Abstract: Actinobacillus pleuropneumoniae (APP) is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Biofilm formation contributes to full virulence in many bacterial pathogens. In our previous STM studies we found that the disruption of the hns gene encoding a histone-like nucleoid structuring protein (H-NS) could enhance biofilm formation of APP. To further investigate the effect of H-NS on APP biofilm formation, the 408 bp complete coding sequence of hns gene was amplified by PCR from the genomic DNA of APP serotype 1 strain 4074 and cloned into the prokaryotic expression vector pET-28c. The resultant recombinant plasmid pET-hns was transformed into Escherichia coli BL21 (DE3). A 19KDa of recombinant protein (rH-NS) was obtained by IPTG induction, and purified using the Ni-NTA agarose columns. Different concentrations of rH-NS were added into the culture medium of the hns transposal mutant strain 1-21 and its parental strain 4074, and biofilms were quantified using the microtiter plate biofilm assay. Without rH-NS in the medium, strain 1-21 formed obvious biofims, but strain 4074 did not. Supplemented with 0.1-0.3μM rH-NS in the culture medium, the biomass of biofilms formed by strain 1-21 continuously decreased, whereas that formed by strain 4074 was increased. No obvious changes could be observed when more than 0.4μM of rH-NS was supplemented in both strains. Our data indicated that H-NS negatively regulates biofilm formation of A. pleuropneumoniae in a dose-dependent manner.

Abstract: From 1999-2002, we had isolated 21 H5N1 subtype avian influenza virus in health ducks from South China, We analyzed the biological properties and the heredity evolution of the H5N1 viruses systematically found that A/Duck/FuJian/01/02(DKFJ) and A/Duck/Guangxi/53/02(DKGX) are genetically similar, which were highly pathogenic to chickens but have different pathogenicity obviously in the BALB/c mice that is a useful mammalian model. DKGX is low pathogenic in mice (MLD50>106.5EID50), the reverse genetics system of DKGX had established in our Laboratory, whereas A/duck/FuJi/01/02 (DKFJ) is highly pathogenic (MLD50<100.5EID50). With the purpose of study the Molecular mechanisms of A duck H5N1 subtype avian influenza Pathogenic difference in mice of the two virus, an eight-plasmid reverse genetics system was established of DKFJ and the reassortant virus was rescued from cell transfection. The rescued virus(R-DKFJ) maintained the same biological properties as the wild virus (W-DKFJ MLD50<100.5). Both of the viruses could be recovered from the brain and spleen and kidney and lung of the Balb/c mice 3 days after inoculated intranasally with 106EID50. The successful establishment of the reverse genetics system of DKFJ will play an important role in the studies of the molecular basis of the evolution and the relationship between the structure and function of H5N1 subtype avian influenza viruses.

Size and nucleotide polymorphisms of intron 1 of chemokine gene SCYA113 were studied in 60 individuals from 3 populations of channel catfish (Ictalurus Punctatus) with DPAGE and DNA sequencing. Results showed that there were 15 haplotypes and 8 length variants (from A to H), ranging from 395 to 443 bp. Sequence analysis of the 8 length variants showed the average composition of nucleotides (A, G, T and C) were 30.01%, 15.43%, 38.37% and 16.19%, respectively, and the GC content (31.62%) was much lower than that of AT content (68.38%). The GT/AG domain was found in exon-intron junctions,which was the 5′ and 3′ splice donor and acceptor sites in higher eukaryotic gene introns. Length variation was mainly due to the variable number of microsatellite TTC and TTA repeats. There were only 6 substitution sites excluding microsatellites: 3 transversions at 266 (T→A), 289 (G→T), 387 (G→C) and 3 transitions at 118 (C→T), 209 (G→A) and 250 (T→C), respectively. And the nucleotide diversity (π) was only 0.004, average number of nucleotide differences (K) was 1.576. The three populations have displayed the genetic diversity. In the P1984 population, eight length types and fourteen genotypes were observed. Eight length types and ten genotypes presented in the P1997 population. In the P2004 population, there were six length types and ten genotypes, and no allele A and B were found. Twenty-two genotypes are found from 60 individuals. All the genotypes are emerging at a lower frequencies (from 0.017 to 0.150), indicating the genetic resources is abundant.

To illuminate the effect of nitric oxide (NO) on Sertoli Cell and provide the base for studying mechanism of spermatogenesis, Sertoli Cell of piglets were chose to study the effect of NO on microfilaments of Sertoli Cell. Sertoli Cells were treated with SNP. Cell viability, antioxygen enzymes activity and changes of p-38MAPKwere detected. The results showed as followed:low level NO could keep Sertoli Cells’ microtubule and cell viability normally,and high level NO could increase the activity of p-38MAPK of cells, decrease antioxygen ability and cell secretion and destroy the structure and distribution of microtubule. The results inferred that SNP resulted in the increase of NO of Sertoli cells, and decreased antioxygen ability. The high level of NO destroyed cell microtubule through activating p38MAPK.

In this study, using AO, Hoechst 33342 and flow cytometry methods, the apoptotic change of pig preadipocyte was investigated after treatment with 9-cisRA (RXRα ligand), and transfection of pRXRα-EGFP and RXRα-siRNA. The results showed that it occurred morphologic change of apoptosis in pig preadipocyte. Cellular shrinkage and round, condensation of chromatin adjacent to nuclear membrane, nuclear fragmentation of apoptotic bodies which were rapidly engulfed by neighboring cells. Moreover, compared with control group, apoptotic rate was significantly decreased in the groups of 10 nmol/L 9-cisRA supplement and pRXRα-EGFP（P0.05）. In contrast, apoptotic rate of the groups with 10 µmol/L 9-cisRA or RXRα-siRNA was remarkably higher than the control group （P0.05）. It suggests that RXRα can inhibit the apoptosis of pig preadipocyte.

2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MCS) is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate (MEP) pathway. A MCS gene was isolated from Hevea brasiliensis by RT-PCR and RACE based on the conserved sequences of plant MCS genes. The cDNA, which designated HbMCS1, consists of 965 bp and encodes a protein of 241 amino acids with a predicted molecular mass of 25.82 kDa. In comparison, this putative HbMCS1 protein shows from 62.2% to 70.8 % identity to other plants MCS. Semi-quantitative RT-PCR analysis indicates that the HbMCS1 is more highly expressed in callius than in latex and leaves. The transcription of HbMCS1 in latex was induced tapping, whereas ethylene had little effect. The present study contributes towards an understanding of the biosynthetic mechanism of natural rubber in Hevea brasiliensis.

Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic diversity and population structure in eight natural populations of Elymus sibiricus L. from the northwestern plateau of Sichuan, part of Qinghai-Tibet Plateau zone. Of the 150 primers screened, 25 produced highly reproducible RAPD bands. Using these primers, 370discernible DNA fragments were generated with 291 (78.65%) being polymorphic, indicating considerable genetic variation at the species level. In contrast, there were relatively low levels of polymorphism at the population level with the percentage of polymorphic loci (PP) ranging from 46.49% to 53.78%. The mean gene diversity (HE) was estimated to be 0.176 within populations (range 0.159 to 0.190), and 0.264 at the species level. A high level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis (32.0%), Shannon’s index analysis (33.7%), Bayesian method (33.5%). The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (59.9%) and among populations (40.1%; P < 0.001). The average number of individuals exchanged between populations per generation (Nm) was 0.503. Populations shared high levels of genetic identity. In addition, a geographical pattern of population differentiation, where the populations from south and north of sampling sites were clearly separated from each other, was revealed by both the cluster and AMOVA analyses. Generally, the result of this study indicates that E. sibiricus contains high molecular variation in its populations. Therefore, we should focus on sampling more individuals from populations with higher genetic diversity for ex-situ conservation of E. sibiricus.

Stat3(signal transducer and activator of transcription 3) gene was amplified from POTB7 plasmid which contained human stat3 gene cDNA fragment, then inserted into pEGFP-C1 vector to construct recombinant plasmid .The recombinant plasmid was transfected into bovine mammary epithelial(BME) cells mediated by LipofectaminTM 2000.The positive cell clones were obtained after selected by G418，The BME cells transfected with stat3cDNA were determined by RT- PCR, Flow cytometry was used to analyze the cell cycles 、multiplication capacity and DNA content. The results indicated that 24h after exposure to the recombinant plasmid the transfection rate of the BME cells was 16%,The strong expression of stat3 in transfected BME cells was confirmed at mRNA levels. After introduction of the recombinant plasmid into BME cells, the capability of proliferation was obviously enhanced and the DNA content was changed, the cell proliferation lifespan was elongated to 13 population doublings to control. Our research indicated that the cell proliferation life-span was elongated invitro with introduction of the recombinant plasmid into BME cells.

The objective of this study was to examine the possibility of using a non-electrophoretic method to detect the TSPY gene for sexing bovine early embryos, which made bovine embryo sexing under farm condition more feasible. Primers were designed to amplify a portion of the TSPY gene and a common gene as an internal control primer. PCR optimization was carried out using a DNA template from bovine whole blood. Furthermore, six embryo samples were diagnosed by this method and the sexing results were contrasted with those of the Loop-Mediated Isothermal Amplification (LAMP) method. The result showed that TSPY was as reliable a sexing method as LAMP. Forty-three morula and blastocyst embryos collected from superovulated donor dairy cattle were sexed by this method, and twenty-one embryos judged to be female embryos were transferred non-surgically to recipients 6 to 8 days after natural estrus. Out of 21 recipients, 9 were pregnant and all delivered female calves. The results showed that the sex predicted accuracy of this protocol was 100%. In conclusion, TSPY gene was a good male specific marker and indicated that a non-electrophoretic method was feasible and accurate to detect TSPY gene for sexing preimplantation bovine embryos.

In this paper, to confirm the bacteriostatic properties， the recombinant human lactoferrin (rhLF) was extracted form the milk of transgenic mice by gel filtration chromatography. The rhLF was also analyzed by SDS-PAGE and ELISA-assay. The bacteriostatic properties of rhLF was tested by agar disc diffusion method and direct counting method. The results showed that the molecular weights of rhLF, both of proteins from the transgenic mice of PCL25 and AP vectors were compared with that of native hLF, and were about 78 KDa. The rhLFs have significant bacteriostatic properties against E.coli and Samonella.

Two DNA fragments encoding N-term of MSTN and INH respectively were incorporated into coding region of hepatitis B virus S gene at C-term and middle(between amino acid position 112 and 113) to construct a myostatin expression vector pVAX-SMI. The plasmid pVAX-SMI was identified by restriction endonuclease digestion and sequencing. pVAX-SMI was transfected into HeLa cells and the recombinant protein was detected by Western Blot. The result showed that the plasmid could express recombinant MSTN and INH with immunogenicity successfully. The pVAX-SMI expression vector was constructed successfully.

Marker-assisted selection (MAS) is one of main techniques, which can greatly accelerate the process and improve the efficiency of plant breeding, but it depends on the data of molecular genetic diversity, especially on the research on target genes and the development of linked marker to target genes. Sucrose content is one of the most important characters, which closely related to the sucrose metabolism genes. In cultivation, cold stress is an important factor that affects the growth of sugarcane. For above, TRAP marker was occupied to study on the genetic diversity of thirty-three important sugarcane clones, including some main cultivars, recent released varieties and some germplasms introduced abroad recently. The total of five genes related to sucrose metabolism plus one related to cold stress were selected as target genes and were used to design the anchored primers for pairing with nine arbitrary primers. In total, there were 90 primer combinations and thirty-four combinations of primers were screened to produce clear banding patterns and polymorphisms. The result showed that a total of 579 bands were scored with the 34 pairs of primed screened, of which 288 taking 49.74% in total were polymorphisms. Thus, the polymorphism results from combinations of forward fixed and arbitrary primers reflected that the exonic regions outside the open reading frames (ORFs) of sugarcane genes have a very high mutation rate and diversity. The cluster analyses using UPGMA revealed a narrow genetic diversity among the entries with genetic distance (GD) ranging from 0.26 to 0.63. At the GD level of 0.52, 33 sugarcane , the cluster analysis based on TRAP analysis with six target-genes, while can’t contribute completely to parentage grouping of the 33 clones, can really reflect the variation degree of these 33 sugarcane clones based on TRAP polymorphism of these key genes. At the same time, some special germplasms were found. Therefore, the data from the TRAP analysis can enhance the accumulation of basic data of parent selection and combination configuration and help in high-sucrose and cold-tolerance breeding practice. Furthermore, it can also be concluded from this study that TRAP marker technique have many other merits, such as good repeatability, high efficiency, and easy operation, which suggests its great potential applicability in sugarcane classification, important trait marker etc. In a word, the results of this study have an important theoretical and practical

To explore an effective and reliable karyptyping method in Brassica crop plants, genomic DNA was isolated from Brassica oleracea genome, labeled as probe with DIG-High Prime Mix kit; genomic DNA was isolated from Brassica rapa genome as block reagent. In situ hybridization to mitotic spreads, specific fluorescent signals were showed on each pair of homologous chromosome. Nine pairs of Brassica oleracea genome were exactly identified. It provided a feasible and accurate method to discriminate species with small chromosomes. And it set up an important found to research localization of self-incompatible genes.

Objective: To construct the prokaryotic expression vector for gcgasa gene encoding Gibberellin-induced cysteine-rich protein, and to express the gene in E.coli BL21(DE3). Material: Gymanadenia Conopsea R.Br. Methods: The primers bearing restriction enzyme site of EcoR I and Hind III were designed and were ultilized to amplify the full-length of ORF and the signal peptide-truncated fragment. The target fragments were then cloned into the pET-32(a) vector to produce the recombinant plasmids pET-32(a)/gcgasa and pET-32(a)/gcgasa. After they were identified by DNA sequencing, the recombinants were transformed into E.coli BL21(DE3) to express the fusion protein with the induction of IPTG. SDS-PAGE, Western blotting, as well as Transmission Electron Micrograph of ultrathin section were done to identify the expression and location of heterogenous protein. Following the solubility analysis, the expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods. Results: DNA sequencing showed that the recombinant plasmids have been successfully constructed. SDS-PAGE and Western blotting analysis revealed that the presence of signal peptide gave rise to the formation of inclusion body, which was located in periplasmic space, however, the absence of signal peptide greatly enhanced the solubility of the target protein. In the latter case, further purification was achieved using Ni2+-NTA and Sephadex G-75 gel filtration column. Conculsion: It was for the first time that the soluble gcgasa protein was successfully obtained from E. coli system with high efficiency. This study will be very fundamental and essential for further investigation on the stucture and function of gcgasa.

Using the polyclonal antiserum against Spodoptera exigua (Hübner) invertebrate intestinal mucin (IIM), a solute carrier gene LstiSLC25 was screened from the cDNA expression library of Loxostege sticticalis midgut. The full-length of LstiSLC25 is 1399bp (GenBank accession no. EU924506) and the open reading frame(ORF) was 897bp, encoding 299 amino acid residues; the predicted MW and pI were 32.1kD and 9.46, respectively. The LstiSLC25 possesses three solcars, which is characterized of solute carrier family. The LstiSLC25 gene has been recombined into pET30 vector. The Western blot analysis demonstrated that LstiSLC25 protein was expressed at low level after being induced by IPTG.

Endoglucanase is one of the most important cellulose enzyme, which cooperates with cellobihydrolase and β-1,4-glucosidase to translate cellulose to glucose completely. Our objectives were to express an endoglucanase gene in Pichia pastoris and find out expression conditions for high-level expression of neutral endoglucanase. According to the sequence of endoglucanase gene (GenBank Accession No.DQ782954) from Bacillus subtilis, a pair of primers was designed and synthesized, and a high-fidelity polymerase (Pfu DNA polymerase) was used for amplifying the mature endoglucanase expression gene. A 1.4kb fragment, which does not contain signal peptide sequence obtained, and was ligated to expression vector pPIC9k. pPIC-End was constructed and transformed to Pichia pastoris GS115. Three transformants named GS115-pPIC-EndⅠ, GS115-pPIC-EndⅦ, GS115-pPIC-EndⅧ were obtained through MD,MM plate screening and enzymatic activity test. The expression conditions containing the initial cell density, pH and methanol concentration were studied in shaking culture. Optimal endoglucanase production was observed when the initial cell density OD600was 5, and the optimal methanol concentration was 0.5%-1%. The endoglucanase production increased obviously when provided adequate aeration, whereas it did not seem to vary when cells were induced at pH4-8. Under the above expression condition, the endoglucanase activity of the three transformants was 860.7U, 760.3U, 786.2U, 13.5 times, 11.9 times and 12.3 times compared with the endoglucanase activity of the original strain (63.78U) respectively. The enzyme maintained over 80% of the original enzyme activity after incubated at 65℃ for 30 min, and the SDS-PAGE showed that The molecular weight is about 79.82KDa.

N-acylhomoserine lactones (AHLs) serve as signal molecules of bacterial quorum sensing system, regulate virulence of several plant pathogens. The N-acylhomoserine lactonase (AHL-lactonase) hydrolyzes AHLs and attenuates the pathogenicity of plant pathogenic bacteria. A plant binary vector carrying aiiA under the control of 35s promoter was constructed into Arabidopsis thaliana by Agrobacterium mediated transformation. The homozygous transgenic plants seeds were botained by resistance screen. The gene transformation and expression was confirmed by PCR and RT-PCR respectively.The aiiA expressing transgenic A. thaliana showed an increased resistance to soft rot disease caused by Erwinia carotovora subsp.carotovora.

Abstract： The genetic polymorphism of GlyCAM1 gene in three different breeds (China Holstein cattle, Luxi Yellow cattle, Bohai Black cattle) were studied by CRS-PCR and PCR-SSCP and DNA sequencing. Sequencing results showed that two polymorphic sites were identified at first, which they were at position (A/C) and (C/T) in the GlyCAM1 gene exon3 and intron3 respectively. In the 2081 site the allelic frequencies of A and B were 0.7525/0.2475、0.6112/0.3888、0.3375/0.6625 respectively in China Holstein cattle, Luxi Yellow cattle and Bohai Black cattle. In the 2417 site the allelic frequencies of A and B were 0.9046/0.0954、0.8383/0.1617、0.7875/0.2125 respectively in three different breeds. χ2 test indicated that the polymorphic sites of intron3 were in accordance with the Hardy-Weinberg equilibrium (P>0.05) in the three different breeds. The polymorphic sites of exon3 were in accordance with Hardy-Weinberg equilibrium (P>0.05) in Luxi Yellow cattle and Bohai Black cattle.But it was not in accordance with Hardy-Weinberg equilibrium (P<0.05) in Chinese Holstein cattle.

The PCR-SSCP polymorphism of IGFBP-3 gene exon 3 and Its Correlation with milk yield and growth traits were studied in 105 Holstein cattle and its second backcross generation . The result showed: there were two alleles and three genotypes in the locus of IGFBP-3 gene exon 3. The Allelic frequencies of A and B were 0.7667 and 0.2333 , and the genotypic frequencies of AA, BB and AB were 0.6000, 0.3333 and 0.0667 respectively. The population was at Hardy-Weinberg equilibrium at this locus. The gene heterozygosity and polymorphism information content were listed for 0.3578 and 0.2937 respectively. The sequencing result showed: The B allele had happened a base transformation of G-A at the 8515th bp contrasting with the AF305712, but the A allele has same sequence in here to the AF305712; In addition, there were a no function mutation of T-C for the A and B alleles at the 8634th bp of AF305712. The study by Least Squares Method showed : the milk yield for 305 days and body weight at 12 mouth old of individuals with genotype AA was highly significantly higher than that of individuals with genotype AB and BB respectively (p<0.01). The body length and height at hip cross of individuals with genotype AA was highly significantly higher than that of individuals with genotype AB(p<0.01)，but no significant difference between BB and AB as well as between BB and AA; The genotype did not affected on heart girth and paunch girth significantly.

Two complementary sense and antisense oligo-nucleotides, encoded Bovine Lactoferricin B Gene (LfcinB), were synthesized based on amino acids sequence of Bovine Lactoferricin B published in the antimicrobial peptide database (APD). After the sense and antisense oligo-nucleotides annealed, a cohesive BamHI site at 5’ terminus and a cohesive XhoI site at 3’ terminus.were formed. The synthetic LfcinB gene was cloned into prokaryotic expression vector pET-32a. The positive bacteria clones were amplified and cultured in terrific broth medium. Then the aimed protein was expressed with the induction of 0.1 mmol/L IPTG.. The result of SDS-PAGE indicated that LfcinB gene was strongly expressed in E. coli BL21, and the expressed recombinant protein was in form of inclusion body. Inclusion bodies of LfcinB recombinant protein were purified. The purified inclusion bodies were renatured by dialysis using TGE dialysate.

Objective: The aim of this work was to purify and to identify active antimicrobial peptides from the heterophils of ostrich blood. Method: Ostrich heterophils antimicrobial peptides were isolated by adding of 0.83% ammonium chlorid solution, sonicatingto release the heterophils granules, suspending in acetic acid and mixing overnight to extract the antimicrobial peptides. The crude extractions were obtained. Iox-exchange chromatography and reversed phase high performance liquid chromatography (RP-HPLC) were used to separate the components present in the crude extract. The radial diffusion plate assay method was used to determine antimicrobial activity and the minimum inhibitory concentration (MIC). The molecular weight and amino acid sequence were determined by MS/MS. Result: The results showed that there were antimicrobial substances in ostrich heterophils, and there were strongly active against Escherichia.coli O78, Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231all the bacteria strains above. The peptides were cationic moleculesand have good character of hot stabilization. Peak 6, 16 and 24 were obtained by RP-HPLC. The MIC of peak 6 against Candida.albicans ATCC10231 was 0.065μg/mL. The molecular weight of perk 16 was 4012.472Da, and its MIC against Escherichia.coli O78 and Staphylococcus.aureus 1056MRSA were 3.971μg/mL and 5.245μg/mL respectively. The molecular weight of fractions 24 was 3542.479Da, and the MIC of fraction 16 against Escherichia.coli O78 and Staphylococcus.aureus 1056MRSA were 26.472μg/mL and 21.561μg/mL respectively. Conclusion: The peptides we obtained displayed stronger activity than ostricacins reported by others. Further work need to determine whether they are same substance.

To examine the bioactivity of rEIL-18 obtained from E.coli, we evaluated if rEIL-18 showed a synergistic effect with rhIL-12 for the induction of EIFN-gamma gene expression from equine PBMCs using by real-time RT-PCR. REIL-18 could induce EIFN-gammamRNA expression from equine PBMCs about 20-fold higher than that of negative control in the presence of rhIL-12. And the effect to induce EIFN-gamma gene expression was dose-dependent with rEIL-18 in a fixed concentration of rhIL-12. We also evaluated if 9 anti-rEIL-18 monoclonal antibodies (mAbs) could neutralize the bioactivity of rEIL-18. One of 9 mAbs could neutralize rEIL-18 bioactivity and its effect was dose-dependent. The results showed that rEIL-18 had bioactivity commensurate with the native molecule, as they were neutralized by mAbs against rEIL-18.

Using phage display technique we constructed a library of repertoire immunoglobulin from mouse immunized with SS. The heavy-chain and light－chain variable region gene (VH and VL) repertoire of immunoglobulin were amplified individually from the spleen cell RNA by RT－PCR and joined by a DNA linker encoding (G1y4Ser)3 as a single chain (ScFv) DNA fragment with overlap extension. These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemid was transformed to susceptible E.coli TGl by electroporation.In the presence of helper phage M13KO7, the ScFv fusion protein were displayed on the surface of recombinant phages. The phage antibody library was generated and its size was determined to be 9.3×107,these results are bases to further phase antibody library construction

52 pigs from F2 population ( Pietrain × Erhualian ) were slaughtered for sampling after 2h transport. Pork quality was characterized by measuring pH45min, pH24h, tenderness, water holding capacity, cooking loss, and color scores- l*, a*, b*. 22 microsatellite DNA loci were selected from the published stress QTL regions in swine. One-way ANOVA was applied to analyze the relation between microsatellite polymorphisms and pork quality. The results revealed that: the allelic number was 3～8, heterozygosity was 0.4155～0.7432 and polymorphism information content (PIC) was 0.3651～0.8150. S0101 had significant effect on meat color-a* (P< 0.01). The effect of SW1023 on water holding capacity (WHC) was significant (P<0.05). The effects of SW1808 on WHC (P< 0.01), meat color-l*(P<0.05) and b*(P<0.05) were significant, respectively. Both S0112 and S0092 had significant effects on pH45min (P<0.05). Our study suggested that these 5 microsatellites in swine stress QTL regions were closely associated with pork quality in response to transport.

A Competitive ELISA kit for detection of Difloxacin（DIF-Kit）was developed with generating hybridoma lines that secrete DIFmonoclonal antibodies（DIF mAb）and the method of ciELISA,its traits were identified. The DIF-Kit had 0.24% cross-reactivity towards Danofloxacin,and little or no cross-reactivity towards other FQNS and sulfamido.The detection limit for DIF was 0.1µg/L and IC50 was 2.2µg/L.The The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15% in range of 0.1～128.0µg/L. The recoveries from milk and chicken were 89.3% and 83.9%,respectively,when 0.4、2.0、10.0、50.0.0µg/L DIF were spiked. The validity of DIF-Kit in 4℃ was above six months.The DIF-Kit possesses high accuracy, good reproducibility,specificity.That is proved to be used for the rapid detection of DIF residues in animal food.

To study the isolation rate and toxin type of Clostridium perfringens in freshwarter fishes. METHODS: Four hundred and twenty fresh intestinal content samples (not including intestinal tissues) of freshwater fishes caught from one water reservoir were examined bacteriologically for the occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (α, β, ε and ι) for classification into toxin types and for genes encoding enterotoxin and the novel β2 toxin for further subclassification. RESULTS: C. perfringens could be isolated in 75 intestinal contents samples (17.9%) from freshwater fish, 58 strains (77.3%) were C. perfringens toxin type C (α and β toxin positive), 13 strains (17.3%) were toxin type A (α toxin positive) and 4 strains (5.3%) were toxin type B (α, β and ε toxin positive). In addition, the gene encoding for β2 toxin was found in all the isolates. These amplified toxin gene fragment were cloned and sequenced and compared with standard strains, the identity varied from 98.15% to 99.29%. CONCLUSION: This is the first report of C. perfringens α, β, ε, β2 toxins in freshwater fish and of β, ε toxins in fish in general, and is the first discovery that the β2 toxin could be detected in strains of type B. The origin of this bacterium and its importance to human food poisoning in freshwater fish is discussed.

According to the sequence of aac gene associated with quorum-quenching of Ralstonia solanacearum, the PCR primers were designed to amplify aac gene, then the PCR fragment was cloned into pGEM-T-easy vector. The recombinant plasmid containing aac gene was mutated by introducing the gentamicin-3-acetyltransferase gene (Gmr gene). Then aac gene containing Gmr gene was subcloned into suicide vector pDS132, named pDS-aac’-Gm. Moreover, GMI 1000 strain was used to construct aac gene mutant by homologous recombination. The marked aac strain inserted with Gmr gene was selected by three-step methods and aac inserted by Gm gene on the genome was determined by PCR. pDS-aac’-Gm constructed from the sequence of R. solanacearum resulted in the construction of the in-frame insertion aac gene mutant of GMI 1001 successfully. The result of tomato inoculation test showed that the GMI 1000-m strain were much less virulent on tomato than the wild-type GMI 1000, which indicated that the aac gene is a very important factor in the pathogenesis of R. solanacearum.

Difference of H-FABP mRNA transcription of Longissimus dorsi in Laiwu pigs and Duroc weighted about 100kg were detected by fluorescence quantitative RT-PCR. And its relationship with meat quality was analyzed. The result indicated that the H-FABP expression of Laiwu pigs was higher 38.60％ than it of Duroc, and perhaps it was related with the fact that IMF of Laiwu pigs were significantly higher than it of Duroc pigs（P<0.01）. The correlation between the H-FABP expression with IMF and pH were positive, with diameter of musclur fiber negative, and with marble score and meat color were not consistent in Laiwu pigs and Duroc. Thereinto, the correlation coefficients between the H-FABP expression with IMF were highest, 0.363 and 0.934, respectively. Therefore study of H-FABP can not only increase the IMF content, but increase pH and decrease diameter of musclur fiber.

Separate Cry1Ac receptor proteins in Helicoverpa armigera Hübner midgut using two dimension electrophoresis (2D electrophoresis) method is very important for further defining kinds of receptors in H. armigera, finding new receptors and searching receptors related to Bt resistance. According to the different mode of isoelectric focus, 2D electrophoresis can be divided into two systerm: ISO-DALT (isoelectric focus-dalton weight) and IPG-DALT (immobilized pH gradient-dalton weight). In this study, combined western blotting, we compared two 2D electrophoresis instruments of ISO-DALT and IPG-DALT for the separation of Cry1Ac receptor proteins in Helicoverpa armigera. Results showed that ISO-DALT got better separation results than IPG-DALT, ISO-DALT costs less money but effect mass spectrum work, IPG-DALT is more expensive but advantage to following work.

Based on the gene sequence of cap of PCV2, a pair of primers was designed and, a 384 bp fraction of the carboxyl end of the capsid protein gene was obtained by PCR amplification, which was then cloned into plasmid pR to yield an integrative plasmid pR-cap. The integrative recombinant plasmid was used to transform E. coli E2 host bacteria, and then this E2 containing the recombinant plasmid was used for homologous recombination with lysozyme gene-deficient T4, thus yielding a recombinant bacteriophage T4-cap. When purified T4-CAP was tested with SDS-PAGE and Western blotting, a 25 Ku protein band was detected in polyacrylamide gel and nitrocelluLose membrane, which could combine specifically with PCV2 antiserum, attesting to the successful display of SOC fusion protein on the T4 bacteriophage surface.

Abstract: It has been almost eight years since the first report of live piglets produce by somatic cell nuclear transfer (SCNT) technology. Although it is more difficult, low efficiency (1~2%), compared with embryonic cell nuclear (ECNT) technology, the cloning pigs with SCNT technology, has been further studied. Some progress has taken in the fields of mechanism and approaches. In this paper, we mainly focus on the key technology and its difficulties of SCNT in cloning pigs, including its applications and approach of increasing the efficiency of SCNT.