Mad cow disease is caused by the prion protein-protease-resistant form (PrPsc), which is an isoform of protease-sensitive isoform of prion protein(PrPc), the aggregation of PrPsc in the central nervous system can result in the transmissible spongiform encephalopathy(TSE). By cutting down PrPc's expression to reach the purpose of decreasing its conversion into PrPsc is an effective way to inhibit TSE. In this article, we used the technology of RNA interference(RNAi) to cut down PrPc's expression, three candidate short hairpin RNAs(shRNAs) (prnp 1, prnp 2 and prnp 3) targeting the coding sequence of bovine prnp gene and a negative control (prnp -NC) were designed, then they were ligated into the vector of pGenesil-1 respectively, and were transfected into bovine fibroblast cells. By the means of Real-time PCR and Western blot, we could examine their suppression efficiency, at the same time, we selected cells in cell culture medium with G418 under a concentration of 700 μg/mL. The results showed that the interference effects of the three shRNA vectors were 51.2%, 85.7% and 74.6% respectively, the negative control's efficiency was 2.05%, so we obtained a more efficacious shRNA, and the positive cell clones were also got. This study provides a way for producing therapeutic transgenic animals by somatic cell nuclear transfer.

PCR primers were designed according to the adipocyte fatty acid-binding protein gene (A-FABP) of cattle (Bos taurus). A 399 bp cDNA fragment (GenBank accession No. EU815937.1) of A-FABP gene was obtained by RT-PCR using total RNA from subcutaneous fat of adult Jiulong yak (B. grunniens). This fragment showed 98.7% nucleotide sequence similarities with cattle, with four amino acid substitutions and resulted in a shift of theoretical isoelectric point. The expression profiling of A-FABP in Jiulong yak tissues were analyzed using semi-quantitative RT-PCR. A-FABP expression was observed in fat, skeletal muscle, heart, liver, kidney and spleen tissues except for lung, respectively. The expression of A-FABP in fat tissue was significantly higher than other tissues studied (P<0.01), and also exhibited high level in liver and skeletal muscle. The expression of A-FABP gene in longissimus muscle of 0.5 and over 9 years old Jiulong yaks was markedly higher than that of the age of 3.5 and 5.5 years old (P<0.05), respecctively. Moreover, its expression showed significant correlation with intramuscular fat content of longissimus muscle.

In the research, microsatellite markers were used to identify individuals of 3 foreign pig breeds (Yorkshire, Landrace and Duroc). 10 littermates were chosen from each breed. Individual variation were analyzed based on SSR loci polymorphisms. The results showed that 20 pig individuals of 2 litters could be differentiated using 7 microsatellite markers; after adding further 7 microsatellite markers, the last 10 pig individuals of another litter could also be differentiated. Traceability from meat to blood using these microsatellite markers was also studied by random selecting 5 pigs from slaughter. Results showed that microsatellite loci information of meat samples were correspondent to which of blood samples. So it is feasible to trace the pigs from meat samples in supermarket to blood samples in slaughter by microsatellite markers.

Adipose triglyceride lipase (ATGL) is a key enzyme in lipid lipolysis. In this experiment, the whole cDNA length ATGL gene with 2 074 bp (GenBank accession No. GQ918145) was isolated and cloned by RT-PCR and RACE (rapid amplification cDNA ends) from Xinong Saanen dairy goats (Capra hircus). ATGL gene contains 141 bp 5'UTR, 472 bp 3'UTR, and 1 461 bp coding sequences (CDS) which encode a protein of 486 amino acid residues (GenBank accession No. ADD25174). AA sequence alignment showed that the AA of goat ATGL had high score similarity, more than 85%, when compared with bovine (Bos taurus), rat (Rattus norvegicus), mouse (Mus musculus), pig (Sus scrofa), and human (Homo sapiens) in GenBank. The protein structure had a Patatin domain, α/β hydrolase folds and GXSXG (Gly-X-Ser-X-Gly) structure. A transmembrane helix was speculated in the N-terminal and no signal peptide found in the entire sequence. The protein molecular weight was 53 243.4 D and the isoelectric point was 7.4. Furthermore, the ATGL gene expressions in various organs and tissues were analyzed by real-time fluorescence quantitative-PCR. The results showed that the ATGL mRNA existed in all of the 12 detected organs and tissues. The most abundant expression was detected in subcutaneous adipose tissue, which was significantly higher than that of the other tissues, and the minimal expression was observed in heart tissue. The higher expression level of ATGL gene in the mammary gland of dairy goats provided the experimental evidence for its function analysis.

Using the technology of gene immunization, 60 sheep(Ovis aries) were immunized with recombinant plasmid of tandem inhibin gene, pcDNA-DPPISS-DINH and pcDNA-DPPISS-DINH-sC3d3, respectively. The P(Positive absorbancy of blood sera)/N(Negative absorbancy of control) values of antibody against inhibin were detected by ELISA(enzyme-linked immunoassay) and the hormone levels were detected at different stage by RIA(radioimmunoassay) after gene immunization of tandem inhibin in sheep. The results showed that the P/N values of antibody against inhibin in gene immunization groups of pcDNA-DPPISS-DINH and pcDNA-DPPISS-DINH-sC3d3 were higher than those in control group(P＜0.05), respectively, and the levels of antibody against inhibin were rose significantly after the third immunization. The levels of follicle stimulating hormone(FSH) in gene immunization groups were higher than those in control group after the first immunization, and the difference was significant after the second and third immunization (P＜0.05), respectively. The levels of LH(luteinizing hormone), E2(estradiol) and P(progesterone) were all higher than those in control group after the second immunization, but there were no significant differences (P＞0.05). After the third immunization, the levels of E2 and P were higher than those in control group(P＜0.05), and the twinning rates were 22.2% and 30.0%, respectively, which were significantly higher(P＜0.05) than that in the control group. All the results showed that the recombinant plasmid of tandem inhibin gene as vaccine for sheep can stimulate FSH secretion, and molecular adjuvant sC3d(sheep complement 3d) can promote immune response, so as to effect follicle development and induce sheep twinning.

To study the expression of microRNA (miRNA) during the early stages of sex differentiation in chicken embryo, and investigate the effect of miRNA on the early development of chicken gonad, six miRNAs (including two chicken novel miRNAs, miR-551b and miR-612) with sexual differential expression were selected based on the results of miRNA chips of chicken (Gallus gallus) embryonic gonads at 3.5~6.5 d and studied by semiquantitative RT-PCR through RNA tailing and primer extension, following validating stem-loop precursor structures of new miRNAs. The results of RNA folding showed that predictive chicken miR-612 and miR-551b possessed stem-loop precursor structures. Semiquantitative results showed the expression of all of the six miRNAs appeared sexual different with varying degrees. Except for 4.5 d, the expression of gga-miR-612 and gga-miR-1456 were higher in females than that in males at other tested stages. The level of gga-miR-551b expression was significantly higher than that in male chicken embryo at 4.5~6.5 d (P<0.05). gga-miR-126 expressed at lower level in female gonads than that in male at 3.5 and 6.5 d, and reversed at 4.5 and 5.5 d. The expression level of gga-miR-1599 was higher in males at all tested stages with significant level at 4.5 d (P<0.01), which was opposite with the expression of gga-miR-10b. Based on the expression results, it is speculated that gga-miR-551b and gga-miR-1599 should have effected on the early process of goand development in chicken embryo.

Troponin CⅢ(TnCⅢ) is one subtype of TnC. In this research, TnCⅢ fusion protein was expressed in Escherichia coli and polyclonal antibody was prepared for further research. A subtype cDNA sequence coding TnCⅢ was obtained from our silkworm(Bombyx mori) pupal cDNA library, named BmTnCⅢ(Bombyx mori TnCⅢ). This cDNA contained a full length of open reading frame(ORF) of BmTnCⅢ coded 153 amino acid residues. According to the cDNA sequence, the primers bearing restriction enzyme sites of BamHⅠand HindⅢ were designed for amplifying the full length of ORF. The intact ORF of BmTnCⅢ(GenBank accession No.DQ889211) was amplified from total RNA of silkworm pupa by RT-PCR, and cloned into a prokaryotic expression vector pET-28a. Following the recombinant plasmid pET-28a-BmTnCⅢ was transformed into E. coli Rosetta (DE3), the fusion protein was observed at approximately 21 kD with the induction of 1 mmol/L IPTG. The interest protein His-Tag BmTnCⅢ was purified by affinity chromatography and used as an antigen to immunize a male New Zealand rabbit(Oryctolagus cuniculus). Then, the polyclonal antibody of the fusion protein was harvested and used in Western blotting analysis. The result indicated that this polyclonal antibody had fairly high specificity and can be used to carry out the researches on tissue distribution and localization of BmTnCⅢ.

Sox2, one of genes which express in embryonic stem (ES) cells, plays an important role in self renewal of ES cells, and is used to make induced pluripotent stem (iPS) cells. In this study, we subcloned mouse (Mus muscules) full-length Sox2 cDNA and inserted Sox2 into pET-41a expression vector. Then the recombinant vector was transformed into Escherichia coli BL21 (DE3) cells to express Sox2 fusion protein. The expressed products were identified by Western blot using anti-GST antibody. The result indicated that the size of Sox2 fusion protein was in 61 kD. And the protein was recovered from the SDS-PAGE. And we used the sox2 fusion protein to prepare polyclonal antibody. The Dot blotting result showed that the anti-Sox2 antiserum had 1∶12,800 titers. The antibody will be useful for studying the function of Sox2 and its role in ES cell self-renewal.

In order to study the function of a putative interferon responsive gene (Ifrg15), the genomic DNA of Kunming White mouse(Mus musculus) was purified and used as the PCR template for amplifying the Ifrg15 coding region. The PCR product was cloned into pGEM-T vector and confirmed by DNA sequencing. The Ifrg15 fragment was subcloned into pET-41(c) vector by EcoR Ⅰ and Xho Ⅰ double-digestion. Escherichia coli BL21(DE3) transformants were then selected and tested by IPTG induction for high level expression of GST&6xHis-Ifrg15 fusion protein. The results showed that mouse Ifrg15 gene was successfully cloned and expressed in the prokaryotic expression system. By using denaturing condition in the affinity chromatographic step, a decent level of purification of GST&6xHis-Ifrg15 fusion protein was achieved.

β-1,3-1,4-endoglucanase is a ubiquitous function enzyme in plant endophytic Bacillus. To evaluate the function of the enzyme, a β-1,3-1,4-endoglucanase gene (bglS) was cloned from endophytic B. amyloliquefaciens strain TB2 and expressed in Escherichia coli. The ORF length of the gene was 732 bp, which encoded 243 amino acids. The theoretical molecular weight of the bglS protein was 27.33 kD and pI was 6.89. The ORF sequence has been registered in GenBank (Accession No. EU368224). The nucleotide and deduced amino acid sequences of the gene shared high identities with that of B. amyloliquefaciens FZB42 β-1,3-1,4-endoglucanase which was associated with plant(The genome accession No. of FZB42 in GenBank is CP000560), and both of the identities were 97%. The ORF and its promoter of bglS were inserted into XbaⅠ/HindⅢ-digested pUC18 vector to construct expression plasmid pUC-bglS which was transformed and expressed in Escherichia coli BL 21. The result of SDS-PAGE showed that about 27 kD protein was expressed in E.coli BL 21::pUC-bglS. The optimum temperature and pH for reaction of the enzyme were 55℃ and 6.2, respectively. The enzyme maintained over 85% of the original enzyme activity between pH 4.4 and pH 6.8 after incubated at 4℃ for 30 min. When the enzyme was stored at temperature below 50℃ for 60 min, it could maintained over 80% of the original enzyme activity. Ca2+ and Fe2+ could enhance the enzyme activity, however the Cu2+, Zn2+, Mg2+ and Mn2+ had an inhibited effect on its activity. The result applies a base for further research on β-1,3-1,4-endoglucanase from endophytic B. amyloliquefaciens TB2.

Streptomyces venezuelae var. qinlingensis is a Zuelaemycin producting strain. For studying on effect of Vitreoscilla hemoglobin gene（vgb） expression in Streptomyces venezuelae var. qinlingensis and on Zuelacmycin production, it expressed through promoter of erythromycin resistance gene, characteristics of engineered strain and original strain growth and metabolism and Zuelaemycin valence were studied in 5 L fermenter. The result showed that: In high dissolved oxygen concentrations, the growth rate and ammonia nitrogen expenditure of engineered strain was slightly lower than that of the original strain, but utilization rate of starch was higher than that of the original strain, Zuelaemycin valence of engineered strain was decreased by 8% than that of original strain; in low dissolved oxygen concentrations, espression of hemoglobin enhanced growth, secondary metabolish and utilization rate of starch, and Zuelaemycin valence of engineered strain was more 45% than that of original strain.

As a member of Gli-Kruppel family, Gli3 can regulate the Sonic hedgehog target genes in the growth and development of animal, especially in the early time. In this study, polymorphisms of four loci(E4, E5, E6 and E7) at the coding region of the bovine Gli3 gene were detected in 187 Nanyang bovine by PCR-SSCP and sequencing method. The fragments showing different SSCP patterns were sequenced, and a total of three SNPs (T123600C, T123696C and A128688G) were found. The E5 locus of Gli3 gene was equilibrium at Hardy-Weinberg, while E4 locus of Gli3 gene was not disequilibrium at Hardy-Weinberg. The association analysis of different genotypes with growth traits showed that the individuals with genotype CC had a larger body weight, chest girth, hucklebone width, average daily gain than that of the individuals with genotypes TT(P<0.05) at six months in E4 locus. It suggests that E4 locus may be one of the marker-assisted selection used as growth traits in Nanyang bovine.

The polymorphisms in exon 17 and 3'UTR of Acyl CoA:diacylglycerol acyltransferase gene (DGAT1) in cattle(Bos taurus) including three Chinese breeds(Luxi, Jinnan and Qinchuan cattle) and four cross-breeding populations (Limousin×Luxi cattle, Simmental×Luxi cattle, Charolais×Luxi cattle and Angus×Luxi cattle) were detected by PCR-SSCP. Five mutations in 3'UTR of DGAT1 gene were found and named 8 402 bp(C→T), 8 414 bp(A→C), 8 445 bp(T→C), 8 465 bp(A→G) and 8 545 bp(G→A), respectively. And the 8 402 bp(C→T) mutation was not reported before. In the studied populations, the locus was at Hardy-Weinberg equilibration (P<0.05 or P<0.01) by χ2 test. Individuals with BB genotype had significantly higher living weight, carcass weight, loin eye area, body condition and average daily gain than those with CC genotype. Individuals with CC genotype had significantly higher marbling score, back fat thickness and intramuscular fat contents than those with BB genotype. In conclusion, B allele of DGAT1 gene is related to the carcass traits such as loin eye area, while C allele of DGAT1 gene was related to meat quality traits such as marbling score, back fat thickness and intramuscular fat contents.

Toll like receptors (TLRs) recognize pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, Chinese holstei and XinJiang brown cattle were used as experimental example. Their genetic polymorphisms of TLR4 gene exon Ⅲ were detected by sequencing first and by PCR-RFLP in the second. Results showed that three poiymorphism loci, TLR4 E3+1656, TLR4 E3+2021 and TLR4 E3+2414,were discovered. Statistical results of χ2 test indicated that two polymorphism sites (TLR4 E3+1656 and TLR4 E3+2021) in the three populations fitted with Hardy-Weinberg equilibrium(P>0.05). The associations were observed between genotypes and SCS by the least square analysis. The results showed that SCS was not significantly affected by different genotypes in TLR4 E3+1656 (P>0.05); the significant different effect between genotype AA and AB was found (P<0.05), the difference was significant between genotype AA and BB was found (P<0.01) and no significant difference were detected between genotype AB and BB(P>0.05) in TLR4 E3+2021 site. In conclusion, the AA genotype in TLR4 E3+2021 site may be the best genotype to resist mastitis, the allele A may play an important role in mastitis resistance in bovine. To research the effect of different genotypes of TLR4 E3+2021 site on SCS, part of the mRNA of the gene expression where in TLRs signaling pathway downstream of Xinjiang brown cattle were detected by Real-Time PCR. The level of Nuclear Factor-kappa b1(NF-κB1), Nuclear Factor-kappa b2(NF-κB2) and Interleukin-1β( IL-1β) transcripts was significantly correlated with genotype of AA and BB in TLR4 E3+2021 (P<0.05), but the level of TLR4, IL-10 and IL-8 transcripts was no significantly correlated with genotype of AA and BB in TLR4 E3+2021(P>0.05).

To make highly expressed swine antimicrobial peptide protegrin-1(PG-1) in prokaryotic expression system, the part of codon of PG-1 was replaced by codon preference of Escherichia coli and a new PG-1 series was designed in this study. Two complementary primers were designed and the whole PG-1 fragment was spliced and amplified by overlap extension PCR. The amplified fragment was inserted into pGEX-4T-1 and the fusion expression plasmid pGEX4T-PG-1 was constructed. The plasmid was transferred into E. coli BL21(DE3) plyS and induced by IPTG (1.0 mmol/L). SDS-PAGE and Western-blotting analysis showed that the recombinant had expressed a 28 kD specific protein in E. coli. By Glutathione Sepharase 4B affinity chromatography, the fusion protein GST-PG-1 was obtained and purified. After cleaved by thrombin, 1.38 mg/L antimicrobial peptide PG-1 was obtained. The recombinant PG-1 showed antibacterial activities against E. coli and Staphylococcus aureus by growth inhibition method.

This study was designed to optimize the factors about onion (Allium cepa) epidermal cells infected by Agrobacterium tumefaciens so as to establish a new transient expression system. Then the new transient expression system was applied to analyze maize In5-2 promoter functional area to clear the position of cis-acting elements responsive to chloroacetanilide. In this experiment onion(Allium cepa) epidermal cells were transformed with beta-glucuronidase(GUS) gene via Agrobacterium tumefaciens. The effects of different concentration of acetosyringone (As), infection period, concentration of Agrobacterium tumefaciens and days of co-culture on the transient expression of GUS gene were analyzed to establish a new transient expression system. The result indicated that a higher transient express of GUS gene could be acquired from onion epidermal cells which were dipped into Agrobaterium suspension (OD600 adjusted to 0.8, concentration of As adjusted to 20 μmol/L) for 15 min, and co-cultured for 3 days. So a new transient expression system was established. Then different length of maize(Zea mays) In5-2 promoter deletions were cloned to vector pCAMBIA1301 and transformed into the new transient expression system to analyze its functional regions. Cis-acting responsive elements to chloroacetanilide could be located within the range of -220 to -143 bp upstream of ATG. The results indicated that the new transient expression system is a fast and effective way to analyze promoter function.

Ochratoxin A(OTA) is a mycotoxin produced by several kinds of fungi growing on food source materials. The main target of OTA is the kidney and liver. However, its plant toxicity is rarely reported. In this paper, we studied the plant toxicity by OTA in Arabidopsis thaliana through the phenomenon and expression of protein during the cell necrosis. The results showed that plant growth was obviously inhibited by OTA, especially the roots and leaves; Trypan blue staining, Evans blue staining and DNA ladder showed that OTA could lead Arabidopsis thaliana cell necrosis, cell membrane integrity damage and DNA crack; Two-dimensional gel electrophoresis maps illustrated that OTA could induce different protein expression in A. thaliana. Some proteins were up regulated, and some others proteins were down regulated, even disappeared. The research indicates the plant toxicity of OTA.

In order to study yellow stripe like from Malus xiaojinensis(MxYSL5) gene expression and regulation, the regulatory sequence(891 bp) of MxYSL5 gene was cloned by Tail-PCR. Silico analysis of sequence suggested that the sequence contained several typical cis-acting elements, including multiple light-responsive elements(LREs), ethylene-responsive element(ERE), gibberellins(GAs)-responsive element(GARE, P-box), TATA-box and CAAT-box. The 891 bp promoter sequence upstream of translation start site of MxYSL5 was cloned and designated as MxYP5-GFP. MxYP5-GFP was constructed and transformed into onion(Allium cepa) epidermal cells by particle bombardment. The result showed that MxYP5 could drive the transient expression of GFP protein in onion epidermal cells, can be used to show the expression and regulation state of MxYSL5.

pFGC5941 is a widely used vector for RNA interference of plant genes, but its spacer is too long, and the restriction sites between the promoter and the spacer are not enough. In this study, a novel spacer based on the 2nd intron of Brassica napus PAP2 (BnPAP2I2) was used to substitute the original PhChsA spacer of pFGC5941, and an AatⅡ site was introduced between the promoter and the spacer, yielding an improved vector pFGC5941M. An RNAi fragment BTT1Ⅰ targeted to Brassica transparent testa 1 (TT1) gene family was cloned. Its antisense and sense fragments were inserted to the promoter-spacer and spacer-terminator cloning sites using NcoⅠ+ AatⅡand BamHⅠ+ XbaⅠ double digestions, respectively. The resulted recombinant vector was pFGC5941M-BTT1Ⅰ(simplified as pBTT1I), which was verified by multiple-PCR checking and successfully transformed into Agrobacterium tumefaciens LBA4404 to form engineering strain. Construction of pBTT1Ⅰ will promote the clarification of the mechanism of development and pigment deposition in the seed coat and molecular breeding of yellow seed trait in Brassica.

The diamondback moth (Plutella xylostella) is the grisly pests for cruciferous vegetables and crops. In this experiment, a cDNA library of the second instar larvae of diamondback moth was built by SMART (switching mechanism at 5' end of RNA transcript) technology. Sequencing and library analysis showed that the original storage capacity of the full-length cDNA library was 1.1×106, storage capacity of the amplified library was 2.6×109, the recombination frequency was 92%, with an average fragment size around 1 kb. All these indicated that the library quality met the requirements. Sequence analysis showed that the library contained some new genes, and some of them have been submitted in GenBank (Accession No. GW883877~GW883893, GU014922 ). Predicate the protein structure of the gene No. 11(GenBank accession No：GU014922) indicate that the gene belonged to gene family of serine protease, which was concerned with important metabolism and immunoreaction. The construction of the library may benefit for finding new gene and lay a foundation for the further genes function analysis of P. xylostella.

The objective of this study is to develop and characterize monoclonal antibodies against duck avian β-defensin 9 (AvBD9). Firstly, the pGEX-duck AvBD9 recombinant protein was used to immunize the female BALB/c Mus musculus (6~8 weeks) three times every two weeks followed by boost immunization 3 d before fusion. Then the duck-AvBD9 gene was subcloned into EcoRⅠand SalⅠrestriction sites of prokaryotic pET-32a vector to construct a recombinant plasmid pET-32a-duck AvBD9. The expression of protein in Escheriohia coli BL21 (JM83-) containing the recombinant plasmid was induced by IPTG followed by SDS-PAGE analysis. The molecular weight of the recombinant protein was 26 kD. The pET-32a-duck AvBD9 protein was purified by nickel affinity chromatography. Mouse antiserum positive against pGEX-duck AvBD9 protein could also recognize pET-32a-duck AvBD9 protein. MAb (monoclonal antibody) against duck AvBD9, designated as 1F11, was finally identified by indirect ELISA and Western blotting assays using the recombinant protein pET-32a-duck AvBD9. The MAb 1F11 could specifically recognize pGEX-duck AvBD9, from pGEX-duck AvBD10 protein, pGEX-chicken AvBD10 protein or pGEX-6p-1 protein. The results of isotype analysis showed that MAb 1F11 belonged to IgG1 subclass and the light chain is the κ chain. The MAb 1F11 with good specificity can be used for further research of duck AvBD9 protein.

Genetically modified corn(Zea mays) MON88017 is developed by U.S. Monsanto Company through recombinant DNA technology, which is characterized by resistances to maize beetles(Diabrotica virgifera) and to glyphosate herbicide. The purpose of this study is to develop and standardize the event-specific qualitative detection method of this line in China. Based on the information of the flanking sequences GM maize MON88017, a series of primer combinations were designed in the left margin of the binding site. The best primer pair was selected and the reaction system was optimized with the best primer pair. The maize MON88017 events-specific qualitative PCR detection method was established. After validated by 7 external laboratories, results showed that this method could specifically detect the samples of MON88017 event with the detection sensitivity about 0.1%, and had a good repeatability and reproducibility，thus suitable for detection of the corn MON88017 and a technical support for the implementation of the biosafety management regulations in China.

In this paper a gene vaccine C1-△CLIP-Ii-F343 containing Newcastle disease virus(NDV) F epitope based on chicken invariant chain(Ii) as carrier and its immune response in mice are studied. First the NDV-F epitope gene vaccine, an endogenous targeting fragment, was constructed by overlap extension method via three cycles of PCR, in which class-Ⅱ associated invariant chain peptide (CLIP) of chicken Ii was substituted with NDV F epitope, F343 (327-359 AA). Secondly the transfection of COS7 cells with C1-△CLIP-Ii-F343 and C1-F343 (no Ii carrier) was observed. And both could efficiently express in the cost cells. Finally twenty-five female Balb/c mice(Mus musculus) of 6~8 weeks old were randomly divided into five groups and injected with C1-△CLIP-Ii-F343 and C1-F343, as experimental controls with C1-△CLIP-Ii, pEGFP-C1 and saline, respectively. After immunizations three times, the humoral immune response was estimated. The C1-△CLIP-Ii-F343 and C1-F343 could induce the mice to produce specific antibody to NDV-F and the titers could reach 1/6400 and 1/1600, respectively. Western blot results showed that the antibodies could bind specifically with NDV allantoic fluid of chicken embryo injected with NDV and PET-NDV-F306 protein of prokaryotic expression. The results suggest that the C1-△CLIP-Ii-F343 epitope vaccine based on Ii as carrier can stimulate specific humoral immune response in mice and it may be a potential candidate gene for NDV vaccine development.

One chromosome with the character of chromosome 4Ag was microdissected from the root tip cell of blue-grained wheat (Triticum aestivum L.(2n=6x=42)×Thinopyrum ponticum Liu & Wang (2n=10x=70)) at mitotic metaphase using glass needle. Then the cell was analyzed by the fluorescent in situ hybridization (FISH). The result showed that the single chromosome left in the cell and the microdissected chromosome were chromosome 4Ag. The isolated chromosome was amplified in vitro by Sau3A linker adaptor-mediated PCR，and from 200 bp to 2 000 bp, especially from 250 bp to 750 bp smear DNA fragments were obtained．Southern hybridization result indicated that DNA from the single chromosome has been successfully amplified. FISH showed that the in vitro amplified fragments of microdissected chromosome were from 4Ag chromosome of the blue-grained wheat．This study broaden the scope of chromosomal mircodissection and provide a useful way to construct specific library and replicate some important genes from a single chromosome．

The hrp genes of Xanthomonas oryzae pv. oryzicola (Xoc) encode type Ⅲ secretion system (T3SS) and deliver T3SS effectors into host cells to trigger hypersensitive response (HR) on nonhost plants tobacco (Nicotiana benthamiana) and pathogenicity on rice (Oryza sativa). Hpa1 protein triggers HR on tobacco and HpaB is an exit protein for T3SS. However, it is still unclear whether hpa1 and hpaB genes together determine Xoc to cause pathogenicity on rice and to trigger HR on tobacco. In this study, we acquired the hpa1 and hpaB single mutant as well as the hpa1hpaB double mutant. In planta assay showed that the hpa1 mutant reduced the virulence of Xoc on rice, whereas still maintained the capacity of triggering HR on tobacco. Both of the hpaB mutant and the hpa1hpaB double mutant induced HR on tobacco, but abolished pathogenicity on rice. These results indicated that other unknown harpin(s) exist(s) in Xoc, which is (are) HpaB-independent effector(s). Immunobloting assay showed that XopQ1Xoc was not secreted through T3SS when hpaB gene was mutated, suggesting that HpaB control the secretion of T3SS effectors that are considered to be pathogenic factors. The above evidences provide the clues to elucidate novel unknown harpin(s) and to analyze the molecular interaction of HpaB-dependent and HpaB-independent effectors with rice.

The aim of this study is to isolate microalgae strains with high lipid productivity from natural water area, and to optimize the fermentation conditions which is the basis for industrial production of biodiesel. Seventeen microalgaes were isolated and cultivated from a variety of fresh habitats, then identified according to their morphological characteristics. A comparison study was made among 11 species based on biomass, lipid content and lipid productivity. Three strains Scenedesmus ovalternus, Chlorella pyrenoidosa and Chlamydomonas reinhardii with highest biomass and lipid content were selected to study the optimal growth conditions including light, pH, temperature, carbon, nitrogen, and combinations of different levels of carbon and nitrogen sources. Our data suggested the three microalgaes grow faster and at the same time had higher lipid productivity when cultured with adding glucose in the dark. The optimal temperature for the three microalgaes was 28℃. The optimal pH for S. ovalternus and C. pyrenoidosa was 7.0, and for C. reinhardii was 9.0. Glucose and urea were the optimal carbon source and nitrogen source for the three algaes, respectively. Considering microalgal lipid productivity, the best carbon and nitrogen sources combinations for S. ovalternus were 30 g/L glucose and 2.1 g/L urea; for C. pyrenoidosa were 40 g/L glucose and 2.1 g/L urea; and for C. reinhardii were: 30 g/L glucose and 1.2 g/L urea. C. reinhardii and C. pyrenoidosa were tested for fermentation in 5-liter fermenter. Compared with the fermentation using shake flask, fermentation time reduced from 7 d to 5 d and cell density of C. reinhardii and C. pyrenoidosa achieved were 61.2 (OD540 nm) in 118 h and 59.86 (OD540 nm) after 5 days. Beside the two microalgae biomass of dry weight increased from 11.2 g/L and 8.8 g/L to 26.58 g/L and 20.19 g/L, respectively; lipid content from 20.3%and 17.2% to 23.2% and 20.1%, respectively; and lipid productivity from 0.3248 g/L/d and 0.2162 g/L/d to 1.2333 g/L/d and 0.8112 g/L/d, respectively. According to the results, through the optimization of fermentation conditions, the lipid productivity of microalgaes Y7, Y9, isolated and screened from natural waters, can reach 1.2333 g/L/d and 0.8112 g/L/d, which are expected to apply in the industrialization for biodiesel production.

Amniotic fluid stem (AFS) cells are the Oct4 positive multipotent stem cells derived from amniotic fluid. These cells possess the robust proliferation, multi-linear differentiation and lowness of immunity and have great promise in different fields of biology and clinical application. In this study, we isolated and characterized the porcine AFS (pAFS) cells devived from different gestational age of porcine fetus, and revealed the correlation between the gestational age of fetus and the proliferation and adhesion rate of pAFS cells. Further more, pAFS cells were able to differentiate into adipocytes when cultured in the special inducing medium. The results showed that pAFS cells derived from the fetus in 40~70 d range obtained the optimal proliferation and adhersion rate. Two porcine AFS cells lines were establishment so far. By FASC and RT-PCR analysis, pAFS cells expressed the following cell surface antigens including CD117, CD44, CD166 and CD90, but negatived for CD34, CD45 and CD54; These cells could be expressed constantly the stem cell special gene markers such as Oct4, Nanog, c-Myc and HLA-abc; The EGPF drived by promoters of Oct4 and Nanog were expressed in pAFS cells when transfected with pOct4-EGFP and pNanog-EGFP; Besides, pAFS cells were successful induced into adipocytes in vitro. This paper presents a method which is useful for the study of AFS cell from domestic animals.

Myostatin(MSTN) is found to be a negative regulator of muscle growth recently, which belongs to transforming growth factor-β(TGF-β) family. Four jlMSTNs genes(jlMSTN1a, jlMSTN1b, jlMSTN2a and jlMSTN2b), with the same genetic structure, were isolated from genome of Cyprinus carpio var. jian using PCR. Homology analysis showed that jlMSTN1a, jlMSTN1b and jlMSTN2a, jlMSTN2b belonged to fish MSTN1 and MSTN2, respectively. The similarities of ORFs between the two paralogs were 96% and 94%, but there were length or sequence differences occurred to the introns. Two introns of jlMSTN2a were obviously longer than that of jlMSTN2b , 1 384 bp, 1 763 bp and 879 bp, 835 bp, respectively. The different expression levels in different tissues and the SNP loci indicated that jlMSTN1s standed higher selection stress than that of jlMSTN2s; jlMSTN2a standed higher selection stress than that of jlMSTN2b. This study also identified that 2 SNP loci associated with body form and ADG (average daily gain) separately, which can be used as markers of assistant breeding.