Bacillus thuringiensis strain WZ-9, isolated from soil in Hebei province, China, was effective against Henosepilachna vigintioctomaculata larvae. The strain presented bipyramidal crystals with a protein band of 130 kDa in SDS–PAGE. The pH changes of the culture media showed important fluctuations during the 24 h growth cycle. The pH varied less in log and stationary phases than it did in the exponential phase. Bioassay results showed that the WZ-9 strain was only harmful to larvae of H. vigintioctomaculata and not to either adults of H. vigintioctomaculata or other several lepidopteran and coleopteran insects. LC50 to second-instar larvae of H. vigintioctomaculata was 2.95×107 cells/ml after 72 h. Genotypic investigations showed that this strain possessed the cry7 gene. Sequence analysis demonstrated that the encoding gene contained an open reading frame (ORF) of 3414 bp and encoded 1138 amino acid residues. The deduced amino acid sequence was 99.65% identical to that of the reported Cry7Ab2 sequences. This gene was designated by the Bt δ-endotoxin nomenclature committee as Cry7Ab3 with accession number BI 1015188 in the GenBank database.

The elements for the Semliki forest virus (SFV) RNA replicon were obtained from the Alphavirus genome. It was designed to overcome the poor efficacy of some current plasmid vectors. Genes coding for viral replicases are preserved while genes coding for structural proteins are replaced by foreign genes in the RNA replicon. High levels of RNA replication and expression of foreign genes in the cytoplasm are regulated by the replicases. To evaluate the effects of the SFV RNA replicon on the improvement of gene expression, a LacZ gene was inserted into pIRES-neo digested by BamHI and dephosphorylated by alkaline phosphatase to construct pIRES-neo-LacZ. The RNA replicon vector pCMV-rep-LacZ and two conventional cytomegalovirus (CMV) promoter-based vectors (pLNCX-LacZ and pIRES-neo-LacZ) were transfected, using Lipofectin, to prepared 293 cells. Growth hormone releasing hormone (GHRH) expression vectors (pCMV-Rep-GHRH, pCDNA3.1(+)-GHRH and pIRES-neo-GHRH) were also tested using the same procedure. Target gene expression was detected with radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR). Results showed that the expression level of the RNA replicon vector was 2–3 times higher than with normal plasmid vectors. This result will help to improve the efficiency of gene expression in eukaryotic cells.

A full-length cry1Ia gene fragment, obtained by polymerase chain reaction (PCR) amplification with a pair of primers designed according to cry1Ia-type gene sequences and DNA from Bacillus thuringiensis as template, was introduced into the expression vector pET-21b and transformed into Escherichia coli BL21(DE3). The molecular weight of the induced expression product was 81.2 kDa. The amino acid sequence of Cry1Ia was very different from sequences of 12 known Cry1Ia-type proteins. This gene was designated as cry1Ia8 by the International B. thuringiensis Insecticidal Protein Nomenclature Committee (accession number AF373207). The bioassay results indicated that Cry1Ia toxin protein showed distinct insecticidal activity against Ostrinia furnacalis and Plutella xylostella with LC50 of 0.268 μg/g and 2.227 μg/ml, respectively. It also had insecticidal activity against Leguminivora glycinivorella, but not against Pyrrhalta aenescens. The novel cry1Ia8 gene will be an important resource in constructing genetically engineered bacteria and transgenic plants for biocontrol of insect pests. It is also available for screening gene stacks to delay pest resistance.

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.

The population doubling number (70–80 times) of human fetal bone marrow mesenchymal stem cells (BMMSCs) is about two times higher than that (30–40 times) of adult BMMSCs, and their differentiation capacity is superior to that of their adult counterparts. In this study, BMMSCs were isolated from long bones of 2- to 3-month-old human abortuses by rinsing and selectively culturing whole marrow cells. Basic medium and serum concentration were optimized and growth curves plotted, both by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] reduction assay. Isolated cells were identified by flow cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and a tumour-forming experiment. The results indicated that lengthwise scissoring of fetal long bones and rinsing of their marrow cells was practical and useful for recovery of BMMSCs from the investigated human abortuses. In this experiment, α-MEM (minimum essential medium, alpha medium)+20% FCS (fetal calf serum) was the best for in vitro culture of BMMSCs. The third-passage BMMSCs expressed Oct4, SSEA3 and SSEA4 besides the surface markers of their adult counterparts. The population doubling time of the BMMSCs of passage 6, 12 and 24 were 34, 36 and 40 h, respectively. Cells in all passages showed a diploid karyotype and formed no tumours in nude mice. The BMMSCs used in this study proved to be biologically safe and ideal seed cells for research on human tissue engineering and regeneration medicine.

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.

The purpose of this study was to evaluate the effects of donor sex, treatments of cell cycle synchronization and donor nuclei obtained from fresh or frozen–thawed conditions on developmental competence of yak–bovine interspecies nuclear transfer embryos. Bovine (Bos taurus) oocytes were used as recipients and yak (Bos grunniens) ear fibroblast cells were used as donors. Results indicated that the development rate of male blastocysts was higher than that of female (56.6% versus 39.5%, P<0.05), whereas cleavage and total cell number showed no difference between the two groups. No significant difference was observed in the development and quality of blastocysts with donor cells treated by serum starvation or contact inhibition, and there was no significant difference in embryo development with fresh or frozen–thawed donor cells, whereas the cleavage rate in the group of frozen–thawed cells was significantly lower than that of the fresh cell group (54.5% versus 78.2%, P<0.05). The results demonstrated that donor sex could impact the developmental competence of yak–bovine interspecies nuclear transfer embryos, whereas different treatments of cell cycle synchronization and freezing had little influence.

Effect of lamb age, transport stimulation and repeated hormone superovulation on the number of collected oocytes were determined. Effect of the culture medium containing ethylenediaminetetraacetic acid (EDTA) on the development of embryos produced in vitro from lamb oocytes was also investigated. Results indicated that the mean numbers of collected and available oocytes (oocytes with compacted cumulus cells, evenly granulated ooplasm and morphologically normal) from 6- to 8-week-old lambs were 60.8±13.9 and 58.2±12.3, respectively. These figures were higher than those obtained from 12- to 14-week-old lambs (27.3±5.1 and 26.0±4.9) (P<0.05). Stimulation by transportation didn't decrease the number of collected oocytes from the superovulated lambs (P>0.05). However, the number of collected oocytes in the repeated superovulation group was significantly reduced, compared with the control group (P<0.05). The embryonic culture medium supplemented with 10 μmol/l EDTA highly improved the development capability of the embryos from lamb oocytes (P<0.05). Healthy lambs were born following embryo transfer.

Using the INRA – University of Minnesota porcine 7000 Rad Radiation Hybrid panel (IMpRH) containing 118 clones, porcine cellular retinol binding protein gene 1 (RBP1) and plasma retinol binding protein gene 4 (RBP4) were assigned to porcine chromosomes 13 and 14, respectively. The mRNA distributions of the two genes in adult Wuzhishan pig tissues (lung, skeletal muscle, spleen, heart, stomach, large intestine, lymph node, small intestine, liver, brain, kidney and fat) were examined. One single nucleotide polymorphism (SNP) was identified in Laiwu, Wuzhishan, Guizhou, Bama pig breeds and a Tongcheng experimental population and analysed using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. Significant associations were found between different genotypes of A/G156 and maximum carcass length (LM), minimum carcass length (LN), haemoglobin (HGB), mean corpuscular volume haemoglobin (MCH), and day to slaughter weight (age) traits in the Tongcheng experimental population. This SNP locus might be used as a genetic marker in pig breeding and production.

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.

Genetic diversity and genetic variation of 240 adult plants of four tea populations in Hunan – Camellia sinensis var. sinensis, C. sinensis var. assamica cv. Duntsa, C. ptilophylla and C. sinensis var. assamica cv. Jianghua – were studied by rapid amplification of polymorphic DNA (RAPD) markers. The results showed 226 loci using 21 random primers (10 bp), of which 201 (88.9%) were polymorphic. The genetic diversity analysis indicated that Shannon's index was 0.43; 74.7% of which was within-population genetic diversity while 25.3% was among-population variation. The gene diversity indexes of total populations (HT), within populations (HS) and among populations (HST) were, respectively, 0.37, 0.28 and 0.09. The coefficient of gene differentiation (GST) among populations was 0.23, indicating a 76.7% variation within populations and 23.3% among populations. These results displayed a rich within-population genetic variation, as in Shannon's diversity index. Interpopulation gene flow (Nm) was 0.74, which indicates the limited genetic exchange between populations.

Activation tagging plays an important role in plant genomics studies. In this paper, an activation tagging library containing 50 000 Basta-resistant lines was constructed by mediated transformation of the mutant bzr1-D of Aribidopsis thaliana. Among these transformants, 47 lines showed obvious phenotypes, including late flowering time, dwarf growth habit, changed leaf shape, longer leaf petiole, leaves lacking trichomes, sterility and no kink between stem/leaf. Some T-DNA flanking sequences were obtained by TAIL (thermal asymmetric interlaced) and nested PCR. Results showed that the mutants contained one to three T-DNA insertions. The insertions were distributed in the first, fourth and fifth chromosomes of the genome.

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.

Intergeneric transfer of plasmid vectors pSET152 and pHL212 from donor Escherichia coli ET12567/pUZ8002 and S17-1 to Streptomyces cinnamonensis was demonstrated and optimized. Assisted by this conjugation system, nsdA gene disruption was achieved through PCR-targeted gene replacement. One AprRKanS exconjugant BIB309 was then isolated and confirmed to be the nsdA null mutant. Compared with the starting strain, monensin production by the nsdA− mutant BIB309 increased 270% in vitro.

The bovine interferon-tau (bIFN-τ) gene, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and subcloned into a pGEM-T vector. After being verified, the fragments, with or without signal sequence, were inserted into the expression vector pET-30a(+). Two recombinant plasmids were induced to express the recombinant proteins by isopropyl β-d-1-thiogalactopyranoside. The results showed that the bIFN-τ gene could be obtained from five bovine blastocysts by PCR without extraction of genomic DNA. It had 99% homology with nucleotides and 97% with amino acids in the GenBank sequence (accession number: XM_593584). The products of recombinant bIFN-τ, minus signal sequence, expressed in pET-30a(+) were analysed by SDS-PAGE. A new 20 kDa protein was detected and its molecular weight was as expected. The antiviral activity of recombinant bIFN-τ was 1×104 IU/mg using a standard cytopathic reduction assay. Marked morphological changes were induced by recombinant bIFN-τ in bovine endometrial epithelial cells. The cell volume was larger than that of controls and a lot of vesicles appeared in the cytoplasm after 24 h culture in the presence of 2.9 μg/ml recombinant bIFN-τ. In conclusion, a purified recombinant, biologically active bIFN-τ was obtained in this experiment.

Progenies derived from rice (Oryza sativa ssp. japonica) plants pollinated by Oenothera biennis exhibited numerous morphological and developmental traits. Some variant individuals appeared in generation D2. From generations D3 to D6, a large number of variants were observed, showing distinct variable traits including giant embryos. Statistical analysis on D6 lines showed significant differences between progenies and their rice parental line in several main traits, including plant height (49.2–164.5 cm), panicle number (12.1–38.2), panicle length (20.3–30.3 cm), length of sword leaf (13.8–57.5 cm), leaf width (11.1–25.2 mm), grain number of main panicle (142.0–367.0), percentage of seed setting (0.8–99.0%), 1000-grain weight (19.7–33.8 g) and time from germination to panicle emergence (90.0–108.0 days). Most of the variable coefficients were above 20% (the highest was 40.8%). Through pedigree selection of these plants, genetically stable lines were obtained, which are useful for rice breeding. Results from amplified fragment length polymorphism (AFLP) analysis showed that several rice lines contained extensive genetic variations, which included disappearance of rice parental bands and/or appearance of novel bands.

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

Polyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products. In this study, wheat PPO sequences (mRNA) were searched/BLASTed in the NCBI database and aligned using DNAMAN software. The results showed that wheat PPO genes could be divided into two clusters (I and II) and that three genes (‘i’) of cluster II seemed not to be located on chromosomes 2A and 2D. Ninety-four single nucleotide polymorphisms (SNPs) were detected between two haplotypes of the PPO gene on chromosome 2D. Eighty of these were found in the coding region (coding (c) SNPs) and 36 were non-synonymous cSNPs, which could affect the PPO amino acid sequence. Primers (STS-H) were designed at some non-synonymous cSNPs sites and were used to investigate the correlations between allelic variants and PPO activity of seeds – a total of 130 common wheat varieties were evaluated in 2 years. The results showed that STS-H could amplify a 460 bp DNA fragment in most cultivars with high PPO activity, while no PCR product was detected in most cultivars with low PPO activity. To improve the selection efficiency of a single dominance molecular marker, the multiplex polymerase chain reaction (PCR) system of STS-H and STS01 markers was also studied, based on the complementary between them.

Using microsatellite markers, the genetic structure of nine domestic duck (Anas platyrhynchos) populations in eastern China was analysed. The results showed that the heterozygosity was high in these populations, ranging from 0.5137 to 0.6055, with an average heterozygosity of 0.5523, reflecting the rich diversity. Considerable breed differentiation was observed and 25.65% of the total genetic variation came from breed differences; this low differentiation result affirmed that each breed has its own genetic diversity. The DA genetic distances suggested that greater differentiation existed between populations. The duck populations were clustered into four groups based on neighbour joining (NJ) clustering, and the clustering results showed relationships with duck breed distributions and economic utilization.