WRKY proteins are a superfamily of plant specific transcription factors and take part in the growth, development and aging process. In our research, the SRAP-cDNA technology was used to scan the genes which have different expression patterns during ovule development between the V. vinifera cv. Thompson Seedless and V. vinifera cv. Pinot Noir. A 311 bp sequence was got which was a part of the predicted sequence of probable WRKY transcription factor 20-like (XM_002272334.2). then, the full-length sequence (GenBank accession No. JQ782602) was cloned from the ovule of the V. vinifera cv. Thompson Seedless by the RT-PCR, named as VvWRKY20, which was 1 796 bp, with the open reading frame of 1 653 bp, encoding 550 amino acids, containing two WRKY conserved domains, two C2H2 zinc finger domains, two DNA binding sites, and belonged to groupⅠof WRKY family. Expasy-ProtParam analysis showed the molecular weight of VvWRKY20 was 60 140.4 D, the isoelectric point was 6.10, the instability coefficient was 51.01, which proved that VvWRKY20 was an unstable protein. The qRT-PCR was used to analyze the expression of VvWRKY20 in the ovule development of V. vinifera cv. Thompson Seedless and V. vinifera cv. Pinot Noir. The expression levels was relatively stable in V. vinifera cv. Pinot Noir, and the ratio of maximum to minimum was 2.52, while the same ratio was 17.56 in V. vinifera cv. Thompson Seedless. The expression level of VvWRKY20 in V. vinifera cv. Thompson Seedless were higher than that in V. vinifera cv.Pinot Noir at each stages. the ratio of expression in V. vinifera cv. Thompson Seedless to that in V. vinifera cv.Pinot Noir was 15.77 at 10 d, 229.12 at 30 d. These results indicated that VvWRKY20 may be involved in the process of grape ovule abortion.

Allopolyploid can be used as a bridge to overcome distant hybridization barriers and a promising approach to improve varieties. In this study, allotriploid and allotetraploid hybrids between Chinese cabbage(Brassica campestris L.ssp.pekinensis(Lour.) Olsson) and cabbage (B. oleracea var. capitata L.) were identified by morphological, anatomical and cytological observation and molecular markers analysis. Results showed that polyploidy plants showed obvious heterosis in plant types, floral organ and stomata sizes and allotetraploid heterosis was more obvious than that of allotriploid generally. The pollen viability and stomata density of polyploids were lower than that of both parents and pollen viability of allotriploid was the lowest of 18.78%, whereas it reached 81.56% in allotetraploid. The seed setting rates in the selfing of allopolyploids were lower than that of both parents, which in selfing of allotetraploid was 0.28%. The seeds setting rate was higher in crossing allotetraploid with Chinese cabbage than that in their inverse cross combinations, which increased by 2.11 times. The seed setting rate of reciprocal crosses between allotriploid and Chinese cabbage was very low, and no seed was obtained in the selfing of allotriploid. The SRAP assay further confirmed that polyploids included the genetic information of their parents and were the true hybrid, while it was hard to distinguish allotriploid from allotetraploid by SRAP. Cytological identification revealed that the allotetraploid had putative chromosomes (2n=4x=38) and the allotriploid had putative chromosom(2n=3x=29). The obtaining and identification of allopolyploids provide a foundation for the improvement of Chinese cabbage cultivars in the near future.

Hybrid production using male sterile in pepper has been made great progress. However, the molecular mechanism of male sterile is still not clear. Using anthers as material, differential expression of genic male sterile-fertile line in pepper (Capsicum annuum L.) was analyzed by cDNA-AFLP. One hundred and sixty-eight transcript-derived fragments (TDFs) were obtained. Most of the TDFs were not found homologous sequence in GenBank, probably standing for new genes. Forty-eighty TDFs were found homologous sequence(GenBankAccession No.: JK993564~JK993611). These genes were involved in primary metabolism (25%), signal transduction (6.3%), plant development (10.4%), stress response (4.2%), transcription (14.6%), etc.. Twenty-four TDFs were chosen for semi-quantitative RT-PCR to detect the expression pattern in various tissues and different development stages of anther in fertile and sterile plants. The results showed that 11 TDFs displayed expression specificity in time and space and might be male sterile related genes. TDF14 and TDF21 were anther specifically expressed genes and TDF39 specifically expressed in fertile anthers and sepals of fertile and sterile plants. These findings can provide a basic data for molecular mechanism research of genic male sterile, subsequent marker assisted selection and cloning of fertility-related genes in pepper.

In plants, plasma membrane H+-ATPase can mediate proton excretion into the rhizosphere to acidify the soil, increase the solubility of iron and be beneficial to the iron absorption for plants. In order to research function of MxHA2 gene in the process of iron absorption in Malus xiaojinensis, which is a high Fe-utility-efficiency apple genotype, the full length of the MxHA2 gene was cloned from Malus xiaojinensis based on homologous sequence from online Blast results. The MxHA2 gene had an completed ORF(open reading flame) of 2 865 bp, encoding 954 amino acids (GenBank accession No. JQ867095). Phylogenetic tree analysis revealed that MxHA2 protein had a closer relationship to PPA2 of Prunus persica. Subcellular localization assay of the MxHA2 protein, which used transient expression of MxHA2::GFP fusion protein in onion (Allium cepa) cells by particle bombardment, revealed plasma membrane localization of the MxHA2 protein. The expression profiles of the MxHA2 gene were analyzed with Real-time PCR in leaves and roots. The results showed that the MxHA2 gene was expressed both in roots and leaves. And the MxHA2 gene expression was increased by 3 and 6-fold in roots after imposing Fe deficiency. While in the leaves, the MxHA2 gene expression was increased by 2-fold after imposing Fe deficiency for 3 d, continued until 6 d. In order to study the function of MxHA2 gene, we transformed MxHA2 gene into Saccharomyces cerevisiae(BJ2168). The plasma membrane H+-ATPase activity of transgenic yeast was increased very significantly in normal medium, and the growth of transgenic yeasts was higher than controls continually in Fe-deficiency medium. The results obtained in this study suggested that MxHA2 protein had H+-ATPase activity, and its high level expression could improve the Fe-deficiency resistance of Saccharomyces cerevisiae. These results image that MxHA2 gene may play an important role in the process of resistance to iron deficiency stresss in Malus xiaojinensis and have an important application prospect for the molecular breeding of resistance to iron deficiency in fruit trees. Besides this study provides foundation for further study of the mechanism of iron efficient in Malus xiaojinensis.

Plant lipoxygenases(LOX) play an important role in plant response to environmental stress, as well as in plant growth and development. A novel LOX gene designated as CaLOX2(GenBank accession number: JQ219046) was isolated using in silico cloning from Capsicum annuum L. and characterized by RT-PCR. The length of the gene was 2 843 bp, which contained a 2 730 bp long open reading frame(ORF), coding a 910 animo acid polypeptide. The deduced amino acid sequence of the gene contained the typical domain of lipoxygenase and putative transit peptides for chloroplast import. Phylogenetic analysis revealed that CaLOX2 clustered together with well-characterized plastidic typeⅡ13-LOXs genes from tomato(Solanum lycopersicum)TomLOXD, potato(Solanum tuberosum) StLOXH3 and tobacco(Nicotiana attenuate) NaLOX3. Real-time quantitative PCR analysis showed that CaLOX2 transcripts were expressed in all tissues of pepper, however the expression patterns of the gene had tissue specificity，the transcripts were most abundantly expressed in the leaf but much less in flower; the expression level of the CaLOX2 mRNA could be up-regulated significantly after inoculation with zoospores of Phytophthora capsici in both incompatible and compatible interaction. But, its expression level was much higher and earlier at time point in the incompatible interaction than that in the compatible interaction., which indicated CaLOX2 was involed in race-specific resistance to Phytophthora capsici in pepper. The gene expression patterns differed in different pepper organs, its expression level was much higher but later at time point in the leaves than that in the roots after inoculated with Phytophthora capsici race ph1. The expression of CaLOX2 was up-regulated after treatment with mechanical wounding and NaCl stress, but down-regulated by low temperature. This gene was highly induced by H2O2, salicylic acid (SA), and methy jasmonic acid (MeJA). The comparison of CaLOX2 with the well-studied LOXs from other plant species in phylogenetic analysis and expression patterns indicated that the most likely biochemical function of CaLOX2 was in the biosynthesis of JA through allene oxide synthase(AOS) pathway but not in the hydroperoxide lyase mediated (HPL-mediated) production of C6-aldehydes. These results indicated that CaLOX2 is probably involved in the disease resistance and defense response to Phytophthara capsici and other environment stress，such as low temperature and high salt, through SA and JA pathways. The study may provide basic information for further functional analysis of the genes involved in resistance to Phytophthara capsici and other environmental stress in pepper.

Research of the induced pluripotent stem cells(iPSCs) by inducing the SIM-6-thiogunanie-oualiain(STO) cells. Lentivirus vector FUW-OSKM which included octamer-binding transcription factor 4(Oct4), SRY (sex determining region Y)-box 2(Sox2), Kruppel-like factor 4 (Klf4) and c-Myc transfected to 293FT cells by calcium phosphate method. Then, using the virus infected STO cells after lentivirus was packaged and the cells were cultured in the ESC media. The clones were isolated on the fifteenth day cultured on feed layer and none feed layer culture dishs.Through the biological characteristics analysis, we found the cells named STO-iPSCs had the typical morphological characteristics of embryonic stem cells, had a positive result by alkaline phosphatase staining,expressed embryonic stem cells marker Nanog and Oct4 by the immunocytochemistry and high quantitys of Nanog and Oct4 mRNAs by qPCR, especially could differentiate to nerve cells in vitro. In conclution, we obtained STO-iPSCs and stablished the STO-iPSCs training system without raising layer.

Osteopontin (osteopontin, OPN) which is a secreted glycosylated phosphoprotein plays an important role mediated bone tissue and bone matrix during the connection process. For the purpose of enhancing the genetic progress and providing theoretical basis for marker-assisted selection (MAS) of production traits of milk goat, this research detected the osteopontin(OPN) gene polymorphisms of lactating ewe of milk goat(Capra hircus) in Laoshan by adopting the DNA pooling technology and high resolution melting curve analysis technology and analysed the association effect with milk and growth traits by adopting the generalized least squares. Two SNPs were detected among the 174 samples, one of which was the C/T represented by C/T which located at locus I355 of the fifth intron and the other was C/T represented by M/N which located at locus E341 of the seventh exon, whose polymorphism information content (PIC) were 0.2773 and 0.3267, respectively. The distribution of their allele and gene frequency was uneven in breeding farm (F) and breeding peasant (P), namely the Group P of locus I355 deviated from the equilibrium state of Hardy-Weinberg (P<0.05) and the rest was in the equilibrium state (P>0.05). During the analysis of association with lactation traits, the phenotypic values of all traits of Group P were extremely significantly (P<0.01) above those of Group F except for the conductance trait; as for the locus I355, the individual of genotype CC was significantly (P<0.05) above that of genotypes CT and TT in fat content. The traits of ash content and freezing point of genotype CC were significantly (P<0.05) above those of genotype CT. The allele N at locus E341 was the favorable allele of six traits such as non-fat material, lactose rate, milk density, milk protein rate, freezing point and ash content, and the difference of all traits was insignificant (P>0.05). During the analysis of association with growth traits, the body weight and body length of Group F were extremely significantly (P<0.01) above that of Group P; the rump width and rump length of Group P were extremely significantly (P<0.01) above that of Group F. As for the locus I355, the individual of genotype TT was extremely significantly (P<0.01) above that of genotype CC in body weight. As for the locus E341, the allele M was the favorable allele of the body height and bust; the individual of genotypes MM and MN was extremely significantly (P<0.01) above that of genotype NN in weight and rump width; the individual of genotype MM was extremely significantly (P<0.01) above that of genotype NN in the body length; the individual of genotype MM was significantly (P<0.05) above that of genotype NN in height and bust. Through this research, it is found that the OPN gene mutation locus I355 is related to the fat and carbohydrate contents in milk and the mutation locus E341 has significant association effect with the growth and development traits of milk goat, which therefore can be adopted for the molecular genetic markers of some growth traits of milk goat.

MicroRNA is a class of noncoding small RNA. It is involed in the regulation of many parts of life processes, and plays an important role in cell proliferation, differentiation and tissue development. In order to study the roles of microRNA-31 in wool follicle development of sheep, we collected wool follicle samples from Tibetan sheep(Ovis aries) during three different phases including anagen, catagen and telogen. The results showed that there was a significance change of the expression of microRNA-31 during wool follicle development by quantitative Real-time (qPCR). Results showed that the expression of microRNA-31 was decreased from anagen to telogen and the expression in anagen was about 1.5 fold than catagen (P<0.01). In addition, we predicted the precursor sequence of sheep microRNA-31 by miRdeep2 software and found that it was located on the negative strand of sheep chromosome 2 from 89 779 078 to 89 779 137 nucleotides (OAR2.0 version) . Furthermore, we predicted the target genes by RNA22 software. We found that two candidate genes, Keratin17(KRT17) and distal-less homebox3 (DLX3), existed potential binding sites of microRNA-31 in sheep. Then we detected the expression variation of KRT17 and DLX3 genes in anagen, catagen and telogen wool follicle samples by qPCR. As a result, the expression of KRT17 was increased from anagen to telogen. The level in telogen was almost 3 fold than anagen (P<0.01). It was a reverse pattern of the expression of microRNA-31. However, the expression of DLX3 in catagen was higher than the other two phases (catagen and telogen)(P<0.01). Conclusion, we suggested that microRNA-31 may play an impotent role during wool follicle development, and KRT17 probably is one of the target genes which are regulated by microRNA-31.

The determination of geographical origin is a demand of the traceability system of import-export food products, and 16S ribosomal rRNA (rDNA) PCR-DGGE fingerprinting based on fish intestinal microbia has been considered to trace the fish sources. For this purpose, PCR-DGGE technology were used to detect the variation in bacterial community structures in the intestinal contents and intestinal wall of Crisp grass carp (CGC)(Ctenopharyngodon idellus) from Zhongshan and Dang-zai grass carp (DGC)(C. idellus) from Maoming district. The V3 region of bacterial 16S rDNA from fish was amplified by PCR and was analyzed by DGGE. It's demonstrated in the DGGE profiles of intestinal contents that there were 17 bands in the CGC and 15 bands in the DGC, 11 bands co-exiting in both CGC and DGC. The results of molecular identification of specific bands showed that specific bacteria of CGC included uncultured bacteria GU301246.1, FJ675051.1 and GU293197.1, specific bacteria of DGC included uncultured bacteria AY578409.1 and GU301246.1. It's demonstrated in the DGGE profiles of intestinal wall that there were 18, 19 and 16 bands in the foregut, midgut and hindgut of CGC, and 18, 13 and 13 bands in the foregut, midgut and hindgut of DGC. There were 6 bands co-exiting in the foregut, midgut and hindgut of CGC and 3 bands co-exiting in the foregut, midgut and hindgut of DGC. The intestinal wall DGGE profiles displayed that similarities between foregut and midgut, midgut and hindgut, foregut and hindgut of CGC were followed respectively by 50.5%, 54.3% and 33.2%, and those of DGC were 36.1%, 47.7% and 15.4%, respectively. The similarity among the foregut, midgut and hindgut in the DGGE fingerprint clustering of CGC was much larger than that of DGC. The results of this paper demonstrated that PCR-DGGE method could be a traceability tool which provides grass carp products with a unique fingerprinting and makes it possible to trace back the CGC and DGC to their original location.

Fish "liver and gall syndrome", characterized by liver (hepatocyte) injury, has become more and more serious in China aquaculture, however no effective methods have been found for the prevention and treatment of this disease. The present study aimed to develop an in vitro model of hepatotoxicity using tert-butyl hydroperoxide (t-BHP) as hepatotoxicant and evaluate the protective effects of Glycyrrhiza glabra extract against t-BHP induced hepatotoxicity in Jian carp (Cyprinus carpio var. jian). Exposure of the hepatocytes to 1 mmol/L t-BHP for 2 h significantly elevated the levels of glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT), lactate dehydrogenase (LDH) and malondialdehyde (MDA), and reduced the cell viability and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Three concentrations of G. glabra extract (0.1, 0.2 and 0.4 mg/mL) were added to the primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) the exposure of the hepatocytes to t-BHP. Results showed that pre- and post-treatment of the hepatocytes with G. glabra extract at 0.1, 0.2 and 0.4 mg/mL suppressed the elevations of LDH, GOT, GPT and MDA, and reversed the reduced activities of GSH-Px and SOD. Pre-treatment of the hepatocytes with 0.4 mg/mL G. glabra extract reduced the increased levels of GPT, GOT, LDH and MDA, and increased the reduced GSH-Px activity. Post-treatment didn't show significant effects expect for the enhanced GSH-Px activity in the cells post-treated with 0.4 mg/mL G. glabra extract. The significantly increased cell viabilities were observed when the cells were pre-treated, post-treated and pre- and post-treated with 0.2 mg/mL and 0.4 mg/mL G. glabra extract. The results also showed that the timing of G. glabra extract treatment substantially influenced the protective efficacy of the extract, the pre- and post-treatment of the hepatocytes with G. glabra extract was better than that of the other two treatment regimes. It can be concluded that G. glabra extract exhibited protective effect against t-BHP-induced hepatotoxicity in fish. Further in vivo studies are needed to provide more evidence for using G. glabra as a hepatoprotective agent for the prevention and treatment of fish "liver and gall syndrome".

Porcine reproductive and respiratory syndrome virus(PRRSV) is a major pathogen of economic losses in pigs(Sus domesticus), causing severe reproductive failure in pregnant sows and boars, and respiratory problems in young pigs. This study was aimed to detect the expression of the recombinant co-expressing GP3 and GP5(N-glycosylation protein) and M(membrane protein) fusion protein of PRRSV in PK-15 cells (Porcine kidney cells), and to detect their immunogenicity. The eukaryotic recombinant plasmid pcDNA3.1-ORF3-ORF5-ORF6 was transfected into PK-15 cells by lipofectamineTM 2000 reagent, the stable expressive cells were selected (72 h) by G418 (μg/mL). We designed three pairs of primers based on the published sequences of PRRSV, the ORF3 and ORF5 and ORF6 was transfected into PK-15 cells, and then detected by RT-PCR, the GP3-GP5-M fusion protein was detected by IFA (indirect immunofluorescence assay). The immunogenicity of GP3-GP5-M was detected by Western blot and ELISA (enzyme linked immunosorbent assay). S/P (the ratio of sample/postive control) reached 1.80 after the second immunization, and lasted for at least 1 monthes. The results showed that the fusion protein GP3-GP5 (N- glycosylation protein)-M (membrane protein) could express in mammalian cells. Moreover, the fusion protein showed good immunogenicity. The recombinant plasmid pcDNA3.1-ORF3-ORF5-ORF6 may be an attractive candidate vaccine for the prevention and control of PRRS.

Plant-growth promoting rhizobacterium (PGPR) Bacillus cereus AR156 is a potential bio-pesticide. In order to study the mechanism of drought tolerance in tomato (Lycopersicon esculentum) induced by B. cereus AR156, we tested relative water content, malondialdehyde(MDA) content, relative electrical conductivity and chlorophyll content of leaves and recovery intension of roots, the antioxidant enzymes activities and the expression of cytosolic ascorbate peroxidase gene (cAPX), monodehydroascorbate reductase gene (MDHAR) and ribulose-1, 5-bisphosphate carboxy/oxygenase(Rubisco) small subunit gene(rbcS) under drought stress condition. Results showed that after treated with culture supernate of AR156, relative water content of leaves was 82% comparing to control group, increased by 44%, recovery intension of roots was 0.336 mg/g/h, increased by 100%, chlorophyll a, chlorophyll b and total chlorophyll were increased by 16%, 20% and 20.2%, respectively, and the expression of rbcS maintained a stable high level, besides MDA content was 2.2×10-3 μmol/g FW, decreased by 21.4%, relative electrical conductivity was 56.0%, decreased by 28.2%, compared with control treatment. What's more, the activities of superoxide dismutase(SOD), peroxidase(POD) and hydrogen peroxidase(CAT) were increased markedly after applying with the culture supernate of AR156, and the expression of cAPX and MDHAR were up-regulated at anaphase of drought stress. Moreover the survival rate of tomato 24 h after re-watering were increased markedly, compared with control group. These results suggested that the culture supernate of AR156 increased drought tolerance of tomatoes by protecting plant cell, maintaining photosynthetic efficiency and increasing some of peroxidases activities. This made a contribution to the studies on the mechanism of systemic tolerance induced by PGPR. These results demonstrate the primary mechanism of drought torelance in tomato induced by AR156, and also provide theoretical basis for the application of AR156 as a good drought tolerance biological inducer.

Yellow peach moth, Conogethes punctiferalis(Guenée) (Lepidoptera: Crambidae), which is a polyphagous insect pests, has become more serious on corn in some corn planting areas in recent years. To investigate the genetic differentiation and phylogeny evolution within and among the different populations of the C. punctiferalis in China, we analyzed mitochondrial cytochrome oxidase Ⅱ (COⅡ) sequences of 24 geographical populations collected from different provinces and cities in China, including Guangxi, Guangdong, Sichuan, Hubei, Zhejiang, Shandong, Hebei, Henan, Beijing and Liaoning. In the total number of 622 C. punctiferalis individuals, 57 variable sites and 53 haplotypes were detected respectively (Genbank Accession numbers: JQ363873~JQ363922). In the nucleotide of C. punctiferalis partial COⅡ gene sequences, the average A+T content was 75.32%, which indicated obvious base A/T bias. In all 53 haplotypes, Hap2 was the most common haplotype, which included 67.36% of individuals tested, while 31 haplotypes were very rare in C. punctiferalis populations which were only appeared once in all collected samples. The average gene flow (Nm) among the 24 populations was 1.09, and the overall fixation index (Fst) was 0.585. It revealed that there was different level of genetic differentiation among the C. punctiferalis populations in China. Fst value of South region (Fst=0.24381) was higher than that of North and East regions (Fst=0.01197) and Southwest region (Fst=0.0133). The result was in accord with gene flow results (North and East regions Nm=41.58, Southwest region Nm=118.90, South region Nm=0.99) which indicated that the level of gene flow was very low with obvious differentiation among the different populations in South region and other regions. However the gene flow among the different populations of C. punctiferalis in North and East regions and Southwest regions were considerably frequent. Those variable sites accounted for 7.87% of all sites analysed. The Neighbor-Joining (NJ) phylogenetic tree and network relationship of the 53 haplotypes revealed that there was long genetic distance among haplotypes between South region populations of Guangdong, Guangxi and other populations, while no remarkable correspondence between haplotypes and geographical distances were found in other populations. Molecular variance (AMOVA) analysis indicated that a high proportion of the total genetic variance was attribute to variations within populations (89.99%); We analyzed the nucleotide mismatch distribution and did neutrality test using Tajima's D and Fu's Fs test method, the value of Taijima's D (-2.5776) was at significant level (P<0.01), and Fu's Fs was below zero. Besides, the mismatch was appeared uneven unimodal pattern. All these result suggested that for C. punctiferalis, there might have been population expansion in history, and the population expansion happened a long time ago (presumptively about 46 700 ~116 800 years ago).

Protocorm-like bodies (PLBs) can be efficiently induced by TDZ from Rosa canina rhizoids. More understandings on the molecular biological mechanism of PLBs induction contributes to the establishment of protocorm-like bodies protocols of cut roses. One important premise of the mechanism study in the molecular biological level is to quickly and accurately clone and screen the key genes during PLBs induction. On the basis of 'conserved sequences' in 5' untranslated regions(5'UTR) of homologous genes with high scores of Malus×domestica, Prunus persica and Fragaria vesca, we designed the forward degenerate primers to directly isolate the cDNA 5'ends of PIN1(pin-formed) and PLT(plethora) of R. canina by PCR amplification using the cDNA template catalyzed by M-MLV reverse transcriptase. The results showed that there were some conserved fragments in the 5' rapid amplification of cDNA ends(5'UTR) of RcPIN1, RcPLT and their homologous genes with high scores of Malus×domestica, Fragaria vesca and Prunus persica, which indicated the above method was practicable. With this method, costly 5'RACE kits were entirely unnecessary because no homopolymer sequence and abridged anchor primers were to be introduced to the 5' ends of cDNA template, for genes from plants of Rosacease family. As a result, this method took advantages of speediness and economy. It is believed that this method will be a practical pathway and worthy reference to identify cDNA 5'ends for some Rosaceae plants.

Porcine transmissible gastroenteritis (TGE) is one important causative enteric infection caused by Transmissible gastroenteritis virus(TGEV). The S and N genes of TGEV have important immunological function respectively, a DNA vaccine co-expressing S and N gene will provide better immune efficacy for controlling. In this research, the S gene fragment (2.1 kb, including A, B, C and D antigenic sites of the S protein) and the full-length N gene(1.2 kb) of TGEV were amplified by RT-PCR, respectively. The N and S genes were respectively inserted into the multiple cloning sites (MCS) of the dural-promoter eukaryotic vector pVAXD to construct the recombinant plasmid pVAXD-N and pVAXD-S firstly. The N gene was subsequently inserted into the recombinant plasmid pVAXD-S to construct the dural-promoter eukaryotic plasmid pVAXD-N/S. The plasmid pVAXD-N/S and the control plasmids(pVAX-S, pVAX-N and pVAXD) were transfected into COS-7 cells to express. The transcription of the S and N genes could be detected by RT-PCR from transfected COS-7 cells. The expression of pVAXD-N/S in COS-7 cells could react with the rabbit anti-S protein serum、the rabbit anti-N protein serum and the rabbit anti-TGEV serum, respectively, by indirect immunofluorescence assay(IFA), the fluorescence intensity detected from the pVAXD-N/S transfected COS-7 cells was consistent with that detected from the pVAXD-N group and pVAXD-S group. The results indicated that the dural-promoter eukaryotic vector pVAXD-N/S could express the S protein and N protein at the same time. Six-weeks-old BALB/c mice(Mus musculus) were immunized with the plasmid pVAXD-N/S and the control groups(pVAXD-N, pVAXD-S and pVAXD). The serum IgG antibody was measured by indirect ELISA. The result showed that specific anti-TGEV serum IgG antibody could be detected at day 14 post-immunization, and the antibody peak was detected at day 35 post-immunization. The IgG antibody level induced by pVAXD-N/S was significantly higher than that induced by the pVAXD-N group or pVAXD-S group(P<0.5), and was consistent with that induced by the group(pVAXD-N+pVAXD-S). This study provides a basic materials for developing a bivalent DNA vaccine of TGEV.