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%.

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.

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.

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.

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.

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.

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.

A full-length cry1Ia gene fragment, obtained by polymerase chain reaction (PCR) amplification with a pair of primers designed according to cry1Ia-type gene sequences and DNA from Bacillus thuringiensis as template, was introduced into the expression vector pET-21b and transformed into Escherichia coli BL21(DE3). The molecular weight of the induced expression product was 81.2 kDa. The amino acid sequence of Cry1Ia was very different from sequences of 12 known Cry1Ia-type proteins. This gene was designated as cry1Ia8 by the International B. thuringiensis Insecticidal Protein Nomenclature Committee (accession number AF373207). The bioassay results indicated that Cry1Ia toxin protein showed distinct insecticidal activity against Ostrinia furnacalis and Plutella xylostella with LC50 of 0.268 μg/g and 2.227 μg/ml, respectively. It also had insecticidal activity against Leguminivora glycinivorella, but not against Pyrrhalta aenescens. The novel cry1Ia8 gene will be an important resource in constructing genetically engineered bacteria and transgenic plants for biocontrol of insect pests. It is also available for screening gene stacks to delay pest resistance.

The purpose of this study was to evaluate the effects of donor sex, treatments of cell cycle synchronization and donor nuclei obtained from fresh or frozen–thawed conditions on developmental competence of yak–bovine interspecies nuclear transfer embryos. Bovine (Bos taurus) oocytes were used as recipients and yak (Bos grunniens) ear fibroblast cells were used as donors. Results indicated that the development rate of male blastocysts was higher than that of female (56.6% versus 39.5%, P<0.05), whereas cleavage and total cell number showed no difference between the two groups. No significant difference was observed in the development and quality of blastocysts with donor cells treated by serum starvation or contact inhibition, and there was no significant difference in embryo development with fresh or frozen–thawed donor cells, whereas the cleavage rate in the group of frozen–thawed cells was significantly lower than that of the fresh cell group (54.5% versus 78.2%, P<0.05). The results demonstrated that donor sex could impact the developmental competence of yak–bovine interspecies nuclear transfer embryos, whereas different treatments of cell cycle synchronization and freezing had little influence.

Using the INRA – University of Minnesota porcine 7000 Rad Radiation Hybrid panel (IMpRH) containing 118 clones, porcine cellular retinol binding protein gene 1 (RBP1) and plasma retinol binding protein gene 4 (RBP4) were assigned to porcine chromosomes 13 and 14, respectively. The mRNA distributions of the two genes in adult Wuzhishan pig tissues (lung, skeletal muscle, spleen, heart, stomach, large intestine, lymph node, small intestine, liver, brain, kidney and fat) were examined. One single nucleotide polymorphism (SNP) was identified in Laiwu, Wuzhishan, Guizhou, Bama pig breeds and a Tongcheng experimental population and analysed using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Significant associations were found between different genotypes of A/G156 and maximum carcass length (LM), minimum carcass length (LN), haemoglobin (HGB), mean corpuscular volume haemoglobin (MCH), and day to slaughter weight (age) traits in the Tongcheng experimental population. This SNP locus might be used as a genetic marker in pig breeding and production.

Genetic diversity and genetic variation of 240 adult plants of four tea populations in Hunan – Camellia sinensis var. sinensis, C. sinensis var. assamica cv. Duntsa, C. ptilophylla and C. sinensis var. assamica cv. Jianghua – were studied by rapid amplification of polymorphic DNA (RAPD) markers. The results showed 226 loci using 21 random primers (10 bp), of which 201 (88.9%) were polymorphic. The genetic diversity analysis indicated that Shannon's index was 0.43; 74.7% of which was within-population genetic diversity while 25.3% was among-population variation. The gene diversity indexes of total populations (HT), within populations (HS) and among populations (HST) were, respectively, 0.37, 0.28 and 0.09. The coefficient of gene differentiation (GST) among populations was 0.23, indicating a 76.7% variation within populations and 23.3% among populations. These results displayed a rich within-population genetic variation, as in Shannon's diversity index. Interpopulation gene flow (Nm) was 0.74, which indicates the limited genetic exchange between populations.

Intergeneric transfer of plasmid vectors pSET152 and pHL212 from donor Escherichia coli ET12567/pUZ8002 and S17-1 to Streptomyces cinnamonensis was demonstrated and optimized. Assisted by this conjugation system, nsdA gene disruption was achieved through PCR-targeted gene replacement. One AprRKanS exconjugant BIB309 was then isolated and confirmed to be the nsdA null mutant. Compared with the starting strain, monensin production by the nsdA− mutant BIB309 increased 270% in vitro.

Polyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products. In this study, wheat PPO sequences (mRNA) were searched/BLASTed in the NCBI database and aligned using DNAMAN software. The results showed that wheat PPO genes could be divided into two clusters (I and II) and that three genes (‘i’) of cluster II seemed not to be located on chromosomes 2A and 2D. Ninety-four single nucleotide polymorphisms (SNPs) were detected between two haplotypes of the PPO gene on chromosome 2D. Eighty of these were found in the coding region (coding (c) SNPs) and 36 were non-synonymous cSNPs, which could affect the PPO amino acid sequence. Primers (STS-H) were designed at some non-synonymous cSNPs sites and were used to investigate the correlations between allelic variants and PPO activity of seeds – a total of 130 common wheat varieties were evaluated in 2 years. The results showed that STS-H could amplify a 460 bp DNA fragment in most cultivars with high PPO activity, while no PCR product was detected in most cultivars with low PPO activity. To improve the selection efficiency of a single dominance molecular marker, the multiplex polymerase chain reaction (PCR) system of STS-H and STS01 markers was also studied, based on the complementary between them.

To identify Porcine haemagglutinating encephalomyelitis virus (HEV) 67N receptor in porcine kidney (PK) cell membranes, the S1 protein of HEV was expressed in Pichia pastoris and purified by Ni2+ affinity chromatograph. Polyclonal antibodies to HEV were prepared by immunizing rabbits by injecting the purified S1 protein four times. After SDS–polyacrylamide gel electrophoresis (SDS–PAGE), the PK cell membrane proteins were transferred on to nitrocellulose membrane. A virus overlay protein binding assay (VOPBA) was performed using the recombinant S1 protein to identify the protein binding receptor, HEV-S1. The result showed that HEV-S1 protein bound to one band (about 90 kDa) in PK cell membranes. This result is very important for the study of the pathogenic mechanism of HEV.