The plant expression vector pCAMBIA1300-AtNHX1-als was constructed by inserting the herbicide resistance gene als of Arabidopsis thaliana into the plasmid pCAMBIA1300-AtNHX1, which contains the AtNHX1 gene encoding the Na+/H+ antiport from the vacuolar membrane of A. thaliana. Transgenic tobacco plants were obtained via Agrobacterium-mediated transformation. PCR and Southern blot assay indicated that genes als and AtNHX1 had been integrated into the genome of the transgenic plants. The herbicide resistance and salt tolerance of transgenic plants increased by about 1000-fold and by 1.5% NaCl concentration, respectively, compared with controls. Herbicide resistance of the T1 progeny was evaluated by spraying transgenic plants with different concentrations of Luhuanglong at the four-leaf stage. Controls gradually died under 100 mg/l Luhuanglong whereas 73% of the T1 plants still survived at 500 mg/l Luhuanglong. Thus the plant expression vector pCAMBIA1300-AtNHX1-als could be used to improve the herbicide resistance and salt tolerance of crops.

Gene expression profiles between submergence rice (Oryza sativa ssp. indica) varieties, tolerant FR13A and sensitive IR39595-503-2-1-2, under submergence stress were identified using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). A total of 1428 bands were amplified with 40 pairs of primers, of which 102 were significantly different between the two lines. The differential display ratio was 7.1%. Among 42 differential display bands derived from the submergence tolerant variety, the expression of seven fragments was confirmed by Northern blot analysis. The analysis of their sequences indicated that four of them showed high homology with genes related to a water stress response: genes encoding ATP-binding protein, isocitrate dehydrogenase, NADH dehydrogenase and terminal acetyltransferase, respectively. The remaining three fragments were novel cDNA fragments.

The black muntjac (Muntiacus crinifrons) is a critically endangered mammalian species, confined to a narrow region of south-eastern China. In the present study, the homogeneous ear fibroblast cells of a black muntjac were respectively cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose), DMEM (high glucose) and RPMI-1640. The population doubling time of subcultured cells was not significantly different between the three different media, but cell growth was greater in DMEM (low glucose). The interspecies embryos were reconstructed using the fibroblasts of black muntjac as donors and enucleated goat or rabbit oocytes as recipients and their blastocyst rates were 0 and 11.5%, respectively. The results demonstrated that the two cytoplasts could support reprogramming of the ear fibroblasts of black muntjac, but the developmental potential of the reconstructed embryos was different.

Fifty-eight actinomycetes were isolated from surface-sterilized tomato (Lycopersicon esculentum) roots and 43 isolates were screened for herbicidal activities. Isolate S5 was found to have potent herbicidal activity against germination of wheat (Triticum aestivum L.), mung bean (Phaseolus radiatus L.) and grass (Paspalum notatum and Cynodon dactylon) seeds. But the metabolites of isolate S5 showed no influence on the growth of wheat seedlings. The S5 strain was identified as Streptomyces lavendulae var. glaucescens based on its morphological characteristics and physiological properties. The highest herbicidal activity was observed when 2% inocula were applied into S medium (containing 1% glucose, 0.3% beef extract and pH 7.0) and incubated at 25°C on a rotary shaker (160 rpm).

Mature porcine oocytes containing first polar bodies (Pb I) were obtained by in vitro culture of follicle oocytes from ovaries obtained from a local abattoir, and zygotes with second polar bodies (Pb II) were grown after in vitro fertilization of the mature oocytes. Extrusion, biological activity and morphology of Pb I and Pb II were statistically analysed. Polar bodies were isolated and collected from oocytes by enzyme digestion or micromanipulation. Their vigour under different preservation conditions was analysed and evaluated using a Trypan blue staining method. The results showed that 66.7% of the oocytes extruded Pb I after 40 h of in vitro mature culture of oocytes, and 49.7% of the zygotes extruded Pb II 20 h after in vitro fertilization. The efficiency of isolation of Pb II by micromanipulation significantly exceeded that by enzyme digestion, the Pb I and Pb II isolated by micromanipulation presenting with good vigour and normal morphology (95.3% versus 58.9%). The survival rates of Pb I and Pb II were 63.3% and 93.1% for 4 h at 39°C, 85.0% and 72.9% for 40 h at 4°C, and over 95.0% and 84.6% for less than 7 days at −20°C. In comparison with the above preservation conditions for Pb I and Pb II, the results for cryopreservation were best, with rates of survival as high as 89.1% for Pb I and 87.9% for Pb II for preservation periods of over a month, and rates of normal morphology of 97.8% and 95.7%, respectively. The Pb I and Pb II could be isolated and preserved effectively, for use in further research on the recombination of oocytes and zygotes.

To illustrate the effect of nitric oxide (NO) on the microtubules of Sertoli cells (SC), SCs of piglets were treated with sodium nitroprusside (SNP). Changes in cell viability, anti-oxidant activity, enzyme activity and p38 mutagen-activated protein kinase (p38MAPK) activation were detected. The results were as follows. A low concentration of NO can keep SC microtubule and cell viability normal, and a high concentration of NO could increase p38MAPK activation, decrease anti-oxidant activity and transferrin secretion, and destroy the structure and distribution of the microtubules. The results suggest that SNP treatment results in an increase in NO in SCs and decreased cell anti-oxidant activity. The high concentration of NO destroys cell microtubules by activating p38MAPK.

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%.

Based on conserved homologous amino-acid sequences of the Gq protein α subunit in arthropods, a pair of degenerate primers were designed to amplify the gene from the English grain aphid (Sitobion avenae), using reverse transcriptase polymerase chain reaction (RT-PCR) and (3′/5′)-rapid amplification of cDNA ends (3′/5′ RACE) techniques. A Gqα protein was obtained from the alate adult aphids. The open reading-frame was 1062 bp, encoding 352 amino-acid residues with a calculated molecular weight of 40.8 kDa. The cDNA sequence was deposited in GenBank with accession no. EF638906. The deduced amino-acid sequence of Gqα shared a high identity (≥82.17%) with reported Gqα from other insects and even vertebrates, and had the typical characteristics of Gqα protein. In order to explore the function of the Gqα gene, a eukaryotic expressional system (baculovirus expression vector system, BEVS) was constructed by TOPO and Gateway techniques. After the recombinant reaction occurring between pUC-Gqα and the Gateway-adapted baculovirus DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV), the construct recombinant viruses containing V5-His6Gqα were transfected singly into the insect cell line of Tn-5B1-4. After collecting the infected cell, detection was conducted by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The result showed that the system comprising recombinant baculovirus and Tn could express Gqα protein successfully.

The structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA of Actinobacillus pleuropneumoniae (APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that the apxIIA gene was expressed in Escherichia coli BL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108 cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

In order to detect the molecular mechanism of heterosis in pigs, an mRNA differential display (DD) technique was performed to investigate the differences in gene expression in the longissimus dorsi muscle tissues from Meishan, Meishan×Large White cross and Large White pigs. Fourteen expressed sequence tags (ESTs), differentially expressed between the hybrid and purebred pigs, were isolated and identified through semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Nucleotide sequence analysis revealed that the 14 ESTs are not homologous to any of the known genes or ESTs. These novel ESTs were then deposited in the GenBank database. Tissue expression profile analysis showed that the ESTs were expressed in most tissues, including heart, spleen, liver, kidney, small intestine, ovary and lung, and this also implied that these genes must be important for the life process. Our results indicate the diversity of differential display of genes between the hybrids and purebreds in the Meishan×Large White cross combination. Results also suggest that heterosis in pigs might be derived from the differential expression of many indispensable genes in specific life phases.

The Dorset ram of the callipyge phenotype presents with muscular hypertrophy in the buttocks, and its inheritance is polar overdominant. A partial DNA fragment of 250 bp was obtained from the goat (Capra hircas) callipyge gene (CLPG; GenBank accession no. EU753362), which shared 96.04% and 88.65% identity with the corresponding regions of ovine (Ovis aries) and porcine CLPG, respectively. A polymorphism in the DNA fragment was detected by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Sequencing results indicated no A→C mutation corresponding to the ovine CLPG gene, although one A→C transversion was located 147 bp downstream from the CLPG site. The polymorphism, named SNP216 after its position (where SNP indicates single-nucleotide polymorphism), was investigated in Boer (n=63), Laiwu Black (n=70), Lubei White×Boer Hybrid (n=40), Lubei White (n=29) and Inner Mongolia Alashan White cashmere (n=115) goat populations. The results indicated that allele A was dominant in four of the goat populations, the Inner Mongolia Alashan White cashmere goats being the exception. The first four populations were in a state of Hardy–Weinberg equilibrium (P>0.05). In Inner Mongolia Alashan White cashmere goats, least-square means of birth weight, production of cashmere and body weight gain from birth to weaning did not differ significantly between the AA and AC phenotypes (P>0.5).

The haemagglutinin (HA) gene of H5N1 Avian influenza virus (AIV) was amplified from the plasmid pGEM-HA and cloned into the baculovirus transfer plasmid pFastBacHT to construct the recombinant transfer vector pFastBacHT-HA. The pFastBacHT-HA was transformed into DH10Bac competent cells, transposed with a shuttle vector (Bacmid) and the transposition rBacmid-HA was constructed. The recombinant baculovirus was harvested from sf9 cells transfected with rBacmid-HA. The expressed HA protein was identified and analysed by SDS–PAGE, Western blot and haemadsorption assay. The 66 kDa protein could only react with chicken serum positive to H5 subtype AIV. The haemadsorption assay showed that the sf9 cells infected with rBacmid-HA baculovirus could absorb chicken red blood cells. These results indicated that the HA protein was successfully expressed in sf9 cells, with reaction specificity to H5 subtype antiserum.

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.

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.

Inter-simple sequence repeat (ISSR) analysis was carried out in Thatcher, 20 near-isogenic lines (NILs) containing respectively different genes conferring resistance against wheat leaf rust (Puccinia recondite f.sp. tritici), three materials carrying Lr37 and three materials without Lr37. All of the 100 ISSR primers showed clear amplification products. Two of them amplified the polymorphic DNA bands in the NILs, Thatcher and Lr37/6*Thatcher. The polymorphic bands were named UBC812-1200 and UBC848-700, respectively. The three materials with and without Lr37 were detected in tests using the two primers UBC812 and UBC848. Results also showed that only band UBC812-1200 was amplified in all resistant and absent in all susceptible materials. This suggests that UBC812-1200 marker is linked to the resistance gene Lr37. The genetic linkage of the polymorphic marker with Lr37 was tested using a segregating F2 population (128 plants) derived from a cross between the leaf rust-resistant Lr37/6*Thatcher and the susceptible cultivar Thatcher. The ISSR marker UBC812-1200 showed co-segregation to the Lr37 resistance gene. It could be used in molecular marker-assisted selection in a wheat breeding programme for leaf rust resistance.

The homology of the sequences, reported and registered in GenBank, of different strains of Avian influenza virus (AIV), Newcastle disease virus (NDV), Classical swine fever virus (CSFV) and Foot-and-mouth disease virus (FMDV), was analysed and compared with each other. According to the properties of these viruses, the conservative domain of the M gene for AIV, the F gene for NDV, the 5' non-coding domain end for CSFV and the 2B gene for FMDV were selected for polymerase chain reaction (PCR) amplification. In order to prevent the formation of conformational dimers between different primers, four pairs of primers designed with the DNAsis system under the condition of G+C (50–60%),18–25 bp in length and Tm (72–85), were analysed using the VNTI5.5 system. The specific fragments amplified were as follows: 141 bp for FMDV, 200 bp for CSFV, 319 bp for NDV and 471 bp for AIV. The optimal conditions of PCR for each virus mentioned above were determined by orthogonal assay, and two or four of the four pairs of primers were then combined and used for amplification trials. The results showed that four specific fragments of different lengths would be successfully amplified in one tube at the same time. The products of PCR were tested to be specific by sequencing. Out of 46 pathological samples detected with the multiple PCR, there were 5 AIVs, 7 NDVs, 15 CSFVs and 6 FMDVs. The amplification above was identified with a single PCR. On the other hand, the results corresponded to those of electronic microscopy, haemagglutination (HA) and enzyme-linked immunosorbent assay (ELISA). The method described here is practicable, sensitive, specific, simple and cheap. It could be used for diagnosing AIV, NDV, CSFV and FMDV in different animals.