binding, enzyme activity, catalytic activity and antioxidant activity. Furthermore, the study had classified the mechanism of antler morphogenesis and offers the experience for further studies. What's more, among the 17 successfully identified proteins, the proteins were classified into 5 categories with different structures and functionsand these proteins were associated with binding, enzyme activity, catalytic activity and antioxidant activity. The study will provide theoretical foundation for revealing growth regulating molecular mechanism of antler development. Furthermore, offer the reference data for the mechanism of organogenesis.

CD9, known as motility- related protein- 1(MRP1), is a tetraspanin transmembrane protein. This kind of protein is related to aggressive behavior of cancer cells. CD9 was initially reported as a tumor metastatic suppressor, and its overexpression suppressed metastasis of cancer cells. However, similar studies reported that CD9 expression was rather intensified in cancer tissue in comparison with normal tissues. The role of CD9 in tumor development is still controversy. Deer antlers are the only mammalian appendages capable of full renewal. Unprecedented growth rate (up to 2.75 cm/d) of antlers provides a rare system where fast cell proliferation is elegantly regulated without becoming cancerous. Previous studies reported that antler stem cells expressed high level of CD9 antigen. CD9 is thought to be involved in cell proliferation, motility, differentiation and signal transduction. To examine the function of CD9 in antler stem cells, Western blot analysis, immunofluorescence and flow cytometry were performed to verify expression of CD9 in antler stem cells (including antlerogenic periosteum cells (APC), pedicle periosteum cells (PPC) and reserve mesenchymal cells (PMC) in antler tips). To estimate the biological function of CD9 in cell motility, Anti-CD9 antibody was used in motility assays. And in cell proliferation, MTT assay with Anti-CD9 antibody was performed. The results showed that all of 3 kinds of antler stem cells expressed high levels of CD9 antigen, and more than 90% of primary cultured antler stem cells were CD9 positive. Neutralization of CD9 in antler stem cells did not significantly affect cell migration. Neutralization of CD9 could significantly promote cell proliferation of pedicle periosteum cells and reserve mesenchymal cells, but not antlerogenic periosteum cells. Most of factors involved in extracellular matrix-integrin and focal adhesion signal path were detected in antler stem cells. The results demonstrated that CD9 could serve as a useful marker for isolation of antler stem cells and might play an important role in antler stem cell proliferation. Revealing the function and regulatory mechanism of CD9 in antler stem cells may contribute to the understanding of the role of CD9 in rapid proliferating cells, especially tumor cells. Overall, deer antlers can be developed into a novel model for research of CD9 function.

Velvet is a natural cell growth factor library. The top tissue of the antler as the most active growth point of the deer body to store a large number of growth factors, they are the biochemical basis of the clinical role of deer antler. A variety of growth factors have important roles in promoting the proliferation of antler. The rapid growth of velvet and the generation of blood vessels in cartilage tissue are inseparable and vascular endothelial growth factor (VEGF) can promote blood cell formation and promote the formation of blood vessels. Therefore, the study of sika deer (Cervus nippon) VEGF will provide a reference for the mechanism of rapid growth of velvet. To obtain the biologically active protein, the pET-30a prokaryotic expression vector and pMD-18T-VEGF were digested with HindⅢ and BamHⅠ. The digested product was recovered separately. VEGF mature peptide gene from Cervus nippon was inserted to expression vector pET-30a by T4 DNA ligase to obtain recombinant plasmid. The recombinant plasmids were identified by PCR and identified by double digestion. The correct recombinant plasmid was made into glycerol. Proteins were expressed (0.6 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 20 ℃) in the host Rosetta cells in this study. The results were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Target protein was purified by Ni-Agarose affinity column and renatured to obtain the biologically active protein. Target protein on the proliferation of mouse embryonic fibroblast cell line (NIH3T3) cell was analysed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT). A specific band of about 519 bp was obtained by double digestion of pET-30a-VEGF with 2 restriction endonucleases BamHⅠ and HindⅢ, which were consistent with the expected results. The PCR results of pET-30a-VEGF were used as templates to show a band corresponding to the size of the target band. The results showed that the VEGF gene of sika deer was successfully inserted into pET-30a vector. Restriction enzyme mapping showed that pET-30a-VEGF expression vector was constructed successfully. The recombinant plasmid pET-30a-VEGF was transformed into Escherichia coli Rosetta competent cells and induced by IPTG. SDS-PAGE results showed that there was a specific band at about 23.4 kD as shown, which was consistent with the expected results. Optimization of the optimal IPTG concentration showed that the protein was highest expressed when the IPTG concentration was 0.6 mmol/L at 37 ℃. The best IPTG induction temperature was optimized, and when the temperature was 20 ℃, the protein was the highest expressed. The induction time was optimized. The results showed that the expression level of recombinant protein was the highest when the induction time was 16 h. Therefore, when the IPTG concentration was 0.6 mmol/L, the induction temperature was 20 ℃ and the induction time was 16 h, the recombinant protein was the highest. The purified VEGF mature peptide appeared at a molecular mass of about 23.4 kD, which was consistent with the molecular weight of VEGF mature peptide. The results of MTT showed that proliferation of NIH3T3 was promoted by different concentration of recombinant protein, and the proliferation rate of the cells increased with the increase of the concentration. Among them, the most significant impact on NIH3T3 cell proliferation was at 48 h, and the final concentration was 200 ng/mL. The results of the study will further explore the function of VEGF in the rapid value-added process of velvet and regulation mechanism to provide experimental basis.

The theory of bioelectric code is a remarkable achievement for life sciences. It is unknown whether this theory could apply to mammals. In the present study, sika deer (Cervus nippon) antlers were used as a model to investigate if morphogenesis of mammalian organs was also related to bioelectricity. Deer antlers are complex mammalian organs. Morphogenetic primordia of deer antlers is antlerogenic periosteum (AP). Deer antler morphogenesis can be altered artificially by manipulating AP conveniently. Ten pieces of APs were collected through surgery from 3 male and 3 female sika deer calves before antler generation. These APs were cultured in sterile Dulbecco's modified eagle medium (DMEM) medium (containing 10% fetal calf serum (FBS) and 2% penicillin-streptomycin) in vitro and treated with four kinds of drugs for 2.5 d respectively. These drugs were ivermectin, MS-222, concanamycin A and SCH-28080 which were known to be able to alter bioelectricity state of tissues/cells through interrupting ion channels. Before the culture, APs were punctuated with a beam of needles to facilitate the drugs to diffuse into the tissue. After the culture, APs were implanted back to their original places. Subsequent development of antlers was observed weekly and photographed when necessary. Two months later, following results were obtained. Irrespective of the depolarized or hyperpolarized status, antler development was affected. The results from male deer were consistent with those from female deer (mechanical stimulation could induce female deer to grow antlers). The role of mechanical stimulation was impaired by drug treatment in female deer in this particular case. For the purpose of testing the effect of drug, AP cells were isolated from AP tissue and cultured in sterile DMEM medium (containing 10% FBS and 2% penicillin-streptomycin) in vitro. MS-222 was selected as a representative drug and added to DMED medium. Three groups of AP cells were designed and treated with MS-222 for 12, 24 and 48 h, respectively. The same treatments were carried out but without MS-222 as controls. The intracellular sodium (Na) ion of AP cells were strained with CoroNa green indicator dye. The difference of intracellular Na ion concentration was analyzed with fluorescent density. Results showed that intracellular Na+ concentration was decreased by MS-222 treatment, indicating that MS-222 played its proper effect on AP cells. Cell growth rate was measured with (3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di- phenytetrazoliumromide) MTT assay. Results showed that growth rate of AP cells was not influenced by MS-222 treatment, indicating that inhibition of antler development was not induced by cytotoxicity of drug but may be interfering polarization state of AP. All results indicated antler development was inhibited by treatment of interfering ion channels of AP cells. Therefore, a specific bioelectricity state of AP may be critical to antler development, as ion channel is the main factor to maintain the bioelectricity state of cells. This study provides theoretical basis for further verification of bioelectricity encoding mammalian organ morphogenesis.

Velvet antler 32 polypeptide of sika deer (Cervus nippon)， which is a kind of peptide extracted from fresh antler, has obvious activity to promote proliferation and induce differentiation for mesenchymal stem cells (MSCs). Both of adipose tissue-derived stem cells (ADSCs) and MSCs come from mesoderm, and they are high-profile in the research of bone injury repair and anti-tumor. In this research, the effect of velvet antler polypeptide on ADSCs proliferation, migration and adhesiveness have been observed. Methyl thiazolyl tetrazolium assay (MTT) showed that OD had no obvious difference between experiment group and control group after 24 h. But experiment group was obviously higher than control group after 48 and 72 h. Wound healing assay showed that the concentration of velvet antler polypeptide was 1 μg/mL, the scratch width was narrower than control group, and when the concentration was 50 or 5 μg/mL, the scratch width was bigger than control group. Trypsinization assay found that adding isotonic trypsin to digest, with the increase in peptide concentration, cell digestion time shortened significantly and cell adhesion decreased gradually. This research showed that antler polypeptide promoted proliferation of ADSCs, a certain dose and time-dependent could promote the proliferation of ADSCs in mouse (Mus musculus), reducing cell adhesion and low concentrations of peptides could promote cell migration and high concentrations were inhibited. The results suggested that velvet peptides could be used to increase the number of seed cells and reduce its adhesion in specific areas. While providing the possibility of facilitating its transfer to the damaged site at the same time, which provides a new idea for bone tumor and injury treatment.

Cell wall invertase(CWI) is the key regulator which forms sugar concentration gradient between source and sink by catalyzing sucrose hydrolysis and promotes sucrose into sink. In order to uncover the important roles of CWI in wheat (Triticum aestivum), bioinformatics were utilized to analyse the protein properties and structure map of wheat TaCWI-B1. The prokaryotic expression vector pET28a-TaCWI-B1 was constructed by inserting the coding region of TaCWI-B1 into pET28a, and transformed into Escherichia coli strain BL21(DE3). The induction conditions, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, culture temperature and induction time, were optimized, and the recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified. After identifying the recombinant protein by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the polyclonal antibody was prepared, its titer was measured by enzyme-linked immunosorbent assay (ELISA) and its specificity was analyzed by Western blot. The results showed that the full-length cDNA of TaCWI-B1 was 1 845 bp and this gene had a ORF of 1 776 bp and encoded a protein of 591 amino acid with molecular mass 65.92 kD and pI 9.26. The analysis based on software of Protscale and Swiss-model showed that TaCWI-B1 was a hydrophilic protein with a structure of N-acetyl glucosamine outside, and it was a major component of cell walls. The prokaryotic expression showed that TaCWI-B1 was expressed abundantly as inclusion bodies, and the optimum condition for the target protein production was 0.2 mmol/L of IPTG and induction at 28 ℃ for 6 h. The His-tag fused TaCWI-B1 protein was purified by Ni-NTA SefinoseTM Resin Kit and the MALDI-TOF-MS analysis showed that the similarity between recombinant protein and TaCWI-B1 was 99.992%, which indicated it was really the cell wall invertae of wheat. ELISA showed that the serum antibody titer of 2 TaCWI-B1 immunized rabbits reached over 1∶50 000. The result of Western blot demonstrated that the TaCWI-B1 polyclonal antibody could specifically recognize not only the recombined protein, but also the target protein from wheat. These results provide basic data for further investigation of TaCWI-B1 gene functions.

Stearoyl-CoA desaturase (SCD), which is also called delta-9 desaturase, is the rate-limiting enzyme of regulating unsaturated fatty acids synthesis in milk, and being as a key enzyme for endogenous synthesis of the conjugated linoleic acid in the meat and milk of ruminants. It has the ability to catalyze saturated fatty acyl coenzyme A to generate monounsaturated fatty acyl coenzyme A. Most importantly, SCD takes an effect on the fatty acids composition and the contents of unsaturated fatty acids in goat (Capra hircus) milk. However, a lack of appropriate tools has hampered further study of SCD. Therefore, goat specific SCD antibody is necessary for the function research of SCD gene fatty acid regulation. The objective of this study was to prepare rabbit (Oryctolagus cuniculus) polyclonal antibodies (pAbs) against SCD through prokaryotically expression and SCD protein purification. The clarified function of the antibody would provide effective tools for the study of SCD functions in fatty acids metabolism. The SCD gene sequence was chosen through bioinformatic analysis mainly using DNAStar software. SCD was amplified by PCR from the cDNA. SCD gene of dairy goat was then sub-cloned into the vector pET32a(+) to construct recombinant plasmid pET32a(+)-SCD which was transformed into Escherichia coli Rosetta (DE3) expression strain. SCD fusion protein was purified by His-tag protein purification kit. Purified fusion protein being mixed with adjuvant was used as antigen to immune 6 months of rabbits to prepare polyclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to detect the titer of the immune serum. The obtained SCD polyclonal antibodies were used for specificity identification. The sequence with a total of 210 bp length at SCD gene N-terminal was amplified by PCR, and the prokaryotic expression vector pET32a(+)-SCD was successfully constructed. Recombinant protein was successfully expressed in E. coli Rosetta (DE3). Western blotting result showed that SCD fusion protein was correctly expressed which had 6×His tag. Recombinant protein molecular weight was about 30 kD, consistent with the expected protein molecular weight. The best expression condition was identified as follows, 25 ℃, 1 mmol/L of (isopropyl-β-d-thiogalactoside, IPTG), induction for 12 h; SCD fusion protein was mainly expressed in soluble form. Then, the fusion protein from supernatant of lysates was purified using His-tag protein purification beads. The concentration of the obtained protein was 2.5 mg/mL determined by bicinchoninic acid (BCA) Protein Assay Kit. The purified protein was used as antigen to immune rabbits and prepare polyclonal antibody successfully. ELISA result suggested the final titer of the SCD pAbs was 1∶64 000. Western blot analysis showed that the SCD pAbs could identify the specific antigen epitope on the SCD protein expressed in the form of fusion protein and the natural protein. In conclusion, the SCD protein was successfully expressed and purified. The polyclonal antibody with high reactivity and specificity was successfully prepared, which could help researchers explore the biological functions of SCD in future studies.

The pineal gland, which is a neuroendocrine organ, is influenced by both the dark-light cycle and the seasonal cycle and plays an important role in the neuroendocrine control of reproductive physiology in a variety of species. This article provides an overview of how photoperiodic information controls seasonal variations in animal reproductive activity through the endocrine activity of the pineal gland and focuses on the role and mode of action of melatonin. Moreover, this review is to update current knowledge about the clock genes in the pineal gland such as period circadian clock (per), cryptochrome (cry), clock circadian regulator (clock) and aryl hydrocarbon receptor nuclear translocator like (arntl or bmal).These clock genes have been found in a variety of vertebrate clock structures including the avian pineal gland. This article provides a better understanding of the molecular mechanisms and physiological correlations of the pineal gland regulates seasonally reproduction.

H3N1 subtype Avian influenza virus (AIV) can infect wild birds, poultry, pigs (Sus scrofa) and dogs (Canis lupus familiaris). In order to understand the biological property of H3N1 subtype AIV, phylogenetic analysis and specific pathogen free (SPF) animal infection experiments were conducted for two H3N1 strains, A/environment/Anhui/S4108/2011 (EN/AH/S4108/2011) and A/duck/Fujian/S2196/2012 (DK/FJ/S2196/2012) which were isolated from Anhui and Fujian province in 2011 and 2012, respectively. The phylogenetic analysis showed that the genetic origin of the two stains were obviously diversified. The neuraminidase (NA) and matrix protein (M) genes of DK/FJ/S2196/2012 and the polymerase basic protein 1 (PB1), NA, and non-structural protein (NS) genes of EN/AH/S4108/2011 were most closely related to those of H5N1 AIVs. Besides, these two viruses shed only through oropharyngeal route of infected chickens (Gallus gallus) and didn't have the ability to horizontal transmission. The two viruses could efficiently replicate in the nasal turbinates and lungs of infected mice (Mus musculus) without prior adaptation. In addition, these two viruses did not cause any obvious clinical symptoms of infected mice, and thereby they were low pathogenic to mice. In conclusion, two H3N1 viruses showed genetic diversity and complexity in their origin and had a low potential risk to infect mammals. This study indicated that H5N1 AIVs in eastern China were involved in reassortment with H3N1 subtype AIVs genome, which provides scientific basis for prevention and control of H3N1 subtype AIVs.

The H5 subtype Avian influenza virus (AIV) has been stably established in nature and gains high pathogenicity to its natural hosts through continual evolution, variation and adaptation. Its prevalence not only causes serious harm for the poultry industry, but also has potential threat to human public health security. To better understand the biological property of H5N3 AIV, phylogenetic analysis, mouse (Mus musculus) infection experiment and receptor binding assay were conducted for a low pathogenic H5N3 AIV strain A/Duck/Jiangsu/S1665/2015 which was isolated from Jiangsu province in 2015. The phylogenetic analysis showed that its eight gene segments (hemagglutinin (HA), neuraminidase (NA), polymerase basic protein 1 (PB1), PB2, polymerase acid protein (PA), nucleoprotein (NP), matrix protein (M) and non-structural protein (NS)) were derived from different ancestors and demonstrated obvious genetic diversity in there origin. The result of mice infection experiment showed that this virus could efficiently replicate in the nasal turbinates and lungs of infected mice without preadaptation. It caused only slight weight loss, and thereby it was low pathogenic to mice. Receptor binding assay showed that the virus could bind both α-2,3-linked sialic acid (SA) (avian type receptor) and α-2,6-linked SA (human type receptor). In conclusion, the present study systematically analyzed the biological properties of A/Duck/Jiangsu/S1665/2015 virus. The findings indicated that the H5N3 AIV could potentially infect mammalian hosts, which provides important data for the prevention and control of H5N3 subtype AIVs.

H4N2 subtype Avian influenza virus (AIV) has potential threat to the health of mammalian. To investigate the roles of inflammation-associated cytokines in pathopoiesis development of H4N2 subtype AIV, mice (Mus musculus) were inoculated intranasally with 106 fifty percent embryo infection dose (EID50) of P20 and P50 strains. At 1, 3 and 5 days after infection, the expression of anti-inflammatory cytokines (interleukin-2 (IL-2), IL-4 and IL-10), pro-inflammation cytokines (IL-1β, IL-6 and tumour necrosis factor-α (TNF-α)), monocyte chemoattractant protein-1 (MCP-1) and interferon-α (IFN-α), IFN-β, IFN-γ in lungs were detected by ELISA and qRT-PCR, respectively. The ELISA results showed that the expressions of IL-1β, IL-6, IL-10, MCP-1, IFN-γ and TNF-α were up-regulated, while the expressions of IL-2, IL-4 and IFN-α/β were down-regulated after the infection of both strains. Compared with P20 group, the expression level of IL-6 in P50 group very extremely significantly increased at 3 and 5 days post infection (P＜0.001), but the expression level of IL-10 was very extremely significantly higher than that in P20 group at 5 days post infection (P＜0.001). MCP-1 expression in P20 group was very extremely significantly lower than that in P50 group at 3 and 5 days post infection (P＜0.001). IFN-γ expression in P50 group was extremely significantly (P＜0.01) or very extremely significantly (P＜0.001) higher than that in P20 group at 1, 3 and 5 days post infection. The mRNA expression of IL-6 in P50 group showed significant increase compared to P20 group at 3 and 5 days post infection (P＜0.01). But the mRNA expression of IFN-γ and MCP-1 in both P20 and P50 groups had no significant changes post infection. To validate the role of IL-6 in pro-inflammation, mice were inoculated intravenously with anti-IL-6 neutralizing antibody after inoculated intranasally with P50 virus. The results showed that the expression levels of IL-6, MCP-1 and IFN-γ in each group were not statistically different, and inflammatory response was not alleviated, and it did not produce a protective effect on mice. This study showed that H4N2 subtype AIV was associated with inflammatory response in mice, but reduction of IL-6 could not alleviate the inflammatory response. The results provide a basis for the development of candidate drugs that can be used to prevent and control influenza A virus infection.

H7N9 Influenza A virus (IAV) is an important pathogen of zoonosis which threatens the public health of human. In the present study, to investigate the H7N9 IAV-mediated innate immune responses, recombinant plasmids expressing the polymerase basic protein 1 (PB1) gene were first constructed. However, to exclude the influence of PB1-F2 that is encoded by PB1 via a frame-shifting manner, two nucleotides at positions of 96 and 129 in PB1 gene were mutated by site-directed mutagenesis, respectively. Subsequently, the results from a dual-specific luciferase reporter assay indicated that PB1 significantly inhibited retinoic-acid-inducible geneⅠ CARD (RIG-ⅠN)-induced interferon-β (IFN-β), typeⅠinterferon (IFN) stimulated response element (ISRE) and nuclear factor kappa-B (NF-κB) activities. Consistently, the qRT-PCR experiment confirmed that the expression of IFN-β and its downstream interferon stimulated gene 56 (ISG56)and ISG15, and C-X-C motif chemokine 10 (CXCL10) were down-regulated at the transcriptional levels, suggesting PB1 inhibits the RIG-Ⅰ-mediated signaling pathway. Furthermore, the co-immunoprecipitation experiment was performed to test the interaction between PB1 with the key molecules in the RIG-Ⅰ signaling pathway. The results indicated that H7N9 PB1 specifically interacted with mitochondrial antiviral signaling (MAVS). In addition, PB1 was co-transfected dose-dependently with RIG-Ⅰ or MAVS into HEK293 cells, the Western blotting data showed that the increased expression of H7N9 PB1 inhibited MAVS expression, but not RIG-Ⅰ. Therefore, the preliminary data in the present study demonstrated that H7N9 PB1 was specifically associated with the important adaptor, MAVS in the RLRs signaling pathway and decreased the MAVS expression, leading to the inhibition of IFNs expression and the blockade of the downstream signaling transduction. Our finding revealed that the target of H7N9 IAV PB1-associating in the RLRs signaling pathway would be helpful for the H7N9 control.

Avain influenza (AI) and Infectious bursal disease (IBD) are harmful to poultry industry, and vaccination is one of the most effective method of controling these two diseases. In order to explore the feasibility of Duck enteritis virus (DEV) vector combined vaccine against AI and IBD that were used in chicken (Gallus gallus), two recombinant viruses that co-expressed hemagglutinin (HA) gene of H5 type Avian influenza virus (AIV) and virus protein 2 (VP2) gene of Infectious bursal disease virus (IBDV) were constructed, and experiments were done to evaluate the immune efficacy of the recombinant viruses. HA gene from A/Chicken/Guizhou/4/2013 (H5N1) (GZ/4) and VP2 gene from IBDV (HLJ-0504) were connected by internal ribosome entry site (IRES) sequence in different orders, HA-IRES-VP2 and VP2-IRES-HA. The two gene fragments were then inserted between unique short region 7 (US7) and US8 genes of DEV genome in fosmid T-us78Kan ccdB which is overlapping fosmid DNAs rescue system. Using this system, the target gene between the US7 and US8 genes of the DEV genome were successfully inserted, and the two recombinant viruses rDEV-HA/VP2 and rDEV-VP2/HA were successfully constructed. The in vitro experiments showed that the inserted gene could be stably inherited and express without affecting replication of virus vector in cells. In order to evaluate the protective efficacy induced by recombinant virus in specific pathogen free (SPF) chicken, groups of ten 3-week-old SPF chicken were immunized with 103 tissue culture infective dose (TCID50), 104 TCID50, 105 TCID50 recombinant virus, and challenged with 100 median lethal dose (LD50) GZ/4 or 100 LD50 very virulent IBDV (vvIBDV) at 14 days post vaccination. Recombinant DEV with only HA gene of GZ/4 inserting between US7 and US8 genes (rDEV H5-8) and commercial IBDV vaccine Gt strain were immunized as control. The result showed that chicken which were immunized with 103 TCID50, 104 TCID50, 105 TCID50 rDEV-HA/VP2 induced low level of hemagglutination inhibition (HI) antibody, and no IBD antibody could be detected. The protective efficiency against H5 AIV was lower than 70%, and the protective efficiency against vvIBDV was lower than 30%, respectively. While, chicken which were immunized with 103 TCID50, 104 TCID50, 105 TCID50 rDEV-VP2/HA could not induce HI or IBD antibody. The protective efficiency against IBDV was lower than 40%, and the protective efficiency against H5 AIV was 0. However, chicken which were immunized with rDEV H5-8 or Gt strain could induce 100% protection against H5 AIV or vvIBDV. In conclusion, the two recombinant viruses rDEV-HA/VP2 and rDEV-VP2/HA could not induce complete protection against AIV or IBDV, and the strategy of using IRES to connect HA and VP2 genes to construct the DEV vector vaccine against AI and IBD was not feasible. This study provides a reference for the construction of DEV vectored multi vaccine in chicken