Lentiviruses as gene transfer vectors have been used successfully to transfect mammal embryonic stem cells and germline stem cells, but this has not been attempted in avian primordial germ cells (PGCs). PGCs were isolated from the gonads of Isa-brown chicken embryos at stage 28 and co-cultured with gonadal stroma cells. A lentiviral vector pLenti-CMV-EGFP was constructed and the virus harvested by cotransfecting 293FT cells with the vector and packaging plasmids. Concentrated lentiviruses were used to transfect chicken PGCs, the transfection efficiency was up to 24.19%.

Recent advances and limitations of using zinc finger nuclease to stimulate a high efficiency of homologous recombination, and chimeric oligonucleotides to promote single base replacement in functional genomic research and plant genetic breeding, are systematically reviewed. Approaches to improve gene targeting efficiency through molecular modification of key pathways in plant homologous recombination are also discussed.

Using 16S rDNA-polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE), electron microscopy and a conventional plating method, the genetic diversity and phenotype polymorphism of the endophytic bacteria within Azolla microphylla were explored. The 16S rDNA-PCR-DGGE profile showed a complex and divergent bacterial community, with Bacillus cereus as the dominant species, within the Azolla–cyanobacteria association. This result was supported by the fact that endobacterial cells exhibited distinct ultrastructural characteristics in vivo and, in vitro, bacteria displayed various colonies with different sizes, shapes and colours. This study demonstrates that the genetic diversity of endophytic bacteria in Azolla can be investigated using the16S rDNA-PCR-DGGE technique.

The cDNA of bovine interleukin-18 (BoIL-18) was subcloned into pGEX6P-1 vector and transformed into Escherichia coli BL21(DE3). The recombinant protein was successfully expressed in E. coli by induction of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 0.3 mmol/l for 8 h. SDS-PAGE indicated that the BoIL-18 fusion protein, 44 kDa, was highly expressed. Densitometric scanning showed that the fusion protein expression was about 31.8% of the total bacterial protein. The biological activity of the chromatographically purified protein was assayed. The peripheral blood mononuclear cells (PBMC) proliferation test indicated that the BoIL-18 fusion protein could enhance PBMC proliferation when its concentration was more than 0.10 mg/l. Enzyme-linked immunosorbent assay (ELISA) showed that the BoIL-18 fusion protein could induce interferon (IFN)-γ production from spleen lymphocytes when it was at a concentration of more than 0.20 mg/l, and that the inducing effect of BoIL-18 fusion protein on IFN-γ was directly proportional to its concentration. This verified that the purified BoIL-18 fusion protein possessed a functional activity and could be applied in further studies.

Efficiency and specificity are key limiting factors for the production of transgenic animals. This review describes the recently developed animal gene transfer techniques, including non-site-specific methods of gene transfer into the testis and ovary for easy production of transgenic animals; gene targeting in embryonic stem cells, somatic cells and primordial germ cells for site-specific methods; methods to improve cloning efficiency in gene targeting; and site- and timing-specific gene targeting and controlled expression of transferred genes. In addition, methods of utilizing newly developed RNA interference, combined with the above techniques for controlling gene expression, to produce transgenic animals to spatio-temporally and reversibly knock down specific genes, are also discussed. The merits and disadvantages of each method are covered, as well as the potential use of these methods to develop transgenic animals for breeding new animal lines, to study disease models and to produce therapeutic medicines.

An optimal protocol for cryopreservation of boar semen was established. First, the boar semen was pre-diluted with ZORLESCO (ZO) solution and pre-equilibrated at room temperature for 1 h. After adding extender I, spermatozoa were equilibrated at 5°C for 1.5 h; then an equal volume of extender II was added and the spermatozoa equilibrated for 2 h. The resulting spermatozoa were loaded into 0.25 ml straws, equilibrated for 10 min at 3 cm above the surface of liquid nitrogen (LN), then promptly submerged into LN. When thawing, straws were incubated in a water bath at 37°C for 30 s. This procedure yielded the highest post-thaw motility of 0.58±0.03 and plasma integrity of 63.2±1.2%, together with a normal acrosome in 51.4±2.6% of spermatozoa. Abnormal spermatozoa after freezing represented only 14.0±3.0%.

A bioassay method was developed to use the parasporal crystal protein of Bacillus thuringiensis against plant-parasitic nematodes. Using this method, the parasporal crystal proteins of ten Bt strains showed activity against plant-parasitic nematodes. The toxicity of YBT-021 against Meloidogyne hapla, Pratylenchus scribneri, Tylenchorhynchus sp., Ditylenchus destructor and Aphelenchoides sp. was also assayed. The resulting LC50 values were 35.62 μg/ml, 75.65 μg/ml, 94.31 μg/ml, 215.21 μg/ml and 128.76 μg/ml, respectively.

Melanocortin-4 receptor (MC4R) is a member of the superfamily of G-protein-coupled receptors (GPCRs), which affects body weight, energy homeostasis and food intake in humans and mice. In this study, the Asp298Asn polymorphism of the MC4R gene was investigated in Laiwu, Yorkshire×Laiwu and commercial cross-bred pig populations using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and the relationship of this mutation with backfat thickness was analysed. The results indicated that only genotype 11 exists in 33 individuals of Laiwu pigs, and three genotypes (11, 12 and 22) were detected in Yorkshire×Laiwu and commercial cross-bred populations. The distributions of allele and genotype frequencies in Yorkshire×Laiwu and commercial cross-bred populations were similar, with the frequency of allele 1 being higher than that of allele 2. In commercial cross-bred pigs, the mean backfat thickness of individuals with genotype 22 was significantly higher than that of individuals with genotypes 12 (P<0.01) and 11 (P<0.05). This study provides evidence that the Asp298Asn polymorphism of the MC4R gene is associated with backfat thickness in commercial cross-bred pigs with Western pigs as parental lines and, therefore, can be used as a DNA marker for breeding in such populations of pigs.

In plants, acid invertases are known to be the key enzymes cleaving sucrose into reducing sugars (RS) (glucose and fructose). To improve the quality of potato (Solanum tuberosum L.) chips, which is largely influenced by RS accumulation in tubers stored at low temperature, a part of acid invertase cDNA with hairpin RNA (hpRNA) structure was transformed into potato cv. N2. Detection of polymerase chain reaction (PCR) amplification and Northern blotting suggested that the RNA interference (RNAi) vector was successfully transformed into cv. N2. The analysis of acid invertase activity in the plantlets and microtubers of RNAi transgenic lines indicated that the expression of the acid invertase was significantly repressed by the activity of RNAi of plantlets by an average 69.8% (with the exception of line Ni-1) with a maximal decrease of 78% (line Ni-4), and the highest decrease of activity in microtubers of 68%. Compared with that of well-inhibited antisense inv transgenic plants, the comparative downregulation of RNAi suggests a distinct alteration of endogenous acid invertase activity and a potential strategy for post-transcriptional gene silencing (PTGS) in modulation of cold-sweetening in potato.

Butyrophilin 1A1 (BTN1A1), which is a member of the Ig superfamily, is highly expressed in the lactating mammary gland and is secreted into milk in association with lipid droplets, and concentrated in the apical plasma membranes of mammary epithelial cells. Because butyrophilin is expressed only during lactation, it appears to be essential for milk fat globule production. However, there is little information about its properties in the dairy goat. The objective of this study was to confirm whether butyrophilin was related to milk changes in different lactation periods of Xinong Saanen goats. Using suppression subtractive hybridization analysis, we identified a significant increase in butyrophilin mRNA in Xinong Saanen goat mammary gland at middle lactation when compared to that of late lactation. The gBTN1A1 complementary DNA has an open reading frame of 1581 nucleotides encoding a putative protein of 526 amino acids. Amino acids 1–26 were the signal peptide domain. The homologies of nucleotide and peptide sequences of goat butyrophilin with bovine (NM_174508), human (NM_001732) and mouse (AK145168) butyrophilin were 97 and 96%, 88 and 84%, 84 and 70%, respectively. The secondary structure, transmembrane domain and hydrophilicity of goat butyrophilin were similar to the those of the bovine, human and mouse protein. In conclusion, we confirmed that the goat mammary gland mRNA level of gBTN1A1 was responsible for goat milk fat globule secretion.

Using reverse transcription polymerase chain reaction (RT-PCR) and gene splicing by overlap extension (SOE-PCR), the DNA sequence encoding the chicken receptor activator of NF-κB ligand (RANKL) was amplified, cloned into the expressing plasmid pET-32a(+) with His-tag, and highly expressed in Escherichia coli. The purified protein was then added to primary cultures of chicken osteoclasts to observe its bioactivity. The results showed that the size of the PCR product was 1200 bp, which was consistent with the expected size, and the relative molecular weight of the induced protein was 64 kDa. Western blotting indicated that induced protein could react with anti-His antibody. It was also found that the induced protein can stimulate mature chicken osteoclasts to resorb bone.

In vitro bulb scales of Lilium longiflorum×L. formosanum were used as explants to develop a highly efficient regeneration system. A high regeneration rate (100%) was reached through organogenesis on basal Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 6-benzylaminopurine (6-BA) and 1.0 mg/l naphthaleneacetic acid (NAA). A genetic transformation system for the lily was developed using an Agrobacterium tumefaciens-mediated method. An improved genetic transformation rate (12‰) was obtained when the explants were pre-cultured for 3 days, immersed in bacterial suspension (OD600≈0.8) for 5 min, and co-cultivated for 5 days. The binary vector pBI121 containing Zm401, a maize pollen-specific gene, was introduced into the Agrobacterium strain LBA4404 and transformed into the explants using the genetic transformation system. Gene integration into the lily genome was confirmed by polymerase chain reaction (PCR) and PCR–Southern analysis. These results could lead to the production of new pollenless lily plants.

The highly efficient plant binary expression vector pGBIF4ABCVHB was constructed using the artificially modified, synthesized VHb gene and fused insectidal gene (GFMcryIA and CPTI). Transgenic tobacco plants were obtained by Agrobacterium-mediated transformation. For 32 plants, the bivalent genes were inserted successfully into the tobacco genome and detected by polymerase chain reaction (PCR) amplification. Southern blot and Western blot analyses showed that VHb gene was expressed in the transgenic plants. Toxicity assay indicated that the fused insectidal gene expressed pesticidal toxin protein. The net weight of transgenic tobacco plants exceeded that of non-transgenic ones by 8%. Compared to non-transgenic tobacco plants, transgenic plants appeared to be high-yielding, insect-resistant varieties.

The hybrid purity of melon (Cucumis melo L.) was tested by polymerase chain reaction (PCR) assay based on simple sequence repeat (SSR) markers in two F1 melon hybrids (‘Dongfangmi 1’ and ‘Dongfangmi 2’) and their parental lines. Twelve pairs of SSR primers for ‘Dongfangmi 1’ and three pairs for ‘Dongfangmi 2’ were selected. Results showed that self-inbred seeds were effectively distinguished from F1 hybrid seeds using these SSR primers, a finding that was consistent with the results recorded from field tests. ‘Dongfangmi 1’ and ‘Dongfangmi 2’ were identified from their parental lines, and seven other uterine hybrid lines by multiplex primers MS48+MS60 and MS4+MS20, respectively. Contamination of F1 hybrid seeds caused by self-inbred and other unknown pollens can be effectively and more reliably detected by PCR assays with multiplex SSR primers.

The gene ap36 encoding a protein elicitor from Alternaria sp. was fused downstream of the slh (S-layer homology) motif of Bacillus thuringiensis S-layer protein gene ctc. The recombinant gene was then transferred into B. thuringiensis plasmid-free derivative strain BMB171. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the SLH–Ap36 fusion protein was expressed in B. thuringiensis BMB171. After tomato (Lycopersicum esculentum) leaves were treated for 90 min with the recombinant strain cultured at 28°C for 24 h, the activity of peroxidase and the amount of proline of tomato leaves were increased to 57.14% and 131.59%, respectively, compared to the control, and after the tomato leaves were treated with the cultured recombinant strain for 4 days, the activity of phenylalanine ammonia lyase was also higher than that in the control. Furthermore, tubers of treated potato (Solanum tuberosum) plants showed higher resistance to rot disease caused by Erwinia corotovora SCG1 compared to the control treatments.

Spermatogonial stem cells (SSCs), which were isolated from chicken (Gallus gallus) embryo testes 16 days after laying, were cultured, subcultured, and induced into adipocytes in vitro. The differentiated cells were identified by oil red-O staining. Dexamethasone, insulin and 3-isobutyl-1-methylxanthine (IBMX) were tested for their induction potential. About 7–21 days after induction, SSCs differentiated into adipocytes, and the resulting adipocytes strongly expressed peroxisome proliferation activation receptor-γ (PPAR-γ). The assay outcome showed that an optimal treatment consisted of dexamethasone, insulin and IBMX application for 3 days and insulin for 1 day (3 cycles), then insulin for 21 days. The differentiation ratio was 85%, better than the combined use of dexamethasone, insulin and IBMX (P<0.01). However, the combination of the three derivatives triggered a stronger induction than any of them used alone (P<0.01). This study has demonstrated the potential of chicken embryonic SSCs to differentiate in vitro into adipocytes.

Eupatorium adenophorum (crofton weed) is one of the most widespread invasive species in China. Its genetic diversity and population structure in China were analysed by amplified fragment length polymorphism (AFLP). Three primer pairs were selected for the analysis and 490 bands were produced from 62 E. adenophorum populations selected from five major geographic areas. A total of 328 of the bands showed polymorphism [percentage of polymorphic bands (PPB)=59.4%]. Diversity levels of populations were relatively high (mean expected heterozygosity=0.154, mean Shannon index=0.241). At the regional level, the AMOVA indicated that about 70.25% of variation in the data set was from genotypic variations within populations, whereas 8.04% of the variation was due to regional differences, and the remaining 21.71% to differences among populations within the provincial regions. Cluster analysis based on the unweighted pair-group method using the method of arithmetic averages (UPGMA) grouped the majority of E. adenophorum populations into four main clusters, which correspond to their geographic regions. It is concluded that E. adenophorum spread mainly by wind or water and its genetic diversity level in newly invaded areas was lower than that in formerly colonized areas.

Intron 5 of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene was cloned and its single nucleotide polymorphism (SNP) was tested by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) in Wanxi White geese (WW) (Anser anser). The carcass, meat quality and feather-down traits of 116 geese from WW males×Sichuan White goose (A. anser) females were assessed and analysed in association with SNP. The results showed that the sequence length of intron 5 of the HMGR gene in geese was 704 bp, and the homology between goose and chicken was 44.0%. A SNP site was tested in intron 5 by PCR-SSCP, and frequencies of alleles A and B at the SNP site were 0.7716 and 0.2284, respectively. The effects of genotype AA were significant, not only on an increase of chest muscle weight, stomach gland weight (P<0.05), fibre diameter of chest muscle and 1000-down weight (P<0.01), but also on a decrease of down water-holding percentage and pH of chest muscle 45 min after slaughterP<0.05). The effect of genotype AB was in the opposite direction to that of genotype AA.

To investigate the wheat transcriptional profile under drought stress, a drought-tolerant variety of wheat (Triticum aestivum), Hanxuan 10, was treated with polyethylene glycol (PEG6000) and samples were collected at 0, 1, 6 and 24 h. Complementary DNA was labelled with fluorescent dye and hybridized with the BGI-RiceChip, a whole genome rice gene chip platform, which contains over 60 000 oligos based on the rice genome sequence. Data analysis detected 166, 207 and 328 differentially expressed genes (DGs), respectively, at 1, 6 and 24 h, indicating that the number of DGs increased with the length of the PEG treatment. Functional category analysis showed that the number of DGs related to energy metabolism pathways increased – 4.2%, 8.2% and 16.8%, respectively, as a proportion of the total number of DGs. Most of the photosynthesis-related genes were up-regulated. It is interesting to note that Psbr and ribulose-bisphosphate carboxylase (Rubisco)-coding genes were down-regulated, suggesting their potential role in the response to drought tolerance.

Using hypocotyls as explants, the adventitious shoots of Euonymus fortunei var. radicans were differentiated directly from basal Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The highest regeneration frequency was obtained with MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP) and 0.01 mg/l α-naphthalene acetic acid (NAA). A regeneration frequency of 92% and 4.2 shoots per explant were obtained after 30 days of culture. The binary vector pBCGm, containing Galanthus nivalis agglutinin (GNA) gene, was introduced into Agrobacterium tumefaciens LBA4404. Hypocotyl segments of E. fortunei var. radicans were infected through A. tumefaciens-mediated transformation. Polymerase chain reaction (PCR) and PCR–Southern blot analysis results confirmed that the GNA gene was integrated into the genome of transgenic plants. The highest transformation frequency was obtained with un-precultured explants infected for 30 min with OD600=0.6 Agrobacterium tumefaciens, and co-cultivated for 3 days.