The effects of some factors on the isolation of protoplasts from Penicillium digitatum were studied, including the appropriate material (young mycelia and generating spores), the concentrations of enzyme and osmotic pressure stabilizers, reaction time and reaction temperature. Results demonstrated that germinating spores were an ideal material resource for the isolation of P. digitatum protoplasts. Highest yield and quality of P. digitatum protoplasts were obtained by shaking germinating spores suspended in a solution of 10 mg/ml Lywallzyme™ dissolved in 0.7 M NaCl as osmotic pressure stabilizer at 80 rev/min and 30°C for 3.0–3.5 h. The regeneration rate of the isolated protoplasts was as high as 24.9% on double-layer Czapek medium containing 0.7 M NaCl. Additionally, observation of the protoplast release pattern showed that the protoplasts of P. digitatum were released primarily from the hyphal apex and occasionally from the subapical or original sites of germinating tubes. The protoplasts of P. digitatum were regenerated in a direct manner of either yeast-like cell development or mycelium formation.

The bone morphogenetic protein receptor IB (BMPR-IB) gene, which controls the fecundity of Booroola Merino ewes, was studied as a candidate gene for the prolificacy of Small Tail Han and Hu ewes. A single nucleotide polymorphism of the BMPR-IB gene was detected in both high (Small Tail Han and Hu) and low (Suffolk and Dorset) fecundity sheep breeds by polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) analysis. The results indicated the presence of the same mutation (A746G) of the BMPR-IB gene in both Small Tail Han and Hu ewes and in Booroola Merino ewes, but not in both Suffolk and Dorset ewes. In Small Tail Han ewes, frequencies of BB, B+ and ++ genotypes were 0.524, 0.383 and 0.093, respectively. In Hu ewes, these frequencies were 0.882, 0.118 and 0.000. The BMPR-IB genotype distributions were significantly different (P<0.001) among high- and low-fecundity sheep breeds. Small Tail Han ewes with genotype BB had 0.92 (P<0.01) and 1.02 (P<0.01) lambs more than those with genotype ++ in the first and second parity, respectively. These results demonstrated that the BMPR-IB gene is a major gene affecting the prolificacy in both Small Tail Han and Hu ewes, and could be used as a molecular genetic marker to select the litter size in sheep.

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.

The Lc regulatory gene affects the formation of anthocyanin in plants. XY355 promoter, a petal-specific promoter, was obtained from the genome of rape (Brassica napus) by polymerase chain reaction (PCR). A plant expression vector, pXY60, was constructed, which contained the maize Lc regulatory gene under the control of the XY355 promoter. The vector was introduced into tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) by an Agrobacterium tumefaciens-mediated method. The flower colour of some transgenic tobacco plants was changed from light red to deep red and that of some transgenic petunia plants had changed from white to light purple.

Two hundred and forty bacterial strains were isolated from three potato (Solanum tuberosum) cultivars from eight different sources. On the basis of antagonistic bacteria examination in vitro, greenhouse and field tests, 22.9% of isolated bacteria were endophytic and antagonistic. Three strains, namely A-10', T3 (Bacillus sp.) and H1-6 (Pseudomonas fluorescens), were chosen for their suppressive effect on bacterial ring rot (Clavibacter michiganensis subsp. sepedonicus) and their growth promotion. The strain A-10' was an endophytic bacterium with the effects of colonization, plant growth promotion and suppression of the pathogenic bacteria, showing good prospects for commercial use.

The genetic diversity of 43 male sterile and low fertility Citrus accessions, as well as 13 fertile ones, were assessed using simple sequence repeat markers (SSRs). Thirty-five polymorphic alleles were generated from eight primers (on average 4.4 alleles per primer). Cluster analysis was performed via unweighted pair group method analysis (UPGMA) using the NTSYS-pc version 2.10. The results showed that the accessions could be classified into three groups: cultivars of mandarin were classified into group 1; those of sweet orange, grapefruit, ponkan or tangor into group 2; and Microcitrus with male sterile cytoplasm into group 3. Cluster analysis also revealed that Satsuma mandarin was more closely related to Bendiguangju mandarin than to Zaoju, Mankieh or Huangyan Bendizao tangerine. The present study on genetic diversity of male sterile and low fertility Citrus will provide useful information for further collection, preservation and utilization of this plant.

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.

Wheatgrass, Thinopyrum Löve (Elytrigia Nevski), is one of the most important alien resources for wheat improvement. To determine the transferability of wheat microsatellite primers to Thinopyrum spp., 40 wheat microsatellite primer pairs were selected to perform PCR analysis on 17 accessions of Thinopyrum spp. and two common wheat (Triticum aestivum) cultivars. Among the 40 primer pairs used, 25 pairs could produce PCR products on all or most of the alien species. Moreover, all of these 25 primer pairs, except Xgwm325, showed polymorphism in the Thinopyrum accessions. In total, 108 alleles were detected by the 25 primer pairs. The number of alleles per primer pair varied from 1 to 8, with an average of 4.3 alleles per primer. Although primer Xgwm325 did not detect polymorphic alleles in Thinopyrum spp., it could be used as a specific SSR marker for them. It amplified four specific DNA fragments in Thinopyrum spp. of length approximately 1400, 440, 120 and 100 bp. To verify the validity of these molecular markers, a further PCR programme with primer Xgwm325 was conducted on a full set of seven wheat–Thinopyrum elongatum disomic addition lines and 15 disomic substitution lines. This showed that only the 100-bp fragment appeared in all materials possessing the alien chromosome, indicating that it was a molecular marker for the entire genome of Th. elongatum, although primer Xgwm325 was specific for chromosome 6D in wheat.

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.

Single-nucleotide polymorphisms (SNPs) of exon 8 of the GHR gene were detected in Chinese Holstein cows by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The results showed that exon 8 of the GHR gene digested by TasI could be divided into two kinds of alleles and three kinds of genotypes. The frequencies of allele A and T were 0.6339 and 0.3661, respectively. The frequencies of genotypes AA, AT and TT were 0.459, 0.350 and 0.191, respectively. Sequencing showed one single nucleotide mutation T→A at 4962 bp of the gene in genotype TT when compared with genotype AA, and this mutation resulted in an amino acid change of phenylalanine (TTT)→tyrosine (TAT). The result of χ2 testing indicated that the genotypic frequency of the GHR gene digested by TasI did not fit with Hardy–Weinberg equilibrium in this population (P<0.05). The least-square mean of the fat percentage of this population was higher for genotype TT than for genotype AA (P<0.05).

GbRac1 gene was cloned from Gossypium barbadense with degenerate primers and 3′-RACE. Northern blot analysis indicated that GbRac1 mRNA was expressed abundantly in G. barbadense seedlings inoculated with Verticillium dehliae compared with mock-inoculated plants. A plant constitutive expression vector pRac harbouring GbRac1 gene was constructed and leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) were transformed with pRac by Agrobacterium-mediated transformation. Disease challenge test of detached leaves of the transgenic plants by inoculation with Alternaria alternata showed that resistance was enhanced dramatically compared with the non-transgenic plants. Results suggest that GbRac1 gene might have potential application in the genetic engineering of plants with enhanced disease resistance.

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.

Three pairs of primers were designed for amplification of the adiponectin gene in geese (Anser demeatica) based on the published sequences of chicken, duck and other adiponectin homologues. The complete 1062 bp sequence of the adiponectin gene in geese, obtained by polymerase chain reaction (PCR) amplification and splicing, contained two exons and one intron. The coding sequence was 738 bp long and included codes for 245 amino acids (GenBank Accession No. EU370686). The coding sequence and the corresponding protein of goose adiponectin shared the highest homology with ducks, 94.85% and 95.51%, respectively; homology with chicken adiponectin was next highest, while the lowest homology was to be found with mammals. The predicted molecular weight (MW) and isoelectric point (pI) were 26,603.3 Da and pH 5.19, respectively, which were highly similar to those of species considered above. Reverse transcription-PCR revealed that goose adiponectin mRNA was highly expressed in skeletal muscle, fatty tissue, heart and stomach muscles. Medium levels of expression were detected in the small intestine, stomach glandular tissue, kidney and lung, while it was only weakly expressed in the liver, spleen, ovaries and diencephalon.

In order to investigate the molecular mechanism of rehydration after serious drought in broomcorn millet (Panicum miliaceum L.), a forward subtracted cDNA library was constructed between normal watered leaves and rehydrated leaves after serious drought conditions, using the suppressive subtraction hybridization (SSH) technique. A total of 60 positive clones were picked out at random from the subtracted library and sequenced, and redundancy sequences were removed after sequence alignment. Based on the results of sequence homologous comparison and function querying, 32 expressed sequence tags (EST) were highly homologous with known ESTs. Most of those sequences were related to either abiotic or biotic stress in plants. Of those sequences, 11 ESTs were homologous with ESTs in rat (Rattus norvegicus) liver after partial hepatectomy. The Blast result of proteins revealed that 28 ESTs were similar to known proteins. The functions of these proteins mainly involve signal transduction, transcription and protein processing. This experiment demonstrated that a range of specific genes was induced and expressed in broomcorn millet during the rehydration stage after serious drought.

A cDNA library was constructed from the heading leaf in the early phase of the heading stage of Chinese cabbage (Brassica rapa L. ssp. pekinensis). By sequencing the randomly selected clones, 1363 sequences longer than 200 bp were found, with better trace data. After removing the poly(A) and contamination sequences, 1162 ESTs longer than 150 bp were obtained, of which 1102 shared significant similarity with known sequences in protein and nucleotide databases of the National Center for Biotechnology Information (NCBI) as revealed by searches using the BLASTX and BLASTN engines. Functional assignment of the ESTs was based on the method used in the Arabidopsis thaliana genome-sequencing project. About 77% of the putative protein sequences with known biological functions best matched with those of A. thaliana deposited in the non-redundant database of NCBI. These data suggest that Chinese cabbage is closely related to A. thaliana. This result is different from that reported in other Brassica species. At nucleotide level, however, 51% of the ESTs were homologous to those deposited for A. thaliana when all ESTs were searched against the est-others database. In addition, 60 ESTs had no homology with any of the plant gene sequences deposited in GenBank. These ESTs are very important for understanding the unique developmental process of Chinesecabbage and elaborating its genetic mapping. Among the genes with assigned functions, the most abundant representatives were those involved in protein synthesis and energy metabolism. With the 1162 ESTs, 895 non-redundant contigs were generated after being aligned using the Seqman II module of DNAStar software at the threshold of more than 80% homology over a minimum of 40 base pairs. Of these, 723 were singletons containing only one EST sequence, indicating that many kinds of such genes are expressed in the heading leaf of Chinese cabbage. An expression profile of Chinese cabbage heading leaf with the 1162 ESTs was therefore acquired in this work. This could be very useful for uncovering the mechanism of the heading process, which is the most obvious characteristic of Chinese cabbage and perhaps other related species, such as Brassica oleracea. This work could accelerate the finding and characterization of genes specifically expressed in the heading stage of Chinese cabbage.

Increasing the growth rate is especially important for low-quality wood applications, so this has become an important goal in poplar breeding. The present study describes the transfer of Vitreoscilla haemoglobin (VHb) gene (vgb) driven by constitutive promoters, by Agrobacterium tumefaciens into poplar (Populus alba×P. glandulosa). From about 450 leaf discs used for transformation, 60 Kan-resistant plants were obtained, and 52 proved to be true transgenic plants. The transgenic nature of these plants was confirmed by polymerase chain reaction (PCR) amplification and Southern dot blot hybridization. The expression of vgb gene in transgenic plants was confirmed by reverse transcriptase-PCR (RT-PCR). The performance of the transgenic lines was evaluated during the first year of growth in a greenhouse. These plants showed no significant stable morphological differences from the untransformed plants. Among them, three vgb-transgenic lines exhibited noticeably higher growth rates in terms of height and diameter.

Amplified fragment length polymorphism (AFLP) markers linked to the stem nematode resistance gene were developed in sweet potato (Ipomoea batatas (L.) Lam.). Using bulked segregant analysis (BSA), 800 AFLP primer combinations were screened in the resistant and susceptible bulked DNA from the 186 progeny of an F1 single-cross population of Xu781 (resistant parent)×Xushu18 (susceptible parent), and 245 of these AFLP primers showed polymorphic bands between resistant and susceptible DNA. Primer combinations detecting polymorphism between the two bulks were used to screen the parents and eight individuals from each of the bulks. The results showed that E2M23 and E33M20 produced a specific band of about 500 bp and 200 bp in length, respectively, in the resistant plants but not in the susceptible plants, suggesting that the markers named E2M23500 and E33M20200 linked to a gene for stem nematode resistance. Amplified analysis of the 186 F1 individuals indicated that the genetic distance between these two markers and the stem nematode resistance gene was 6.9 cM and 11.1 cM, respectively, measured with Mapmaker 3.0. These two AFLP markers were used to identify ten sweet potato varieties planted widely in China and the results were consistent with those of conventional resistance identification, indicating that the two markers can be used in molecular marker-assisted breeding for stem nematode resistance in the sweet potato.

Pancreases obtained from 1–9-day-old rabbits were digested with 1 mg/ml ?? collagenase and cultivated with low-glucose Dulbecco's modified Eagle's medium (DMEM). The cells were subcultured almost until confluence. They were identified as pancreatic stem/progenitor cells using two methods: an immunohistochemical staining technique and the response of glucose-stimulated insulin secretion after β-cell induction. Results showed that the cells of rabbits acquired strong proliferation ability, and expressed the markers of pancreatic stem/progenitor cells: PDX-1, CK19 and nestin. After β-cell induction for 2 days, 20% of cells were dithizone (DTZ) positive, rapidly increasing to 80% after 6 days. Insulin secretion was stimulated by a high glucose concentration (26 mmol/l). These powerful tests demonstrated that isolated and cultivated cells in rabbits were pancreatic stem/progenitor cells. This result has not yet been reported elsewhere.

Epidermal growth factor receptor (EGFR) is becoming a perfect target for killing carcinoma cells, especially because of its overexpression on the surface of these cells. Cationic antimicrobial peptides (CAP) have their own special mechanism of membrane-lytic cytotoxicity. In this study, a membrane-lytic immunotoxin (IT), chimeric protein MEGFMEL, was constructed to kill carcinoma cells with EGFR overexpression. This protein is composed of mouse (Mus musculus) epidermal growth factor (MEGF), as the target part, and melittin (MEL), as the cytotoxic part. Using Escherichia coli BL21 and pET30a as expression strain and vector, respectively, 63.45 μg/ml of MEGFMEL (68% purity) was obtained through low-temperature induction of expression and a thawing-freezing purification procedure (without cytolysis). In vitro activity measurement showed that this MEGFMEL significantly induced a lethal effect on A431 carcinoma cells overexpressing EGFR on the surface, with an LD50 value 52.6 μg/ml. The results suggest that the use of CAP as the toxin in the construction of unique membrane-lytic ITs aimed at EGFR is feasible.

The testosterone-inducible regulator (teiR) gene was cloned from Comamonas testosteroni chromosomal DNA, and introduced into plasmids pKtac2 (containing a tac promoter) and pK18 to yield plasmids pKtac2-teiR and pKteiR100. The recombinant plasmids were transformed into competent Escherichia coli HB101 and total protein was extracted to detect the TeiR protein expression level using enzyme-linked immunosorbent assay (ELISA). E. coli transformed by pKtac2-teiR and pKteiR100 produced 6.65 and 5.93 μg/mg of TeiR protein, respectively. Recombinant plasmids were also co-transformed into competent E. coli HB101 with plasmid p6 [containing hsdA gene (3α-HSD/CR, 3α-hydroxysteroid dehydrogenase/carbonyl reductase encoding gene)] to reveal the relationship between 3α-HSD/CR and TeiR by ELISA. The amounts of TeiR protein expressed by E. coli containing pKtac2-teiR and pKteiR100 were 5.94 μg/mg and 5.33 μg/mg, respectively, and these increased up to 6.81 μg/mg and 6.10 μg/mg after inducing with 1 mmol/l isopropyl-β-d-thiogalactoside (IPTG). Interestingly, 3α-HSD/CR protein expression level, after co-transformation with plasmids pKtac2-teiR and p6, was lower than that observed in the co-transformation with pKteiR100 and p6. The first co-transformation induced 1.20 μg/mg 3α-HSD/CR protein and the second 1.71 μg/mg. These values rose to 1.42 and 1.80 μg/mg, respectively, after treatment with 1 mmol/l IPTG. Our results proved that the tac promoter was more efficient than the lacZ promoter and that the teiR gene could act as an activator for hsdA gene expression.