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.

Transgenic cotton expressing the Cry1Ac toxin from Bacillus thuringiensis has been planted widely in China since 1997, and reached 70% of the total cotton area in 2006. The results of monitoring the environmental impact of Bt cotton commercialization indicated that the target pests, cotton bollworm (Helicoverpa armigera) and pink bollworm (Pectinophora gossypiella), were effectively controlled while the mirids (Hemiptera: Miridae) evolved to be key pests in the cotton system. There were no significant changes in resistance gene frequency of field populations of cotton bollworm, but a shift toward increased tolerance was apparent in the area of intensive planting of Bt cotton, indicating that the potential risk of resistance of the target pest has become a major threat for sustainable planting of Bt cotton. Considering the factors associated with the evolution of resistance, risk management strategies are discussed in this paper.

Alternative splicing is an important cellular mechanism that increases the diversity of gene products. The study of alternatively spliced genes reported so far in plants is far less documented than that in mammals, but considerable results have been reported, showing the role of these genes in regulating mechanisms, influencing factors, and specificities and function of alternative splicing in plants. This review summarizes briefly the major progress made on alternative splicing in plants.

The genetic diversity of 43 male sterile and low fertility Citrus accessions, as well as 13 fertile ones, were assessed using simple sequence repeat markers (SSRs). Thirty-five polymorphic alleles were generated from eight primers (on average 4.4 alleles per primer). Cluster analysis was performed via unweighted pair group method analysis (UPGMA) using the NTSYS-pc version 2.10. The results showed that the accessions could be classified into three groups: cultivars of mandarin were classified into group 1; those of sweet orange, grapefruit, ponkan or tangor into group 2; and Microcitrus with male sterile cytoplasm into group 3. Cluster analysis also revealed that Satsuma mandarin was more closely related to Bendiguangju mandarin than to Zaoju, Mankieh or Huangyan Bendizao tangerine. The present study on genetic diversity of male sterile and low fertility Citrus will provide useful information for further collection, preservation and utilization of this plant.

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.

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.

A rapid and accurate real-time quantitative polymerase chain reaction (real-time PCR) method with SYBR Green I was established for detecting Escherichia coli O157:H7. A pair of primers were designed to amplify the eae gene. The dissociation curves showed that the amplification product was very specific. The optimal conditions and standard curve were established. The result indicated that real-time PCR was 1000 times more sensitive than ordinary PCR.

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.

Endophytic bacteria isolated from Guangdong bamboo (Bambusa blumeana) plants were obtained under aerobic and anaerobic cultural conditions. Using the acetylene reduction assay (ARA), 40 strains were identified as diazotrophs. Diazotrophic isolates were grouped into five clusters (I, II, III, IV and V) by SDS-PAGE whole-cell protein patterns. The analysis of full-length 16S rDNA sequences of the representative strains of each group indicated that the endophytic diazotrophs showed great diversity and belonged to the genera Azospirillum (α-subclass of proteobacteria), Escherichia and Pseudomonas (γ-subclass of proteobacteria), and Aquaspirillum (β-subclass of proteobacteria).

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.

To evaluate the effects of H5 subtype avian influenza DNA vaccine with HA gene and expressive vector optimization, groups of 3-week-old specific pathogen free (SPF) chickens were inoculated intramuscularly with a single dose of 100 μg or 10 μg of pCIHA5, pCAGGHA5, pCIoptiHA5 or pCAGGoptiHA5 in 200 μl volume. Another group of chickens was injected with 200 μl phosphate-buffered saline (PBS) as controls. Sera were collected every week after vaccination to detect the HI and agar-gel precipitin (AGP) antibodies. Four weeks after the single vaccination, all chickens were challenged with 100LD50 of the highly pathogenic A/Goose/GuangDong/1/96(H5N1) [Gs/GD/1/96(H5N1)]. Oropharyngeal and cloacal swab specimens were collected from all chickens 3, 5 and 7 days after inoculation for titration of virus in eggs. Chickens were observed daily for 2 weeks for signs of disease and death. Results showed that in the groups vaccinated with 100 μg pCAGGoptiHA5 and pCAGGHA5 chickens were completely protected from virus challenge (no disease signs, no virus shedding and no deaths), while only partial protection occurred in chickens vaccinated with 100 μg pCIoptiHA5 (75%) and pCIHA5 (50%). In groups vaccinated with 10 μg pCAGGoptiHA5 and pCAGGHA5 chickens were protected from virus challenge (no disease signs and no deaths). In the group vaccinated with 10 μg pCIoptiHA5 the protection ratio was 75%, while chickens vaccinated with 10 μg pCIHA5 all died after challenge. Results indicated that codon usage optimization of the HA gene and β-actin promoter enhanced the protection efficiency of H5 subtype avian influenza DNA vaccine and the construction pCAGGoptiHA5 could protect chickens from lethal H5N1 virus challenge even at the low dose of 10 μg, implying the possibility for the commercialization of an avian influenza DNA vaccine in the near future.

Combining the long serial analysis of gene expression (LongSAGE) and the generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification (GLGI) technique, a new strategy called modified GLGI (M-GLGI) was developed to isolate unknown 3′ expressed sequence tags (ESTs) and discover novel genes. A 17 bp LongSAGE tag was used as sense primer instead of a 10-base SAGE tag; PCR reaction was performed under an appropriate annealing temperature for each tag; universal DNA polymerase was used in PCR amplification instead of Pfu enzyme; a common cloning strategy using pMD-18T vector and Escherichia coli DH5α cells were used instead of a special vector and competent cells. Moreover, ESTs isolated by M-GLGI had 3′ ends with the polyadenylation signals and poly(dA) tails. This method is more sensitive for identifying genes expressed in low abundance than conventional EST sequencing.

Target region amplified polymorphism (TRAP) was used to compare genetic structures among three populations of grass carp (Ctenopharyngodon idella) – one wild and two cultured populations. Seven out of 15 primer combinations produced good amplification patterns and provided 103 amplified loci from the three populations. Numbers of polymorphic loci in the wild population were higher, indicating a decrease in genetic polymorphism in the two cultured populations. Compared with the wild population, only 39.98% loci gene frequency remained unchanged in the cultured samples, showing a genetic structure change in cultured populations. The genetic distances between wild and cultured populations were 0.0421 and 0.0809. With primer combination Ga5-800-E5, we detected a region in the electrophoretic pattern in which the number of amplified loci apparently decreased in cultured populations. These results establish a good scientific basis for developing molecular markers that can help in distinguishing wild from cultured populations.

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.

This trial was aimed at investigating the effect of different doses of progesterone (P4) and types of vaginal bolts on new follicular wave induction, oestrus synchronization and superovulation in Holstein heifers. Results showed that the interval after prostaglandin F2α (PGF2α) application and oestrus occurrence between the sponge blot (SP) group (48.3 h) and the progestin release device (PRD) group (39 h) was significantly different (P<0.05). The average number of embryos obtained in the SP group (13.2) was significantly higher than that (8.7) in the PRD group (P<0.05). The same trend was observed for the number of usable embryos (5.7 and 3.3 for SP and PRD groups, respectively). The injection of 50 and 100 mg of P4 efficiently induced a new follicular wave, whereas no significant difference (P>0.05) was recorded on oestrus synchronization and superovulation between the two groups.

A total of six amplified fragment length polymorphism (AFLP) primer combinations were used to detect genetic variation of pooled DNA in a sample of 12 chicken breeds indigenous to China, and AFLP DNA fingerprinting of each chicken breed was constructed. Polymorphic bands, specific bands and genetic similarity coefficients of 12 chicken breeds were calculated from AFLP data. A total of 279 polymorphic bands were generated by the six primer combinations, giving, on average, 46.5 polymorphic markers detected per primer combination. Nine specific bands were produced in the pooled DNA of Jiuyuan black and Dongxiang black chickens. However, one specific band was produced in the pooled DNA of Wenchang and Xingyi bantam chickens. An unweighted-pair-group method using average linkages (UPGMA) cluster analysis revealed that the 12 chicken breeds could be divided into three groups. Genetic similarity coefficients and the UPGMA tree of the 12 chicken breeds were consistent with their breeding history as well as their geographical distribution. Based on AFLP DNA fingerprinting, genetic diversity, genetic relationship and identification of chicken breeds can be analysed.

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.

This research focused on the effect of distant hybridization to improve the flesh quality of offspring from Oreochromis aurea (♀)×Siniperca chuatsi (♂). The proportions of different tissues and percentages of nutrient components were compared between fish of the F3 generation and O. aurea and S. chuatsi. The results showed that the proportion of flesh in F3 hybrids was significantly lower than that in O. aurea. Crude lipid content was lower and crude protein content significantly higher in the F3 generation. Concentrations of four kinds of amino acids important for flavour (DAA) were higher in the F3 generation than in O. aurea, and the total DAA was significantly higher. Furthermore, the other amino acid contents and essential amino acid index of the F3 generation were intermediate between O. aurea and S. chuatsi. We conclude that some effects of hybridization have induced flesh quality improvement.

A yellow leaf curl disease with chlorotic and yellowish leaves, upward leaf curling and stunting symptoms was observed on tomato in Shantou city of Guangdong province. A virus isolate BS was obtained from a diseased tomato plant. The complete DNA-A sequence of the virus isolate BS was determined to be 2740 nucleotides long, with all the characteristic features of begomovirus genome organization. BS DNA-A encoded six potential open reading frames (ORFs), with two (AV1 and AV2) in virus sense and four (AC1, AC2, AC3 and AC4) in complementary sense, and contained an intergenic region of 269 nucleotides. The results of BLAST searches showed that BS DNA-A had higher sequence identity with reported begomoviruses in Asia than with those in America and Africa. Further sequence comparisons indicated that BS was most closely related to the isolate of Tomato leaf curl Taiwan virus (ToLCTWV-[Taiwan]) with a sequence identity of 97.7%. Nucleotide sequence identities of AV1, AV2, AC1, AC2, AC3, AC4 and intergenic region (IR) between BS and ToLCTWV-[Taiwan] were 98.6, 98.0, 98.0, 97.5, 96.3, 98.6 and 96.6%, respectively, while that of the six ORF-encoded proteins between BS and ToLCTWV-[Taiwan] were 97.7, 99.1, 97.5, 95.6, 91.8 and 99.0%, respectively. Phylogenetic analysis based on the DNA-A sequences has also indicated that BS is most closely related to ToLCTWV-[Taiwan], forming a branch with ToLCTWV-[Taiwan], Tomato leaf curl Guangdong virus and Tomato yellow leaf curl Guangdong virus. The above results demonstrate that BS is an isolate of ToLCTWV.

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.