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.

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%.

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.

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.

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.

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.

Bacillus thuringiensis strain WZ-9, isolated from soil in Hebei province, China, was effective against Henosepilachna vigintioctomaculata larvae. The strain presented bipyramidal crystals with a protein band of 130 kDa in SDS–PAGE. The pH changes of the culture media showed important fluctuations during the 24 h growth cycle. The pH varied less in log and stationary phases than it did in the exponential phase. Bioassay results showed that the WZ-9 strain was only harmful to larvae of H. vigintioctomaculata and not to either adults of H. vigintioctomaculata or other several lepidopteran and coleopteran insects. LC50 to second-instar larvae of H. vigintioctomaculata was 2.95×107 cells/ml after 72 h. Genotypic investigations showed that this strain possessed the cry7 gene. Sequence analysis demonstrated that the encoding gene contained an open reading frame (ORF) of 3414 bp and encoded 1138 amino acid residues. The deduced amino acid sequence was 99.65% identical to that of the reported Cry7Ab2 sequences. This gene was designated by the Bt δ-endotoxin nomenclature committee as Cry7Ab3 with accession number BI 1015188 in the GenBank database.

The elements for the Semliki forest virus (SFV) RNA replicon were obtained from the Alphavirus genome. It was designed to overcome the poor efficacy of some current plasmid vectors. Genes coding for viral replicases are preserved while genes coding for structural proteins are replaced by foreign genes in the RNA replicon. High levels of RNA replication and expression of foreign genes in the cytoplasm are regulated by the replicases. To evaluate the effects of the SFV RNA replicon on the improvement of gene expression, a LacZ gene was inserted into pIRES-neo digested by BamHI and dephosphorylated by alkaline phosphatase to construct pIRES-neo-LacZ. The RNA replicon vector pCMV-rep-LacZ and two conventional cytomegalovirus (CMV) promoter-based vectors (pLNCX-LacZ and pIRES-neo-LacZ) were transfected, using Lipofectin, to prepared 293 cells. Growth hormone releasing hormone (GHRH) expression vectors (pCMV-Rep-GHRH, pCDNA3.1(+)-GHRH and pIRES-neo-GHRH) were also tested using the same procedure. Target gene expression was detected with radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR). Results showed that the expression level of the RNA replicon vector was 2–3 times higher than with normal plasmid vectors. This result will help to improve the efficiency of gene expression in eukaryotic cells.

The population doubling number (70–80 times) of human fetal bone marrow mesenchymal stem cells (BMMSCs) is about two times higher than that (30–40 times) of adult BMMSCs, and their differentiation capacity is superior to that of their adult counterparts. In this study, BMMSCs were isolated from long bones of 2- to 3-month-old human abortuses by rinsing and selectively culturing whole marrow cells. Basic medium and serum concentration were optimized and growth curves plotted, both by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] reduction assay. Isolated cells were identified by flow cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and a tumour-forming experiment. The results indicated that lengthwise scissoring of fetal long bones and rinsing of their marrow cells was practical and useful for recovery of BMMSCs from the investigated human abortuses. In this experiment, α-MEM (minimum essential medium, alpha medium)+20% FCS (fetal calf serum) was the best for in vitro culture of BMMSCs. The third-passage BMMSCs expressed Oct4, SSEA3 and SSEA4 besides the surface markers of their adult counterparts. The population doubling time of the BMMSCs of passage 6, 12 and 24 were 34, 36 and 40 h, respectively. Cells in all passages showed a diploid karyotype and formed no tumours in nude mice. The BMMSCs used in this study proved to be biologically safe and ideal seed cells for research on human tissue engineering and regeneration medicine.

Effect of lamb age, transport stimulation and repeated hormone superovulation on the number of collected oocytes were determined. Effect of the culture medium containing ethylenediaminetetraacetic acid (EDTA) on the development of embryos produced in vitro from lamb oocytes was also investigated. Results indicated that the mean numbers of collected and available oocytes (oocytes with compacted cumulus cells, evenly granulated ooplasm and morphologically normal) from 6- to 8-week-old lambs were 60.8±13.9 and 58.2±12.3, respectively. These figures were higher than those obtained from 12- to 14-week-old lambs (27.3±5.1 and 26.0±4.9) (P<0.05). Stimulation by transportation didn't decrease the number of collected oocytes from the superovulated lambs (P>0.05). However, the number of collected oocytes in the repeated superovulation group was significantly reduced, compared with the control group (P<0.05). The embryonic culture medium supplemented with 10 μmol/l EDTA highly improved the development capability of the embryos from lamb oocytes (P<0.05). Healthy lambs were born following embryo transfer.

Activation tagging plays an important role in plant genomics studies. In this paper, an activation tagging library containing 50 000 Basta-resistant lines was constructed by mediated transformation of the mutant bzr1-D of Aribidopsis thaliana. Among these transformants, 47 lines showed obvious phenotypes, including late flowering time, dwarf growth habit, changed leaf shape, longer leaf petiole, leaves lacking trichomes, sterility and no kink between stem/leaf. Some T-DNA flanking sequences were obtained by TAIL (thermal asymmetric interlaced) and nested PCR. Results showed that the mutants contained one to three T-DNA insertions. The insertions were distributed in the first, fourth and fifth chromosomes of the genome.

The bovine interferon-tau (bIFN-τ) gene, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and subcloned into a pGEM-T vector. After being verified, the fragments, with or without signal sequence, were inserted into the expression vector pET-30a(+). Two recombinant plasmids were induced to express the recombinant proteins by isopropyl β-d-1-thiogalactopyranoside. The results showed that the bIFN-τ gene could be obtained from five bovine blastocysts by PCR without extraction of genomic DNA. It had 99% homology with nucleotides and 97% with amino acids in the GenBank sequence (accession number: XM_593584). The products of recombinant bIFN-τ, minus signal sequence, expressed in pET-30a(+) were analysed by SDS-PAGE. A new 20 kDa protein was detected and its molecular weight was as expected. The antiviral activity of recombinant bIFN-τ was 1×104 IU/mg using a standard cytopathic reduction assay. Marked morphological changes were induced by recombinant bIFN-τ in bovine endometrial epithelial cells. The cell volume was larger than that of controls and a lot of vesicles appeared in the cytoplasm after 24 h culture in the presence of 2.9 μg/ml recombinant bIFN-τ. In conclusion, a purified recombinant, biologically active bIFN-τ was obtained in this experiment.

Progenies derived from rice (Oryza sativa ssp. japonica) plants pollinated by Oenothera biennis exhibited numerous morphological and developmental traits. Some variant individuals appeared in generation D2. From generations D3 to D6, a large number of variants were observed, showing distinct variable traits including giant embryos. Statistical analysis on D6 lines showed significant differences between progenies and their rice parental line in several main traits, including plant height (49.2–164.5 cm), panicle number (12.1–38.2), panicle length (20.3–30.3 cm), length of sword leaf (13.8–57.5 cm), leaf width (11.1–25.2 mm), grain number of main panicle (142.0–367.0), percentage of seed setting (0.8–99.0%), 1000-grain weight (19.7–33.8 g) and time from germination to panicle emergence (90.0–108.0 days). Most of the variable coefficients were above 20% (the highest was 40.8%). Through pedigree selection of these plants, genetically stable lines were obtained, which are useful for rice breeding. Results from amplified fragment length polymorphism (AFLP) analysis showed that several rice lines contained extensive genetic variations, which included disappearance of rice parental bands and/or appearance of novel bands.

Using microsatellite markers, the genetic structure of nine domestic duck (Anas platyrhynchos) populations in eastern China was analysed. The results showed that the heterozygosity was high in these populations, ranging from 0.5137 to 0.6055, with an average heterozygosity of 0.5523, reflecting the rich diversity. Considerable breed differentiation was observed and 25.65% of the total genetic variation came from breed differences; this low differentiation result affirmed that each breed has its own genetic diversity. The DA genetic distances suggested that greater differentiation existed between populations. The duck populations were clustered into four groups based on neighbour joining (NJ) clustering, and the clustering results showed relationships with duck breed distributions and economic utilization.