Nucleotide sequence of full-length cDNA of peanut stunt virus (PSV) Mi strain RNA3 was determined and compared with those of PSV-ER and -J (subgroup I) and PSV-W (subgroup II), strains of cucumber mosaic virus (CMV) and tomato aspermy virus (TAV). PSV-Mi RNA3 consists of 2170 nt and has two open reading frames, encoding a putative movement protein (3a protein) and a coat protein (CP). PSV-Mi RNA3 is 77.7% and 78.5% identical to those of PSV-ER and -J, whereas it shares 76.6% identity with PSV-W. Nucleotide identity of 3a and cp genes between PSV strains Mi and ER, J and W was 78.3–79.3% and 74.4–77.8%, respectively. Amino acid identity of 3a and CP between PSV-Mi and -ER, -J and -W was 73.9–77.4% and 64.8–77.5%, respectively. RNA3 of PSV-Mi (GenBank accession no. AY775057) had a varied intercistronic and 5′-untranslated region compared with those of PSV strains ER, J and W. Results indicate that PSV-Mi represents a new PSV subgroup from China, designated as subgroup III.

Four variant lines, HB1, HB2, HB3 and HB4, were derived from progenies of upland cotton (Gossypium hirsutum) cultivar Yumian 17 that had been transformed with genomic DNA from sea island cotton, Gossypium barbadense, using the pollen tube pathway (PTP) method. They showed significant differences from the wild-type recipient in terms of fibre quality, lint percentage, boll weight and other agronomic traits. The four variant lines also differed from the wild-type recipient in the amplification products of two SSR loci, indicating that the DNA of G. barbadense had been introduced into the genome of the recipient and was inherited stably. On the other hand, the agronomic traits of another three lines (HB5, HB6 and HB7) derived from the same transformation experiment were identical to those of the wild-type recipient, indicating that G. barbadense DNA had not integrated into the genome of these lines. A combination of the analyses on agronomic traits and SSR markers indicated that transgenic upland cotton strains produced by the PTP method could become homozygous in only a few generations and the foreign DNA introduced during the transformation experiments could be inherited stably in the transgenic progenies.

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.

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.

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.

Interferon (IFN)-α genes were cloned from genomic DNA of Fuan and Fuzhong water buffaloes by PCR, and the PCR products were inserted into a pQE30 vector to construct recombinant expression plasmids. Sequence analysis showed that both clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids (aa). They were defined as two new subtypes, with 91.6–94.2% identity at the amino acid level by comparison with eight previously published bovine IFN-α subtypes. Results of SDS-PAGE and Western blotting showed that each of the recombinant proteins was expressed in inclusion bodies in Escherichia coli with molecular weight of 20 kDa and the recombinant proteins were 25% of the whole proteins. Inclusion bodies were denatured and renatured with urea and the antiviral activities of the recombinant buffalo IFN-α (rBuIFN-α) were 105 U/mg and 106 U/mg in CEF/VSV and MDBK/VSV cell lines, respectively. Additionally, rBuIFN-α had good effects against challenge by infectious bovine rhinotracheitis virus. The rBuIFN-α are potential biological agents for the prevention and treatment of various kinds of bovine viral disease.

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.

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.

A pollen cDNA library of Japanese pear (Pyrus pyrifolia) from the family Rosaceae was constructed and a cDNA (bgn-1) of 1408 bp, which encodes a protein (BGN-1) secreted from growing pollen tubes, was cloned and sequenced. bgn-1 cDNA is composed of a 5′-untranslated region of 47 bp, an open reading frame of 1194 bp encoding 397 amino acid residues and a complete 3′-untranslated region of 167 bp. Alignment of the deduced amino acid sequence of bgn-1 with that of barley (Hordeum vulgare) β-1,3-glucanase (GII) showed 39.7% amino acid identity. Several residues that were critical for the function of GII were conserved in the deduced BGN-1 polypeptide. Moreover, hydrophobic cluster analysis (HCA) showed an overall HCA homology score of 87.1% and analysis of BGN-1 with the 3D-PSSM program predicted a three-dimensional structure of BGN-1 highly homologous to that of barley GII with ≥95% certainty. These results suggest that the cloned bgn-1 cDNA encodes a β-1,3-glucanase-like protein in Japanese pear. The predicted mature protein (375 amino acids) has a theoretical molecular mass of 40 723 Da, a basic pI of 9.59 and a diagnostic amino acid residue mode of D, L, S and L, which is very similar to that of growth-related subfamily D (D, L, S and Q) in cereals. A ProXXPro repeat is also found between positions 352 and 367 in the C-extension region.

Upstream open reading frames (uORFs) in 5′-untranslated regions (5′-UTRs) of eukaryotic mRNAs play an important role in translation efficiency. Computational analysis of the upstream ATG (uATG) and uORFs of 5′-UTRs of plant mRNAs, adopted from the nucleotide sequence databank, was carried out. Statistical analysis revealed that up to 18% of 5′-UTRs contain uATG, which is much higher than the earlier estimate. Among them, about 50% of the genes have one uATG and nearly 20% of them have two uATGs. About 85% of uORFs are non-overlapping. Thirty per cent of uORF peptides comprise 1–5 aa, and about 80% of uORFs fall in the range of below 20 aa. Sequences flanking the uATG codon differ strikingly from the functional initiation codon and the uATG triplet is more frequently located in a non-optimal context. Consensus sequences of the ATG codon context of mRNA with and without uATG are similar, whereas the ATG codon context of mRNA without uATG is more frequently located in an optimal context than is mRNA with uATG. Most mRNAs with uATGs are possibly related to regulatory functions. In addition, most mRNA uORFs have no similarity between plant species whereas sequences of a few uORFs are highly conserved. For example, mRNA uORFs encoding S-adenosyl-l-methionine decarboxylase (AdoMetDC) share 75–100% homology between plant species, which is much more conserved than AdoMetDC protein.

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.

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.

mRNA differential display was used to analyse differences of gene expression between cross combinations, EC and CE, and their parental inbreeds, White Plymouth Rock (EE) and Silkies (CC). Three genes specifically expressed and one gene over-expressed in hybrids were found. By cloning, sequencing and BLAST analysis, one specific expression cDNA band was identified as the cytosolic isocitrate dehydrogenase gene, two were new genes of unknown function, and the over-expressed one was a proteosome subunit p112 gene. The results imply that functional and regulatory genes, expressed or over-expressed in chicken hybrids, are related to heterosis of some carcass traits.

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.