The effects of different concentrations of tumour necrosis factor-α (TNF-α) on rat adipocyte apoptosis were detected by optical microscopy, agarose gel electrophoresis and flow cytometry methods. The morphological changes of rat adipocyte apoptosis induced by TNF-α correlated linearly with the concentration of TNF-α, ranging from 5 to 20 ng/ml. High concentrations of TNF-α induced more obvious apoptosis. Significant morphological changes of rat adipocytes treated with 5 ng/ml TNF-α were noticed, but DNA ladders did not appear in the DNA electrophoresis analysis, i.e. morphological changes occurred earlier than the biochemical changes. TNF-α induced apoptosis in the rat adipocyte in a dose-dependent manner. The induced apoptotic effect of 5, 10, 15 and 20 ng/ml TNF-α was significantly different (P0.01), but the effect among 10, 15 and 20 ng/ml TNF-α treatments was not significantly different (P0.05). Thus the optimum concentration of TNF-α for inducing apoptosis was 10 ng/ml.

To amplify the transferrin-binding protein 2 (Tbp2) gene from Actinobacillus pleuropneumoniae (App) by polymerase chain reaction (PCR), a pair of primers were designed according to sequence Z46774 of A. pleuropneumoniae serotype 5. The amplified DNA fragment (1642 bp) was cloned into pMD18-T and sequenced. The sequencing result showed that the homology was 99.8% compared with the reference sequence. The target fragment of the positive clones was inserted into pET-32a and transformed in Escherichia coli BL21 (DE3). After its induction, the fragment was expressed in E. coli. The Western blotting results were positive.

The homology of the sequences, reported and registered in GenBank, of different strains of Avian influenza virus (AIV), Newcastle disease virus (NDV), Classical swine fever virus (CSFV) and Foot-and-mouth disease virus (FMDV), was analysed and compared with each other. According to the properties of these viruses, the conservative domain of the M gene for AIV, the F gene for NDV, the 5' non-coding domain end for CSFV and the 2B gene for FMDV were selected for polymerase chain reaction (PCR) amplification. In order to prevent the formation of conformational dimers between different primers, four pairs of primers designed with the DNAsis system under the condition of G+C (50–60%),18–25 bp in length and Tm (72–85), were analysed using the VNTI5.5 system. The specific fragments amplified were as follows: 141 bp for FMDV, 200 bp for CSFV, 319 bp for NDV and 471 bp for AIV. The optimal conditions of PCR for each virus mentioned above were determined by orthogonal assay, and two or four of the four pairs of primers were then combined and used for amplification trials. The results showed that four specific fragments of different lengths would be successfully amplified in one tube at the same time. The products of PCR were tested to be specific by sequencing. Out of 46 pathological samples detected with the multiple PCR, there were 5 AIVs, 7 NDVs, 15 CSFVs and 6 FMDVs. The amplification above was identified with a single PCR. On the other hand, the results corresponded to those of electronic microscopy, haemagglutination (HA) and enzyme-linked immunosorbent assay (ELISA). The method described here is practicable, sensitive, specific, simple and cheap. It could be used for diagnosing AIV, NDV, CSFV and FMDV in different animals.

Tyrosinase (TYR) is a key enzyme of melanin biosynthesis. Single-strand conformation polymorphism (SSCP) analysis was applied to detect the single nucleotide polymorphisms (SNPs) in the upstream regulating region from -641 to -2125 bp of the TYR gene. Three SNPs were found in this region. Correlations were obtained between genotypes of the SNP sites and pigment traits in chicken parental and F2 generations of the Chinese Agricultural University (CAU) resource population. The chi-square test indicated that these mutations were significantly related to shank and body skin colours.

Two hundred and forty bacterial strains were isolated from three potato (Solanum tuberosum) cultivars from eight different sources. On the basis of antagonistic bacteria examination in vitro, greenhouse and field tests, 22.9% of isolated bacteria were endophytic and antagonistic. Three strains, namely A-10', T3 (Bacillus sp.) and H1-6 (Pseudomonas fluorescens), were chosen for their suppressive effect on bacterial ring rot (Clavibacter michiganensis subsp. sepedonicus) and their growth promotion. The strain A-10' was an endophytic bacterium with the effects of colonization, plant growth promotion and suppression of the pathogenic bacteria, showing good prospects for commercial use.

A trivalent vector containing genes of Watermelon mosaic virus (WMV) coat protein (CP), and replicase genes of Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV), was constructed for transformation of watermelon (Citrullus lanatus) plants, mediated by Agrobacterium tumefaciens. The integrated foreign genes were identified in the regenerated progenies by polymerase chain reaction (PCR) and Southern blots. The transformation efficiency was about 1.7‰. Resistance to virus infection was determined by mechanical inoculation in the greenhouse and by field trials. The transgenic watermelon lines showed different phenotypes of susceptible, resistant, immune or recovery from virus infections in the late growth stage. A relatively high level of resistance was shown by T3 plants of the line BH1-7. This result indicates the possibility of creating, by transgenic protocols, new varieties of watermelon resistant to viral infection.

In order to investigate the effects of DNA methylation on pig carcass traits, the methylation-sensitive amplified polymorphism technique (MSAP) was adopted to amplify DNA from blood samples taken from 77 F1 hybrids of Large White×Meishan crosses. A total of 15 carcass traits were tested. Results showed that means of internal fat rate (IFR), lean meat percentage (LMP) and ratio of lean meat versus fat meat (RLF) were significantly different (P<0.05) among five levels of general individual methylation difference (GIMDP); means of carcass weight (CW), IFR, backfat thickness at buttock (BFT3), average backfat thickness (ABF), carcass length to the first rib (CLR) and carcass length to the first neck vertebra (CLN) were also significantly different (P<0.05) among five levels of special individual methylation percentage difference (SIMDP), while means of 15 carcass traits were not significantly different (P>0.05) among five levels of neutral individual methylation percentage difference (NIMDP). Of all traits that were significantly affected by SIMPD, CW, ABF and BFT3 increased, IFR and backfat thickness at shoulder (BFT1) decreased, while CLR and bone percentage (BP) fluctuated with the SIMDP increase. The regressions between SIMDP and IFR, BFT1, BFT3 and ABF were significant (P<0.05). It is concluded that DNA methylation can be applied as a marker to related studies in pigs; positive methylation sites were superior to negative methylation sites in predicting hybrid performance, and methylation differences should be maintained at specific levels for different traits to improve the productivity of pigs.

In order to probe the effect of methylation on heterosis, the methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) technique was adopted to amplify pig genome DNA with 40 single arbitrary primers. The material involved parental lines and F1 hybrids of Large White×Meishan crosses. Nineteen differentially methylated sites with RsaI+HpaII digestion and 14 differentially methylated sites with RsaI+MspII digestion between parental lines and the hybrid were found. All fragments detected in this study were grouped into four classes: (1) the same level of methylation in both parental lines and the hybrid; (2) the same level of methylation in one parent and the hybrid; (3) an increased level of methylation in the hybrid compared to the parents, and (4) a decreased level of methylation in the hybrid. Five sites had significant effects on seven traits (P<0.05). Sequence analysis showed that three sequences had a high-identity match in GenBank (greater than 87%) and two sequences had no match in the database. The percentage of G+C in three sequences was over 50, and the observed/expected CpG of all sequences was above 0.6. Furthermore, one sequence contained G/C boxes. This study demonstrated that the sites in CpG islands within a gene promoter were differentially methylated in the hybrid compared to parental lines; methylated sites contributed differentially to F1 performance, showing that heterosis could benefit from either expression or repression of some genes.

Inverted repeats of the partial movement protein gene (ΔMP) of Tobacco mosaic virus (TMV) were introduced into the plant expression vector pBin438, and the recombinant plasmids were transformed into Agrobacterium tumefaciens EHA105 by a tri-parental mating method. Fifty transgenic plants of Nicotiana tabacum cv. Yunyan 87 were obtained after Agrobacterium-mediated transformation. PCR and Southern blot analyses showed that the target gene had integrated into the tobacco plant genome. When transgenic tobacco plants were challenged with TMV, symptoms expression and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that transgenic plants had three kinds of phenotypes in response to the virus infection: immune (10%), resistant (4%) and susceptible (86%). Northern blot analysis showed that the levels of target mRNA accumulation varied among transgenic tobacco lines and a negative correlation between target mRNA accumulation and virus resistance was observed.

Genomic RNA was extracted from the subcutaneous adipose tissue of piglets (Duroc×Landrace×Tai-hu) at 1, 14, 28, 42 and 56 days of age, and obese gene (ob) mRNA was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). A DNA fragment of about 504 bp was obtained and the PCR product was cloned into a pGEM-T vector. The ob gene was isolated and sequenced from the positive clones screened. Sequence analysis suggested that this fragment was a partial sequence of ob cDNA, coding 167 amino acid residues, which constituted the major part of leptin mature protein. The gene homology of the fragment obtained in this study compared to the reported ob cDNA sequence in adipocytes of pig was 99.405%, and amino acid homology was 98.94%. Based on the ob gene clone, we successfully constructed an optimal semi-quantitative RT-PCR method. Using β-actin as the internal control, we investigated the difference of ob gene expression at different ages of piglets. Results showed that ob mRNA levels increased steadily at postnatal days 1–28 (preweaning), peaked at postnatal day 28, when piglets were weaned, and decreased from day 28 to 56.

The RNA-dependent RNA polymerase (RdRp) gene of Bombyx mori cypovirus 1 (BmCPV-1) (China strain) was cloned in three fragments using step-by-step reverse transcriptase-polymerase chain reaction (RT-PCR). The whole RdRp gene of 3691 bp was sequenced (GenBank accession number: AY496445). Vector pET-28b(+) was used to construct the expression vector pET28b–RdRp which was expressed in Escherichia coli BL21 (DE3) induced with isopropyl β-d-thiogalactoside (IPTG). The molecular weight of the expression product was about 138 Da. The primary antibody employed was rabbit antibody against BmCPV–RdRp recombinant protein and the secondary antibody was 15 nm immunogold-labelled goat anti-rabbit IgG. Immunogold was mostly bound to the virions which were dispersed in a virus generation matrix and polyhedron in the columnar cells of the midgut of the silkworms, and the average marking ratio was about 35%. This result demonstrated that BmCPV–RdRp complexes are indeed located at the BmCPV capsid.

Research into, and the environmental release of, transgenic rapeseed in China are overviewed and the environmental risks are assessed, focusing on competitive survival ability, gene dispersal and biodiversity impact of transgenic rapeseed. It is concluded that transgenic rapeseed has a higher probability of gene dispersal when compared with other major crops. Brassica napus may transfer genes through pollen and seeds to vegetables and wild species of B. rapa and B. juncea, for which China is the biodiversity centre and also the country of highest consumption. It is considered that the risk of gene dispersal is present, but can be reduced to an acceptable limit. Commercialization of transgenic rapeseed should not be stopped, but should be built on a safe and sound basis by building a reasonable management system. Awarenes of biosafety considerations for transgenic rapeseed should be strengthened, and a technical platform of genetically modified organism (GMO) detection and monitoring should be properly established. Countermeasures against environmental risks are also discussed.