Bacillus cereus can produce α-amylase, which has important industrial production value and can endure high temperatures. The enzymatic characteristics of two α-amylases from B. cereus (B905 and B904) were studied. The results showed that both preserved their activity at 90–100°C. Their thermal stability and enzymatic activity did not depend on Ca2+. However, their protein molecular weights were obviously different. The structural genes of amy905 and amy904 were cloned successfully using the polymerase chain reaction (PCR) and expressed in Escherichia coli. The amy904 and amy905 genes were 1362 bp and 1761 bp long and their protein molecular weights were about 55 kDa and 68 kDa, respectively.

A rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) <2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

With the rapid development of nanotechnology, the security of nanomaterials has been an increasing cause for concern. In this study, the impact of titanium dioxide nanoparticles (nano-TiO2) on the phyllosphere bacterial community were analysed by both a culturable-dependent method and a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) method. The quantity of culturable phyllosphere bacteria was significantly reduced with an increased concentration of nano-TiO2. With increasing concentrations from 0.002 to 20 mg/ml of nano-TiO2, the quantity of culturable phyllosphere bacteria decreased from 1.8×106 to 3.1×105 cfu/g. The phyllosphere bacteria community was analysed by PCR-DGGE, and when the concentrations of nano-TiO2 were higher than 0.02 mg/ml, the DGGE bands were significantly lower than in the control. Sequencing results of the bands from the DGGE gel showed that there were at least seven genera in the phyllosphere bacteria. Only one uncultured bacterium was unaffected by the concentration of nano-TiO2.

The Dorset ram of the callipyge phenotype presents with muscular hypertrophy in the buttocks, and its inheritance is polar overdominant. A partial DNA fragment of 250 bp was obtained from the goat (Capra hircas) callipyge gene (CLPG; GenBank accession no. EU753362), which shared 96.04% and 88.65% identity with the corresponding regions of ovine (Ovis aries) and porcine CLPG, respectively. A polymorphism in the DNA fragment was detected by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Sequencing results indicated no A→C mutation corresponding to the ovine CLPG gene, although one A→C transversion was located 147 bp downstream from the CLPG site. The polymorphism, named SNP216 after its position (where SNP indicates single-nucleotide polymorphism), was investigated in Boer (n=63), Laiwu Black (n=70), Lubei White×Boer Hybrid (n=40), Lubei White (n=29) and Inner Mongolia Alashan White cashmere (n=115) goat populations. The results indicated that allele A was dominant in four of the goat populations, the Inner Mongolia Alashan White cashmere goats being the exception. The first four populations were in a state of Hardy–Weinberg equilibrium (P>0.05). In Inner Mongolia Alashan White cashmere goats, least-square means of birth weight, production of cashmere and body weight gain from birth to weaning did not differ significantly between the AA and AC phenotypes (P>0.5).

The genetic diversities of 72 individuals from three wild Lenok populations of Mudanjiang River (MD), Yalujiang River (YL) and Wusulijiang River (WSL) in the northeast of China were analysed using amplified fragment length polymorphism (AFLP) markers. The results showed that 541 polymorphic loci out of 559 were amplified by 12 primer pairs and the percentage of polymorphic loci was 96.78%. Shannon indices for the MD, YL and WSL populations were 0.3988±0.2913, 0.3254±0.3037, 0.2125±0.2862, respectively, and Nei's gene diversity indices were 0.2737±0.2062, 0.2229±0.2129, 0.1446±0.1985, respectively. The average total genetic diversity (Ht) was 0.3512±0.0.0208 and the average genetic diversity within populations (Hs) was 0.2137±0.0152. Among the three populations, the average genetic distance (Dst) was 0.1375 and the gene differentiation coefficient (Gst) was 0.3914. The genetic diversity was 60.85% within populations and 39.15% among populations. The gene flow index (Nm) was 0.7776. The analysis of molecular variance (AMOVA) indicated that the average fixation index (Fst) was 0.55336. The variance was 55.16% within populations and 44.84% among populations. The highest polymorphism ratio was in the MD group and the lowest in the WSL group.

A rapid and sensitive real-time polymerase chain reaction (PCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from the TRBIV major capsid protein (MCP) gene was involved in the real-time PCR (RT-PCR) assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescent dye, SYBR Green I, which exhibited fluorescence enhancement when bound to double-stranded (ds) DNA. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid, pUCm-T/TRBIV MCP, containing the target sequence, was quantified to make a standard curve for sample detection after serial tenfold dilution. Linear coefficient correlations between the cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (R2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of virus in different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively), while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has become epidemic in turbot farms located along the Shandong peninsula. The virus number varied from 1.27×102 to 2.33×106 viral genome copies/mg tissue in spleens of infected turbot. These results suggest that the RT-PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.

The spatio-temporal expressed profiles of two kinds of chemical communication-related protein genes, the odorant-binding protein of Ac-ASP2 and chemosensory protein of Ac-ASP3, were identified by real-time polymerase chain reaction (PCR). Results obtained using the 2−ΔΔCt method showed that Ac-ASP2 was a gene coding antenna-specific protein that did not express in larvae and pupae, but had discontinuous high abundance periods at 1, 9, 15, 27 and 30 days. The expressing abundance at such periods was at least ten times higher than that at other periods. From the distribution of Ac-ASP3 mRNA observed in different tissues, the transcript levels seemed to be higher in the wings, abdomen and thorax (of order ~106), and lower in the legs, antennae and head (of order ~105). From highest to lowest, the original copy number was found in the various body parts in the following order: wings, abdomen, thorax, legs, antenna, and head. The results suggest that Ac-ASP3 has an intimate relation with the chemosensory behaviour of wings and abdomen in Apis cerana cerana.

The DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA of Pseudoalteromonas atlantica 19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed in Escherichia coli ER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.

The recombinant PET-N plasmid, which includes the N gene of the Transmissible gastroenteritis virus (TGEV), was transformed into Escherichia coli BL21 (DE3), and induced at 37°C with 1.0 mmol/l IPTG (isopropyl β-d-1-thiogalactopyranoside). An indirect enzyme-linked immunosorbent assay (ELISA) for detecting the TGEV nucleocapsid protein antibody was developed after the reactogenicity of the recombinant protein was demonstrated by Western blot. The operating conditions for the ELISA, an antigen concentration of 15 μg/ml, serum dilution of 1:40, blocking solution of 0.5% fetal bovine serum (FBS), serum sample incubation for 90 min, a concentration of horseradish peroxidase (HRP)-spa of 1:5000, incubated for 60 min, incubation of the substrate at room temperature for 10 min, and a cutoff value for the ELISA OD450⩾0.35, were carried out using a checkerboard titration method. The sensitivity and specificity of this method relative to the Svanova TGEV/PRCV antibody diagnosis kit were 93.5% and 93.8%, respectively.

Macrophages from the head kidney (HK) and peripheral blood leucocytes were isolated fromCyprinus carpio by Percoll gradient density centrifugation, cultured in vitro and exposed to different concentrations of the immunomodulator lentinan. To evaluate the immunostimulating effects of lentinan, proliferation of the peripheral blood leucocytes, respiratory burst of macrophages and interleukin-1β (IL-1β) gene mRNA expression of macrophages were investigated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], NBT (nitroblue tetrazolium) reduction, Griess reaction and real-time polymerase chain reaction (PCR). Results showed that proliferation of peripheral blood leucocytes after 24 h incubation by the induction of lentinan at 100 and 1000 μg/ml was markedly stimulated. Lentinan at 1, 100 and 1000 μg/ml could significantly induce superoxide anions in macrophages. Production of nitric oxide by HK macrophages after 96 h incubation by lentinan showed that it had no conspicuous effect on nitrogen burst activity of macrophages. Moreover, it inhibited the nitrogen burst activity at higher doses. The expression of IL-1β in the HK macrophages after 24 h of polysaccharide stimulation showed that lentinan stimulated IL-1β expression in the head kidney in carp. Lentinan can modulate the immune response of C. carpio.

Amniotic fluid stem (AFS) cells isolated from human amniotic fluid were cultured and the factors affecting the primary culture of these cells were investigated. The isolated AFS cells express both embryonic stem cell markers, such as Oct-4, and adult stem cell markers, such as CD29, and possess a high proliferation capability. Moreover, isolated AFS cells were able to differentiate into Nestin-positive and α-actin-positive cells. There were several factors, including the date of gestation, the level of blood contamination and the volume of amniotic fluid, which could significantly affect the attachment time and numbers of AFS cells in primary culture. The results showed that the cell attachment time (4.7±0.6 days) during the second trimester of gestation was significantly different from that during the third trimester of gestation (6±0.5 days) (P<0.05), suggesting that cells collected from the fluid during the third trimester of gestation needed a longer attachment time. Blood contamination could significantly affect the cell attachment time. The attachment time of the brown-coloured fluid group (10.8±0.3 days) was significantly different from the blood-cell-free fluid group (6±0.5 days) and the blood-cell group (6.3±0.6 days) (P<0.05). The volume of amniotic fluid influenced, to some extent, both cell attachment numbers and time. With the increase in amniotic fluid volume, cell attachment numbers significantly increased (P<0.05), and cell attachment time extended, but not significantly (P>0.05). The present studies systematically examined the factors that influence the primary culture of human AFS cells and provided useful data on AFS cell research. Additionally, the isolated AFS cells maintain the capacity for differentiation into other cell types and are able to become seed cells for clinical applications.

To verify its antibacterial activity, recombinant human lactoferrin (rhLF) was extracted from the milk of transgenic mice (Mus musculus) (PCL25 and AP) by gel filtration chromatography and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and enzyme-linked immunosorbent assay (ELISA). In addition, its bacteriostatic properties were tested using the agar disc diffusion method. ELISA analysis showed that the concentration of rhLF in the milk of transgenic mice ranged from 7 to 8 mg/ml, and the recombinant protein expressed in the milk had the same molecular weight as the native protein (~78 kDa), indicating that the rhLFs had a strong antibacterial activity on Escherichia coli and Salmonella.

Simple sequence repeats (SSR) and random amplification of polymorphic DNA (RAPD) molecular markers were used to assess the genetic diversity of 80 isolates of Phytophthora infestans in potato (Solanum tuberosum) from Fujian, Heilongjiang, Hebei and Inner Mongolia Provinces in China. Polymorphism was identified by 13 SSR primers and 14 RAPD primers in the isolates of P. infestans in potato. A total of 76 bands were amplified by SSRs, with the percentage of polymorphic bands (PPB) being 78.9% and the similarity coefficient ranging between 0.00 and 0.42. A total of 189 bands were amplified by RAPDs, with the percentage of polymorphic bands being 95.2% and the similarity coefficient ranging between 0.04 and 0.66. Analysis of genetic diversity showed that there exists higher genetic variation in the Fujian population in comparison to the populations of Heilongjiang, Hebei and Inner Mongolia. Nei's genetic identity analysis indicates that the genetic similarity between populations of Heilongjiang and Inner Mongolia is the highest and that between Fujian and Hebei is the lowest. A cluster analysis revealed that isolates from Fujian, in the south of China, are distantly related to those from Heilongjiang, Hebei and Inner Mongolia in the north, and the Fujian population is distributed among more groups than the other three, exhibiting a higher genetic diversity.

BalB/C neonate mice and adult BalB/C mice were vaccinated using BCG (Bacille Calmette–Guerin). The pathogenic growth characteristics of BCG in in vitro culture on spleen cells (SPC) were observed and changes in induced expression of IFN (interferon)-γ and IL (interleukin)-4 in SPC were detected using the ELISPOT assay. The results showed that a low dose of BCG (2×103 cfu) exerted 100% immunoprotection on 7-day-old neonate mice and a high dose of BCG (4×104 cfu) exerted 75% immunoprotection. A low dose of BCG (2×103 cfu) exerted 67% immunoprotection on 35-day-old mice. It is also shown that Th1-type cell immunity dominated by IFN-γ was enhanced significantly in the neonate mice injected with a low dose of BCG (2×103 cfu), and Th2-type cell immunity dominated by IL-4 was depressed at the same time. IFN-γ and IL-4 induced by a high dosage of BCG (4×104 cfu) in neonate mice were both increased. IFN-γ and IL-4 induced by a low dose of BCG (2×103 cfu) in 35-day-old mice were also increased. The results indicate that there exists a marked correlation between immunoprotection by BCG in mice and both the immunizing dose and age of the immune animals, which might be relevant to the changes induced by BCG on Th1- and Th2-type cell immunity.

This study aimed to explore high-throughput cDNA array monitoring technology and to apply it to the gene expression spectrum analysis of salinity-challenged tobacco plants. A Nicotiana tabacum cDNA library was sequenced and found to consist of 5927 high-quality sequences (GenBank accession nos CV015900-CV021826). By analysing the expressed sequence tags (ESTs), the proportion of N. tabacum genes was identified at the EST level. A cDNA array was constructed based on the tentative unique transcripts (TUTs) derived from EST assembling results. A total of 42 differentially expressed genes were identified, including plasma membrane intrinsic protein 2a, ethylene-responsive proteinase and pre-mRNA splicing factor prp31 gene, suggesting that there was a complicated biological response in N. tabacum under saline stress.

To illustrate the effect of nitric oxide (NO) on the microtubules of Sertoli cells (SC), SCs of piglets were treated with sodium nitroprusside (SNP). Changes in cell viability, anti-oxidant activity, enzyme activity and p38 mutagen-activated protein kinase (p38MAPK) activation were detected. The results were as follows. A low concentration of NO can keep SC microtubule and cell viability normal, and a high concentration of NO could increase p38MAPK activation, decrease anti-oxidant activity and transferrin secretion, and destroy the structure and distribution of the microtubules. The results suggest that SNP treatment results in an increase in NO in SCs and decreased cell anti-oxidant activity. The high concentration of NO destroys cell microtubules by activating p38MAPK.

The F1 hybrid offspring of the distant hybridization between Cichlasoma trimaculatum (male) and Heros managuense (female) were successfully obtained through artificial fertilization, and the chromosome specimen of kidney cells of the two parents and their offspring were prepared using a low colchicine concentration air drying method, Howell and Black's (1980) silver–nucleolar organizer region (Ag–NOR) staining method and Sumner's (1972) C-band staining method. The karyotypes, Ag–NORs and C-bands of C. trimaculatum, H. managuense and their offspring were observed. The results showed that the diploid numbers of the two parents and the F1 hybrid offspring were all 2n=48. For C. trimaculatum, the karyotype formula was 2n=2m+8sm+26st+12t, NF=58. Two Ag–NORs were seen on the tips of the short arm of chromosome st7, and the entire chromosomes of st9 and st13 and the centromeres of the other chromosomes had positive C-bands. For H. managuense, the karyotype formula was 2n=2m+6sm+28st+12t, NF=56. Two Ag–NORs were located on the tips of the short arm of chromosome sm2 and positive C-bands were found in the centromere of all chromosomes. In the F1 hybrid offspring, the chromosome formula was 2n=2m+8sm+30st+8t, NF=58. Two Ag–NORs were seen on the tips of the short arm of chromosome sm2; all chromosomes presented positive C-bands in the centromere. No heteromorphic sex chromosome was found. After comparison with the two parents, the F1 hybrid offspring was suggested to be the filial generation of the two hybrid parents rather than the gynogenesis of H. managuense induced by heterogeneous sperm of C. trimaculatum.

Six transgenic maize lines with the fully modified gene GFM Cry1A were obtained and polymerase chain reaction (PCR) analysis of their T5 generation plants indicated that foreign genes could be stably inherited. Three hybrids (Simi25Bt, Tongdan24Bt and Jidan209Bt) were created using the transgenic lines and combined with regular maize inbred lines. The results of resistance identification of the transgenic inbred lines and hybrids showed that the difference of resistance among transgenic lines was very significant; there also existed a difference among individuals of the same line and among the three hybrids. Compared with the control inbred line (CK), the average of four transgenic inbred lines in the number of tunnels per stalk, larval tunnel length per plant and length of surviving larvae were decreased by 70%, 80% and 70%, respectively. Enzyme-linked immunosorbent assay (ELISA) testing showed that the average of Bt Cry1A protein expression level for six transgenic inbred lines was 0.16% total protein and the highest expression level was 0.19%. At maturity, compared with the control variety (CK), the larval tunnel length per plant of the three hybrids (Simi25Bt, Tongdan24Bt and Jidan209Bt) was decreased by 39.97%, 36.20% and 53.83%, respectively, which was a decrease of 43.36% on average. The investigation of agronomic characters showed that there was no significant difference between the improved hybrids and the control in plant height, ear length, row number per ear, kernel number per row and 100-kernels weight. It is thought that the GFM Cry1A gene can be applied to improve resistance to corn-borer, and maize inbred lines with the Bt gene can be directly applied to conventional maize breeding.

Primers bearing restriction enzyme sites for EcoR I and Hind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of the gcgasa gene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation into Escherichia coli BL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.