Recent advances and limitations of using zinc finger nuclease to stimulate a high efficiency of homologous recombination, and chimeric oligonucleotides to promote single base replacement in functional genomic research and plant genetic breeding, are systematically reviewed. Approaches to improve gene targeting efficiency through molecular modification of key pathways in plant homologous recombination are also discussed.

Efficiency and specificity are key limiting factors for the production of transgenic animals. This review describes the recently developed animal gene transfer techniques, including non-site-specific methods of gene transfer into the testis and ovary for easy production of transgenic animals; gene targeting in embryonic stem cells, somatic cells and primordial germ cells for site-specific methods; methods to improve cloning efficiency in gene targeting; and site- and timing-specific gene targeting and controlled expression of transferred genes. In addition, methods of utilizing newly developed RNA interference, combined with the above techniques for controlling gene expression, to produce transgenic animals to spatio-temporally and reversibly knock down specific genes, are also discussed. The merits and disadvantages of each method are covered, as well as the potential use of these methods to develop transgenic animals for breeding new animal lines, to study disease models and to produce therapeutic medicines.

In plants, acid invertases are known to be the key enzymes cleaving sucrose into reducing sugars (RS) (glucose and fructose). To improve the quality of potato (Solanum tuberosum L.) chips, which is largely influenced by RS accumulation in tubers stored at low temperature, a part of acid invertase cDNA with hairpin RNA (hpRNA) structure was transformed into potato cv. N2. Detection of polymerase chain reaction (PCR) amplification and Northern blotting suggested that the RNA interference (RNAi) vector was successfully transformed into cv. N2. The analysis of acid invertase activity in the plantlets and microtubers of RNAi transgenic lines indicated that the expression of the acid invertase was significantly repressed by the activity of RNAi of plantlets by an average 69.8% (with the exception of line Ni-1) with a maximal decrease of 78% (line Ni-4), and the highest decrease of activity in microtubers of 68%. Compared with that of well-inhibited antisense inv transgenic plants, the comparative downregulation of RNAi suggests a distinct alteration of endogenous acid invertase activity and a potential strategy for post-transcriptional gene silencing (PTGS) in modulation of cold-sweetening in potato.

In vitro bulb scales of Lilium longiflorum×L. formosanum were used as explants to develop a highly efficient regeneration system. A high regeneration rate (100%) was reached through organogenesis on basal Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 6-benzylaminopurine (6-BA) and 1.0 mg/l naphthaleneacetic acid (NAA). A genetic transformation system for the lily was developed using an Agrobacterium tumefaciens-mediated method. An improved genetic transformation rate (12‰) was obtained when the explants were pre-cultured for 3 days, immersed in bacterial suspension (OD600≈0.8) for 5 min, and co-cultivated for 5 days. The binary vector pBI121 containing Zm401, a maize pollen-specific gene, was introduced into the Agrobacterium strain LBA4404 and transformed into the explants using the genetic transformation system. Gene integration into the lily genome was confirmed by polymerase chain reaction (PCR) and PCR–Southern analysis. These results could lead to the production of new pollenless lily plants.

The gene ap36 encoding a protein elicitor from Alternaria sp. was fused downstream of the slh (S-layer homology) motif of Bacillus thuringiensis S-layer protein gene ctc. The recombinant gene was then transferred into B. thuringiensis plasmid-free derivative strain BMB171. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the SLH–Ap36 fusion protein was expressed in B. thuringiensis BMB171. After tomato (Lycopersicum esculentum) leaves were treated for 90 min with the recombinant strain cultured at 28°C for 24 h, the activity of peroxidase and the amount of proline of tomato leaves were increased to 57.14% and 131.59%, respectively, compared to the control, and after the tomato leaves were treated with the cultured recombinant strain for 4 days, the activity of phenylalanine ammonia lyase was also higher than that in the control. Furthermore, tubers of treated potato (Solanum tuberosum) plants showed higher resistance to rot disease caused by Erwinia corotovora SCG1 compared to the control treatments.

Intron 5 of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene was cloned and its single nucleotide polymorphism (SNP) was tested by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) in Wanxi White geese (WW) (Anser anser). The carcass, meat quality and feather-down traits of 116 geese from WW males×Sichuan White goose (A. anser) females were assessed and analysed in association with SNP. The results showed that the sequence length of intron 5 of the HMGR gene in geese was 704 bp, and the homology between goose and chicken was 44.0%. A SNP site was tested in intron 5 by PCR-SSCP, and frequencies of alleles A and B at the SNP site were 0.7716 and 0.2284, respectively. The effects of genotype AA were significant, not only on an increase of chest muscle weight, stomach gland weight (P<0.05), fibre diameter of chest muscle and 1000-down weight (P<0.01), but also on a decrease of down water-holding percentage and pH of chest muscle 45 min after slaughterP<0.05). The effect of genotype AB was in the opposite direction to that of genotype AA.

Using hypocotyls as explants, the adventitious shoots of Euonymus fortunei var. radicans were differentiated directly from basal Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The highest regeneration frequency was obtained with MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP) and 0.01 mg/l α-naphthalene acetic acid (NAA). A regeneration frequency of 92% and 4.2 shoots per explant were obtained after 30 days of culture. The binary vector pBCGm, containing Galanthus nivalis agglutinin (GNA) gene, was introduced into Agrobacterium tumefaciens LBA4404. Hypocotyl segments of E. fortunei var. radicans were infected through A. tumefaciens-mediated transformation. Polymerase chain reaction (PCR) and PCR–Southern blot analysis results confirmed that the GNA gene was integrated into the genome of transgenic plants. The highest transformation frequency was obtained with un-precultured explants infected for 30 min with OD600=0.6 Agrobacterium tumefaciens, and co-cultivated for 3 days.

A 5′-flanking region and partial open reading frame (ORF) of the β-actin gene (GenBank accession No. EF026001) of Nile tilapia (Oreochromis niloticus) was cloned by polymerase chain reaction (PCR) amplification. The segment included a 1643 bp regulatory sequence and a 90 bp partial ORF which encoded a 30-amino-acid peptide. The regulatory sequence comprised a 108 bp 5′ proximal promoter, the first untranslated exon and the first intron of the β-actin gene. The proximal promoter region contained elements that were critical for transcription activity, including a CCAAT, TATA and CArG box located at –92, –29 and –62 bp upstream of the transcription initiation site, respectively. The regulatory sequence was inserted into the promoterless pDsRed2-1 vector to construct the expressing vector pNA-DsRed. The linearized pNA-DsRed was microinjected into the fertilized eggs of Tanichthys albonubes. The expression of the DsRed2 gene in transgenic fish could be observed under the microfluoroscope and anatomical lens. The results showed that the β-actin gene promoter possessed effective transcription activity.

Six silver birch (Betula platyphylla) parents of diverse provenances were crossed according to Griffing 4 diallel design, and the plant height and diameter at ground level (DGL) of their F1 progeny were observed. The analysis of general combining ability (GCA) and specific combining ability (SCA) showed that two phenotypes were simultaneously controlled by additive and non-additive effects. The GCA between two phenotypes in a parent, and GCA of the same phenotype among different parents displayed significant differences: Q2 from Qingyuan was the best of the parents, followed by M2 from Maoer-mountain. On the other hand, the SCA of two phenotypes in a cross and the SCA of a phenotype among different crosses also varied considerably: the cross between E8 from Finland and Q1 from Qingyuan showed the highest SCA of plant height and DGL. The heritability of plant height and diameter at ground level was strong (over 60%), suggesting that these phenotypes can be used for early selection.

A parent strain of Aspergillus niger LW-1 producing β-mannanase, preserved in our laboratory, was isolated. A strain, N-9, was screened out and further treated with vacuum microwave irradiation and ethyl methane sulphonate (EMS). A mutant strain, E-30, producing a high and stable yield of β-mannanase was obtained through screening by solid-state cultivation on the basic fermentation medium and several generations of bevel subculture. Its enzyme activity (36 675 U/g) was increased by 2.15 times compared to that of A. niger LW-1 (17 048 U/g). The production of high-yield β-mannanase by E-30 remained stable when maintained at 4°C for 2 months.

Nine prepubertal gilts (JunMu No. 1) were randomly allocated into three groups (n=3) and fed with a high-energy diet (Group H), a low-energy diet (Group L), or a moderate-energy diet (Group M) for 14 days. Free access to water was provided throughout the research period. Ovaries and uteri were collected after the energy treatments, and processed for determination of the absolute quantities of insulin-like growth factor receptor (IGF-1R) and epidermal growth factor receptor (EGFR) mRNA, using real-time polymerase chain reaction (PCR). The expression of IGF-1R and EGFR mRNA in ovaries and uteri was significantly ranked as: Group H>Group M>Group L (P<0.05). This result suggests that high energy intake markedly enhanced the ovarian and uterine expression of IGF-1R and EGFR in prepubertal gilts, whereas insufficient energy intake markedly inhibited such expression. IGF-1R and EGFR may be involved in mediating the effects of energy intake on the development of the reproductive system in prepubertal gilts.

HbUEP, an ubiquitin extension protein gene from latex of the rubber tree (Hevea brasiliensis) was cloned and sequenced using a differentially ethphon-induced expressed cDNA subtraction library. The cDNA had 771 bp nucleotides, comprising a 226 bp 3′ untranslated region (UTR), 77 bp 5′UTR and a 468 bp open reading frame encoding a 156 amino acid peptide. Southern blotting analysis showed that this gene was a low copy number gene in the H. brasiliensis genome. Within 24 h after application of ethphon, the gene was expressed weakly in both control and latex sampled at 6 h, and strongly in latex sampled at 12 h, showing that this gene expression could be regulated by ethphon. Ethphon could increase the latex yield in H. brasiliensis. It is suggested that the HbUEP gene may be involved in the regulation of ethphon-induced high latex yield in H. brasiliensis.

The developmental functions of oocytes of three strains of mice (Kunming, ICR and C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb) recombined with the nuclei of first polar bodies (Pbs I) were explored. Cumulus oocyte complexes (COCs) from the mice were collected after superovulation, then Pbs I were obtained from the COCs by 2% pronase treatment. The survival of Pbs I under different temperatures was identified by morphology and trypan blue staining. Later, the polar body I (Pb 1) nucleus and a little cytoplasm was injected into each oocyte, the nuclei of which had been enucleated by micromanipulation. Oocytes recombined with Pbs I were fertilized, then cultured in vitro in order to observe their further development. The results showed that the vigour of Pbs I was maintained for 12–14 h after superovulation, and was still maintained after 48 h at 4 °C. A total of 13 out of 117 recombined oocytes from Kunming and ICR mice, as well as 3 out of 38 recombined oocytes from C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb mice, developed into two-cell embryos. The experiments confirmed that mouse oocytes recombined with the nuclei of Pbs I could maintain fertilization and development. These results present valuable references for further utilization of genetic resources for farm animals

A total of 791 microsatellites (SSRs) were isolated from 7055 Panax ginseng expressed sequence tags (ESTs). According to primer design criteria, 68 primer pairs for EST-SSR were designed. Under an appropriate polymerase chain reaction (PCR) system, all EST-SSR primer pairs were screened against genomic DNA of Ji'anchangbo and Fusong'ermaya from Panax ginseng, and 43 EST-SSR primer pairs out of the above 68 resulted in PCR products. Then, all 43 pairs were detected in nine P. ginseng, two Panax quinquefolius and two Acanthopanax senticosus cultivars for polymorphisms, and 26 pairs (60.47%) were found to be polymorphic, accounting for 38.23% of the total number of designed primer pairs. These results demonstrate the possibility of developing EST-SSR markers using P. ginseng ESTs.