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.

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.

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.

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.

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.

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.

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.

Seminiferous tubule tissues from goat fetal testis were treated using four methods. One of the treatments, with 0.1% collagenase for 15 min and then with an enzyme mixture (containing 0.25% trypsin, 10 μg/ml DNase I, 0.04% EDTA) for 5 min showed ideal cell dispersal after centrifugation and washing. The in vitro culture of the cells separated with a different medium system showed that mulberry-shaped male germ-line stem cell (mGSC) clusters and a single layer of Sertoli cells appeared as the original generation in 120 h. The mGSC clusters developed in suspension and were distributed in different locations from the Sertoli cells in the plate. The cells in the marginal area of incompletely digested mGSCs clusters, and the single cells from them, were obviously homogenized after culture for 5 days in a fibre cell feeding layer from fetal mice (MEF feeding layer), whereas identically treated mGSCs clusters co-cultured with Sertoli cells in the same culture system did not displayed clear homogenization in marginal cells of the clusters. Also, the first generation of mGSC clusters co-cultured with Sertoli cells could form the same morphological properties with the original generation; cells in clusters were tight and mGSCs divided more slowly than those in the MEF feeding layer.

The bone morphogenetic protein receptor IB (BMPR-IB) gene, which controls the fecundity of Booroola Merino ewes, was studied as a candidate gene for the prolificacy of Small Tail Han and Hu ewes. A single nucleotide polymorphism of the BMPR-IB gene was detected in both high (Small Tail Han and Hu) and low (Suffolk and Dorset) fecundity sheep breeds by polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) analysis. The results indicated the presence of the same mutation (A746G) of the BMPR-IB gene in both Small Tail Han and Hu ewes and in Booroola Merino ewes, but not in both Suffolk and Dorset ewes. In Small Tail Han ewes, frequencies of BB, B+ and ++ genotypes were 0.524, 0.383 and 0.093, respectively. In Hu ewes, these frequencies were 0.882, 0.118 and 0.000. The BMPR-IB genotype distributions were significantly different (P<0.001) among high- and low-fecundity sheep breeds. Small Tail Han ewes with genotype BB had 0.92 (P<0.01) and 1.02 (P<0.01) lambs more than those with genotype ++ in the first and second parity, respectively. These results demonstrated that the BMPR-IB gene is a major gene affecting the prolificacy in both Small Tail Han and Hu ewes, and could be used as a molecular genetic marker to select the litter size in sheep.

A multifunctional-engineering strain was constructed by fusing the insecticidal bacterium (Bacillus thuringiensis) with vip3A gene and the endophytic bacterium (B. subtilis) protoplasts. Results of polymerase chain reaction (PCR) showed that the insecticidal vip3A gene of B. thuringiensis was present in the fusant. The fusant's antagonism to Fusarium oxysporum f. sp. cucumerinum, the adjusted mortality to Plutella xylostella and the endophytic characters were detected. The result showed that the antagonism of the fusant was against the F. oxysporum f. sp. cucumerinum; the adjusted mortality to P. xylostella reached 59% and it had insecticidal characteristics.

The construction of two cDNA libraries from young shoots of tea plant [Camellia sinensis cv. Longjing 43 and Anji Baicha], the sequencing of Longjing 43 cDNA clones and the analysis of expressed sequence tags (ESTs) are reported. Totally, 4320 clones from the cDNA library of Longjing 43 were sequenced, and 2963 useful sequences were obtained, corresponding to 68.6% of clones. A total of 416 clones shorter than 150 bp and 863 repeated clones were excluded, the first 1684 valid tea plant ESTs were generated. Most of the ESTs were between 300 and 700 bp, with an average of 478 bp. Six hundred and seven ESTs with known function or putative function were identified by BlastN searches against the National Center of Biotechnology Institute (NCBI) non-redundant nucleotide databases, corresponding to more than 130 functional genes in the tea plant. The rest, 1077 ESTs, were novel gene partial or full sequences. The results indicated that EST sequencing was a rapid and effective approach to identification of novel functional genes for tea plant.

Interferon, an important cytokine, is an immunomodulator and possesses antiviral and anti-tumour activity. In vitro, it can be administrated in the treatment of diseases alone or with genetically engineered vaccine to enhance the immune effect of the latter. The recombinant transferring vector pSY681–ChIFN-γ was obtained in this study by inserting the chicken type II interferon (ChIFN-γ) gene into the Fowlpox virus (FPV) transferring vector pSY681. The resulting plasmid was then transfected into chicken embryo fibroblast (CEF) cell cultures pre-infected with the parental FPV S-FPV-017. Finally, the recombinant Fowlpox virus (rFPV) expressing ChIFN-γ (rFPV–ChIFN-γ) was produced by homologous recombination with the FPV gene in CEF. rFPV-positive plaques were verified by polymerase chain reaction (PCR), restriction analysis and indirect immunofluorescence assays. The rFPV–ChIFN-γ supernatants, cultured in CEF for 72 h and inoculated into rat fibroblasts (L929), had an inhibitory effect on the replication of Rous sarcoma virus (RSV) with an antiviral titre of 2048 U/ml.