In order to protect and develop valuable snappers (Lutjanus spp.), genetic diversity and molecular markers of five species (Lutjanus vitta, L. fulvus, L. fulviflamma, L. sebae and L. stellatus) were detected and analysed using random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) techniques. The polymorphic loci ratio (P) (86.00–92.11%), the mean intraspecies genetic distances (D) (0.1775–0.3431) and the intraspecies genetic diversity indexes (Hi) (0.1022–0.1634) were calculated using the RAPD technique. The genetic diversities of L. fulviflamma and L. vitta were richest in terms of P, and D and Hi, respectively. The results of SSR showed that low effective numbers of alleles (1.7893–3.6591), medium average heterozygosities (0.332–0.676) and medium polymorphism information contents (PIC) (0.302–0.641) occurred in five species of snappers, indicating comparatively rich genetic diversity among these fish. Nine molecular markers in the products amplified by primers OPA8 and OPP10, and six molecular markers in 11 microsatellite loci were found to be useful as specific markers to identify five species of snappers. Two neighbour-joining (NJ) dendrograms based on the results of RAPD and SSR suggested that L. stellatus and L. sebae are closely related and clustered in one branch, with L. vitta, L. fulviflamma and L. fulvus in the other.

The spotted longbarbel catfish (Mystus guttatus) is a member of the family Bagridae and is mainly distributed in the Pearl River Valley and Nandujiang River of Hainan Island. It is one of the four most famous fishes in the history of Pearl River Valley. Nowadays, M. guttatus has become a potential target for aquaculture in China due to its high nutritional value, but its genetic background is still unknown. This study was carried out to estimate its population genetic diversity in Pearl River. The random amplified polymorphic DNA (RAPD) technique was performed on 30 samples, using 20 random primers of length 10 nucleotides. The results showed that each primer amplified 3–9 bands per sample. Twenty primers produced 3210 well-amplified DNA fragments with reproducible band patterns and the average number of amplified DNA fragments reached 107 in each individual. Broadly speaking, 48 (44.9%) of 107 loci were polymorphic. Only the S30 primer did not produce polymorphic loci. The genetic distance of individuals ranged from 0.0467 to 0.2804. The average genetic distance among the 30 individuals was 0.1526±0.037. Genetic data in this study could be useful in genetic resource assessment as well as in wild population conservation and exploitation of M. guttatus in the future.

Based on quantitative trait locus (QTL) mapping, the gene for porcine uncoupling protein 3 (UCP3) was chosen as a candidate gene for pig fat deposition and meat quality traits. In this study, a partial coding region of the UCP3 gene was sequenced and one single nucleotide polymorphism (cSNP) was found at 395 bp. The mutation was G→A and resulted in the amino acid change from glycine to arginine. This site was also recognized by restriction endonuclease SmaI. The UCP3 SmaI polymorphism was analysed among 186 individuals of Large White×Meishan F2 progeny using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). The genotypes of the UCP3 SmaI polymorphism were AA, AB and BB. The frequency of A and B alleles was respectively 0.56 and 0.44. Statistical analyses showed that the SmaI polymorphism in the F2 population was significantly associated with back-fat thickness at thorax–waist and buttock, as well as with intramuscular fat, drip-loss rate and water-holding capacity. The additive effect of UCP3 SmaI was clearly shown. The genotype AA reduced back-fat thickness and drip-loss rate, increased water-holding capacity, and decreased the intramuscular fat. The effect of the pig UCP3 SmaI polymorphism needs to be analysed in other populations using larger samples.

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.

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 oocyan (O) gene determines blue shell pigmentation of domesticated chicken. The O locus is closely linked to ALEV1 on chromosome 1 with a genetic distance of 2.3 cM. Blue eggshells contain a protoporphyrin, biliverdin and zinc biliverdin chelating complex, which belongs to the porphyrin metabolic pathway. A bio-informatic approach was first used to BLAST for the genes of the enzymes in the porphyrin pathway; no gene was detected to specify the O locus. Due to a limited number of simple sequence repeat (SSR) markers around the O locus, polymerase chain reaction–single-strand conformational polymorphism (PCR-SSCP) was then employed to search for candidate single nucleotide polymorphism (SNP) markers. The sequence bearing a marker was found to consist of two transversions at Chr1:61754089T→A and Chr1:61754175A→T, respectively. Population screening showed that 81% of homozygous blue-shelled layers (OO) were classified as AA genotype at the SNP loci, while 89% of heterozygous blue-shelled layers (Oo) were AB genotype and 93% of tinted layers (oo) were BB genotype. The results indicated that there is a close association between O locus and SNP locus, suggesting that the marker locus could be used as a molecular marker in breeding for blue-shelled chickens.

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.

Using hypocotyl segments of aseptic seedlings of cabbage (Brassica oleracea var. capitata) as explants, regenerated plants with kanamycin resistance were obtained mediated by Agrobacterium tumefaciens (strain LBA4404). The transformed plants with the CryIA(c) (Bt) gene were confirmed by Southern blotting analysis, indicating the integration of the transgene into the cabbage genome. The majority of the transgenic plants had only a single copy of the inserted CryIA(c) gene. Leaf section bioassays showed that resistance against larvae of diamondback moth in CryIA(c) transgenic cabbage was significantly enhanced. The inheritance patterns of the transgene in T1 offspring of transgenic cabbage were investigated using polymerase chain reaction (PCR) analysis and a kanamycin resistance test on the leaves of young seedlings. The results showed that dominant gene loci, CryIA(c) or neomycin phosphotransferase gene (NPTII), followed Mendelian inheritance, with a ratio of 3:1 segregation in T1 populations.

The Lc regulatory gene affects the formation of anthocyanin in plants. XY355 promoter, a petal-specific promoter, was obtained from the genome of rape (Brassica napus) by polymerase chain reaction (PCR). A plant expression vector, pXY60, was constructed, which contained the maize Lc regulatory gene under the control of the XY355 promoter. The vector was introduced into tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) by an Agrobacterium tumefaciens-mediated method. The flower colour of some transgenic tobacco plants was changed from light red to deep red and that of some transgenic petunia plants had changed from white to light purple.

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.

cDNA of the porcine small adipocyte factor 1 (SMAF1) gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate primers designed according to the conserved sequences between human and mouse genes. The cDNA (GenBank accession No. DQ191892) containing a complete encoding region was 256 bp in length, sharing 86 and 78% identity with that of human and mouse, respectively. Comparison of the deduced amino acid sequence between porcine SMAF1 and the human, mouse, cattle and rat protein showed that the amino acid similarity was 81, 67, 84 and 70%, respectively. The results of semi-quantitative RT-PCR showed that the porcine gene was expressed abundantly in adipose tissue, and at a significantly lower level in lean-type pigs (Large White pigs) than in lard-type pigs (Meishan pigs) at the age of 4 months (P<0.05). The results suggest that the porcine SMAF1 gene may have similar functions as in other species, that is, it may regulate adipogenesis and/or adipocyte function.

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.

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.

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.

A real-time fluorescent polymerase chain reaction (PCR) assay was established to detect Streptococcus suis serotype 2. Primers and Taqman probe were designed according to cps2I (capsular polysaccharide 2I) gene using bio-software Primer Express2.0 and Oligo6.0. An 81 bp DNA fragment was amplified from S. suis serotype 2 genomic DNA, and the PCR product was cloned into pMD18-T vector and confirmed by DNA sequencing. The real-time fluorescent PCR amplification curve on a Lightcycler® showed that the method is accurate and specific for S. suis serotype 2 amplification, whereas reference bacteria S. suis, Escherichia coli, Salmonella sp., Staphylococcus aureus, Shigella sp., Listeria monocytogenes strains and a blank control were all negative. Tenfold serial dilutions of S. suis serotype 2 were used to measure the sensitivity of real-time fluorescent PCR: ten copies of bacteria could be detected in one PCR reaction and only 30 min were required for a single test. To examine the stability of the real-time fluorescent PCR, the positive control was detected at two different times. The threshold cycle (Ct) values showed no statistical differences (P>0.05). Thus, this method was stable and repeatable. These results indicate that this real-time fluorescent PCR technique could be applied for epidemic supervision in entry–exit inspection and quarantine.

The methylation levels of genomes were compared in swine, cattle, sheep, rat, chicken and duck, using the methylation-sensitive amplification polymorphism technique (MSAP). The results showed that the methylation levels in genomes of the species investigated were mostly about 40–50% (except cattle); the methylation level varied in different species; the methylation pattern in various tissues of each species was specific; for the same species, the methylation level of the tissue genome was mostly higher than that of the blood genome; the difference of methylation level between birds and mammals was not significant, however mammals appeared to have a lower hemi-methylation frequency and higher full methylation frequency than birds.

The isozymes of 10 enzymes connected with energy metabolism in Tegillarca granosa were analysed by vertical polyacrylamide gel electrophoresis. Esterase and α-amylase are enzymes related to energy intake, their activities were high in the digestive gland. Malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, succinate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase (G-6-PDH) and adenosine triphosphatase (ATPase) are enzymes related to energy metabolism. The main energy supply of T. granosa comes from aerobic respiration; anaerobic metabolism and the pentose phosphate pathway take an auxiliary role in energy metabolism. The high activity of G-6-PDH in T. granosa might mean a considerable proportion of carbohydrates metabolized through this pathway. This reaction could provide abundant NADP for metabolism in T. granosa. Compared with other shellfish, T. granosa had lower activity of ATPase, which might have some relationship with the amnicolous life and low motility of this animal.

To slow down the accumulation of reducing sugar in potato tubers exposed to low-temperature storage, an expression vector, pBICNI, including Nt-VIF (Nicotiana tabacum vacuolar inhibitor of beta fructosidase, a vacuolar invertase inhibitor from Nicotiana tabacum) gene regulated by potato tuber specific promoter class I patatin promoter (CIPP) was constructed and transformed into potato (Solanum tuberosum L.) cultivar E-potato 3 (E3). Detection by polymerase chain reaction (PCR), Northern and Southern hybridizations indicated that the full-length Nt-VIF cDNA was transformed successfully into cv. E3. After storing potato tubers of 14 transgenic lines at 4 or 20°C for 30 days, their activities of vacuolar invertase (VI) and reducing sugar (RS) content were analysed. The results showed that there were no significant differences in RS content between transgenic and untransformed (control) tubers stored at 20°C. However, RS content of transgenic lines was obviously reduced at 4°C compared to the control, from 34.0% (line B-13) to 76.8% reduction (line B-1), implying that VI activity was inhibited by Nt-VIF cDNA expression and RS content was reduced. Further analysis revealed a positive linear relationship between VI activity and RS content (VI=0.308RS+0.067), and lines B-1, B-2, B-6, B-9 and B-14 could meet the requirements of potato chips in terms of their low RS content after cold storage.

A complete chitin deacetylase (CDA) complementary DNA (cDNA) from Mucor racemosus was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification cDNA end (RACE) with gene special conserved primers. The cDNA sequence was submitted to GenBank (DQ538514). The complete cDNA with full-length of 1506 bp contained a 67 bp 5′-untranslated region, an open reading frame of 1344 bp and 95 bp 3′-untranslated region including tailing site AATAAA. The gene encoded a sequence of 448 amino acid residues and consisted of core nucleotides encoding a polysaccharide deacetylase domain covering 32% of the entire sequence. The CDA gene shared sequence homology with those of several fungi. The corresponding homology of the deduced amino acid sequences varied from 21 to 69%. Phylogenetic analysis according to the deduced amino acid sequences matched the classical fungi taxonomy. The three-dimensional structure of this protein was predicted. The protein had a whole CDA functional domain and a polysaccharide deacetylase domain.