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.

Analysis of mating type can provide an evaluation of the population genetic diversity of the rice blast fungus, Magnaporthe grisea. According to the sequences of MAT1-1 and MAT1-2 genes of the fungus, two pairs of PCR primers specific to the MAT1-1 and MAT1-2 alleles were designed, and the PCR thermal profile was also optimized. To confirm its application in mating type assessment, 10 tester isolates were tested by PCR. The PCR amplification pattern of these tester isolates corresponded to their known mating type. Furthermore, 150 rice-field isolates from Fujian Province were mated with tester isolates GUY11 and KA3 side by side and also tested by PCR. Results showed that 95.1% of 123 fertile isolates were the same in mating type as determined by both PCR-amplified allele-specific fragments and mating with GUY11/KA3. Among 27 sterile isolates determined by GUY11 and KA3, seven were MAT1-1 and 20 were MAT1-2 as determined by PCR. This study indicates that PCR is applicable in assessing M. grisea mating type and especially is capable of predicting the potential mating type of sterile isolates in the natural population of the fungus.

GbRac1 gene was cloned from Gossypium barbadense with degenerate primers and 3′-RACE. Northern blot analysis indicated that GbRac1 mRNA was expressed abundantly in G. barbadense seedlings inoculated with Verticillium dehliae compared with mock-inoculated plants. A plant constitutive expression vector pRac harbouring GbRac1 gene was constructed and leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) were transformed with pRac by Agrobacterium-mediated transformation. Disease challenge test of detached leaves of the transgenic plants by inoculation with Alternaria alternata showed that resistance was enhanced dramatically compared with the non-transgenic plants. Results suggest that GbRac1 gene might have potential application in the genetic engineering of plants with enhanced disease resistance.

An antisense class I patatin gene under control of the CaMV 35S promoter was introduced into potato (Solanum tuberosum) cultivar E-potato 3 using the Agrobacterium tumefaciens system. PCR amplification and PCR–Southern blot analysis indicated that the antisense class I patatin gene had been integrated into the potato genome. Northern hybridization analysis showed that the antisense gene transcribed normally in the transgenic potato plants and resulted in a reduction of endogenous class I patatin mRNA. Total soluble protein content and lipid acyl hydrolase activity of microtubers, derived from transformed plants, decreased by a maximum of 36.4% and 31.4%, respectively, compared with control plants. The expression of this antisense gene also resulted in reductions of the plantlets forming tubers, tubers per plantlet and the effective tubers (≥50 mg) of the transformed plants.

To differentiate pigs infected with Pseudorabies virus (PrV) from pigs vaccinated with gE-PrV, a glycoprotein E enzyme-linked immunosorbent assay (gE-ELISA) based on recombinant glycoprotein E (gE) (which was expressed by Escherichia coli, purified, denatured and renatured) was developed. By testing 115 serum samples, the diagnostic specificity and sensitivity of the developed gE-ELISA were evaluated to be 94.5% and 96.7%, respectively. Five serum samples were tested with plates from five lots, and the results had a coefficient of variation of less than 10%, showing good reproducibility of gE-ELISA. This gE-ELISA was compared with a commercial blocking ELISA by testing 356 serum samples. The agreement rate of the two assays was 92.13% (328/356). These results suggested that the gE-ELISA developed in our laboratory could be used in differentiating PrV-infected and gE-PrV-vaccinated pigs.

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.

Eleven isolates within four species of the entomogenous fungus Aschersonia from China, the USA, Japan, the Philippines, Malaysia and Columbia were characterized using 17 RAPD primers. Genetic diversity among these strains of Aschersonia was found. The clustering results showed that the genetic variability among interspecies was more than that among intraspecies of Aschersonia. In the constructed phylogenetic tree, these isolates were not clustered according to their geographic origins or hosts. Furthermore, sequences of the divergent domain at the 5′-end of the large subunit (LSU) in nuclear rRNA from the mitosporic entomogenous fungi were employed to analyse the phylogenetic relationships of 11 Aschersonia isolates. The relationships of interspecies or intraspecies shown in the phylogenetic tree were almost consistent with the results of the morphological study. Different species isolated from different geographic origins could be clearly distinguished in the tree. But there were no close relationships among species isolated from the same family or order of insect hosts. The tree indicated that isolate Aa, belonging to A. aleyrodis, was the same as Aa992 and Aa3.4485. Moreover, results of RAPD analyses were consistent with those of LSU nuclear rDNA analysis for the same isolates tested, which consequently indicates that both methods can be independently applied for classification and identification of Aschersonia.

To control the spread of bovine spongiform encephalopathy and scrapie, a method was developed for the simultaneous detection of bovine- and sheep-derived materials in feedstuffs. Chelex-100 was adopted for the fast extraction of animal genomic DNA. A pair of universal primers, designed on the basis of homology in sequences of bovine and sheep mtDNA, was used for PCR. This method is of great value to quarantine departments for its simplicity and rapid detection.

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.

Six DNA components of Banana bunchy top virus (BBTV) were amplified by polymerase chain reaction (PCR) using total DNA from banana tissues having the typical BBTV symptoms as template. The sequence analysis indicated that the six fragments (1104 nt, 1067 nt, 1059 nt, 1045 nt, 1014 nt and 1081 nt) were all full-length components of BBTV. The viral sequences from different countries were compared. Results revealed that the sequence of component 1 was more conservative than other components, DNA1 of the Hainan isolate shared 95.4% homology with that of the Vietnam isolate, while sequences of both components 2 and 4 showed less homology. DNA2 of the Hainan isolate only shared 83% homology with that of the Guangzhou isolate. According to the results of the DNA1 sequence comparison, the BBTV Hainan isolate should belong to the Asian sub-group.

A 1009 bp promoter sequence of cab gene, which encodes chlorophyll a/b binding protein belonging to a class of light-inducible proteins, was cloned from Gossypium arboreum. Sequence analysis showed that it had no obvious homology with previously published cab promoters. The full-length Gacab promoter and 5′ deletions with length of 197, 504 and 779 bp were fused with gus (uid A) gene, respectively, and plant expression vectors were used for transformation of Nicotiana tabacum cv. NC89. β-Glucuronidase (GUS) histochemical assay of transgenic tobacco plants showed that GUS was expressed specifically in leaves and young green tissues. GUS was not detected in the leaves of transgenic plants grown in the dark for 6 days. However, it was highly expressed in the leaves of these plants after induction with light for another 6 days, demonstrating that the full-length Gacab promoter is a light-inducible promoter. Transient GUS expression in rice calli indicated that the expression level of Gacab504::gus was the highest and stronger than that of the CaMV 35S promoter, while expression was reduced for Gacab197::gus, Gacab779::gus and Gacab1009::gus constructs. This suggests that −197 bp to −1 bp is a basic promoter of Gacab, some positive regulatory elements may exist in −504 bp to −197 bp, and the fragment −1009 bp to −504 bp may contain negative elements.

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.

The yolk proteins of the silkworm, Bombyx mori, are important sources of nutrition and energy during embryo development. By analysing the yolk proteins from silkworm eggs, the composition and the metabolism of yolk proteins during embryo development stages can be studied at the proteome level. Yolk proteins were obtained from unfertilized eggs and eggs at different embryo development stages by dissection and then separated and analysed by two-dimensional electrophoresis and its corresponding image analysis system. It was discovered that over 200 proteins exist in mature silkworm eggs. Some specific spots of yolk proteins were found in different silkworm strains, while few differences in yolk proteins appeared during different development stages in the same silkworm strain. This result indicates that the silkworm embryo does not utilize yolk proteins selectively by absorbing one or some special proteins, but by absorbing the yolk protein grains one by one during development.

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.

Pseudomonas fluorescens P5 was isolated in Yunnan Province and proved to be an effective biocontrol agent of various soil-borne plant diseases. The aim of the study was to enhance the biocontrol effect of this strain by introducing a chitinase gene. A 6.5 kb DNA fragment containing the chitinase gene chiB was inserted into pDSK519 vector to construct a new plasmid, pDSK51965. This plasmid was introduced into P. fluorescens P5 and resulted in a new strain named P5-1. Restriction enzyme digestion and chitin plate culture confirmed that P5-1 contained a functional chitinase gene. Compared with wild-type P5 in pot experiments, P5-1 had an increased effect against rice sheath blight and cotton damping-off caused by Rhizoctonia solani. It also increased the effect on suppression of wheat take-all caused by Gaeumannomyces graminis var. tritici.

To evaluate the potential adjuvant effect of chicken interferon-γ (IFN-γ) expressed by recombinant fowlpox virus (rFPV–IFN-γ), SPF chickens and commercial chickens with (MAb+) and without (MAb−) maternal antibodies were immunized with rFPV–IFN-γ plus either H5 subtype avian influenza vaccine based on recombinant fowlpox virus (rFPV–HA) or conventional inactivated vaccine. In the assay of humoral responses, rFPV–IFN-γ plus vaccines induced a slightly lower titre of haemagglutinin inhibition (HI) antibody response than either rFPV–HA or inactivated vaccine alone, in both MAb− and MAb+ chickens. As for protective efficacy, the combinations of rFPV–IFN-γ and both vaccines induced 95–100% protection against lethal challenge with highly pathogenic avian influenza virus in SPF chickens and MAb− chickens, which were equal to those induced by either vaccine alone. However, immunization of MAb+ chickens with 106 pfu rFPV–HA plus 105 pfu rFPV–IFN-γ showed higher protection efficacy (33.5%) than that (11.4–15.8%) induced by treatments with other dosage combinations. These findings show that IFN-γ expressed by recombinant fowlpox virus could not promote HI antibody response induced by H5 avian influenza vaccines in chickens. The results also suggest that rFPV–IFN-γ enhances the protection of chickens in a dose-dependent and immune status-dependent manner.

The β-glucosidase gene has important effects on alcoholic aroma precursors and insect resistance of the tea plant [Camellia sinensis (L.) O. Kutze]. The complete cDNA sequence of β-glucosidase of the tea plant was cloned; its full length was 1475 bp, and shared 40–60% similarity with corresponding parts of the nucleotide sequence of β-glucosidase gene from other plants. Its secondary structure contains 14.33% α-helix, 25.43% β-pleated sheet and many functional amino acid domains. The β-glucosidase gene was cloned into the pET-32a expression system and expressed at high-efficiently in Escherichia coli BL21 (DE3); the molecular weight of expressed fusion protein was 63 kDa. The results of enzymic reaction showed that the fusion protein possessed normal bioactivity, and it could catalyse the dehydration of the glycosidic bond. The soluble fusion protein was expressed mainly in the cytoplasm.

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.

GP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) of Spodoptera litura nucleopolyhedrovirus (SpltMNPV) gp41 gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). The gp41 gene was recombined in vitro with prokaryotic expression vector pQE30 and transformed into Escherichia coli M15 [pREP4]. The M15 [pREP4] strain, containing gp41 recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.