This study aims to obtain the transcription regulatory region of Nramp1 gene which is tissue-specifically expressed in porcine pulmonary alveolar macrophages. The study identified the transcription start site of pig(Sus scrofa) Nramp1 gene using 5'-RACE. Eight expression vectors, pLUC1430, pLUC1136, pLUC840, pLUC751, pLUC487, pLUC379, pLUC274 and pLUC179, with serial deleted upstream fragments were constructed by cloning different deletion fragments of －1 327 bp to ＋86 bp region into pGL3-Basic vector, respectively. The expression vectors were co-transfected with Renilla luciferase vector into porcine pulmonary alveolar macrophages, kidney cells, preadipocytes and fetal fibroblast cells, respectively. Expression activities were analyzed using dual luciferase reporter system for different fragments in order to identify the core regulatory region. Our result showed the core regulatory region of Nramp1 gene ranged from －386 bp to －173 bp. The transcripion activities of pLUC487[－386~＋86] region were further compared among porcine pulmonary alveolar macrophages, kidney cells, preadipocytes and fetal fibroblast cells, in which Nramp1 promoter exhibited significantly higher activity in porcine pulmonary alveolar macrophages(P<0.01). We confered that Nramp1 promoter was specifically expressed in porcine alveolar macrophages. Our study gained a transcription regulatory element specifically expressed in pulmonary alveolar macrophages, which facilitates the tissue-specific expression of foreign genes in porcine pulmonary alveolar macrophages.

Transmembrane protein 18 gene(TMEM18) is expressed in the central nervous system that has recently been linked to human obesity and body mass index (BMI) in genome wide association studies (GWAS). In this study, direct DNA pool sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were used to genotype novel SNPs in TMEM18 gene, and then analyzed associations between different genotypes and growth traits in Jiaxian Red cattle(Bos taurus). Three novel SNPs were detected in Jiaxian Red cattle TMEM18 gene: NW_003099175.1: g. 2303G>A, g. 3835 G>A and g. 3865 A>G(aa. Gly>Ser). Significant association analysis revealed that the individuals with genotype AA had a larger body height, body length, heart girth and body weight than that of the individuals with genotypes GG at g.2303 A>G locus(P<0.05), The result also suggested that the individuals with genotype AG had a larger body weight and heart girth than that of the individuals with genotype AA at g. 3835 G>A locus (P<0.05). Therefore, these two loci can be used as body size and body weight candidate markers to breed Jiaxian Red cattles.

The chicken Major histocompatibility complex(MHC) is composed of a group of closely linked, highly polymorphic loci composition, which has an extremely important role in the body's immune response and regulation. This experiment was conducted to study the association between immune traits(Sheep red blood cell(SRBC), Avian influenza(AI) and Newcastle disease(ND)) and Major histocompatibility complex(MHC) B-F gene in Shandong local chickens(Gallus domesticus) breeds(Luqin chicken, Shiqiza chicken and Langya chicken), The results of comparison of AI, ND and SRBC titers among the three breeds showed that the antibody titers of SRBC, ND, and H9 in Shiqiza chicken were significantly higher than the two breeds(P＜0.01); but the antibody titers of H5 in Shiqiza chicken were significantly lower than the two breeds(P＜0.01); The Luqin chicken had the closest genetic relationship to Langya chicken, the antibody titers of them were basically the same and no significant difference with the other two populations(P>0.05). A 396 bp sequence including exon 2 of chicken MHC B-F gene was assessed in this study by the PCR-SSCP. The results indicated that 49, 45 and 41 SNPs were respectively detected in Luqin chicken, Shiqiza chicken and Langya chicken. Among them, 40, 37 and 32 nucleotide variations affected amino acid variations. Based on the three varieties, six, nine and four SNPs were respectively found significantly effect on partial immune traits(P＜0.05). The Luqin chicken had the closest genetic relationship to Langya chicken, the genetic variation of MHC B-F and antibody titers of them were basically the same. Both in Luqin chicken and Langya chicken, a total of 5 identical variants were found significant correlation with immune traits. The loci G232A and A217G were significantly associated with Newcastle disease antibody titers(P＜0.05) and the A217G was significantly associated with avian influenza H5 antibody titers(P＜0.05). Locus A211G was significantly associated with avian influenza H9 antibody titers(P＜0.05). Locus G232A was significantly associated with Sheep red blood cell antibody titers(P＜0.05). The results revealed the correlation between the different varieties of MHC B-F and immune traits in different varieties, which suggested the results had an important role in guiding the clinical use of vaccines.

Lysostaphin is a single chain protease containing zinc which can kill staphylococcus aureus effectively. Endolysin which is the peculiar of the double-stranded DNA bacteriophages is a murein hydrolytic enzyme, it has a wide range of antibacterial effect. Lysostaphin and Endolysin have the high synergistic effect. In this study, the vectors pBC1-seq2+seq3-EGFP-neo containing Endolysin and Lysostaphin genes and two other marker genes of enhenced green fluorescent protein(EGFP) and neomycin(neo) were transfected into bovine(Bos taurus) fetal fibroblast by electroporation and nucleofector of AMAXA. Stable transfected monoclonal cells which were identified to be the positive-cells in the way of PCR technique were obtained through fluorescence and G418 selection. Using transfected cells as the donor, transfected embryos were produced with somatic cell nuclear transfer. we used different conditions of AMAXA nuleofecor(A-023,V-013,V-023 and T-016) to transfect bovine fetal fibroblast, the results showed the suitable program was T-016. There were 5 times transfection efficient of AMAXA nuleofecor (20.11%) than it of electroporation. The blastocyst developed normally and the rate was of it 20.08%. In our study, we built up bovine fetal fibroblast cell line, sought out transfection parameter of high transfection efficiency, and acquired transgenic cell lines and transgenic blastocyst containing Lysostaphin and Endolysin genes. In conclusion, the results can provide technology supporting for producing anti-mastitis transgenic bovine and searching the new therapy way of mastitis.

Estrogen plays an important role for mammalian in interim physiology period. The objective of this study was to reserch the effects of estradiol (E2) on cells proliferation and antioxidation in vitro bovine(Bos taurus) mammary epithelial cells (BMEC). Experimental groups were divided intoⅠ~Ⅴ, which were treated with E2 at 0.5, 1, 5, 10 and 100 nmol/L, respectively, the control group(CK) received the vehicle only. After culturing for 4, 8 and 12 h, the viability of mammary epithelial cells was assessed by MTT assay. The 12 h culture medium and cells were used to determin the content of malonaldehyde(MDA), the activities of Lactate dehydrogenase (LDH) and superoxide dismutase (SOD) by colorimetric method. HSP70(heat shock protein 70) mRNA expression were assayed by Real-time fluorescent quantitative PCR. The results showed: (1) Compared with the control group, Ⅰ~Ⅲ E2-treated groups showed markedly increased cell viabilities (P<0.05). ⅣandⅤE2-treated groups decreased cell viabilities. (2)Ⅰ~Ⅳ E2-treated groups showed a decrease in LDH activity in culture medium, HSP70 mRNA expression and SOD activity were significantly higher than those in the control group(P<0.05) but lipid reaction was reduced.ⅤE2-treated group showed a significant increase in LDH activity in culture medium(P＜0.05), a decrease of HSP70 mRNA expression and SOD activity (P<0.05) but lipid reaction was enhanced. The conclusion of this study: Low concentrations (0.5, 1.0 and 5.0 nmol/L) E2 could promote the proliferation of mammary epithelial cells, induced expression of HSP70 mRNA, which might play antioxidant role by increasing intracellular SOD activity. High concentration (100 nmol/L) of E2 could attenuate the proliferation and antioxidation of mammary epithelial cells, causing cell damage with long-term stimulation. This study provided some theoretical basis that exogenous estrogens can increase the antioxidant activity of bovine mammary tissue.

Valproic acid (VPA) as a histone deacetylase inhibitor, can specifically inhibit the histone deacetylase activity and increase histone acetylation, and can affect gene expression in somatic cells. In this research, the effect of VPA on bovine fetal fibroblast cell growth, cell cycle and gene expression were studied. The bovine (Bos taurus) fetal fibroblasts cells were treated with different concentrations (0.8, 1.0 and 2.0 mmol/L) of VPA for 24, 48 and 72 h, respectively, then cell proliferation and cell cycle were examined by flow cytometry, and the expression of transcriptional factors such as octamer binding factor 4 (Oct4)，Nanog homeobox (Nanog), and caudal-related homeodomain protein 2 (Cdx2) were detected by Real-time PCR. The results showed that cell growth gradually decelerated with the increase of VPA concentration, cells were mostly arrested at gap0/gap1(G0/G1) phase after treatment. The expression of Oct4 and Nanog genes were improved, while Cdx2 gene expression was reduced. This study showed that VPA treatment affected cell growth, cell cycle, and gene expression of bovine fetal fibroblast cells, which could be benefit to the study on reprogramming of somatic cell after nuclear transfer and embryo development.

Current classification within Rhizoctonia solani is largely based on grouping of the population into anastomosis groups(AG), but does not always appear to accurately reflect the genetic variability among population. Hyphal anastomosis reactions and internal transcribed spacer(ITS) sequences were integrated to evaluate the genetic diversity of isolates Rhizoctonia solani AG2-1 and AG3 from potato(Solanum tuberosum). Anastomosis reactions indicated that the complete fusion reaction occurred among the different isolates of AG3. Either the complete fusion reaction or incomplete one occurred among the traditionally defined isolates of AG2-1. Therefore, AG2-1 was grouped into two sub-groups by incomplete fusion reaction. Phylogenetic tree constructed by ITS sequences showed that the AG3 formed only one separated clade, whereas AG2-1 clustered into the two different sub-clades. The horizontal lengths of the branches indicated that AG2-1 has been appearing obvious genetic differentiation because the relative genetic diversity of AG2-1 or the two subgroup AG2-1-1 and AG2-1-2 were higher than that of AG3. Based on ITS sequences, the two sub-clades within AG2-1 were consistent with the two sub-groups based on the incomplete anastomosis reaction. So the incomplete fusion reaction can be used as a referent character for genetic differentiation within an anastomosis group of Rhizoctonia solani.

Insulin-like growth factors play an important role in animal growth and nutrition metabolism. In order to research the effects of IGF-1 receptor(IGF-IR) gene on the skeletal muscle in early development stage of duck, the IGF-IR mRNA expressions on the skeletal muscle during Gaoyou duck and Jinding duck (Anas platyrhynchos domestica) embryonic stage on day 13, 17, 21, 25, 27 and 1 week after patch were detected using quantitative RT-PCR method in this experiment. The result indicated that the profiles of IGF-IR mRNA in muscle in two duck breeds were relatively consistent during early development which showed "wave" trend in pectorales and "L" trend in legs. Expression levels of IGF-IR gene on pectorals at 13 and 21 embryo age were higher, and the subsequent expression levels were somewhat lower at 25 and 27 embryo age and its expression levels in the newborn period(7 days) were slowly upward; the expression of duck IGF-IR gene on legs showed that the highest level appeared at 13 embryo age, and subsequently expression levels decreased to maintain a relatively low expression levels. Within the same breed, the expression levels of IGF-IR mRNA in pectorals were significantly higher than that in legs except for 13 embryo age(P<0.01). The result of bivariate correlate analysis showed that the expression of IGF-IR gene and weight development in pectorales and legs were negatively correlated and the correlate coefficient in legs was stronger. These results indicated that IGF-IR gene involved in the skeletal development, and different expression patterns in the pectorals and legs suggested IGF-IR gene may be related to the chest and leg muscle development and differentiation, and the expression of IGF-IR gene in muscle was important in the early growth and development in ducks. The analysis of IGF-IR gene in early development of skeletal muscle in Gaoyou duck and Jinding duck will provide theoretical basis for studing hormone regulation mechanism in duck.

The polymorphisms of 13 microsatellite loci of 116 Gansu Alpine Merino sheep(Ovis aries) was detected using multiplex polymerase chain reaction(PCR) and DNA sequencing technology, and the paternity testing was studied combined with the parents of this record. The purpose is to supply effective microsatellite locus for germplasm resourses evaluation and paternity testing of sheep. Results demonstrated that 13 microsatellite loci altogether discovered 111 alleles. The number of alleles was most in DRB1-INTRO2(14), and the least in SRCRCP5(5). The heterozygosity was highest in ILSTS011(0.888), while the lowest was MAF214(0.500). The other 12 microsatellite loci were highly polymorphic except MAF214 which was moderate polymorphism. The mean observed heterozygosity(Ho), the expected heterozygosity(He), and the polymorphism information content (PIC) were 0.743, 0.727, and 0.689, respectively. All above results suggested that there were a relatively high genetic diversity in Gansu Alpine Merino. The calculate exclusion probability (CPE) of the 13 locus was 0.999 94 using Cervus 2.0 software. The CPE of the 11 locus which were highly polymorphic were 0.999 85, and 12 locus were 0.999 91. MAF214 had a smaller effect on CPE, and DRB1-INTRO2 had a higher effect on CPE. It could improve the accuracy requirements of paternity, make the operation more convenient, reduce costs, and be more suitable for practical production.

To explore the effect of transfection of the transposon-mediated technology in the chick embryo, the single plasmid of Sleeping Beauty(SB) transposable vector pT2-SV40-EGFP-SB11 was injected into 36 h incubated chicken(Gallus domesticus) blastoderm with eggs windowing, at the same time window non-injection group and non-window group were set as control group. Stereological fluorescence microscopy and RT-PCR were used to detect and compare the expression of enhanced green fluorescent protein gene(EGFP), hatching rate, survival rate and egg weight change at different stages of embryonic. The results showed that the transposon-mediated EGFP in chicken embryos obtained a higher expression rate, in embryos of early development(6 d: 89%, 10 d: 71%), but decreased slightly during later period (14 d: 41%, 18 d: 45%, 21 d: 40%); The difference of three groups of eggs weight change on 7 d and 12 d ,was not significant(P>0.05), the egg weight ratio of the window injection group on 17 d and 21 d, was significantly lower than that of non-window group(P<0.05); the difference of survival rate among three groups on 2 d was not significant(P>0.05); window injection group on the 4 d was significantly lower than that of the window non-injection group(P<0.05), while the window non-injection group was significantly lower than that of the non-window group(P<0.05); from 6 d to 21 d, the survival rate and hatching rate among the three groups showed significant differences(P<0.01). The preliminary experiments proved that the improved egg fenestration can be used for the chick embryo transgenic research and initially establish the method of the transfection exogenous gene in chick embryo mediated with SB transposon.

Overexpression of the stress-inducible transcription factor gene AtDREB1A in Chrysanthemum conferred strong drought stress tolerance. Taking A-112, A-128 and A-136 as experimental materials, which are the three excellent hybrid offspring plants of the ground cover Chrysanthemum fall color transgenic line 1805 with drought stress tolerance harboring Arabidopsis thaliana dehydration response element-binding protein 1A(AtDREB1A) and the Asia Winter Light with better phenotype, we analyzed the stability and expression of AtDREB1A under water stress condition by RT-PCR, and determined the wilting index, survival rate, proline(Pro) content and superoxide dismutase(SOD) activity under drought stress condition. The results demonstrated that the exogenous gene AtDREB1A was transferred into the hybrid offspring plants by sexual propagation and it could be expressed in offspring plants under drought condition. Compared with the control plants, the hybrid offspring plants had obviously stronger drought stress tolerance, and their Pro content and SOD activity were obviously raised under drought condition. These results indicated that the AtDREB1A gene which is transferred into the hybrid offspring plants functions in enhancing drought stress tolerance. This research plays a role in breeding new Chrysanthemum cultivars with drought stress tolerance by genetic engineering combined with traditional hybrid breeding.

Cysteine protease(CP) is one of the important hydrolysis protease, which widely participates in a variety of physiological processes of plants. The FaCP gene cDNA was cloned from strawberry(Fragaria × ananassa) using RT-PCR and RACE techniques. The cDNA sequence was 1 338 bp (GenBank accession number: JN979371), including 1 065 bp of open reading frame (ORF), which encoded a protein of 354 amino acids with a predicted molecular weight of 39 130 D and a hypothetical pI (isoelectric point) of 4.93. Homology analysis showed that the deduced CP protein was highly homologous to other CP proteins from different plant species. Phylogenetic analysis indicated that FaCP was more related to castor, trichocarpa, kiwi and grape. Real-time PCR analysis revealed FaCP could be expressed in different strawberry tissues including root, stem, leaf, calyx and fruit，of which the highest expression level in fruit. The FaCP expressed continuously during the whole period of strawberry fruit development and reached the maximum at the pink ripening stage and decreased slightly at the red ripening stage. The expression level of FaCP gene in leaves was gradually increased with leaf senescence，which the expression in the young leaves stage (leaf unfolding within 15 days) was low, but in the old leaves (leaf unfolding for more than 45 days), the FaCP had a higher expression. This result showed that FaCP may play a role in the fruit ripening and leaf senescence.

Phytic acid and its metabolic intermediates have irreplaceable biological functions. Phytic acid is abundant in cereal grains and soilseeds such as maize and soybean. Phytic acid is an anti-nutritional factor, but it is also an important healthy factor for human. Knowledge about its synthesis and its functions is poorly understood yet, though we know its existence early. For the past few years, a few researches focusing on the metabolism of phytic acid have been reported. Breeders have selected a series of low phytic acid mutant lines in maize(Zea mays), soybean(Glycine max) and rice(Oryza sativa) for future research, which would meet the forage application to enhance the animals' nutrition and to reduce the phosphoric pollution. The earlier research implied that there were two phytic acid synthesis pathway in plants, the phosphatidylinositol dependent pathway and the phosphatidylinositol independent pathway. Direct proofs implied that the phosphatidylinositol independented pathway makes the main contribution to the seeds' phytate phosphorus accumulation. Maize is the main food and feed crop in China, focusing on the phytic acid's metabolism in maize is benefit to the bio-fortification and the health of the environment. Here, we describe the functions of phytic acid and the progress concerning its metabolism research; describe and analyze its pathway in plant; and review the previous research toward its metabolism, as an reference to the successive research.

Interactions among proteins are important for the majority of biological functions. MADS-box genes exert their functions through formation of homo- or/and hetero- dimer/tetramer. Rice OsMADS15 is a member of APETALA1/SQUAMOSA(AP1/SQUA) subfamily, which plays important roles in biological processes such as flowering time regulation, adventitious roots development, stem growth and palea development. But little information was known for its interaction partners. In this study, we screened its interaction partners using yeast two hybrid system with a rice(Oryza sativa ssp. japonica) cDNA library. A bait plasmid pGBKT7-OsMADS15 was constructed, and because the OsMADS15 was predicted as a transcription factor, the bait plasmid pGBKT7-OsMADS15 was verified in advance that it did not autonomously activate the reporter gene HIS3. The bait plasmid and pGADT7-cDNA library were simultaneously transformed to yeast (Saccharomyces cerevisiae)AH109. After selection of HIS3, ADE2 and LacZ reporter genes subsequently agaist the positive (pCL1+BD and AD-T+BD-53) or negative controls (AD-T+BD-LamC), thirty-three positive colonies were obtained and the insertion cDNAs on pGADT7-cDNA library plasmids were PCR amplified and sequenced. Removing the redundant sequences, we finally got 25 unigenes. We identified three MADS-box genes as well: OsMADS1, OsMADS8 and OsMADS14. OsMADS1 and OsMADS8 were E-class genes, while OsMADS14 was an AP1/SQUA subfamily gene. BIFC assay was applied to confirm the interactions in vivo between OsMADS15 and the three candidate interactors (OsMADS1, OsMADS8 and OsMADS14) in tobacco(Nicotiana benthamiana) epidermal leaf cells. The results showed the nYFP (which linked to OsMADS15) and cYFP (which linked to OsMADS1, OsMADS14 and OsMADS8, respectively) could complement to emit fluorescence in the nucleus, indicating the fused proteins could interact with each other in the plant cells. The results might implicate that OsMADS15 function in tetramer complexes with OsMADS1, OsMADS8 and OsMADS14, as described in floral quartet model. These studies provide experimental data for further researching the functions of OsMADS15 in rice flower development and vegetative organs growth and development.

Cinnamate 4-hydroxylase (C4H) is the second enzyme of phenylpropanoid metabolic pathway in plant, and its expression level affects the contents of many secondary metabolites such as flavonoid and lignin. In order to learn more about the molecular mechanisms of flavonoids biosynthesis, a cDNA encoding C4H was cloned through the methods of RT-PCR and RACE from tartary buckwheat(Fagopyrum tararicum). The results showed that the FtC4H cDNA with 1 515 bp in full-length encoded 504 amino acids including all the active sites of C4H. The semi-quantitative RT-PCR analysis showed that the expression level of FtC4H was improved in both cotyledon and hypocotyl (P<0.05) under UV-B stress. Statistical analysis indicated that both of the expression levels of FtC4H in the cotyledon and hypocotyl were significantly associated with the flavonoid contents in the relative tissues, and their correlation coefficients were 0.945 and 0.768, respectively. Our results can provide useful information to understand the relationship between expression level of FtC4H and flavoniod content induced by environmental factors in tartary buckwheat. Further more, this study indicated that FtC4H can be a new candidate target gene for developing high flavoniod tartary buckwheat by secondary metabolic engineering.

Dairy product authenticity is presently a subject of great concern to food authorities, as the incorrect labelling of foodstuffs can present a commercial fraud. Many consumers are concerned about dairy products they eat and accurate labelling is important to inform consumer choice. Thus, the detection of milk species is important in cheese making, not only with regards to accurate consumer information and legal aspects (concerned with labeling and guarantee requirements) but also to medical requirements, food allergies or religious practices. Specific primers and Taq Man-MGB probe were designed according to highly conserved sequence of mitochondrial cytochrome b(Cytb) gene of buffalo(Bubalus bubalus) to develop a Real-time fluorescence polymerase chain reaction (Real-time PCR) assay for rapid detection of buffalo ingrediens. The primers and probes could specific identify 5 kinds of buffalo with 47 kinds of samples. The detection limit of the method was 0.001 ng/μL DNA concentration and 0.01 %(W/W) buffalo milk. The commercial samples were detected by Real-time PCR and the detection results showed that buffalo ingredients were detected well. All the results confirm that the Real-time PCR assay is a sensitive and specific method for the detection of buffalo materials in dairy products.