The localization of heat shock protein 70 (HSP70) and HSP70 mRNA in the heart, liver, lung, kidney, spleen, thymus and cloacal bursa in broilers that were heat stressed for 6 h was conducted using immunohistochemistry and in situ hybridization techniques. Positive HSP70 mRNA signals were detected in the liver and lung, especially in the vessel walls. A weak presence was found in the myocardial cells. No significant signals were observed in spleen, thymus and cloacal bursa. HSP70 was observed in the vessel walls of all investigated broiler tissues. Localizations of HSP70 and HSP70 mRNA suggest that HSP70 could be correlated with cardiovascular function.

A polymerase chain reaction–enzyme-linked immunosorbent assay (PCR-ELISA) method was set up to detect Mycoplasma in live vaccines and to establish the optimal experimental conditions. According to the 16S rRNA gene sequences published in GenBank, which include Mycoplasma gallisepticum of chicken and M. hyopneumoniae, M. hyosynoviae and M. flocculare of swine (submitted nos: Mg AY744942, Mhp AE017244, Mhs AY973563 and Mf X63377), PCR primers, labelled with digoxin (Dig), and probe, labelled with biotin, were designed using the software DNAstar and Primer Premier 5.0. The system and conditions of PCR–ELISA were optimized using purified genomic DNA extracted from Mycoplasma as a template. Samples of 30 batches of Newcastle disease live vaccines (I strain), 30 batches of Newcastle disease live vaccines (La Sota strain) and 30 batches of swine fever live vaccine were tested by PCR-ELISA. Results showed that the detection rate of Mycoplasma contamination from different vaccines was higher (36.5%) with this technique than with PCR (24.4%). The PCR–ELISA appeared to be a simple, fast and reliable method for qualitative and quantitative analysis of Mycoplasma. The practical use of PCR–ELISA as a kit to detect Mycoplasma contamination in live vaccine seems very promising.

A 1018 bp fragment of the ACO gene cDNA sequence was cloned from tomato (Lycopersicon esculentum) leaves incubated with a pathogen mixture using reverse transcriptase-polymerase chain reaction (RT-PCR) with two PCR primers designed according to the sequence of a tomato cDNA clone (E11). A BLAST search showed the sequence presenting a very high match with the ACO genes in other plants, with 83–99% homology. Using this sequence, an RNA interference (RNAi) transformation vector (pD311) was constructed and transformed into tomato. Twenty-seven regenerated plants with kanamycin resistance were obtained, showing that the transgene was integrated into the tomato genome; this was confirmed by PCR and Southern blotting. Ethylene production by the RNAi transgenic tomato plants was measured by gas chromatography, and showed that ethylene evolution was specifically inhibited in leaves and fruits of the transgenic plants.

The development of near-isogenic lines (NILs) is an important fundamental step in the cloning of quantitative trait loci (QTL) and molecular marker assisted breeding. In the present study, NILs for two major QTLs of basal root thickness (BRT) and 1000-grain-weight (TGW) were obtained by molecular marker-assisted selection (MAS) through foreground selection for target QTL and background selection in three backcross generations (BC1F1, BC2F1 and BC3F1). After phenotypic evaluation in the BC3F2 generation, nine BRT QTL-NILs with BRT ranges of 1.07–1.16 mm (6.11–15.18% greater than the recurrent parent) and an average recovery ratio of genetic background (RRGB) of 97.22%, and 11 TGW QTL-NILs with ranges of 21.25–26.25 g (7.05–32.16% greater than the recurrent parent) and 95.97% of RRGB, were selected.

Duck interleukin (IL)-18 mature protein gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from Ma duck (Tadorna ferruginea) splenocytes. The PCR product was cloned into pGEM-T Easy vector for sequencing. The result revealed that the nucleotide sequence of duck IL-18 mature protein gene (mDuIL-18) consisted of a 513 bp band. A prokaryotic plasmid of mDuIL-18, pQE30-mDuIL18, was obtained by subcloning the encoding region of the DuIL-18 mature peptide into pQE30. pQE30-mDuIL18 transformed Escherichia coli M15. The expression of mDuIL-18 gene was identified by SDS-PAGE and Western blotting. Its molecular weight was 19.76 kDa, and could be specifically recognized by rabbit sera to chicken IL-18. The expressed products existed as inclusion bodies. After being degenerated, then renatured, the activities of the inclusion bodies were detected by methyl thiazolyl tetrazolium (MTT) assay. In ducks injected intramuscularly with mDuIL-18 protein (150 ng or 200 ng per duck) and Avian influenza virus (AIV) vaccine 2 weeks after immunization, the average titres of haemagglutination inhibition (HI) antibodies to AIV reached 7.5–7.7 log2, while those of HI antibody ranged between 6.3 and 6.6 log2 in ducks vaccinated with AIV vaccine only or with 100 ng mDuIL-18 and AIV vaccine. The results clearly showed that 150 ng mDuIL-18 per duck strengthened the in vivo immune responses induced by the inactivated oil emulsion AIV vaccine.

After seven generations of self-crossing assisted by β-glucuronidase (GUS) detection, 26 homozygous transgenic rice lines were obtained. Transgene effects on iron content, agronomic traits, rice quality and stress tolerance were studied. The results indicated that the average iron content in the milled grain of the transgenic (pea ferritin, Fer) rice (Oryza sativa) was 10.37 μg/g among all the homozygous lines, and only four lines (Fer34, Fer36, Fer39 and Fer65) had significantly higher iron contents than that of the control, Xiushui11 (6.46 μg/g). Significant differences in main agronomic traits, such as days from sowing to heading, plant height, main panicle length, total grains of main panicle, seed setting rate, 1000-grain weight, and yield per plant were observed among some homozygous lines, but similar flag leaf length and number of panicles per plant were found. This showed that insertion of the foreign gene (Fer) had a slight impact on the main agronomic traits, but that the transgene had no negative effects. The quality traits of homozygous rice lines, such as the percentage of brown rice, milled rice and full milled rice, grain length, grain width, ratio of grain length and width, and amylose content were all similar to those of the control. However, variations were noticed in chalkiness, translucency, alkali spreading value and gel consistency among partial homozygous lines. Responses of homozygous lines to low and high temperatures were not significantly correlated with the iron contents, and resistance to rice blast (Pyricularia oryzae), bacterial blight (Xanthomonas oryzae) and brown planthopper (Niaparvata lugens) were enhanced with increased iron contents.

The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying H5N1 Avian influenza virus (AIV) haemagglutinin HA1 on the cell surface of B. thuringiensis. Two recombinant plasmids, pCTC-HA1P and pCSHA1P, were constructed by replacing the central part below the surface anchor sequence slh of S-layer protein gene ctc with part ha1 gene (ha1p). pCTC-HA1P harboured the fusion gene ctc-ha1p and pCSHA1P the fusion gene csa-ctc-ha1p, csa representing the csaAB operon (very important in anchoring S-layer protein on the bacterial cell surface). Two recombinant B. thuringiensis strains were constructed by electrotransferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. Strains obtained were CH (bearing pCSHA1P) and BCCH (bearing pCTC-HA1P as well as the csaAB operon-carrying plasmid pMIL-CSA). The vegetative cells of CH and BCCH were used as antigens in haemagglutination (HA) and haemagglutination inhibition (HI) assays. HA assay showed recombinant HA1 proteins successfully displayed on the cell surface of CH and BCCH. HI assay showed that these recombinant HA1 proteins were specific to standard positive HI (haemagglutination inhibition test) serum of subtype H5 AIV. After immunization of mice with vegetative cells, both CH and BCCH elicited a humoral response to HA1 and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that CH exhibited a higher immunogenicity than BCCH. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine against AIV with the B. thuringiensis S-layer protein CTC surface display system.

Three Luxi adult Yellow steers were used to isolate and culture intramuscular pre-adipocytes in vitro as well as to examine factors influencing their proliferation and differentiation. The intramuscular pre-adipocytes were taken from adipose tissues within muscles between the sixth and seventh rib and cultured after digestion with collagenase I. The results showed that the separated cell populations were highly homogeneous, proliferative and doubled within 62 h. When the confluent pre-adipocytes were treated with 10 μg/ml insulin and 0.25 μmol/l dexamethasone, small lipid droplets appeared on day 2 and the number of lipid droplets rapidly increased around the nuclei on day 6. Their dynamic morphological changes, growth curve, Oil Red O staining, and reaction to insulin and dexamethasone all verified their pre-adipocyte identity. Under controlled conditions, the intramuscular pre-adipocytes resumed proliferating and differentiating in vitro. Interestingly, the proportion of cultured diploid pre-adipocytes reached more than 90% after six repeated cultures. This study confirms the existence of functionally active pre-adipocytes within the muscles of Chinese adult local breed cattle. These cell strains are a potentially useful model for understanding further the mechanism of intramuscular adipose deposition in tissues, in order to improve beef quality based on Chinese local breed beef cattle.

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.

To understand the genetic basis of seed setting rate (SSR) in rice, two breeding lines (Oryza sativa ssp. indica), T226 with higher and stable SSR and T219 with lower and fluctuating SSR from different natural conditions, were used for constructing recombinant inbred lines (RILs). Genotype by environment (G×E) interaction and quantitative trait loci (QTL) for SSR were analysed using a population with 202 RILs under eight differing environments. A significant G×E interaction for SSR was detected in rice using the Additive Main Effects and Multiplicative Interaction (AMMI) statistical model, and the IPCA1 and IPCA2 of the G×E interaction accounted for a variation of 57.6%. QTL controlling the SSR were detected by the method of interval analysis. Seventeen QTL on nine chromosomes were identified across eight environments, totally explaining the phenotypic variances from 4.6 to 35.7%. Most of the QTL, each explaining a small part of the phenotypic variances and interacting with environments, were detected in one or two environments, and their alleles for increasing the SSR were derived from T226. However, the QTL (MRG5959–MRG2180) on chromosome 3 was detected across six different environments. It explained maximum phenotypic variances in each detected environment and its allele for increasing the SSR was derived from T226. Another QTL, mapped between markers RM592 and RM169 on chromosome 5, was detected in five various environments and its allele increasing the SSR was derived from T219.

The functional mitochondrial protein alternative oxidase (AOX) gene was used as a marker to analyse the phylogenetic relationship between Cryptosporidium isolates. This gene was characterized, and the phylogentic tree was established from Cryptosporidium isolates and compared to those generated from 18S rRNA and heat-shock protein 70 (HSP70) gene sequences. The present trial aimed at finding out whether the AOX gene is suitable for phylogenetic analysis of the Cryptosporidium genus. The results revealed that the genus Cryptosporidium contained the phylogenetically distinct species C. parvum, C. hominis, C. suis and C. baileyi, which were consistent with the biological characterization and host specificity reported earlier. Cryptosporidium species formed two clades: one included C. hominis, C. suis, C. parvum cattle genotypes and C. parvum mouse genotype; and the other comprised C. meleagridis and C. baileyi isolates. Within C. parvum, both the mouse genotype and the pig genotype I (also known as C. suis) isolates differed from cattle and human (also known as C. hominis) genotypes, based on the aligned nucleotide sequences. The sequence identity of the AOX gene was higher between C. meleagridis and C. baileyi than between C. meleagridis and C. parvum. The phylogenetic trees showed that C. meleagridis was closer to C. baileyi than to C. parvum. This result was inconsistent with the phylogenetic analysis deduced from 18S rRNA and HSP70 gene sequences, respectively. The present results suggest that the AOX gene is not only equally suitable for the phylogenetic analysis of Cryptosporidium, but also provides an outstanding and new approach in determining Cryptosporidium heredity.

The genetic diversity of dinitrogen-fixing bacteria associated with rice (Oryza sativa) was assessed by a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) approach on the nifH gene amplified directly from DNA extracted from washed rice roots and rhizospheric soil. Restriction digestion with the enzymes MnlI and HaeIII was performed to characterize 54 cloned nifH PCR products. RFLP profiles were clustered and analysed with the UPGMA program. Eight pairs of similar RFLP patterns (similarity>50%) and two pairs of homologous RFLP patterns (100% identity) were found from the washed roots and the rhizospheric soil, respectively. Three specific diazotrophic patterns were found from rhizospheric soil and rice roots. The analyses have revealed the presence of different nifH types, which appear to be significant components of the diazotrophic community in paddy fields, indicating that some of the diazotrophs may colonize the inside and the surface of the rice roots.