Abstract The salt overly sensitive (SOS) signal-transduction pathway is a control ion balance signal pathway. This pathway plays a key role in discharge Na+ and ion homeostasis on cellular lever and improves plant salt resistance. It has some realistic significance on enhanced salt tolerance in alfalfa (Medicago sativa), which transformed with muti-gene mediated by Arabidopsis thaliana SOS1, SOS2, SOS3, SOS3-like calcium binding protein 8 (SCaBP8) and bialaphos resistance (Bar) 5 genes of polygenic plant expression vector pSOS. The Agrobacterium-mediated transformation method was used, "Golden Empress" cotyledonary nodes of alfalfa were selected as explants. The best concentration of glufosinate herbicide was identified as 0.6 mg/L, the best concentration of bacteriostat cefotazime was identified as 300 mg/L. 25 independent sources of transformed plants were obtained through screening after the herbicides selection. These plants applied PCR amplification and molecular detection, which showed that there were 6 transformants with the special bands. The positive rate was 24%. Four of positive transgenic plants were randomly selected Southern hybridization identification, and the results showed that there were 3 hybridization signal, and copy number was 1 or 2. The result of RT-PCR analysis demonstrated that the same as expected electrophoretic bands size of Bar gene (463 bp) and SOS1 gene (700 bp) expressed in transgenic plants. It was proved initially that multiple exogenous salt-tolerant genes have been integrated into the transgenic plant genome and expressed at the transcriptional level. After treated with 250 mmol/L NaCl stress, the growth of wild type plants and transgenic plants were inhibited, but the plant height of the transgenic plants were significantly higher than the wild type, the leaf area of the transgenic P1 strain and P2 strain were obviously higher than the control group, the fresh weight of 3 strains were significantly higher than controls, leave chlorophyⅡ content of the transgenic P2 strain and P4 strain were apparently higher than the control group, which demonstrated overexpression of SOS pathway genes could improve the salt tolerance in transgenic alfalfa. The successful construct of transgenic alfalfa provides theoretical basis for selection for saline-alkali soil planting the salt-tolerant alfalfa varieties and alfalfa salt tolerance identification and salt tolerance mechanism research.

Abtract Microbial activity plays very important roles in the formation of the characteristics and quality of Pu-erh tea. Among these microorganism, Bacillus-like species are the dominant group at the late fermentation stage of Pu-erh tea. The present paper reported the distribution and diversity of Bacillus-like species in the different year-old fermented Pu-erh tea. Sixteen Pu-erh tea samples, including Longma Tongqinghao tea, Dayouqing tea and Junyixiang tea, were collected for the investigation. These tea samples were produced by sun-dried green tea leaves of big-leaf species of tea trees (Camellia sinensis) in the certain regions of Yunnan province at different years. The Bacillus-like strains in the samples were isolated by dilution plate method and were preliminarily identified by 16S rRNA gene sequencing analysis. The results showed that 153 Bacillus-like strains were isolated and were classified into 8 genera, 36 species. Among these strains, Bacillus were the most dominate genera (with a number of 20 Bacillus-like species and 87 strains) and was followed by the genera Lysinibacillus (6 species, 20 strains) and Brevibacillus (4 species, 20 strains). The other 5 genus were Fictibacillus, Oceanobacillus, Ornithinibacillus, Paenibacillus and Virgibacillus. Fictibacillus and Ornithinibacillus were first reported and isolating from the fermented Pu-erh tea. Generally, Bacillus-like species and quantities were different in the different tea samples. The numbers of Bacillus-like strain and quantity isolated from the Zhongcha (Hongyin Lvzi) tea were maximum, which were 19 and 3.76×105 cfu/g, respectively. There were 12 Bacillus-like species colonizing in the Wenge Chazhuang (Hongqi Gongshe) tea, which was the most among these tea samples. The quantity of Bacillus-like isolated from the Jingchanghao tea was minimum, which was 7.62×103 cfu/g. The distributions of different Bacillus-like species in these 16 tea samples were different. Ba. cereus and Ba. shackletonii were isolated from 9 tea samples and were the most ubiquitous species. Ba. oleronius was secondly ubiquitous, which was isolated from 8 tea samples. The quantities of the same Bacillus-like species colonizing in different tea samples were different, and the quantities of different Bacillus-like species colonizing in the same tea sample were also different. In conclusion, the results indicated that the Bacillus-like community colonized in the different year-old fermented Pu-erh tea was abundant, but there were differences of Bacillus-like species and quantity among these Pu-erh tea samples. These results may provide material and theoretical reference for exploring the action mechanism of Bacillus-like species on the characteristics of Pu-erh tea, the correlation between Bacillus-like species and the health functions of Pu-erh tea.

Abstract Macrophages are key cells associated with innate immunity, pathogen containment and modulation of the immune response and also the key regulators of tissue repair, regeneration and fibrosis. Commonly used model systems for studying macrophage biology have centered on macrophage-like leukemic cell lines, primary macrophages derived from model organisms and primary macrophages differentiated from blood monocytes. Although these cells have provided important insights into macrophage-associated biology, there are issues that need consideration. The primary drawback of understanding macrophage biology is that reliable and scalable macrophage models for cellular and genetic studies is scarce, limiting their utility in genetic studies. Based on these, in order to elucidate the role and mechanism of guinea pig macrophages in tuberculosis immunization, in the present study, guinea pig peritoneal macrophages were isolated and characterized by using the following methods: Morphological observation was by phase contrast microscope, cell viability analysis was performed using Trypan blue staining method, phagocytosis assay was carried out using the method of swallowing ink particles, the expression of intracellular enzyme was detected using α-naphthol acetate esterace (α-NAE) staining method, and the expression of cell surface specific antigen analysis was detected by flow cytometry. Furthermore, in order to study the effect of Bacillus Calmette-Guérin (BCG) on guinea pig macrophage apoptosis and its production of nitric oxide (NO), guinea pig macrophage was infected by BCG in vitro. The results showed that the guinea pig macrophage performed multiple cell morphology and maintained the characteristic of macrophage-specific morphology, which resembled round shape, oval shape, spindle shape and irregular shape under the microscope. Pseudopodium and bulge were observed in some of the cells. The cells had a large amount of cytoplasm and large nuclei. The result of Giemsa staining showed that the nuclei of macrophage was darker and horseshoe shaped, round or irregular shape, locating at one side of the cells. The macrophage had the ability of favourable phagocytosis, and the phagocytic rate was (94.3±1.04)%. The results of nonspecific esterase staining showed that the rate of positive staining cells was (94.3±1.44)%, which indicated that the purity of the isolated macrophages was higher. Meanwhile, flow-cytometric surface phenotyping of guinea pig macrophage revealed expression of classical macrophage markers such as CD14, CD40 and CD68, and the percentages of positive staining of CD14, CD40 and CD68 were 97.6%, 96.9% and 92.1% respectively. Moreover, the results showed that the production of NO in guinea pig macrophage which were infected by BCG was significantly increased (P＜0.01) compared with the control group. Meanwhile, the results of apoptosis analysis by FITC Annexin V Apoptosis Detection Kit assay combined with fluorescence activated cell sorter (FACS) showed that the ratio of apoptosis of the BCG infected group was (14.4±2.86)% which was significantly higher than that of the control group (4.26±1.06)% (P＜0.01). These results initially confirmed that pre-stimulation of starch broth solution could extract a large number of guinea pig peritoneal macrophages, and confirmed that the guinea pig macrophage had the function in tuberculosis immunity. And the present results may provide new experimental material and data support for the research on interaction between macrophage and Mycobacterium tuberculosis.

Abstract Betaine aldehyde dehydrogenase (BADH) is the key enzyme in the synthesis process of glycine betaine (GB). To explore the expression difference of BADH gene in different tissues of cassava (Manihot esculenta) and the expression under drought and salt stress, bioinformatics approaches were used to identify and characterize MeBADH genes. Using computational methods, these genes were localized on cassava genome chromosome and stress-responsive cis-elements within their 5' upstream regions were identified. Expression profiles of these genes in different tissues were detected by qRT-PCR, at the same time, the differential expression of MeBADH genes under PEG-6000 stress, salt stress and natural drought stress were investigated in cassava. The GB content in cassava was determined by ELISA. The results showed that MeBADHs genes expressed in all tissues (root, root tuber, stem, leaf). The expression in root tuber was significantly higher than that in root (P＜0.05). The expression of MeBADH1 and MeBADH2 in leaves, stems, root and root tuber in 1~2 h changed a little compared with 0 h under PEG- 6000 treatment. Under NaCl treatment, the expression of MeBADH1 in leaf at 8 h was the highest, and was extremely significantly higher than that at 0 h (P＜0.01); the expression of MeBADH2 did not change much at different times. In stems and roots, the expressions of MeBADH1 and MeBADH2 at 1 h were significantly higher than that at 0 h (P＜0.01). Under natural drought treatment, the expressions of MeBADH1 and MeBADH2 in stem and root at 6 and 18 d were significantly higher than that at 0 d (P＜0.01), respectively. Under PEG-6000 treatment, the GB content was fluctuant and slightly increased. The GB content under NaCl treatment showed stable expression in the early stage, and then occurred intense response at 8 h (appeared downward trend), and the GB content at 24 h was significantly lower than that at 0 h (P＜0.05). Under natrual drought stress, the GB content gradually increased at beginning, reached he peak at 9 and 15 d, and was higher than that st 0 d (P＜0.05). This study provides foundation for further research on the molecular mechanisms of cassava resistance to drought stress and breeding drought-resistant new cultivars.

Abstract Osmotin (OSM) is a multifunction protein present in wide plant species. It is inferred that OSM regulates plant adaption to stress through involving in plant growth and development, However, direct evidence linking between OSM and growth is rare. Based on StOSM-3b cDNA of potato (Solanum tuberosum) isolated in previous study, an antisense StOSM-3b vector, aiming to inhibit OSM expression, was constructed in this study. The antisense StOSM-3b was transformed into potato by Agrobacterium-mediated approach. A positive transgenic line was screened by β- glucuronidase (GUS) staining. Analysis on data assayed showed that accumulations of StOSM-3b mRNA and OSM contents by enzyme linked immunosorbent assay (ELISA) in the transgenic potato leafs was 25% and 52% of that in control, respectively. This indicated that the antisense StOSM-3b partially inhibited expression of StOSM-3b and down-regulated OSM accumulation in the transgenic potato. OSM down-expression led to a tardy growth, dwarfing and no tuber-formation phenotype with disorder in growth rhythm. The results provide a direct evidence to indicate that osmotin involves in plant growth and development.

Abstract Glutamate decarboxylase (GAD) mainly distributes in the cytoplasm and catalyzes the reaction of glutamate decarboxylation as a kind of intracellular enzyme found in many organisms. The metabolite of γ-aminobutyric acid (GABA) has the function of regulating plant growth, and development and improving the resistance to abiotic stress. This study cloned the gene of GAD in pakchoi (Brassica campestris ssp. chinensis) cultivar, cv. Wuyueman by the homologous cloning technology, namely BcGAD (GenBank accession No. KP852557). In addition, the effect of exogenous GABA on growth, expression of BcGAD gene and protein as well as enzyme activity were detected in pakchoi plants treated with application or soaking seeds of GABA under rhizosphere high nitrogen level. The results showed that the BcGAD gene contained a 1 485 bp ORF encoding 494 amino acids, which included a conserved domain of pyridoxal phosphate dependent aspartate aminotransferase superfamily. The homology analysis of amino acid sequence showed that the the similarity of amino acid sequences of BcGAD gene and GAD2 gene in B. rapa reached 100%, which demonstrated that there existed the closest homology between them. Moreover, the amino acid sequence of BcGAD was similar to GAD2 of B. napus, Raphanus sativus and B. juncea with the similarities of 99%, 99% and 98%, respectively. The treatments of soaking seeds at 3.75~7.5 mmol/L and application in nutrient solution at 1.25~5 mmol/L significantly promoted the growth supraterraneous compared with higher nitrogen treatment(P＜0.05). qRT-PCR and enzyme linked immunosorbent assay (ELISA) analysis showed that exogenous GABA increased the BcGAD gene transcriptional level and protein expression in pakchoi leaves treated with applying in nutrient solution and soaking seeds at different GABA concentration under the higher level of rhizosphere NO3-. BcGAD gene expression in leaves was significantly increased treated with soaking seeds (2.5~10 mmol/L) and applying GABA in nutrient solution (1.25~5 mmol/L) than that treated with high nitrogen condition. BcGAD protein expression in leaves enhanced accordingly treated with GABA of application in nutrient solution (3.75~10 mmol/L) and soaking seeds (1.25~5 mmol/L). Meanwhile, the GAD activities were significantly increasing by applying 5~6.25 mmol/L GABA in nutrient solution or soaking seeds with 2.5~3.75 mmol/L GABA correspondingly. In general, the treatments of soaking seeds at 3.75~6.25 mmol/L and applying solution at 2.5~3.75 mmol/L showed obvious effects among the different GABA concentration treatments(P＜0.05). Above all, the improved effects were dominant of soaking seeds at 5 mmol/L GABA and application GABA at 2.5 mmol/L. The results primarily confirmed that the growth, BcGAD, BcGAD protein and GAD activity were all affected by exogenous GABA concentration, application method and higher nitrogenlevel of pakchoi rhizosphere, which provides a theoretical basis for further utilization of BcGAD gene and exogenous GABA to improving the quality of leafy vegetables.

Abstract Expansin (EXP) is a class of proteins that causes cell wall relaxation in plants, which play an important role in petal cell expansion during flower opening. In order to explore the expression characteristics of EXP genes in Osmanthus fragrans, 12 unigene with high homology to plant EXP genes were identified from the transcriptome database of floral buds of O. fragrans in this study, and their cDNA sequences were cloned from the petals of the full flowering stage of O. fragrans. According to the sequence alignment in the NCBI database, 12 unigene sequences were named OfEXPA1~OfEXPA8, OfEXPB1~OfEXPB2, OfEXLA1, and OfEXLB1(GenBank No.: KY968700~KY968711), respectively. BLASTX search and comparison with ORF Finder tool analysis revealed that except OfEXPA1, OfEXPA5 and OfEXLB1 genes, whose 5' cDNA sequences were incomplete, the other 9 EXP genes had the full-length cDNA sequences. Sequence analysis showed that the deduced amino acid residues of 9 genes with full-length cDNA sequences were ranging from 240~274, the predicted molecular weights were ranging from 26.06 to 29.7 kD, and pI were ranging from 4.77 to 9.74. The deduced amino acid sequences of the OfEXPA1~OfEXPA8 and OfEXPB1~OfEXPB2 included the typical structural characteristics of all expansins, with eight typical Cys residues in the N-termini of the proteins, four Trp residues near the C-termini and most of the EXPA and EXPB proteins contained histidine domain motif sequence HFD. And OfEXLA1 had the characteristic motif of CDQC, and the OfEXLB1 motif had not been reported. The results of qRT-PCR analysis found that all EXP genes were expressed in different flower bud developmental stages of O. fragrans ‘Yanhong Gui’. The overall expression pattern of OfEXPA1, OfEXPA3, OfEXPA5, OfEXPA6, OfEXPA7, OfEXPA8, OfEXPB1, OfEXPB2 and OfEXLB1 was similar, the expression in the former stage of flower bud development (B1-B6) was significantly higher than that of petal expansion stages (B7-FF), and the expression decreased gradually in these stages; While OfEXPA2, OfEXPA4 and OfEXLA1 were lowly expressed in the former stages, but rapidly increased in the obvious expansion period of petals (B6), and presented high expression in petal expansion stages. The phylogenetic tree analysis showed that OfEXPA4 and OfEXPA2 had a close relationship with GgEXPA1 in gladiolus (Gladiolus grandiflorus) and RhEXPA1 in rose (Rosa chinensis), respectively, and the expression patterns of OfEXPA2 and OfEXPA4 were similar to those of GgEXPA1 and RhEXPA1 genes, suggesting that these two genes were the key members of expansin gene family related to the process of flower opening of O. fragrans; According to the characteristics of OfEXLA1 expression, it was speculated that it was also involved in the regulation of flower opening. The results provide a theoretical basis for the comprehensive understanding of the mechanism of flower opening and the function of EXP genes.

Abstract DNA methylation is a relatively stable epigenetic modification, and it plays critical roles in plant growth and development as well as stress resistance. Currently, much is understood about DNA methylation processes, but the understanding of DNA demethylation processes remains as yet incomplete. To better unravel the molecular mechanisms of DNA demethylation, this study performed mutant screening and consequently isolated a low luminescence mutant, referred to as rll2 (for reduced LUC luminescence 2), from an ethyl methanesulfonate (EMS)-mutagenized M2 population derived from a Col-LUC transgenic line that carries a 2×35S:LUC (LUC is the abbreviation of luciferase) reporter and emits high LUC luminescence in regular growth conditions. The wild-type gene defined by the mutation was hence termed as RLL2. Compared to the Col-LUC, the rll2 mutant emitted quite low luminescence, and exhibited obvious dwarfism. Genetic analysis revealed that the rll2 mutant carried a single nuclear-encoded recessive mutation that proved to be responsible for the low LUC phenotype. The RLL2 gene was mapped on chromosome 5 and then narrowed down the rll2 mutation to a small region between the markers CL506-B14M1 and CL507-B6M1 which were located at BAC(bacterial artificial chromosome) clones F5O24 and T6G21, respectively. Chop-PCR results demonstrated that several genomic loci were hypermethylated in the rll2 mutant, and reverse transcription PCR(RT-PCR) data indicated the expression levels of a few endogenous genes targeted by RNA-directed DNA methylation (RdDM) pathway were decreased to varying extents in such a mutant. In summary, in this research a low luminescence mutant rll2 was obtained, and the RLL2 gene was mapped on chromosome 5 by map-based cloning strategy. This study revealed that RLL2 is presumably involved in the DNA demethylation processes, which is going to further contribute to our understanding of the molecular mechanisms of such DNA demethylation processes.

Abstract Estrogen receptorα (ERα) gene plays an important role in the process of animal breeding. It has been considered as the main gene affecting the litter size of swine, but it is hardly studied in goat (Ovis linnaeus). In order to reveal the effects of ERa gene on reproductive traits of Guizhou local goat, the polymorphism of ERα gene 8 exons was detected by direct sequencing and the ERα mRNA expression in Qianbei Ma goat hypothalamus, pituitary and gonadal axis was detected by qRT-PCR. The results showed that C127T existed in the eighth exon of ERα gene of Qianbei Ma goat and Guizhou black goat. The C127T was a missense mutation and caused the change of mRNA secondary structure and encoding protein of ERα gene. CC, CT and TT genotypes were detected at C127T locus of the eighth exon of ERα gene. The Qianbei Ma goat with CC achieved significant difference level compared to CT and CC genotype. But the Guizhou Black goat did not reach significant differences. qRT-PCR results showed that the ERα gene in 2 groups (single lamb group and more lamb group) were expressed in 5 tissues of the Qianbei Ma goat. The highest in ovaries, the lowest in the fallopian tube, and the mRNA expression in the two groups of North Guizhou sheep tissues did not reach significant levels (P＞0.05). These results indicated that C127T mutation of ERα gene might have an effect on reproduction traits of local goats in Guizhou province. This study provided the theoretical basis for fostering the high fecundity lines of local goats in Guizhou.

Abstract Raffinose is ubiquitously occurred in higher plant as an important soluble carbohydrate, which is known to be involved in various abiotic stresses. Raffinose synthase (RS, EC2.4.1.82) is the key enzyme that catalyses the reversible galactosylation, yielding raffinose and myo-inositol. In this study, the raffinose synthase gene TaRS of wheat (Triticum aestivum) was cloned by homologous alignment. The TaRS was assigned to wheat chromosome 3B. Sequence analysis revealed that TaRS had a whole open reading frame (ORF, 2 349 bp) and belonged to the glycoside hydrolase super family (GH-D) which contained 2 conservative motifs, KxD and RxxxD. In evolutionary relationship, TaRS shared the highest homology with the indicated RS from Aegilops tauschii (XM020298559.1). Southern blot assay showed that there existed at least 4 allelic copies of TaRS in Chinese spring wheat genome. Subcellular localization analysis revealed that TaRS protein was localized at the cell membrane in wheat protoplasts. Tissue specific analysis showed that TaRS expressed in root, stem, leaf and seed, but had the highest expression level in leaves. Heterologous expression was conducted in Escherichia coli, and the crude extract was able to utilize sucrose and galactinol as substrates to synthesis raffinose in vitro based on the result of high performance liquid chromatography (HPLC). Furthermore, TaRS displayed an optimum activity at about pH 8.0. Expression pattern analysis under multiple abiotic stresses indicated that the expression of TaRS was induced by dehydration, high temperature, salinity, and low temperatur, and reached the highest expression level at 12, 1, 1, 48 h, respectively. These results indicated that TaRS may play critical roles in wheat tolerance of abiotic stresses and provides theoretical basis for further studying in wheat abiotic stress tolerance breeding.

Abstract Antifreeze proteins (AFPs) is a kind of special small molecule protein produced by cold-resistant organisms for adapting low temperature environment, and it can decrease ice point and inhibit ice recrystalization. In this research, the expression level of DcAFP from carrot (Daucus carota) driven by environment inducible promoter Prd29A (responsive to desiccation 29A) and constitutive promoter Cauliflower mosaic virus 35S (CaMV35S) in transgenic tobacco (Nicotiana tabacum) were detected. The results showed that the DcAFP gene has been integrated into tobacco genome and could express stably in progeny. The phenotype of inflorescence in T1 transgenic tobacco was dysplasia, and its capsule was easily to detach from the carpopodium, and few fruits could develop normally. After 4 ℃ acclimation for 10 d, -1 ℃ low temperature treatment for 24 h and 25 ℃ recover for 7 d, the survival rate of T1 tobacco plants transformed with Prd29A:AFP was 34.6% higher than that transformed with CaMV35S:AFP, and was 61.1% higher than wild type tobacco plants. T1 tobacco plants transformed with Prd29A:AFP grew well, while wild type tobacco was most dead. The chlorophyll fluorescence parameters (Fv/Fm) of all kinds of tobacco plants were decreased under cold stress, but after 25 ℃ recover, the Fv/Fm in Prd29A:AFP transgenic tobacco restored to the normal level, but the Fv/Fm in CaMV35S:AFP transgenic tobacco was just a little higher than that in wild type tobacco. Compared with wide type tobacco, proline content, soluble sugar content and soluble protein content in the two DcAFP transgenic tobacco under cold acclimation at 4 ℃ were increased, and the activity of superoxide dismutase (SOD) and peroxidase (POD) enhanced, and isozyme bands were increased. At the same condition, proline content, soluble sugar content, soluble protein content as well as SOD and POD activity in Prd29A:AFP transgenic tobacco were 45.1%, 12.1%, 20%, 19.7% and 8.6% higher than that in CaMV35S:AFP transgenic tobacco, respectively. The results demonstrated that the expression of DcAFP driven by Prd29A promoter could significantly enhance the cold tolerance of transgenic tobacco and had fewer effects on plant growth. These results have great reference value for breeding new cold tolerant transgenic plant varieties.

Abstract As an endogenous small non-coding RNA (ncRNA), microRNAs (miRNAs) regulate gene expression mainly at post-transcriptional level. In recent years, numerous studies have demonstrated that miRNA could have an impact on adipocyte differentiation by modulating the expression of adipogenic transcription factors and signaling molecules. This study showed that the expression of miR-26a gradually rose on day 4 and reached the maximum level on day 8 during 3T3-L1 cell differentiation. In order to investigate the regulatory effects and mechanism of miR-26a on adipogenesis in 3T3-L1 preadipocyte of mouse (Mus musculus), the miR-26a agomir and antagomir were transfected into 3T3-L1 cells to perform miR-26a overexpression and knockdown, respectively. The result showed that miR-26a was significantly overexpressed following agomir transfection on day 2 of differentiation, and the elevated miR-26a expression was maintained until the eighth day after differentiation (P＜0.01). In contrast, miR-26a was effectively inhibited on day 2, 4 and 8 after induction when the antagomir was transfected into 3T3-L1 cells (P＜0.01). And overexpression of miR-26a significantly accelerated the relative mRNA expression of genes associated with adipogenesis such as peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and lipoprotein lipase (LPL) (P＜0.01, P＜0.05) on day 2, 4 and 8, and also increased the protein level of PPARγ and FAS (P＜0.05) on day 8 after differentiation. MiR-26a overexpression also led to a notable increase in lipid accumulation. In contrast, inhibition of miR-26a expression decreased the relative mRNA expression (on day 2, 4 and 8) and the protein level (on day 8) of PPARγ and FAS (P＜0.01, P＜0.05) after differentiation. Although there was no significant change in LPL mRNA on day 8 compared to ago-NC group, a significant decrease was detected on day 2 and 4 after differentiation (P＜0.05). Photomicrograph and quantitative analysis of Oil Red O on day 8 of differentiation also revealed that knocking down miR-26a decreased the formation of neutral lipid droplets. Next, to further determine the function of miR-26a, bioinformatics analysis was carried out to predict direct targets of miR-26a, the result showed that the 3' UTR of phosphatase and tensin homolog (PTEN) contained the target sites for miR-26a. By luciferase reporter assay, the firefly luciferase activity was reduced when co-transfection of pmirGLO-PTEN-3' UTR and miR-26a agomirs (P＜0.01), but remained unchanged in other combinations. To better understand the mechanism of miR-26a during adipogenic differentiation in 3T3-L1, the effect of miR-26a on the expression of PTEN had been further validated and the result revealed that overexpression of miR-26a lead to an obvious decrease in both the mRNA (P＜0.01) and protein levels (P＜0.05) of PTEN on day 8 of differentiation. In contrast, when the endogenous miR-26a was knocked down with the synthetic miR-26a antagomirs, the PTEN mRNA (P＜0.01) and protein (P＜0.05) levels were increased compared to the ant-NC group. Together, it was concluded that miR-26a positively regulated 3T3-L1 cell differentiation by directly inhibiting PTEN. Therefore, miR-26a and its target genes may play a role in the pathological progression of obesity, which may provide a novel research direction for biological therapy of obesity-related diseases.

Abstract Chicken (Gallus gallus) interleukin-4 (IL-4), which is a key indicator for Th2 type immune response in poultry, is very important in immune response and immune function analysis of chickens (Gallus gallus). Due to the lack of effective assays, the study of chicken IL-4 (ChIL-4) is still lagged behind other cytokines. To prepare recombinant ChIL-4 with high bioactivities, molecular biotechnology was used to construct recombinant vector pBac-ChIL-4, and then pBac-ChIL-4 expressed in Spodoptera furgiperda 9 (Sf9) cells. Firstly, ChIL-4 cDNA fragment was digested from plasmid pVAX1-ChIL-4 constructed previously with restriction enzymes and then subcloned to vector pFastBac1 to construct a new plasmid pFast-ChIL-4. With site-specific transposition, ChIL-4 cDNA fragment was transformed to vector bacmid in the baculovirus vector system to form recombinant plasmid pBac-ChIL-4. Secondly, pBac-ChIL-4 was transfected to Sf9 cells to generate recombinant ChIL-4 protein (rChIL-4 or Bac-ChIL-4). Finally, infected Sf9 cells were identified by immunofluorescent assay (IFA) for its expression of ChIL-4, and the supernatants of Sf9 cells infected were harvested and further analyzed by indirect enzyme-linked immuno sorbent assay (ELISA) and lymphocytes proliferation assay. The results of IFA indirect ELISA showed that rChIL-4 successfully expressed in the baculovirus vector system with bright green fluorescence observed in transfected Sf9 cells, polyclonal antibody against ChIL-4 generated previously could only reacted with rChIL-4 (Bac-ChIL-4, His-ChIL-4), but not other irrelative proteins (bovine IL-4, chicken IFN-γ) revealed by ELISA. Splenocytes proliferation assay demonstrated that rChIL-4 had good bioactivity in stimulating and activating T lymphocytes. This study not only facilitates the structural and functional analysis of ChIL-4, but also provides helpful reference value for the expression of other cytokines.

Abstract Hypoxia is one of the major stresses in aquaculture animals. Bighead carp (Hypophthalmichthys nobilis) is one of the most important aquaculture fish in China, which is sensitive to hypoxia stress, and the related genetic research is rarely reported. In this study, six hypoxia-sensitive samples (S group) and six hoypoxia-tolerant samples (T group) from a hypoxia stress experiment of bighead carp were selected, and differentially expressed genes between the two groups were analyzed using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. A total of 10 571 transcript-derived fragments (TDFs) were obtained from 256 primer combinations of the EcoRⅠand MseⅠ AFLP system, and the number of TDFs produced by a single primer combination ranged from 21 to 63, with an average of 41. Four types of differential straps were screened in the hypoxia stress experiment of bighead carp, including A (up-regulated band), B (down-regulated band), C (the fragment only expressed in one sample) and D (the fragment expressed in all samples), and bands of types A and B were chosen as target bands. Among those 221 differentially expressed TDFs between two groups, 137 (62%) were up-regulated, and 84 (38%) were down-regulated. 70 TDFs were successfully cloned and sequenced, and the length of fragments ranged from 96 to 402 bp. The results of sequence blast against public databases revealed that 29 TDFs (TDF42~TDF70) had no hits to any known homologous sequences, 9 TDFs (TDF33~TDF41) were unknown functional sequences, and 32 TDFs (TDF1~TDF32) showed homology to genes of known functions. Among the 32 differentially expressed TDFs, TDF29 and TDF32 sequences were different but both encoded the same protein (S- adenosylmethionine synthase). These functional genes participated in such physiological pathways as transcriptional regulation (16%), stress and signal transduction (9%), energy metabolism (7%), protein synthesis (4%), immune defense (4%) and cell proliferation (6%). Four TDFs with the genes of known functions (ANXA6, TRIM25, Tnp and RBAT) were randomly selected for verification by qRT-PCR analysis. For ANXA6, TRIM25 and Tnp, the expression patterns from qRT-PCR were consistent with those from AFLP analysis, while the expression pattern of RBAT was different in two methods. The results of this study would facilitate revealing molecular mechanism of genetic regulation responsible to hypoxia stress in bighead carp, and they would also provide useful information for potential gene (marker)-assisted selection towards hypoxia-tolerant variety in aquaculture of this species.

Abstract Zera tag allowed its recombinant fusion antigens to form fully active protein microparticles in planta and markedly enhanced the immune response. Thus Zera tag-induced microparticles may be regarded as promising production and delivery vehicles for subunit vaccines. To test this hypothesis, Zera-GFP sequences were synthesized in vitro, and pJ Zera-GFP plant expression vector was constructed by subclone technology. The vectors were co-inoculated on Nicotiana benthamiana plants with pCBNoX HC-Pro vector, a plant binary vector expressing the RNA silencing suppressor of HC-Pro protein, through agroinfiltration. The epidermal cells expressing Zera-GFP in inoculated Nicotiana leaves were then surveyed by confocal laser scanning microscopy, and the relative expression level of Zera-GFP were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot respectively. All data were collected for the assessment of the potential of Zera tag-induced microparticles as delivery vehicles for subunit vaccines. And a simple purification technique for Zera tag-induced microparticles was established. The effect of Agrobacterium tumefaciens strains on the expression level of pJ Zera-GFP vector was tested firstly and the better results were obtained when using the EHA105 strains instead of GV3101strains. It was also found that Zera-GFP was expressed in the form of protein particles and they were not found to be toxic to the N. benthamiana tissues. The morphological characteristics of Zera-GFP particles in tobacco leaf cells were further observed. The particles were composed of small round spheres with different size and clear edge. The diameter of the sphere particles was between 0.5~2.5 μm, the spheres were close to each other and were closely clustered together. Based on the analysis of the factors such as particle size, morphology, uniformity and antigen content of Zera tag-induced particles, it is suggested that the particles had the potential to be a delivery vehicles of subunit vaccine. Then Zera-GFP particles were purified by sucrose density gradient and sucrose cushion centrifugation. The results showed that Zera-GFP particles could be purified from the inoculated tobacco leaves by both centrifugal technology and 60% sucrose cushion was more suitable for the purification of Zera-GFP particles. Otherwise, we speculated that the formation of Zera tag-induced particles in the plant cells was a dynamic process from soluble to aggregation. The results of this study provide a theoretical and practical basis for the study of the novel particulate vaccine delivery systems based on Zera tag.

Abstract Primordial germ cells (PGCs) are precursor cells of gonocyte, as a kind of pluripotent stem cell,PGCs have a similar morphology and multilineage potential with the embryonic stem (ES) cell. It can be cultured and subcultered in vitro and maintain its undifferentiated state and pluripotency under certain conditions. At present, PGCs are often cultured with feeder cells and serum, which causes difficulties and confusion for the later research. In order to establish a feeder-free culture system for PGCs in poultry, PGCs were isolated and cultured in vitro by improving the culture system in this study. The results showed that the PGCs with no feeder layer were 6~8 μm in diameter and the growth curve showed "S" shape. The population doubling time was (3.75±0.15) d. In the course of in vitro culture, PGCs could have better self-renewal and proliferation in the absence of feeder layer system conditions. PGCs could be cultured in vitro and maintain their undifferentiated state. Compared with similar studies this study not only maintained PGCs differentiation potential, but also obtained a large number of cells. The absence of feeder layer system could make PGCs maintain good growth status and had strong cell viability. The specific genes stage-specific embryonic antigen-1 (SSEA-1) and deleted in azoospermia-like (DAZL) of PGCs were identified by immunofluorescence, and the results showed that both of them were expressed in PGCs, the cells stained by chemical immunization could observe the green fluorescence. The purified PGCs were labeled with PKH26 and cultured by feeder-free layer,then they were injected into the dorsal aorta of recipient chicken embryo that was hatched to the 17~20 stage until 5.5 d. The results showed that the PGCs which were isolated from chicken embryo had the same characteristics of itself after PKH26 labeling, which could be colonized in chicken embryo gonads with the blood circulation of chicken embryo. PGCs in vitro culture system had been optimized to ensure the undifferentiated state of it,since in vitro culture of PGCs was easier to differentiate. At present, the cultivation of PGCs was mainly based on Dulbecco's Modified Eagle Medium (DMEM) or Tumor Conditioned Medium (TCM), while a certain proportion of fetal bovine serum, amino acids and cells growth factors had been added on the basis of the feeder cells. But both the demanding preparation and a variety of cytokines or other components of the feeder cells had turned into a great obstacle for the research on PGCs proliferation, migration and other molecular mechanisms. This study optimized culture conditions of PGCs in vitro and broke the traditional culture method with serum and feeder layer and established a serum-free culture system without feeder layer. This culture system could avoid the adverse effects of the heterologous cells as feeder layer and the damage to cells from the drug residues of feeder layer. The composition of the culture medium was clear and it was convenient to study the molecular mechanism of cell differentiation and provides important technical support for application of PGCs in PGCs-based modern preservation technology, transgenic technology and developmental biology of poultry.

Abstract Orf virus (ORFV) is the pathogen of Contagious ecthyma (CE). Mycoplasma ovipneumoniae (Mo) is the main pathogen of Mycoplasma pneumonia of goats and sheep (MPGS). In recent years, the mixed infection of ORFV and Mo is increasing. Currently, the main detection method is single PCR, it is imperative to develop a method for rapid and simultaneous detection of ORFV and Mo in clinics. Thus, two pairs of specific primers were designed according to the major envelope protein B2L gene of ORFV and membrane protein P80 gene of Mo. The results showed that the assay could amplify 402 bp for ORFV and 700 bp for Mo simultaneously, but other common pathogen's DNA could not amplify, indicating good specificity. The detection limits of the method was 1.1×103 copies/μL for ORFV and 3.47×103 copies/μL for Mo, respectively, and these limits were 10 times more sensitive than the single PCR assay. The duplex PCR and single PCR were simultaneously performed to detect 83 clinical samples from Fujian, and the results showed that the positive rate of ORFV and Mo were 33.7% and 39.8%, respectively, co-infection rate of ORFV and Mo was 10.8%. The date indicated that the duplex PCR established in this study can be used for clinical rapid detection of ORFV and Mo and epidemiological investigation of CE and MPGS.

Abstract In the qRT-PCR analysis, selection of suitable reference genes is a prerequisite for the guarantee of accurate result. As the lack of genome information, the reference gene selection is particularly important for Chinese milk vetch (Astragalus sinicus). In this study, expression of seven commonly used housekeeping genes such as GAPDH, Cons7, ELF1B, Tubulin, Ubiquitin E2, Actin A and Actin 20 were assessed by qPCR in different tissues (roots,stems,leaves, flowers and pods) and different treatment (drought stress, waterlogging stress, salt stress, abscisic acid (ABA) stress) of Astragalus sinicus from NingBo. To determine the expression stability of these genes, qRT-PCR data were determined by three software tools (GeNorm, NormFinder and BestKeeper). The results showed that the highest expression stability candidate reference genes of Chinese milk vetch had many differences in the various experimental conditions. Cons7 could express stably under salt stress and waterlogging stress, and Tubulin, Actin 20, GAPDH were top ranked under different tissues, ABA treatment and drought stress respectively. In different tissues, Tubulin could be the best housekeeping gene. Cons7 was considered as the most appropriate housekeeping gene of Chinese milk vetch among abscisic acid treatment, salt stress and flooding stress, while GAPDH could be chosen as the best housekeeping gene under drought stress. The reference genes selected in this study will be helpful for improving the quality of gene expression studies under various stresses in Chinese milk vetch