The expression patterns of Cry1Ac protein and the resistance to striped stem borer Chilo suppressalis (Walker) of cry1Ac/CpTI transgenic rice (Oryza sativa L.) were investigated by ELISA and laboratory bioassay. ELISA results showed that Cry1Ac protein could be detected in all organs during the development process of cry1Ac/CpTI transgenic rice. The content of Cry1Ac protein varied markedly in different tissues at different stages: it was higher in the earlier stage than in the later stage. The highest level of Cry1Ac protein in terminal leaves of the main stem was found at the jointing stage. Thereafter, it decreased at the booting stage, and a gradual reduction to the lowest level was recorded from the flowering to the filling stage, before a slight increase was shown at the maturing stage. In addition, higher concentrations of this protein were detected in the main stem at the seedling, jointing and flowering stages than at other stages. Relatively lower levels of Cry1Ac protein were found in seeds. The results from laboratory bioassay showed high resistance of terminal leaves of the main stem in all developmental stages to C. suppressalis larvae. Corrected mortality values of C. suppressalis at 4 days after feeding on the detached leaves of cry1Ac/CpTI transgenic rice reached 90% before the filling stage and decreased to nearly 60% after the filling stage. Interestingly, results of whole plant bioassay coincided with those obtained in the detached leaf bioassay. The tendency of larval morality in the detached leaf bioassay and whole plant bioassay was consistent with that of Cry1Ac protein content in the different growing periods and organs.

Sox9 is one of the important genes related to sex differentiation and determination. Two partial cDNAs encoding sox9 were derived from brain, testis and ovary of yellow catfish Pelteobagrus fulvidraco using reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis revealed that the ovarian sox9a2 was different from sox9a1 derived from brain and testis. The identity rate of cDNA was 77%, that of amino acids was 84%, and the different nucleotides were distributed in different sites all over the cDNA. This finding confirmed that there were transcripts from two genes. Based on the homology of the amino-acid sequences for sox9 of other animals, the similarity for sox9a1 from yellow catfish and sox9 of other animals averaged 86%. The similarity for sox9a2 reached a mean of 83%. The phylogenetic analysis revealed yellow catfish sox9a1 and sox9a2 were clustered into the fish branch. And yellow catfish sox9a2 was different from the other fish sox9. There was no significant branch including all sox9 derived from testis and brain or including all ovarian sox9. The genetic distance was the same with the results of the traditional taxonomy. RT-PCR analysis indicated that sox9a1 was expressed in male and female yellow catfish forebrain as well as in gonads, whereas sox9a2 was only expressed in ovaries. The function of sox9a2 in female sex formation and ovary development needs to be studied in depth.

With the rapid development of nanotechnology, the security of nanomaterials has been an increasing cause for concern. In this study, the impact of titanium dioxide nanoparticles (nano-TiO2) on the phyllosphere bacterial community were analysed by both a culturable-dependent method and a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) method. The quantity of culturable phyllosphere bacteria was significantly reduced with an increased concentration of nano-TiO2. With increasing concentrations from 0.002 to 20 mg/ml of nano-TiO2, the quantity of culturable phyllosphere bacteria decreased from 1.8×106 to 3.1×105 cfu/g. The phyllosphere bacteria community was analysed by PCR-DGGE, and when the concentrations of nano-TiO2 were higher than 0.02 mg/ml, the DGGE bands were significantly lower than in the control. Sequencing results of the bands from the DGGE gel showed that there were at least seven genera in the phyllosphere bacteria. Only one uncultured bacterium was unaffected by the concentration of nano-TiO2.

A rapid and sensitive real-time polymerase chain reaction (PCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from the TRBIV major capsid protein (MCP) gene was involved in the real-time PCR (RT-PCR) assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescent dye, SYBR Green I, which exhibited fluorescence enhancement when bound to double-stranded (ds) DNA. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid, pUCm-T/TRBIV MCP, containing the target sequence, was quantified to make a standard curve for sample detection after serial tenfold dilution. Linear coefficient correlations between the cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (R2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of virus in different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively), while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has become epidemic in turbot farms located along the Shandong peninsula. The virus number varied from 1.27×102 to 2.33×106 viral genome copies/mg tissue in spleens of infected turbot. These results suggest that the RT-PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.

We have developed and applied an immunoassay method to detect genetically modified (GM) rape containing phosphinothricin acetyltransferase (PAT). The purified PAT was identified by Western blotting and enzymic activity analysis. The polyclonal antibody against purified PAT protein was obtained and purified by both a saturated ammonium sulphate method and protein A-Sepharose 4B. The sensitivity and cross-reactivity of the polyclonal antibody has been demonstrated in an enzyme-linked immunosorbent assay (ELISA). The result of the ELISA for antiserum sensitivity was about 2×10−5 mg/ml and the cross-reactivity determined experimentally showed a high degree of specificity for the antiserum used, because values were all less than 0.1%. Detection of transgenic plants was evaluated using two transgenic rape lines (MS1/RF1 and MS8/RF3) which could be easily distinguished by ELISA.

Genetic diversity and relationships among 21 accessions of Secale L., including three species and 10 subspecies, were evaluated using RAMP markers. Forty-one out of 80 (50.5%) RAMP primers, which produced clear and polymorphic bands, were selected for PCR amplification of genomic DNA. A total of 446 bands were amplified from the 41 primers, and 428 of these bands (about 96%) were polymorphic. Three to 19 polymorphic bands could be amplified from each primer, with an average of 10.4 bands. The RAMP-based genetic similarity (GS) values among the 21 Secale accessions ranged from 0.266 to 0.658, with a mean of 0.449. A high level of genetic variation was found between or within the wild populations and the cultivars. Based on the GS matrix, a dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA). All 21 accessions could be distinguished by RAMP markers. Clustering results showed that the genetic diversity of Secale based on RAMP markers was correlated with geographical distribution. Six rye cultivars, originating from Poland, Portugal, Mexico, Hungary, Armenia and Ukraine, were clustered into one group. The six countries are all located in the transitional region of broad-leaf forests between maritime and continental temperate zones, with narrow latitude span. In comparison, the other five cultivars from countries scattered over a region with large latitude span were distributed within different groups or subgroups. Genetic relationships based on RAMP markers had great deviation from the original taxonomy. Some subspecies of the same species were distributed within different groups, while some accessions of different species were closely clustered into one subgroup. These results suggest that RAMP markers could be an effective technique for detecting genetic diversity among Secale and give some useful information about its phylogenic relationships.

This study aimed to explore high-throughput cDNA array monitoring technology and to apply it to the gene expression spectrum analysis of salinity-challenged tobacco plants. A Nicotiana tabacum cDNA library was sequenced and found to consist of 5927 high-quality sequences (GenBank accession nos CV015900-CV021826). By analysing the expressed sequence tags (ESTs), the proportion of N. tabacum genes was identified at the EST level. A cDNA array was constructed based on the tentative unique transcripts (TUTs) derived from EST assembling results. A total of 42 differentially expressed genes were identified, including plasma membrane intrinsic protein 2a, ethylene-responsive proteinase and pre-mRNA splicing factor prp31 gene, suggesting that there was a complicated biological response in N. tabacum under saline stress.

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.

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.

Tests were conducted to highlight the effects of donor cell treatment (bovine G0- and non-G0 phase somatic cells refrigerated for 1–5 or 12–14 days at 4°C), fusion solution (with or without cytochalasin B) and age of the rabbit (young, 3–5-months old; or multiparous, 10–12 months old) as oocyte donor on the fusion and mortality rates of bovine–rabbit interspecies reconstructed eggs. Results showed that there were no significant differences (P>0.05) in fusion and mortality rates of eggs reconstructed with G0 cells or non-G0 cells as nuclear donor somatic cells. The same was also true when nuclear donor somatic cells (G0 cells or non-G0 cells) were treated at low temperature (4°C) for 1–5 or 12–14 days. The fusion rate of reconstructed eggs did not change significantly (P>0.05) when adding cytochalasin B into the fusion solution, but the mortality rate increased significantly (P<0.05). No significant effect (P>0.05) was found in fusion rate of the eggs reconstructed with young rabbits' and multiparous rabbits' oocytes as recipient, but the mortality rate decreased significantly (P<0.01) for young rabbit oocytes compared with multiparous rabbit ones.

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.

A trivalent vector containing genes of Watermelon mosaic virus (WMV) coat protein (CP), and replicase genes of Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV), was constructed for transformation of watermelon (Citrullus lanatus) plants, mediated by Agrobacterium tumefaciens. The integrated foreign genes were identified in the regenerated progenies by polymerase chain reaction (PCR) and Southern blots. The transformation efficiency was about 1.7‰. Resistance to virus infection was determined by mechanical inoculation in the greenhouse and by field trials. The transgenic watermelon lines showed different phenotypes of susceptible, resistant, immune or recovery from virus infections in the late growth stage. A relatively high level of resistance was shown by T3 plants of the line BH1-7. This result indicates the possibility of creating, by transgenic protocols, new varieties of watermelon resistant to viral infection.

The β-glucosidase gene has important effects on alcoholic aroma precursors and insect resistance of the tea plant [Camellia sinensis (L.) O. Kutze]. The complete cDNA sequence of β-glucosidase of the tea plant was cloned; its full length was 1475 bp, and shared 40–60% similarity with corresponding parts of the nucleotide sequence of β-glucosidase gene from other plants. Its secondary structure contains 14.33% α-helix, 25.43% β-pleated sheet and many functional amino acid domains. The β-glucosidase gene was cloned into the pET-32a expression system and expressed at high-efficiently in Escherichia coli BL21 (DE3); the molecular weight of expressed fusion protein was 63 kDa. The results of enzymic reaction showed that the fusion protein possessed normal bioactivity, and it could catalyse the dehydration of the glycosidic bond. The soluble fusion protein was expressed mainly in the cytoplasm.

A complete chitin deacetylase (CDA) complementary DNA (cDNA) from Mucor racemosus was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification cDNA end (RACE) with gene special conserved primers. The cDNA sequence was submitted to GenBank (DQ538514). The complete cDNA with full-length of 1506 bp contained a 67 bp 5′-untranslated region, an open reading frame of 1344 bp and 95 bp 3′-untranslated region including tailing site AATAAA. The gene encoded a sequence of 448 amino acid residues and consisted of core nucleotides encoding a polysaccharide deacetylase domain covering 32% of the entire sequence. The CDA gene shared sequence homology with those of several fungi. The corresponding homology of the deduced amino acid sequences varied from 21 to 69%. Phylogenetic analysis according to the deduced amino acid sequences matched the classical fungi taxonomy. The three-dimensional structure of this protein was predicted. The protein had a whole CDA functional domain and a polysaccharide deacetylase domain.

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).

To investigate the wheat transcriptional profile under drought stress, a drought-tolerant variety of wheat (Triticum aestivum), Hanxuan 10, was treated with polyethylene glycol (PEG6000) and samples were collected at 0, 1, 6 and 24 h. Complementary DNA was labelled with fluorescent dye and hybridized with the BGI-RiceChip, a whole genome rice gene chip platform, which contains over 60 000 oligos based on the rice genome sequence. Data analysis detected 166, 207 and 328 differentially expressed genes (DGs), respectively, at 1, 6 and 24 h, indicating that the number of DGs increased with the length of the PEG treatment. Functional category analysis showed that the number of DGs related to energy metabolism pathways increased – 4.2%, 8.2% and 16.8%, respectively, as a proportion of the total number of DGs. Most of the photosynthesis-related genes were up-regulated. It is interesting to note that Psbr and ribulose-bisphosphate carboxylase (Rubisco)-coding genes were down-regulated, suggesting their potential role in the response to drought tolerance.

An antisense class I patatin gene under control of the CaMV 35S promoter was introduced into potato (Solanum tuberosum) cultivar E-potato 3 using the Agrobacterium tumefaciens system. PCR amplification and PCR–Southern blot analysis indicated that the antisense class I patatin gene had been integrated into the potato genome. Northern hybridization analysis showed that the antisense gene transcribed normally in the transgenic potato plants and resulted in a reduction of endogenous class I patatin mRNA. Total soluble protein content and lipid acyl hydrolase activity of microtubers, derived from transformed plants, decreased by a maximum of 36.4% and 31.4%, respectively, compared with control plants. The expression of this antisense gene also resulted in reductions of the plantlets forming tubers, tubers per plantlet and the effective tubers (≥50 mg) of the transformed plants.

A multifunctional-engineering strain was constructed by fusing the insecticidal bacterium (Bacillus thuringiensis) with vip3A gene and the endophytic bacterium (B. subtilis) protoplasts. Results of polymerase chain reaction (PCR) showed that the insecticidal vip3A gene of B. thuringiensis was present in the fusant. The fusant's antagonism to Fusarium oxysporum f. sp. cucumerinum, the adjusted mortality to Plutella xylostella and the endophytic characters were detected. The result showed that the antagonism of the fusant was against the F. oxysporum f. sp. cucumerinum; the adjusted mortality to P. xylostella reached 59% and it had insecticidal characteristics.

The yolk proteins of the silkworm, Bombyx mori, are important sources of nutrition and energy during embryo development. By analysing the yolk proteins from silkworm eggs, the composition and the metabolism of yolk proteins during embryo development stages can be studied at the proteome level. Yolk proteins were obtained from unfertilized eggs and eggs at different embryo development stages by dissection and then separated and analysed by two-dimensional electrophoresis and its corresponding image analysis system. It was discovered that over 200 proteins exist in mature silkworm eggs. Some specific spots of yolk proteins were found in different silkworm strains, while few differences in yolk proteins appeared during different development stages in the same silkworm strain. This result indicates that the silkworm embryo does not utilize yolk proteins selectively by absorbing one or some special proteins, but by absorbing the yolk protein grains one by one during development.