With the development of plant genetic engineering, many transformation methods can be used to transfer heterogeneous genes into plants to develop genetic crops. However, a lot of research results have shown that transgene expression remains largely unpredictable and there is great variation of expression level in different transgenic plant lines. Plant genetic engineering research is reviewed in the present paper. We analysed the reasons why low efficiency of heterogeneous gene expression has happened in transgenic plants in terms of DNA modification, localization of proteins and methods of transformation used. Some strategies for improving heterogeneous gene expression in transgenic plants are also discussed.

The indica rice Xieqingzao eB1 (XQZ eB1), a mutant with an elongated uppermost internode, was produced by mutagenesis from elite maintainer line Xieqingzao B (XQZ B) using γ-radiation. Compared with the wild-type XQZ B, plant height and the length of the rice panicle neck, second internode and third internode from the panicle were elongated significantly in XQZ eB1. Genetic analysis indicated that the trait for uppermost internode elongation of XQZ eB1 is controlled by a single recessive gene eui1, which is allelic to that of IR50eui (derived from 76:4512). Further studies were carried out using the bulk segregated analysis approach. An F3 population, derived from the cross XQZ eB1×Aijiaonante, was used to map the mutant gene eui1. Two SSR makers, RM164 and AC9, were identified to link to eui1, their genetic distances to eui1 being 18.4 and 7.9 cM, respectively. The eui1 gene was also mapped to the long arm of chromosome 5 in this research.

An amplification fragment length polymorphism (AFLP) molecular linkage map with a relatively high density for location of quantitative trait loci (QTLs) controlling the quantitative traits of silkworm (Bombyx mori) cocoons, was constructed using 91 individuals of the F2 generation. Among the 692 effective loci, 550 were allocated to subgroups a and b, of which 21 linkage groups in subgroup a had 233 molecular markers and 28 linkage groups in subgroup b had 317 markers. The number of markers on each linkage group in subgroups a and b ranged from 4 to 43 and 3 to 35, respectively. The total length of linkage groups for subgroup a was 1868.10 cM, and 2677.50 cM for subgroup b. The length of linkage group varied from 22.3 to 424.3 cM in subgroup a and from 2.4 to 366.5 cM in subgroup b. The average variation in the distance between markers was 3.39–17.43 cM in subgroup a and 0.8–26.96 cM in subgroup b. The average distance between the markers was 8.81 cM in subgroup a and 9.26 cM in subgroup b. There were 14 linkage groups, with an average distance below 10 cM, in subgroup a and 18 linkage groups in subgroup b. There were seven linkage groups with an average distance between 10 and 20 cM in subgroup a and ten linkage groups in subgroup b. Each linkage group in subgroup a had 11.1 loci on average, while there were 11.31 loci on each linkage group on average in subgroup b. The mean length for linkage groups in subgroups a and b was 89 and 95.6 cM, respectively. The total average length for both a and b subgroups was 2272.8, and 9.06 cM for the average marker distance, fulfilling the basic known requirements for locating QTLs.

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.

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.

Primordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage and purified by Ficoll density-gradient centrifugation. PGCs were co-cultured with somatic cells in preliminary culture and subcultured. Identification of PGCs was carried out by histochemical methods, including alkaline phosphatase (AKP) and periodic acid–Schiff (PAS). The proliferating activity of PGCs in subculture was demonstrated by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Meanwhile, proliferating PGCs were compared under different culture conditions of 5–20% fetal cattle serum (FCS), insulin–transferrin–selenite (ITS) medium, conditioned medium (CM), 15% FCS+ITS, 15% FCS+40% CM. The results showed that the cultured PGCs were positive for AKP and PAS staining and displayed intensive proliferating activity by PCNA. The PGCs without centrifugation grew better than those with centrifugation. The PGCs formed larger colonies in media with 5% FCS or ITS than other media, indicating that 5% FCS or ITS supplemented media could be an ideal culture system for PGC proliferation in the PGC-somatic cell co-culture, in addition to the embryonic fibroblast feeder layer.

Research into, and the environmental release of, transgenic rapeseed in China are overviewed and the environmental risks are assessed, focusing on competitive survival ability, gene dispersal and biodiversity impact of transgenic rapeseed. It is concluded that transgenic rapeseed has a higher probability of gene dispersal when compared with other major crops. Brassica napus may transfer genes through pollen and seeds to vegetables and wild species of B. rapa and B. juncea, for which China is the biodiversity centre and also the country of highest consumption. It is considered that the risk of gene dispersal is present, but can be reduced to an acceptable limit. Commercialization of transgenic rapeseed should not be stopped, but should be built on a safe and sound basis by building a reasonable management system. Awarenes of biosafety considerations for transgenic rapeseed should be strengthened, and a technical platform of genetically modified organism (GMO) detection and monitoring should be properly established. Countermeasures against environmental risks are also discussed.

A 1009 bp promoter sequence of cab gene, which encodes chlorophyll a/b binding protein belonging to a class of light-inducible proteins, was cloned from Gossypium arboreum. Sequence analysis showed that it had no obvious homology with previously published cab promoters. The full-length Gacab promoter and 5′ deletions with length of 197, 504 and 779 bp were fused with gus (uid A) gene, respectively, and plant expression vectors were used for transformation of Nicotiana tabacum cv. NC89. β-Glucuronidase (GUS) histochemical assay of transgenic tobacco plants showed that GUS was expressed specifically in leaves and young green tissues. GUS was not detected in the leaves of transgenic plants grown in the dark for 6 days. However, it was highly expressed in the leaves of these plants after induction with light for another 6 days, demonstrating that the full-length Gacab promoter is a light-inducible promoter. Transient GUS expression in rice calli indicated that the expression level of Gacab504::gus was the highest and stronger than that of the CaMV 35S promoter, while expression was reduced for Gacab197::gus, Gacab779::gus and Gacab1009::gus constructs. This suggests that −197 bp to −1 bp is a basic promoter of Gacab, some positive regulatory elements may exist in −504 bp to −197 bp, and the fragment −1009 bp to −504 bp may contain negative elements.

Regeneration of sweet potato (Ipomoea batatas cv. Lizixiang) transgenic plants with oryzacystatin-I (OCI) gene was achieved using an Agrobacterium tumefaciens-mediated method. A. tumefaciens strain LBA4404, harbouring a binary vector pBinh with neomycin phosphotransferase II and OCI genes, was used. After 3 days of subculture, Lizixiang embryogenic suspension cultures were co-cultivated with LBA4404 (OD600nm=0.5) for 4 days. Next, the infected suspension cultures were first cultured for 5 days in MS medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/l carbencillin (Carb) but without kanamycin (Kan) and then transferred to MS medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb for the selection culture. Four weeks after selection, 200 Kan-resistant cell aggregates (∼1 mm in size) from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb, and eight embryogenic calluses were obtained. After transferring to MS medium supplemented with 1 mg/l ABA, 50 mg/l Kan and 100 mg/l Carb, these embryogenic calluses formed 13 plantlets via somatic embryogenesis. PCR and PCR–Southern blot analysis indicated that seven of the 13 plantlets were transgenic.

Interferon, an important cytokine, is an immunomodulator and possesses antiviral and anti-tumour activity. In vitro, it can be administrated in the treatment of diseases alone or with genetically engineered vaccine to enhance the immune effect of the latter. The recombinant transferring vector pSY681–ChIFN-γ was obtained in this study by inserting the chicken type II interferon (ChIFN-γ) gene into the Fowlpox virus (FPV) transferring vector pSY681. The resulting plasmid was then transfected into chicken embryo fibroblast (CEF) cell cultures pre-infected with the parental FPV S-FPV-017. Finally, the recombinant Fowlpox virus (rFPV) expressing ChIFN-γ (rFPV–ChIFN-γ) was produced by homologous recombination with the FPV gene in CEF. rFPV-positive plaques were verified by polymerase chain reaction (PCR), restriction analysis and indirect immunofluorescence assays. The rFPV–ChIFN-γ supernatants, cultured in CEF for 72 h and inoculated into rat fibroblasts (L929), had an inhibitory effect on the replication of Rous sarcoma virus (RSV) with an antiviral titre of 2048 U/ml.

Eupatorium adenophorum (crofton weed) is one of the most widespread invasive species in China. Its genetic diversity and population structure in China were analysed by amplified fragment length polymorphism (AFLP). Three primer pairs were selected for the analysis and 490 bands were produced from 62 E. adenophorum populations selected from five major geographic areas. A total of 328 of the bands showed polymorphism [percentage of polymorphic bands (PPB)=59.4%]. Diversity levels of populations were relatively high (mean expected heterozygosity=0.154, mean Shannon index=0.241). At the regional level, the AMOVA indicated that about 70.25% of variation in the data set was from genotypic variations within populations, whereas 8.04% of the variation was due to regional differences, and the remaining 21.71% to differences among populations within the provincial regions. Cluster analysis based on the unweighted pair-group method using the method of arithmetic averages (UPGMA) grouped the majority of E. adenophorum populations into four main clusters, which correspond to their geographic regions. It is concluded that E. adenophorum spread mainly by wind or water and its genetic diversity level in newly invaded areas was lower than that in formerly colonized areas.

Macrophages from the head kidney (HK) and peripheral blood leucocytes were isolated fromCyprinus carpio by Percoll gradient density centrifugation, cultured in vitro and exposed to different concentrations of the immunomodulator lentinan. To evaluate the immunostimulating effects of lentinan, proliferation of the peripheral blood leucocytes, respiratory burst of macrophages and interleukin-1β (IL-1β) gene mRNA expression of macrophages were investigated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], NBT (nitroblue tetrazolium) reduction, Griess reaction and real-time polymerase chain reaction (PCR). Results showed that proliferation of peripheral blood leucocytes after 24 h incubation by the induction of lentinan at 100 and 1000 μg/ml was markedly stimulated. Lentinan at 1, 100 and 1000 μg/ml could significantly induce superoxide anions in macrophages. Production of nitric oxide by HK macrophages after 96 h incubation by lentinan showed that it had no conspicuous effect on nitrogen burst activity of macrophages. Moreover, it inhibited the nitrogen burst activity at higher doses. The expression of IL-1β in the HK macrophages after 24 h of polysaccharide stimulation showed that lentinan stimulated IL-1β expression in the head kidney in carp. Lentinan can modulate the immune response of C. carpio.

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.

Simple sequence repeats (SSR) and random amplification of polymorphic DNA (RAPD) molecular markers were used to assess the genetic diversity of 80 isolates of Phytophthora infestans in potato (Solanum tuberosum) from Fujian, Heilongjiang, Hebei and Inner Mongolia Provinces in China. Polymorphism was identified by 13 SSR primers and 14 RAPD primers in the isolates of P. infestans in potato. A total of 76 bands were amplified by SSRs, with the percentage of polymorphic bands (PPB) being 78.9% and the similarity coefficient ranging between 0.00 and 0.42. A total of 189 bands were amplified by RAPDs, with the percentage of polymorphic bands being 95.2% and the similarity coefficient ranging between 0.04 and 0.66. Analysis of genetic diversity showed that there exists higher genetic variation in the Fujian population in comparison to the populations of Heilongjiang, Hebei and Inner Mongolia. Nei's genetic identity analysis indicates that the genetic similarity between populations of Heilongjiang and Inner Mongolia is the highest and that between Fujian and Hebei is the lowest. A cluster analysis revealed that isolates from Fujian, in the south of China, are distantly related to those from Heilongjiang, Hebei and Inner Mongolia in the north, and the Fujian population is distributed among more groups than the other three, exhibiting a higher genetic diversity.

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.

Using 16S rDNA-polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE), electron microscopy and a conventional plating method, the genetic diversity and phenotype polymorphism of the endophytic bacteria within Azolla microphylla were explored. The 16S rDNA-PCR-DGGE profile showed a complex and divergent bacterial community, with Bacillus cereus as the dominant species, within the Azolla–cyanobacteria association. This result was supported by the fact that endobacterial cells exhibited distinct ultrastructural characteristics in vivo and, in vitro, bacteria displayed various colonies with different sizes, shapes and colours. This study demonstrates that the genetic diversity of endophytic bacteria in Azolla can be investigated using the16S rDNA-PCR-DGGE technique.

The cryptogein (Crypt) gene was obtained by PCR amplification of genomic DNA from Phytophthora cryptogea and confirmed by DNA sequencing. A promoter of the rice phenylalanine ammonia-lyase (PAL) gene was used to regulate the expression of Crypt, because this promoter has a low level of constitutive expression, is strongly induced by pathogen infection and is expressed in epidermal tissues. These promoter characteristics may be suitable for potential inhibition of pathogen attack on epidermal tissues. For functional interaction of Crypt with its outer plasma membrane binding sites, Crypt was led by a signal sequence of the extracellular pathogenesis-related protein (PR1b) of tobacco (Nicotiana tabacum). The fused gene was constructed into a binary vector and the final plasmid (CHF-PAL::Crypt) was transformed into tobacco using the Agrobacterium-mediated transformation method. Twenty-two lines of transformants were obtained from selection medium containing 100 mg/l of kanamycin. Results from PCR amplification and Southern blot analysis demonstrated that Crypt was integrated into the tobacco genome. In the assay of pathogen challenge, nearly two-thirds (68.2%) of the transgenic plants showed significantly enhanced resistance against black shank fungal (Phytophthora parasitica var. nicotianae), brown spot fungal (Alternaria alternata) and wild fire bacterial (Pseudomonas syringae pv. tabaci) diseases. This observation indicates that low-level constitutive expression of Crypt gene in tobacco could have potential use in generating broad-spectrum disease-resistant plants.

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.

Inverted repeats of the partial movement protein gene (ΔMP) of Tobacco mosaic virus (TMV) were introduced into the plant expression vector pBin438, and the recombinant plasmids were transformed into Agrobacterium tumefaciens EHA105 by a tri-parental mating method. Fifty transgenic plants of Nicotiana tabacum cv. Yunyan 87 were obtained after Agrobacterium-mediated transformation. PCR and Southern blot analyses showed that the target gene had integrated into the tobacco plant genome. When transgenic tobacco plants were challenged with TMV, symptoms expression and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that transgenic plants had three kinds of phenotypes in response to the virus infection: immune (10%), resistant (4%) and susceptible (86%). Northern blot analysis showed that the levels of target mRNA accumulation varied among transgenic tobacco lines and a negative correlation between target mRNA accumulation and virus resistance was observed.

The hybrid purity of melon (Cucumis melo L.) was tested by polymerase chain reaction (PCR) assay based on simple sequence repeat (SSR) markers in two F1 melon hybrids (‘Dongfangmi 1’ and ‘Dongfangmi 2’) and their parental lines. Twelve pairs of SSR primers for ‘Dongfangmi 1’ and three pairs for ‘Dongfangmi 2’ were selected. Results showed that self-inbred seeds were effectively distinguished from F1 hybrid seeds using these SSR primers, a finding that was consistent with the results recorded from field tests. ‘Dongfangmi 1’ and ‘Dongfangmi 2’ were identified from their parental lines, and seven other uterine hybrid lines by multiplex primers MS48+MS60 and MS4+MS20, respectively. Contamination of F1 hybrid seeds caused by self-inbred and other unknown pollens can be effectively and more reliably detected by PCR assays with multiplex SSR primers.