The bovine interferon-γ gene cloned from total RNA of peripheral blood lymphocytes stimulated with concanavalin A (ConA), using the reverse transcriptase-polymerase chain reaction (RT-PCR), was cloned into pGEM T-easy vector and sequenced. The mature peptide without signal peptide was then subcloned and constructed into the expression plasmids pET28a/BoIFN-γ and pPICZα/BoIFN-γ, respectively. The former was highly expressed, induced by isopropyl β-d-thiogalactoside (IPTG), in Escherichia coli BL21, with 30% of bacterial proteins in inclusion bodies. The latter was expressed in Pichia pastoris GS115 with methanol induction. The expressed products were secreted directly into cultural supernatant at concentrations of 1.0 g/l. The same recombinant proteins had antiviral activities 10 times higher in an MDBK (Madan-Darby bovine kidney)/VSV (Vesicular stomatitis virus) cell line than in a CEF (chicken embryo fibroblast)/VSV) cell line, and the antiviral activities expressed in P. pastoris were higher than those expressed in E. coli in the same cell line.

Genetic diversity in 122 accessions of pummelo (Citrus grandis Osbeck) and its related varieties was assessed using simple sequence repeat (SSR) markers. Thirty-one pairs of SSR informative primers generated a total of 335 alleles. The average number of alleles per locus was 9.85. The value of allelic polymorphism information content (PIC) ranged from 0.1939 to 0.9073, with an average of 0.7085 per primer. The 122 accessions of pummelo and its related varieties could be clustered into seven groups by the unweighted pair-group method arithmetic average (UPGMA), in which the 110 pummelo accessions could be divided into 18 subgroups at similarity coefficient of 0.712. These subgroups were mainly composed of the Shatian pummelo variety group, the Wendan variety group and many of the hybrid pummelo groups. The classification method can be used in targeting many varieties in order to widen the genetic background of pummelo.

In order to reveal the molecular basis of heterosis, Large White (LW), an introduced European pig breed, and Meishan (MS), a Chinese indigenous pig breed, were selected to hybridize directly and reciprocally in the present experiment. mRNA differential display (DD) technique was performed to identify genes that were differentially expressed in the backfat tissues of hybrids (LW×MS, MS×LW) and purebred (LW×LW, MS×MS) pigs. The ten anchor primers in combination with ten arbitrary primers (100 sets in total) were used and nearly 1500 reproducible bands were observed in polyacrylamide gels. The 40 differentially displayed bands were selected for cloning and sequencing. Thirty-six out of 40 expressed sequence tags (ESTs) proved to be novel and the sequences were submitted to GenBank (accession No. CV507051-CV507087); the other four showed similarity to known genes published in GenBank. Three among 36 novel ESTs were chosen for further identification with semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The result showed that two ESTs were differentially expressed, and the third showed no obvious difference between hybrids and purebreds. In order to reduce the percentage of false-positive DD, RNA pools of four types of pigs were constructed, by mixing samples from six pigs of the same genotype, and subjected to DD. Stringent annealing temperature was applied and only bands that could be repeated in duplicate PCR were used for further study. The results showed that the expression pattern of these 36 ESTs differed among the four genotypes of pigs, suggesting that the genes corresponding to these differentially expressed ESTs might be related to the heterosis occurring in fat tissue.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHA biosynthesis is type II PHA synthase. The gene phaC2 encoding type II PHA synthase was placed under the control of psbA-pro and psbA-ter of rice (Oryza sativa) to construct a phaC2 cassette, which was ligated with the screening marker gene aadA cassette (prrn–aadA–TpsbA-ter). These recombined fragments were cloned between the plastid rbcL and accD genes for targeting to the large single copy region of the chloroplast genome. A chloroplast transformation vector, pTC2, was constructed and introduced into the tobacco (Nicotiana tobacum) chloroplast genome by particle bombardment. PCR and Southern blot analysis confirmed stable integration of phaC2 into the chloroplast genomes of T0 and T1 transgenic plants, and T1 transgenic plants exhibited homoplasmy. The expression of phaC2 at transcription level was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation of phaC2 encoding type II PHA synthase in tobacco via chloroplast genetic engineering.

The genetic variation of isozymes [esterase (EST), superoxide dismutase (SOD), malate dehydrogenase (MDH), malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PD)] extracted from two different adult tissues (adductor muscle and gill) of Pteria penguin R. and offspring from two stocks of Pinctada martensii D. were analysed by vertical polyacrylamide gel electrophoresis. The result showed an obvious and stable variation in the isozyme phenotypes in two different pearl oyster species. The SOD and EST isozymes from gill and MDH, ME and G6PDH from adductor muscle were species-specific. The electrophoretograms of these isozymes could be used as markers to differentiate the two pearl oysters. Between inbreeding and hybridized stocks of P. martensii, the electrophoresis patterns of isozymes were alike, but the similarities of electrophoretograms in the inbreeding progenies were higher than in the hybridized ones. The characteristic electrophoretograms of isozymes could be applied as molecular markers in the breeding of pearl oysters.

Genetically modified organisms (GMOs) containing anti-herbicide genes account for more than 75% of all GMO and the proportion is increasing. The polymerase chain reaction (PCR) method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material in foodstuffs and PCR screening for the presence of transgenic components in food is becoming a routine method in modern food analysis. In this study, the five kinds of the anti-herbicide genes (bar, pat, cp4-epsps1, cp4epsps2 and gox) were examined by PCR in four kinds of genetically modified crops (soy, maize, cotton and rape). Results indicate that the method was sensitive, specific and credible, and that all genetically modified and approved crops containing the anti-herbicide genes can be examined by this method.

The highly conserved DNAs of Equine herpesvirus-1 (EHV-1), Equine arteritis virus (EAV), Equine influenza virus (EIV), Equine infectious anaemia virus (EIAV) and Eastern equine encephalomyelitis virus (EEEV) were acquired by molecular cloning, and spotted on the diagnostic gene chip. The cDNAs reverse-transcribed from RNAs of samples were labelled with Cy5-dUTP/Cy3-dUTP as fluorescent probes. Following specific hybridization of the deposited gene chip and labelled probes, fluorescence signals were scanned by laser scanner and the resulting image was analysed by QiamtArray software on a digital computer. The results showed that the prepared gene chip could detect and distinguish the five equine viruses. Its sensitivity was about 25 viral genome copies. The hybridization specificity was confirmed by the presence of red fluorescence signals on the corresponding sites with samples from the five relevant viruses in horses and by the absence of positive signals with the specimens from irrelevant viruses from other animals. Peripheral blood leucocytes (PBL) from some seropositive horses in post-arrival quarantine were negative according to virus isolation, but were positive for EHV-1 and EAV according to the gene chip technique. The overall results suggest that gene chips, which are quick, specific, sensitive and reliable, can provide a practical alternative for screening quarantined animals, and will be able to deal with a large number of animal samples within a very short period of time.

Two microsatellite markers OarHH35 and BMS2508, closely associated with high levels of reproduction in sheep, were analysed for polymorphisms in Xiangdong black goat, Nanjiang brown goat, Guizhou black goat and Boer goat. The number of alleles for OarHH35 was 11, 8, 7 and 10, respectively, in the four goat breeds. That for BMS2508 was 9, 5, 6 and 7. The polymorphic information content (PIC) values for OarHH35/BMS2508 in the above-mentioned goats were respectively 0.8294/0.8047, 0.8399/0.6894, 0.7985/0.6910 and 0.8582/0.7688. By analysing the dominant effect of the two microsatellite markers on litter size of Xiangdong black goat, it was shown that microsatellite markers BMS2508 and OarHH35 significantly influenced (P<0.05) litter size of goats. At the OarHH35 locus, the least squares means (LSM) of genotype 135/135 bp was the highest (3.67 number born/litter). Moreover, LSM of genotypes 123/135 bp and 125/125 bp also reached 3.00 kids born/litter, indicating that alleles 135 bp and 125 bp exerted a significant positive effect on the litter size in Xiangdong black goat. At the BMS2508 locus, LSM of genotypes 132/145 bp and 93/132 bp for litter size was 4.00 and 3.25 kids born/litter respectively, but that of genotype 122/122 bp for litter size was only 1.00 kid/litter, so alleles 145 bp and 93 bp had significant positive effects on the litter size of Xiangdong black goat, and the allele 122 bp had a significantly negative effect.

Primary adipocytes from subcutaneous adipose tissue samples obtained from 7-day-old Yorkshire×Landrace crossbreed piglets were exposed to 0–400 nmol/l of insulin for 48 h. The accumulated triglyceride was measured through Oil Red O staining and the cumulative glycerol released was determined to assess lipolytic activity in adipocytes. Transcription levels of sterol regulatory element binding protein (SREBP)-1c, carbohydrate response element binding protein (ChREBP), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) were assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that ChREBP and ACC1 mRNA levels were not influenced by insulin alone under low glucose (5 nmol/l). FAS mRNA level was markedly stimulated by all doses of insulin except 200 nmol/l, and SREBP-1c mRNA level increased with 100–300 nmol/l insulin. High insulin doses (300 and 400 nmol/l) increased the HSL mRNA level as well as lipolytic activity.

Increasing the growth rate is especially important for low-quality wood applications, so this has become an important goal in poplar breeding. The present study describes the transfer of Vitreoscilla haemoglobin (VHb) gene (vgb) driven by constitutive promoters, by Agrobacterium tumefaciens into poplar (Populus alba×P. glandulosa). From about 450 leaf discs used for transformation, 60 Kan-resistant plants were obtained, and 52 proved to be true transgenic plants. The transgenic nature of these plants was confirmed by polymerase chain reaction (PCR) amplification and Southern dot blot hybridization. The expression of vgb gene in transgenic plants was confirmed by reverse transcriptase-PCR (RT-PCR). The performance of the transgenic lines was evaluated during the first year of growth in a greenhouse. These plants showed no significant stable morphological differences from the untransformed plants. Among them, three vgb-transgenic lines exhibited noticeably higher growth rates in terms of height and diameter.

To analyse the influence of genetically modified (GM) rice (Oryza sativa) containing the exogenous choline oxidase (codA) gene on physiological metabolism and genetic horizontal transformation in the fed rats, the animals were divided randomly into two groups. One group was fed with the feedstuff containing 30% of transgenic rice. The remaining (control) group was fed with the feedstuff containing 30% of non-transgenic rice. After 30 days, growth, physiological metabolism parameters such as growth hormone (GH), insulin, glucagon, 3,3′,5-triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), and the genetic horizontal transformation were analysed. Results indicated that the weight of all rats from the two groups was similar (P>0.05). Also, there was no significant difference in the level of GH, insulin, glucagon, T4, T3 and TSH between the treated and untreated rats. The serum urea nitrogen (SUN), glucose, the glutamate–pyruvate transaminase (GTP), as well as the total cholesterol, of treated rats were almost identical to those of the control (P>0.05). Moreover, no target DNA sequence of rice endogenous sucrose phosphate synthase (SPS) gene or exogenous choline oxidase (codA) gene was detected in organs (liver, muscle, pancreas, etc.) except the stomach, gastrointestinal and rectum contents, and no genetic horizontal transformation was observed in rats fed with transgenic rice.

Recombinant Bac-GV2 DNA was obtained by inserting a fused gfp gene with the Bacillus thuringiensis vip2A(c) gene encoding a possible enzymatic component under the control of the polyhedrin gene promoter of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). The Trichoplusia ni cell line TnHi5 was transfected with Bac-GFP and Bac-GV2 DNAs respectively. Fluorescent cells expressing the fusion protein GV2 were much fewer than those expressing green fluorescent protein (GFP) alone, and did not obviously increase in number from 2 to 5 days after transfection. This result showed that the Vip2A fusion protein might have an ADP-ribosylating activity on cell skeleton actin, exerting an influence on the production and diffusion of the budded virus from insect cells.

The Arabidopsis homeotic gene AP2 is essential for floral meristem development and organ specification. In this study, we report the isolation and sequence comparison of the AP2 homologue of Brassica rapa (BAP2). The results showed that the BAP2 DNA sequence was 2138 bp in length and contained nine introns. It shared 90% identity with AP2 in the coding region. The putative BAP2 polypeptide contained a nuclear localization signal (NLS) sequence and two copies of highly conserved AP2 domain, suggesting that BAP2 may function similarly to AP2. Two nucleotide variations were detected in DNA sequences from wild-type and apetalous B. rapa plants at the BAP2 locus, while the putative polypeptides were identical. We propose that BAP2 is not likely to be responsible for the apetalous mutation in B. rapa.

Primary culture of goat mammary gland cells was achieved by outgrowth of migrating cells from fragments of tissue. The fibroblast and epithelial cells were purified according to their different sensitivity to trypsin and the characteristics of goat mammary epithelial cells were observed under a light microscope. The results showed that the purified goat mammary epithelial cells retained normal characteristics until the 15th passage. The purified mammary epithelial cells propagated and formed a dome-like structure that resembled a nipple and was called a ‘milk orb’. The mammary epithelial cells could produce and secrete milk. There were different cell types: the majority were short shuttle-like or polygons, resembling a beehive; while some were large, flat and round; and others were elongated. The enhanced green fluorescent protein (EGFP) gene was transferred successfully into the goat mammary epithelial cells using electrotransfection, and its expression was observed under a fluoroscope.

This study used three winter wheat (Triticum aestivum L.) genotypes (H6756, H311 and SP8581) to compare the effects of sampling time, callus induction media, differentiation media and rooting media on in vitro culture of young spikes in wheat. In all these three genotypes, the frequencies of green plantlet differentiation were high when their young spikes were cultured between the stages of protective glume primordium formation and pistil and stamen primordium formation, but low at other stages. The optimum medium for callus induction was Murashige and Skoog (MS) medium+2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The optimum green plantlet differentiation medium was MS medium. Some abnormal plantlets regenerated from calli. When these plantlets were transferred to another differentiation medium [MS+1.0 mg/l 1-naphthaleneacetic acid (NAA)+0.2 mg/l 6-benzylaminopurine (6-BA)], shoot formation and elongation were induced. This allowed 90.91% of them to develop into normal green plantlets. The optimum rooting medium was 1/2MS+0.2 mg/l 3-Indolylacetonitrile (IAA)+80 g/l sucrose. An efficient regeneration system for young spike culture of wheat was set up based on such methods. Using this wheat-regeneration system, young spikes and immature embryos of 17 genotypes of wheat were in vitro cultured to study and compare the callus induction frequencies and green plantlet differentiation frequencies. The results of two successive years showed that in 15 out of the 17 genotypes (88.24%) the green plantlet differentiation frequencies were higher than those of immature embryos by 6.2–65.1%. These results showed that the regeneration system established in this trial for young spike culture of wheat was effective.

According to the sequence of Rccn4, which is highly similar to the nucleotide-binding site (NBS) coding region of the cereal cyst nematode resistance gene, Cre3, three 3′ nested primers were designed to amplify its 3′ flanking region through single oligonucleotide nested polymerase chain reaction (SON-PCR). One 1264 bp band, Rccn-L, was amplified from E-10, a wheat–Aegilops variabilis translocation line containing the cereal cyst nematode resistance gene from Ae. variabilis. Sequence analysis showed that Rccn-L possesses the 3′ flanking sequence of Rccn4 and contains a 55 bp-sized consensus sequence with Rccn4. The coding region was 1026 bp, consisting of an incomplete open reading frame, a terminator codon and no initiation codon and intron; it encoded a peptide of 342 amino acid residues and shared 86% nucleotide sequence identity with Cre3. The peptide had a conserved leucine-rich repeat (LRR) domain, containing the imperfect repeats, XXLXXLXXL, comprising 17% leucine residues, and shares, respectively, 89% nucleotide sequence and 78% amino acid sequence identity with the LRR sequence of the Cre3 locus. In the present study, SON-PCR was used successfully, for the first time, in plant genome research and proved to be a valuable method in plant gene cloning. The acquirement of Rccn-L established the foundation for obtaining the complete Rccn gene and further structural and functional investigations.

In order to investigate the molecular mechanism of rehydration after serious drought in broomcorn millet (Panicum miliaceum L.), a forward subtracted cDNA library was constructed between normal watered leaves and rehydrated leaves after serious drought conditions, using the suppressive subtraction hybridization (SSH) technique. A total of 60 positive clones were picked out at random from the subtracted library and sequenced, and redundancy sequences were removed after sequence alignment. Based on the results of sequence homologous comparison and function querying, 32 expressed sequence tags (EST) were highly homologous with known ESTs. Most of those sequences were related to either abiotic or biotic stress in plants. Of those sequences, 11 ESTs were homologous with ESTs in rat (Rattus norvegicus) liver after partial hepatectomy. The Blast result of proteins revealed that 28 ESTs were similar to known proteins. The functions of these proteins mainly involve signal transduction, transcription and protein processing. This experiment demonstrated that a range of specific genes was induced and expressed in broomcorn millet during the rehydration stage after serious drought.

The molecular genetic characteristics were evaluated, and the genetic effects of marker loci on heterosis of three traits (birth weight, BWT; average daily gain, ADG; and feed and meat ratio, FMR) were analysed in the experimental pig populations: Yorkshire (Y, n=34), Landrace (L, n=46) and Meishan (M, n=55); Yorkshire×Landrace (YL, n=32) and its reciprocal (LY, n=36), Yorkshire×Meishan (YM, n=82) and its reciprocal (MY, n=47), by 39 microsatellite markers selected from pig chromosomes SSC4, 6, 7, 8 and 13. The results indicated that observed alleles ranged from 2 to 6, average 4.13, observed heterozygosity varied from 0.39 (Y) to 0.58 (YM+MY) and average polymorphism information content (PIC) varied from 0.33 (Y) to 0.5 (YM). There were two loci (sw2155 and sw1037) at which the alleles were fixed in Y and L, and there were three loci (sw2409, sw2454 and sw1691) at which the alleles were fixed in M. The results from heterozygosity and kinship analysis revealed an intrinsic genetic relationship among the seven populations. Furthermore, the results on genetic effect analysis indicate that several marker loci had a significant effect on heterosis of the three traits in the two different F1 crossbred populations (P⩽0.01), for example s0161, swr1130 and sw1856 for BWT, sw1856 and swr2036 for ADG, and sw1302 and swr2036 for FMR. The significant marker loci implied a deep genetic relationship between molecular marker loci and heterosis.