The levels of drought tolerance and nucleotide polymorphism at the CBF4 locus were examined in a world-wide sample of 17 core accessions of Arabidopsis thaliana. The results showed that different accessions exhibited considerable differences in adaptation to drought stress. Compared with Columbia accession, the frequency of nucleotide polymorphism at the CBF4 locus of 25av, 203av and 244av accessions, including single nucleotide polymorphism (SNP) and insertion/deletion (Indel), was high, on average 1 SNP per 35.8 bp and 1 Indel per 143 bp. No significance in all regions of Tajima's D test indicated that the neutral mutation hypothesis could explain the nucleotide polymorphism in this CBF4 gene region. The higher polymorphism was the result of purification selection. Nucleotide polymorphism in the non-coding region was three times higher than in the coding region. This might indicate a recent relaxation of selection pressures on the non-coding region of CBF4 gene. In the coding region of CBF4, SNP frequency was 1 SNP per 96.4 bp and one non-synonymous mutation was detected from 25av, 203av and 244av accessions: the amino acid variation gly↔val at position 205, caused by the nucleotide variation G↔T at position 1034 (corresponding to the nucleotide at position 19 696 of GenBank accession no. AB015478 as 1). Furthermore, four differential SNPs were discovered in haplotype 6 constituted by 203av, one of them located in the 3′ non-coding region (A↔C at position 1106) and the others in the 5′ non-coding region (A↔G, A↔C and G↔A at positions 27, 129 and 171, respectively). The drought tolerance assay indicated that accession 203av was the best at tolerating water deficiency. We propose that haplotype 6 is consistent with its drought tolerance.

The cloning and sequencing of two specific molecular markers linked to grape (Vitis vinifera) seedless gene indicated that the full lengths of the markers, 39970524-5-564 (GenBank accession number: AY327513) and 39970524-6-1538 (GenBank accession number: AY327514), were 564 bp and 1538 bp, respectively. The restriction sites of sequences 39970524-5-564 and 39970524-6-1538 were analysed with Wingene231 software. The results indicated that there were 29 and 130 restriction sites in 39970524-5-564 and 39970524-6-1538, respectively, digested by restriction enzymes with recognition sites of six or more than six bases. There was no cleavage site in 39970524-5-564 and there was one site at 135 bp of 39970524-6-1538 when these two markers were digested by EcoRI. Both markers can not be digested by HindIII. Therefore, the full length of 39970524-5-564 and of the two bands of 39970524-6-1538 (1400 bp and 135 bp) could be cut out from the pGEMR-T Easy Vector by EcoRI. The labelled DNA of 39970524-5-564, used as a probe for Southern blotting with genomic DNA from Red Globe, Flame Seedless, Thompson Seedless, four seedless hybrids and three seeded hybrids, showed a unique hybrid band present in all seedless individuals, but absent in all seeded individuals. It was further indicated that the specific marker 39970524-5-564 was derived from the genome of seedless grapes.

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.

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.

To differentiate pigs infected with Pseudorabies virus (PrV) from pigs vaccinated with gE-PrV, a glycoprotein E enzyme-linked immunosorbent assay (gE-ELISA) based on recombinant glycoprotein E (gE) (which was expressed by Escherichia coli, purified, denatured and renatured) was developed. By testing 115 serum samples, the diagnostic specificity and sensitivity of the developed gE-ELISA were evaluated to be 94.5% and 96.7%, respectively. Five serum samples were tested with plates from five lots, and the results had a coefficient of variation of less than 10%, showing good reproducibility of gE-ELISA. This gE-ELISA was compared with a commercial blocking ELISA by testing 356 serum samples. The agreement rate of the two assays was 92.13% (328/356). These results suggested that the gE-ELISA developed in our laboratory could be used in differentiating PrV-infected and gE-PrV-vaccinated pigs.

A rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) <2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

Target region amplified polymorphism (TRAP) was used to compare genetic structures among three populations of grass carp (Ctenopharyngodon idella) – one wild and two cultured populations. Seven out of 15 primer combinations produced good amplification patterns and provided 103 amplified loci from the three populations. Numbers of polymorphic loci in the wild population were higher, indicating a decrease in genetic polymorphism in the two cultured populations. Compared with the wild population, only 39.98% loci gene frequency remained unchanged in the cultured samples, showing a genetic structure change in cultured populations. The genetic distances between wild and cultured populations were 0.0421 and 0.0809. With primer combination Ga5-800-E5, we detected a region in the electrophoretic pattern in which the number of amplified loci apparently decreased in cultured populations. These results establish a good scientific basis for developing molecular markers that can help in distinguishing wild from cultured populations.

The construction of two cDNA libraries from young shoots of tea plant [Camellia sinensis cv. Longjing 43 and Anji Baicha], the sequencing of Longjing 43 cDNA clones and the analysis of expressed sequence tags (ESTs) are reported. Totally, 4320 clones from the cDNA library of Longjing 43 were sequenced, and 2963 useful sequences were obtained, corresponding to 68.6% of clones. A total of 416 clones shorter than 150 bp and 863 repeated clones were excluded, the first 1684 valid tea plant ESTs were generated. Most of the ESTs were between 300 and 700 bp, with an average of 478 bp. Six hundred and seven ESTs with known function or putative function were identified by BlastN searches against the National Center of Biotechnology Institute (NCBI) non-redundant nucleotide databases, corresponding to more than 130 functional genes in the tea plant. The rest, 1077 ESTs, were novel gene partial or full sequences. The results indicated that EST sequencing was a rapid and effective approach to identification of novel functional genes for tea plant.

Melanocortin-4 receptor (MC4R) is a member of the superfamily of G-protein-coupled receptors (GPCRs), which affects body weight, energy homeostasis and food intake in humans and mice. In this study, the Asp298Asn polymorphism of the MC4R gene was investigated in Laiwu, Yorkshire×Laiwu and commercial cross-bred pig populations using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and the relationship of this mutation with backfat thickness was analysed. The results indicated that only genotype 11 exists in 33 individuals of Laiwu pigs, and three genotypes (11, 12 and 22) were detected in Yorkshire×Laiwu and commercial cross-bred populations. The distributions of allele and genotype frequencies in Yorkshire×Laiwu and commercial cross-bred populations were similar, with the frequency of allele 1 being higher than that of allele 2. In commercial cross-bred pigs, the mean backfat thickness of individuals with genotype 22 was significantly higher than that of individuals with genotypes 12 (P<0.01) and 11 (P<0.05). This study provides evidence that the Asp298Asn polymorphism of the MC4R gene is associated with backfat thickness in commercial cross-bred pigs with Western pigs as parental lines and, therefore, can be used as a DNA marker for breeding in such populations of pigs.

To understand the genetic basis of seed setting rate (SSR) in rice, two breeding lines (Oryza sativa ssp. indica), T226 with higher and stable SSR and T219 with lower and fluctuating SSR from different natural conditions, were used for constructing recombinant inbred lines (RILs). Genotype by environment (G×E) interaction and quantitative trait loci (QTL) for SSR were analysed using a population with 202 RILs under eight differing environments. A significant G×E interaction for SSR was detected in rice using the Additive Main Effects and Multiplicative Interaction (AMMI) statistical model, and the IPCA1 and IPCA2 of the G×E interaction accounted for a variation of 57.6%. QTL controlling the SSR were detected by the method of interval analysis. Seventeen QTL on nine chromosomes were identified across eight environments, totally explaining the phenotypic variances from 4.6 to 35.7%. Most of the QTL, each explaining a small part of the phenotypic variances and interacting with environments, were detected in one or two environments, and their alleles for increasing the SSR were derived from T226. However, the QTL (MRG5959–MRG2180) on chromosome 3 was detected across six different environments. It explained maximum phenotypic variances in each detected environment and its allele for increasing the SSR was derived from T226. Another QTL, mapped between markers RM592 and RM169 on chromosome 5, was detected in five various environments and its allele increasing the SSR was derived from T219.

The conditions for protoplast isolation and regeneration from Trichoderma viride were studied. Protoplasts were optimally isolated when mycelia of T. viride that had been cultured for 24 h were digested with 4 mg/ml Glucanex in phosphate buffer (pH 6.98) for 4 h at 30°C, resulting in a protoplast yield of 4.7×107 cfu/mg. The maximum regeneration ratio (14.5%) was obtained in mycelia culture medium containing 0.3 mol/l KCl and 0.3 mol/l inositol. In addition, a plasmid pCSSNCC1 carrying a hygromycin resistance gene and an elicitor-producing gene was transformed into T. viride protoplasts, with an efficiency of 1–2 transformants/μg DNA. The hygromycin-resistant transformants were determined by PCR and the elicitor protein was detected by ELISA. The results indicate that the elicitor protein was expressed stably in T. viride.

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 rice cDNA library was screened using OSK3 protein kinase as bait in a yeast two-hybrid system. The gene encoding oxalate oxidase was one of the positive clones interacting with OSK3 protein kinase. The interactions were verified by detecting expression of the reporter gene lacZ. The results suggest that oxalate oxidase is a downstream element in the disease resistance signal cascade mediated by OSK3 protein kinase in rice.

Analysis of mating type can provide an evaluation of the population genetic diversity of the rice blast fungus, Magnaporthe grisea. According to the sequences of MAT1-1 and MAT1-2 genes of the fungus, two pairs of PCR primers specific to the MAT1-1 and MAT1-2 alleles were designed, and the PCR thermal profile was also optimized. To confirm its application in mating type assessment, 10 tester isolates were tested by PCR. The PCR amplification pattern of these tester isolates corresponded to their known mating type. Furthermore, 150 rice-field isolates from Fujian Province were mated with tester isolates GUY11 and KA3 side by side and also tested by PCR. Results showed that 95.1% of 123 fertile isolates were the same in mating type as determined by both PCR-amplified allele-specific fragments and mating with GUY11/KA3. Among 27 sterile isolates determined by GUY11 and KA3, seven were MAT1-1 and 20 were MAT1-2 as determined by PCR. This study indicates that PCR is applicable in assessing M. grisea mating type and especially is capable of predicting the potential mating type of sterile isolates in the natural population of the fungus.

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.

The expression of the YUCAA1 gene and the amount of endogenous indole acetic acid (IAA) in rice (Oryza sativa subsp. japonica) plants and rice suspension cells infected by Rice stripe virus (RSV) were investigated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and high-performance liquid chromatography, respectively. The results showed that the expression of the YUCAA1 gene and the amount of endogenous IAA increased at various times (16, 32, 48 and 64 h) after infection of rice suspension cells by RSV. In rice plants infected with RSV, the expression of the YUCAA1 gene and the amount of endogenous IAA increased, in comparison with healthy rice plants, at 4–8 days after infection, and decreased at 12 and 16 days. These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally, the expression of the RSV gene CP increased 2.9 times in rice plants after they were treated with a KPSC buffer to deplete the endogenous auxins, and decreased 45% after treatment with 30 μmol/l IAA. All of these results suggest that auxin may play a role in RSV replication in rice plants.

The DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA of Pseudoalteromonas atlantica 19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed in Escherichia coli ER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.

Seven isoenergetic semi-purified test diets containing graded levels of protein ranging from 20 to 50% were formulated using fish meal and casein as the protein sources. Test diets were fed to triplicate groups of Barbodes caldwelli juveniles with initial body weights of 1.26±0.02 g for eight weeks. The results indicated no significant effect of dietary protein levels on survival rate, relative weight of the viscera and relative weight of the liver in the juvenile fish. The weight gain and specific growth rate of the fish were found to be greater as dietary protein levels increased from 20 to 35%, but were not affected significantly as dietary protein level increased from 35 to 50%. Feed efficiencies did not differ significantly when fish were fed diets with protein levels from 30 to 50%, but were significantly higher than those of fish fed diets with protein levels of 20 and 25%. The protein efficiency ratio (PER) was negatively correlated with diet protein level (x) (PER=3.006−0.03251x, R=0.9366). There was no significant effect of dietary protein levels on carcass moisture, crude protein and ash. However, carcass lipid levels (L) decreased with an increase in dietary protein level (x) (L=8.2169−0.0458x, R=0.8551). There was no significant variation in hepatopancreas protease activity among the tests. Intestine protease activity and hepatopancreas amylase activity were increased to some extent, and then decreased as dietary protein levels continued to rise. The intestine amylase activity (IAA) of the juveniles was negatively correlated with dietary protein level (x) (IAA=84.625−0.9147x, R=0.8463). It was estimated that the suitable protein level for the B. caldwelli juvenile is 34% (the broken-line model was used to regress the relationship of the weight gain of the juvenile and dietary protein level).

Butyrophilin 1A1 (BTN1A1), which is a member of the Ig superfamily, is highly expressed in the lactating mammary gland and is secreted into milk in association with lipid droplets, and concentrated in the apical plasma membranes of mammary epithelial cells. Because butyrophilin is expressed only during lactation, it appears to be essential for milk fat globule production. However, there is little information about its properties in the dairy goat. The objective of this study was to confirm whether butyrophilin was related to milk changes in different lactation periods of Xinong Saanen goats. Using suppression subtractive hybridization analysis, we identified a significant increase in butyrophilin mRNA in Xinong Saanen goat mammary gland at middle lactation when compared to that of late lactation. The gBTN1A1 complementary DNA has an open reading frame of 1581 nucleotides encoding a putative protein of 526 amino acids. Amino acids 1–26 were the signal peptide domain. The homologies of nucleotide and peptide sequences of goat butyrophilin with bovine (NM_174508), human (NM_001732) and mouse (AK145168) butyrophilin were 97 and 96%, 88 and 84%, 84 and 70%, respectively. The secondary structure, transmembrane domain and hydrophilicity of goat butyrophilin were similar to the those of the bovine, human and mouse protein. In conclusion, we confirmed that the goat mammary gland mRNA level of gBTN1A1 was responsible for goat milk fat globule secretion.

cDNA of the porcine small adipocyte factor 1 (SMAF1) gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate primers designed according to the conserved sequences between human and mouse genes. The cDNA (GenBank accession No. DQ191892) containing a complete encoding region was 256 bp in length, sharing 86 and 78% identity with that of human and mouse, respectively. Comparison of the deduced amino acid sequence between porcine SMAF1 and the human, mouse, cattle and rat protein showed that the amino acid similarity was 81, 67, 84 and 70%, respectively. The results of semi-quantitative RT-PCR showed that the porcine gene was expressed abundantly in adipose tissue, and at a significantly lower level in lean-type pigs (Large White pigs) than in lard-type pigs (Meishan pigs) at the age of 4 months (P<0.05). The results suggest that the porcine SMAF1 gene may have similar functions as in other species, that is, it may regulate adipogenesis and/or adipocyte function.