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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Avian influenza virus is acquiring the ability to cross the species barrier between birds and mammals. However, the genetic mechanism of this ability is not well known. We successfully rescued an influenza A virus A/Chicken/Guangdong/03 (H5N1), which is highly pathogenic to both SPF chickens and BALB/c mice, by plasmid-based reverse genetics. The rescued virus, R-A/Chicken/Guangdong/03 (R-CG), and the wild-type A/Chicken/Guangdong/03 (W-CG) were found to share similar biological properties, such as in titres of 50% egg infectious dose (EID50), 50% tissue culture infectious dose (TCID50) and intravenous pathogenicity index (IPVI). R-CG, like W-CG, is highly pathogenic in mice following natural route infection. Both R-CG and W-CG viruses can be isolated from many organs of mice such as brain, lung, kidney and spleen. As a result, the constructed R-CG reverse genetic system can be used as a tool in the investigation of the molecular mechanism of infection of the mammalian host by avian influenza virus.