Abstract: To exploit the porcine circovirus type 2 (PCV2) DNA vaccine to prevent the PCV2 disease, the recombination chimeric gene of PCV2-linker-PoIL-2 constructed by PCV2 ORF2 gene linked porcine interleukin-2 (PoIL-2) mature peptide gene via a 15-amino acid glycine-rich linker [linker,（G4S）3] by SOE-PCR (splicing by overlap extension-PCR) method was cloned into pGEM-T Easy vector and subsequently sub-cloned into eukaryotic expression vector pcDNA3.1(+). The positive recombination expression plasmid of pcDNA3.1/PCV2-linker-PoIL-2 (rpcDNA3.1/PCV2-linker-PoIL-2) was selected and transfected into COS-7 cells by lipofectamine. The bioactivities of the expressed recombinant fusion protein (rCap-linker-PoIL-2 protein) in COS-7 cells were tested by the methods of indirect immunofluorescent assay (IFA) for detection PCV2 antigen and porcine IL-2 ELISA assay for detection PoIL-2 protein, respectively. The immune effects in Balb/c mice induced by rpcDNA3.1/PCV2-linker-PoIL-2 and recombinant expression plasmid of pcDNA3.1/OFR2 (rpcDNA3.1/OFR2) were evaluated by ELISA assay for detection PCV2 antibody. The result showed that the chimeric gene of PCV2-linker-PoIL-2 and the rpcDNA3.1/PCV2-linker-PoIL-2 were successfully obtained and constructed, respectively. The rCap-linker-PoIL-2 protein, which was existed in the cytoplasm of COS-7 cells and expressed by rpcDNA3.1/PCV2-linker-PoIL-2, displayed the specific immune response for anti-PCV2 and anti-PoIL-2 antibody by IFA and ELISA assay, respectively, which indicated the rCap-linker-PoIL-2 protein had the duplex bio-activity of Cap protein and PoIL-2 protein. Although the specific antibody response to PCV2 in mice could be induced by being intramuscularly immunized with ether rpcDNA3.1/PCV2-linker-PoIL-2 or the rpcDNA3.1/OFR2, the antibody titer induced by former was much higher than that caused by the latter after the second and third immune. Our study laid the good foundation for further PCV2 DNA vaccine development.

According to the antigenic analysis of duck plague virus (DPV) gB protein , one pair of primers were designed, with which the gene fragment coding the high antigenic domain of DPV N-terminal gB protein was amplified from the DPV genome. The segment was cloned into pET32a vector to obtain the recombinant pET-gB1 plasmid. The recombinant plasmid was transformed into E. coli BL21, and expressed in very high level as inclusion body after induced with IPTG. The expressived product analyzed by SDS- PAGE and Western blotting. The result indicated that the fusion protein (pET-gB1) has specific antigenicity. The inclusion body was solubilized in urea (8mol/L) . The purified protein was obtained by use of His•Bind affinity chromatography. Mice were inoculated transcutaneous three times at a two-weeks interval with fusion protein and normal saline. To evaluate the prophylaxtic efficacy of fusion protein , Titers of antibodies was detected by ELISA and proliferation of lymphocytes were determined by MTT. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses.

A rapid real-time polymerase chain reaction (RT-PCR) for detection of WNV was established in this study. Primers were designed according to capsid protein gene with Primer Premier5.0. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. Amplifying curve showed that this method could successfully amplify WNV gene, nevertheless reference Japanese encephalitis and blank control were all negative. 10-fold dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The newly-built real-time PCR has high sensitivity, good specificity, reliable stability, so it has potential to apply in inspection and quarantine of WNV.

Malus xiaojinensis is iron-efficient genotype in the genus Malus. We isolated and cloned full-length cDNA sequence of MxFRO from Malus xiaojinensis , Semiquantitative RT-PCR was conducted, and MxFRO was found to be induced by iron-deficiency stress in roots and leaves. Yeast was transformed with MxFRO gene, and the ferric reductase activity in transgenic yeast was 2.8-fold that of controls.We concluded that MxFRO possessed ferric reductase activity.

A rapid and sensitive real-time PCR assay using the Roter Gene 3000 sequence detection system coupled with SYBR Green І chemistry was developed for the quantitative detection of turbot reddish body iridovirus (TRBIV) isolated from farmed turbot, Scophthalmus maximus. The assay involved the amplification of a 152 bp DNA fragment from a conserved region of TRBIV MCP gene. The PCR mixture contains a fluorescence dye, SYBR Green І, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid pUCm-T/TRBIV MCP containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (Ct ) value and logarithmic positive plasmid concentration were close to one (r2=0.99806) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23106 and 2.18106 viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed large number of cultured turbots were infected and TRBIV has been epidemic widely in turbot farms locating at Shandong peninsula. These results suggested that the real-time PCR assay reported here could be used as a rapid, sensitive and quantification method for TRBIV.

The GST-pMGA was expressed in E. coli BL21 with the pGEX fusion expression system. GST-pMGA expressed in supernatant was purified using GST·Bind Resin and a plenty of soluble protein was obtained, Overnight incubate GST-pMGA with thrombin protease, the pMGA was obtained and that showed well immunologic competency by Western-blot analysis. Indirect immunofluorescent assay was used to test interaction between pMGA and the tissue paraffin sections of trachea, heart, lung, liver, kidney, thymus, bursa of fabricius, spleen, proventriculus and the duodenum of SPF chick embryo; The results showed that pMGA and the tissues of chick embryo had a specific binding except the spleen, this binding indicated receptor of pMGA on the tissues of chick embryo. This will supply a foundation for the further study in receptor of pMGA.

Fast skeletal myosin light chain 2 (HUMMLC2B or MYLPF) is a Ca-binding protein which participate in many vital movements, and it has become a hot spot in the myosin studies in recent years．Using PCR-RFLP，the distributes of MspⅠ polymorphisms (T613C) in intron 1 of HUMMLC2B gene in seven pig populations were analyzed，and the relationship between the HUMMLC2B genotypes and pork quality and carcass traits was evaluated．The results showed the frequency of allele B were predominant in all other breeds except for Landrace, in which the ratio pf allele A and B was 1:2 and the homozygote of allele A was found. In six detected pig populations, very significant effects of this locus on loin eye width, loin eye area, intramuscular and water moisture (P<0.01) were found, and this locus was significantly associated with lean meat percentage and skin percentage (P<0.05). The expression of HUMMLC2B in the tissues also was examined. It needs further studied whether the polymorphic site is to be used as the candidate locus affecting carcass and meat quality traits in markers assisted selection.

The present study is to construct prokaryotic expression vector of bovine Nanog gene and to induce its expression in E.coli (JM109). By means of reverse transcription –polymerase chain reaction(RT-PCR), cDNA of bovine Nanog gene was amplyfied from the tissues of fetal primodial genital ridges at age of six weeks. It was inserted into PMD18-T vector , then subcloned into vector pGEX-KG by gene recombination technique. After being confirmed by restriction enzyme digestion and sequencing, the recombinant plasmid pGEX-KG -Nanog was induced by IPTG（0.1-2m M/L）to express in JM109 at 25, 30, and 37℃. The results showed that（1）IPTG concentration and culture temperature exerted little impact on the expression yield of GST- Nanog fusion protein in JM109；(2) as indicated by western blotting , expressed efficiently in JM109 and reactivated with GST antibody, the fusion protein is about 60KD, which can be used to prepare polyclonal antibody of bovine Nanog gene.

Abstract：【OBJECTIVE】To establish the method of in situ hybridization (ISH) with digoxin-labeled probe and detect the expression of FcRn in the small intestine of piglets .【METHOD】A digoxin-labeled single-strand cDNA probe for ISH detection of FcRn mRNA was plotted according to the conserved region of the gene for α chain of porcine FcRn. The ISH method was optimized through a series of trials such as the elimination of endogenous HRP, fixing agent, cellular membrane proteopepsis agent , hybridization solution, time and temperature of hybridization treatments. 【RESULTS】①The optimal thickness of the porcine small intestine tissue slice was 14 μm; ② Endogenous HRP was present in the intestine tissues and could be eliminated by the solution of 3％ H2O2 , whereas this step of treatment was proved unnecessary in this research because the enzyme did not apparently interfere with the ISH detection effect; ③The limpid hybridization signals of bright brown particles were detected in the intestinal glands and submucosa of duodenum, jejunum and ileum.【CONCLUSION】The established ISH method was proved to be effective. FcRn mRNA was expressed in the tissues of duodenum, jejunum and ileum of piglets, which indicated the correlation of the presence of FcRn and IgG transportation in the intestine.

Vibrio parahaemolyticus has been considered as one of the most important foodborne bacterial pathogens. In this paper, the loop-mediated isothermal amplification (LAMP) that amplifies DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of this pathogen for the first time. A set of four primers, two outer and two inner primers, was designed specifically to recognize the thermolabile hemolysin gene (tlh) of V. parahaemolyticus in this study. Genomic DNAs from 28 bacterial strains including 14 V. parahaemolyticus strains were amplified using LAMP, and no amplicon was observed in other bacterial strains. The detection limit of this LAMP assay was around 90 fg/LAMP Mixture of V.parahaemolyticus genomic DNA, 24 colony forming units for pure cultures, 89 cfu/g for non-cultured artificially contaminated food samples. In addition, 40 seafood samples were tested, and 8 samples were found V. parahaemolyticus positive by LAMP. Among the tested positive samples, 6 samples were detected positive by traditional culture methods. These results suggested that detection of V. parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid detection and identification of V. parahaemolyticus.

Abstract: 【Objective】Using Real time PCR method detect three functional bacterias’ 16S rDNA copies in rumen liquid such as Anaerovibrio lipolytica, Fibrobacter succinogene, Ruminococcus flavefaciens, and building the method to reflect these bacteria quantities.【Method】At the same diet conditions ,collecting the rumen liquid microbe of different cows at the same time and the same cow at the different time. And extracting their total DNA. Design the special primers according to 16S rDNA. Then using real time PCR detect the different quantities of three functional bacteria.【Result】Three functional bacteria quantities at the same time have little different, however, there are obviously different about the same cow at different time point (P＜0.05). The main reason perhaps were the error on progress or method of collecting rumen bacteria samples.

In vitro mature culture, collection with isolation and preservation of first polar bodies (PbΙ) in porcine oocytes were introduced in this study in order to supply some essential reference for recombination of porcine oocytes by the nucleis of PbΙ. Mature oocytes were obtained by in vitro culture of the follicle oocytes in ovary from abattoir, Expulsion, activity and morph of PbΙ were statistically analyzed. PbΙ was isolated and Collected by two methods of enzyme digestion or micrergy, their survival vigor under the different temperature preservation were approached and evaluated by Trypan blue staining method. The results showed that lots of high vigor PbΙ could be found by in vitro mature culture after 40 hours. The effect on isolation of PbΙ by micrergy outstripped enzyme digestion significantly, the isolated PbΙ by micregy existed ideal vigor and normal morph. Most of PbΙ survived during 4 hours under 39 ℃, rate of survivors was 85.0 % during 40 hours under 4 ℃, and over 95.0 % during a week under -20 ℃. The effect of cryopreservation was best for PbΙ, the rates of survivors and their normal morph were as high as 89.1% and 97.8%, respectively.

Abstract: In order to prepare the nuclear donor cells for bovine transgenic cloning, bovine fibroblast cells were isolated by attaching tissue explants from ear skin of a bovine. Then the cells were purified and the cell growth curve was plotted. The purified plasmid containing two genes of hLYZ and EGFP was transfected by lipofectmine 2000 into bovine fibroblast. The cells were checked 24h hours after transfection under fluorescence microscopy for EGFP expression, and G418 selection (500μg/ml) was applied since then. After 2 weeks, positive colonies were maintained in culture medium containing 250μg/ml G418 for 2 - 3 passages. A small part of the positive cells were analyzed by PCR for gene integration. PCR detection demonstrated that the foreign gene was integrated into the genome. The results indicated that the isolated bovine fibroblast cells might be competent for transgenic cloning.

Objective The effects of the characteristics of amniotic fluid on the culture of human amniotic fluid stem cells were discussed in this study. Method Amniotic fluid cells were cultured to examined the effect of different factors on the culture of human amniotic fluid stem cells as measured by the numbers of the attachment cells and the attachment time. Result The attachment time was significantly influenced by the gestational age (p<0.05), and the time was longer when the amniotic fluid was collected at the third trimester of gestation. The attachment time was also affected by the blood pollution level. The volume of the amniotic fluid influenced the numbers of the attachment cells and the attachment time, while the size of the cell pellet had no distinct effect on it. Conclusion The gestational age, the blood pollution level and the volume of the amniotic fluid could influence the numbers of the attachment cells and the attachment time at a certain extent.

Abstract: Use microsatellite technology to study Genetic diversity of wild populations (HYE, XYE,ZYE) and cultured Populations(XYA,XYA,ZYA) of Lutjanus erythopterus and Lutjanus stellatus and Lutjanus argentimaculatus from South China sea. The number of total alleles of the ten microsatellite loci is 78, the average number allele of every locus is 1~8. Mean observed heterozygosity (Ho) of six populations is:ZYE(0.9550)>ZYA(0.8900),HYE(0.8950)>HYA (0.8400)，XYE(0.8450)>XYA(0.8100); mean polymorphism information content (PIC) range form 0.3648 to 0.7964, the number of PIC of wild populations is greater than cultured populations. Genetic distances between wild populations and cultured populations of three species in respective snapper is 0.1029，0.0371，0.0135 and coefficients of gene differentiation is 0.0371，0.0211，0.0352. Study of population genetic structure shows that genetic diversity of three species of snappers is abundant, genetic diversity declines in cultured populations. faint genetic Differentiati- on exists between wild populations and cultured populations.

The channel catfish (Ictalurus punctatus) was introduced from America to China in the 1984. It has been cultured on a large scale in 20 provinces of China. EST-SSRs was used to analyze the genetic diversity of domestic stocks of channel catfish (Ictalurus punctatus).The four populations were the population in Fuzhou province and Hubei province which introduced in 1984(P1984), the population in Fuzhou province and Liaoning province which introduced in 1997(P1997), the population in Hunan province which introduced in 2004(P2004),and the population in Fuzhou province which was cross breeding between P1984 and P1997(P8497). EST-SSR marker is a new kind of SSR markers which is developed from the ESTs. It not only act as genetic markers, but also reveal differences in related gene expression. In this study, two gene sites were found whose functions were clear. Thirty individuals from each population were analyzed．Amplifications were performed in a PCR reactor for the genetic diversity with 10 EST-SSRs markers. All the primer sets produced discernible DNA fragments, of which 9 primer showed polymorphism. In this study, 32 alleles were obtained．The number of alleles of each locus ranged from 2 to 4, and allele size ranged from 100 to 212 bp. The mean number of alleles (A) ranged from 3.0000 to 3.4000. The mean number of effective alleles (ae) ranged from 1.9455 to 2.3024. The average observed heterozygosity (Ho) for the four populations was 0.5334, 0.4768, 0.5982, and 0.5092, respectively. The average expected heterozygosity(He) for the four populations was 0.4943, 0.5695, 0.4913 and 0.5091, respectively. PIC (Polymorphism Information Content) per locus ranged from 0.4113 to 0.4829. The difference in genetic diversity among the four populations is not significant, and the genetic diversity of four populations was in moderate level. According to gene differentiation (Gst), genetic distance (D), Genetic similarity (S) and UPGMA, we found that the relationship between P1984 and P8497 was nearest, and the relationship between populations P1984 and P2004 was farthest, which proves the order that introduced into China among the four populations. The P value test of Hardy-Weinberg equilibrium showed that 18.75% loci departed from Hardy-Weinberg equilibrium. The results of the F-statistics suggested the surplus heterozygosity for P2004 and P8497, and the absent heterozygosity for P1984 and P1997, and low genetic differentiations among the four populations，and mainly differentiations due to individual difference.

N6 medium, MS medium and BN medium were adopted in tissue culture of Nipponbare (Japanica) Scutellum for performance comparison. Results indicated that, among the three kinds of culture mediums, NB medium worked best in the calli induction and gene transformation and was most suitable for tissue culture of Nipponbare scutellum. Based on this, elicitor-encoding gene pemG1 from Magnaporthe grisea was introduced into the genome of Nipponbare via Agrobacterium-mediated method. Transgenic rice plants were obtained. The integration, transcription and expression of pemG1 in rice were respectively confirmed by PCR, Northern blot and Western blot. Genetic analysis showed that the transgenes were segregated normally in the progenies.

Bt gene (GFM CrylA) was induced into immature embryos derived from Tie7922, a Chinese northeast spring maize inbred line, by particle bombardment. Transgenic lines with stable resistance to corn-borer were gained by the inoculation of corn-borer eggs in field and the experiment for leaves fed on corn-borer indoor for three succession years. Three hybrids (Simi25Bt, Tongdan24Bt, Jidan209Bt) were created by the hybridization of transgenic lines and common lines, which belonged to improved Simi25, Tongdan24, Jidan209. The results of resistance identification showed that the difference of resistance among transgenic lines was very significant; it had the difference among individuals of the same line and among three hybrids, too. The resistance of hybrids derived from transgenic lines was more than that of common hybrids (CK). The investigation of agronomic characters showed that it had no significant difference between improved hybrids and CK in plant height, ear length, row number per ear, kernel number per row and 100-kernels weight. The paper thought that GFM CrylA (Bt) gene could be applied in the improvement of resistance to corn–borer and maize inbred lines with Bt gene could directly be applied in conventional breeding.

AFLP markers linked to stem nematode resistance gene were developed in sweetpotato (Ipomoea batatas (L.) Lam.). Using bulked segregant analysis (BSA), 800 AFLP primer combinations were screened in the resistant and susceptible DNA bulks from the 186 progenies of F1 single-cross population of Xu781 (resistant parent) × Xushu 18 (susceptible parent), and 245 of them showed polymorphic bands between the two bulks. Primer combinations detecting polymorphism between the two bulks were used to screen the parents and eight individuals from each of the bulks. The results showed that E2M23 and E33M20 produced a specific band of about 500 bp and 200 bp in length, respectively, in the resistant plants but not in the susceptible plants, suggesting the markers named E2M23500 and E33M20200 linked to a gene for stem nematode resistance. The amplified analysis of the 186 F1 individuals indicated that the genetic distance was 6.9 cM and 11.1 cM, respectively, between the two markers and the stem nematode resistance gene with Mapmaker 3.0. These two AFLP markers were used to identify 10 sweetpotato varieties widely planted in China and the results were consistant with those of conventional resistance identification, indicating that the two markers can be used in molecular marker-assisted breeding for stem nematode resistance of sweetpotato.

All experiment methods were performed according to the standard of molecular clone technology. The DNA fragment of GFP gene was inserted into the pBI121-Lyz to get the recombined eukaryotic expression vector pBI121-Lyz-GFP. Polymerase chain reaction (PCR) was used to proliferate GFP gene from pBI121-Lyz-GFP plasmid, producing an approximate 750bp band with restriction enzyme SmaI and BamHI at both sides of the DNA. The eukaryotic expression vector was also verified by nucleotide sequencing. Plasmid pBI121-Lyz-GFP was introduced into agrobacterium tumenfaciens strain LBA4404. Discs of HeTian alfalfa were transformed with LBA4404 strain. Kanamycin resistance was selected and induced to regenerate alfalfa callus. The observation of GFP expression in HeTian alfalfa callus with slides was carried out under the fluorescent microscope upon excitation of blue light. PCR amplification and the results of observation showed that the recombined plasmid was transformed into HeTian alfalfa callus successfully.

AtNHX1 antiporter gene from Arabidopsis was successfully introduced into white clover plants through their cotyledons, mediated by Agrobacterium tumefaciens. Transgenic plants resistant to Kanamycin were obtained after selection and regeneration. All of the transgenic plants were tested with PCR, Southern blot and Northern analysis, which indicated that AtNHX1 gene has already been integrated into white clover genome and expressed. Indoor salinity test showed that transgenic plants expressing AtNHX1 gene had significantly higher total leaf area and aboveground dry weight compared with non-transgenic controls, illuminating that vacuolar AtNHX1 gene can help to increase salt tolerance in white clover.

The plant expression vectors pCAMBIA1301PMI and pBIPMI were constructed by substituting Escherichia coli phosphomannose- isomerase (PMI) gene for hpt gene of pCAMBIA1301 and GUS gene of pBI121. Epicocyl explants of ‘Xuegan’ sweet orange were inoculated with EHA105- pCAMBIA1301PMI and EHA105- pBIPMI and subsequently selected on medium supplemented with a combination of 25 g•L-1 mannose and 5 g•L-1 sucrose as a carbon source. The transformation efficiency rate was 27.7% when transformed by pCAMBIA1301PMI and 12.7% by pBIPMI .Genetic transformation was confirmed by Chlorophenol Red assay and PCR. A new method for obtaining transgenic ‘Xuegan’ plants was developed using PMI/mannose selection system.

The DNA and cDNA fragments of SRK kinase domain(named as SRKE1) was amplified from stigma mRNA of Brassica oleracea L. by PCR and RT-PCR. Their lengths were 1241 bp and 1711 bp respectively. Sequence analysis indicated that the SRKE1 contained six exons and five introns，the SRKE1 encoded 407 amino acids. The coding sequence of SRKE1 were inserted into vector pET43.1 and pPIC9K to be the recombinant plasmid pET43.1-SRKE1 and pPIC9K-SRKE1. The recombinant protein were expressed in E. col(BL21) and Pichia pastoris GS115 repectively. When the SRKE1 was incubated with THL1 which we had expressed, SRKE1 could bind with THL1 and formed a stable complex.

ABSTRACT: Azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] is an important legume crop grown for its protein rich grains. Firstly, 45 microsatellite DNA markers were used to amplify genomic DNAs from 80 accessions of azuki bean, including 53 accessions of Chinese azuki bean and 27 accessions of Japanese azuki bean in this work. As a result, 18 pairs of SSR primers could amplify polymorphic single-locus SSR from all of these materials, and these polymorphic markers were then used to analyze genetic diversity of azuki bean. A total of 92 alleles was detected, among them 89 and 74 alleles were detected in 57 accessions of Chinese azuki bean and 27 accessions of Japanese azuki bean respectively. The average number of alleles per locus was 5.4 for all the accessions, 4.9 for Chinese azuki bean, and 4.1 for Japanese azuki bean. The polymorphic information content (PIC) of each SSR locus varied from 0.23 to 0.83 with an average of 0.64 in all the accessions, from 0.23 to 0.86 with an average of 0.63 in the Chinese azuki bean, and from 0.20 to 0.81 with an average of 0.61 in the Japanese azuki bean. Genetic differences between the Chinese and Japanese azuki bean were detected for specific alleles, PIC and pairwise genetic similarity. UPGMA cluster analysis of the similarity data basically separates the eighty accessions into five groups. A dendrogram based on the microsatellite analysis generally agreed with the pedigree of the azuki bean accessions. These results indicated that Chinese and Japanese azuki bean germplasm could be used for broadening genetic base of current cultivars, and the SSR markers should be useful for genetic mapping, genotype identification, and marker-assisted selection of azuki bean.

As an excellent heterologous gene expression system, Pichia pastoris has been utilized wildely. According to the biased codes which are used by pichia pastoris, we optimized the gene of Mn-SOD which is from Bacillus cerues M22 and synthesize a new sequence on the premise of no modification of the amino acid sequence. A recombinant plasmid pPICZαA/Mn-SOD-2 was constructed and integrated into the genome of pichia pastoris GS115 by electroporation after linearization. The positive transformant was filtrated by zeocin resistance and PCR with specific primers. The recombinant Mn-SOD-2 protein was successfully expressed in Pichia pastoris based on the evidences that the obvious activity of SOD existed in Native-PAGE and a relative molecular weight about 24 kD appeard in SDS-PAGE as expected. The maximum activity of the recombinant with codons optimized reached 228 U/mL after being induced with 72h, which was nearly 3.2 times of the original strain. Additionally, the recombinant showed the upstanding expression stability.

S. roseoflavus Men-myco-93-63 isolated from potato scab decline soil is an antagonistic strain. The strain and it’s fermentation can inhibit many phytopathogenic fungi and control the related important plant disease such as cotton verticillium wilt, cumber powdery mildew, and so on. The total protein of S. roseoflavus Men-myco-93-63 and its high-yield antibiotic strain D12-3 were analyzed by two-dimensional gel electrophoresis. As the results, a modified 2-DE protocol and modified electrophoresis program suitable for Men-myco-93-63 were developed in the study. Four different expression protein spots were isolated and identified by Matrix-Assisted Laser Desorption Ionization (MALDI) Time-of-Flight (TOF) mass spectrum. The results show the proteins are PhaP, Beta-lactamase, methyl-accepting chemotaxis protein, and ABC transporter, respectively, which perhaps are related with the biosynthesis of antibiotic of S. roseoflavus Men-myco-93-63.

In order to detect the interaction between Rice stripe virus (RSV) CP, SP and NSvc4, the HA-tagged or c-Myc-tagged CP, SP and NSvc4 of RSV were amplified by PCR, and then inserted into plant expression vector pEGAD or pKYLX71:35S2. The recombinant plasmids were transformed into Agrobacterium tumefaciens EHA105, and the EHA105 containing different recombinant was injected into Nicotiana benthamiana. The total proteins were isolated from Agrobacterium-infiltrated N. benthamiana leaves, and co-immunoprecipitated with HA or c-Myc antibody. The immunocomplexes were detected by Western blot with c-Myc or HA antibody to identify the interaction between these proteins. The results showed that RSV CP can interact with itself, but no interaction between other proteins was detected.

Abstract: In order to elucidate the potential relationship between persistent infection of foot-and-mouth disease virus and FMDV genetic variation, to further investigate the variance of VP1 and 3ABC gene of the persistent infection isolates. With a dose of 104ID50/ml of O/Akesu/58 FMDV strain, five Chinese yellow cattle were inoculated on their tongue. Following clinical or sub-clinical signs, they became asymptomatic FMDV persistently infection state. Monthly, with probang to scrape esophageal-pharyngeal（O/P）fluids from experimental cattle, then inoculated into baby hamster kidney cell line(BHK-21). There are 12 persistent FMDV strains isolates. VP1 and 3ABC gene were amplified by RT-PCR. Sequence analyses revealed VP1 gene had 16 identical mutations in nucleotide sequences compared with their parental strain, only two point mutation led to amino acid change(I56→T,A210→E),and there were transversion mutations of 4 nucleotide and 3 amino acid among the iaolates. In 3ABC gene there were only 13 nucleotide transversion mutations and 5 deduced amino acids mutations. The results indicated FMDV persistent infection is no direct and close related to genetic variation of VP1 and 3ABC gene, maybe the gene related to persistent infection is gone beyond VP1 and 3ABC regions.

Development of biotechnique, especially molecular marker technique, lays the foundation for R gene cloning. Cloning and Identification of R genes promote the research on the molecule mechanism of interaction of host and pathogen. This paper summarized the strategy of cloning, structure and function of R proteins, molecule mechanism of these R genes and R gene distribution. Research prospect of R genes was also discussed．

Abstract: The genetic diversity of wild and cultured Exopalaemon modestus in Taihu Lake was analyzed by random amplified polymorphic DNA (RAPD) technique . 23 random primers were selected from 60 random primers ,which could amplify 105 fragments in total with the fragment lenth varing from 200 to 2000 bp. The resuts using PopGen1.32 showed the proportion of polymorphic loci of wild and cultured Exopalaemon modestus was 50.47% and 43.33%,respectively. The intra-population genetic similarity of wild and cultured population was 0.8635 and 0.8795 respectively ,while inter-population genetic distance between the two populations was 0.0361. The mean Shannon index H0 was 0.2830 in wild population ,which was slightly higher than the value 0.272 in cultured population.

Coreoperca is a group of freshwater fishes endemic in East Asia. To study the genetic structure of different geographical populations of C. whiteheadi in China, the genetic variation of 65 samples, collected from Yangtze River (YZ), Qiantangjiang River (QT), Xijiang River (XJ) and Nandujiang River (ND), was analyzed by the AFLP technique. 10 primer combinations generated 1131 bands with 603 polymorphic bands; the average proportion of polymorphism was 53.32%. However, the proportion of polymorphism, genetic diversity and Shannon index within the YZ, QT, XJ and ND population was quite low. The genetic distances among the mainland populations of YZ, QT and XJ were small (0.098～0.189), whereas the distances between ND with the mainland populations were large (0.507～0.568). The analysis of molecular variance (AMOVA)showed the genetic ifferentiation among these populations was significant (FST = 0.972). NJ tree suggested that 4 populations could be divided into two clades: one clade included YZ, QT and XJ populations, another clade comprised of ND population. C. whiteheadi lives in the upper section of streams with a small population, the poverty of its gene bank may result in low genetic diversity within population. Meanwhile, lack of genetic flow among different populations and occurrence of random genetic drift within population have caused their divergence.

White spot syndrome virus (WSSV) , Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and and Taura syndrome virus (TSV ) are responsible for significant economic loss in the shrimp industry. In order to simultaneously and massively identify WSSV , IHHNV and TSV, three pair of primers and three TaqMan probes were designed and synthesized according to the conserved gene sequences of WSSV (AF369029) , IHHNV (AF218226) and TSV(NC003005) in GenBank. The reaction parameters such as the concentration of three pair of primers, three TaqMan probes and the reaction buffer were optimized to develop a multiplex real-time PCR assay for the rapid detection of WSSV , IHHNV and TSV. The multiplex real-time PCR assay was found to be specific and to be able to detect and differentiate WSSV , IHHNV and TSV, and no positive results were observed when nucleic acid from Vibrio and Streptococcus were used as multiplex real-time PCR templates. The developed multiplex real-time PCR assay was compared with that of routine PCR. The sensitivity of multiplex real-time PCR assay was 20000, 20 and 20000 template copies for WSSV , IHHNV and TSV respectively, and its sensitivity was 10, 1000 and 10 times higher than that of the routine PCR. The samples were examined using the multiplex real-time PCR repeatedly and the results indicated that the multiplex real-time PCR was reproducible. Different concentrations of WSSV , IHHNV and TSV when mixed together still could be identified by this assay, which implied the assay could be applied to clinical confirmation for simultaneous infection of WSSV , IHHNV and TSV. The multiplex real-time PCR results of 45 routine PCR positive samples showed that one specific amplified curve was displayed when shrimp was infected by only one of these three viral pathogens, whereas two or three specific amplified curves were displayed when shrimp was infected by two or three viral pathogens. The result indicated that multiplex real-time PCR was able to detect and differentiate the presence of each pathogen in infected clinical shrimp. This multiplex real-time PCR assay was a quick, sensitive, specific and quantitative tool for detection of WSSV , IHHNV and TSV, and will be useful for the control of WSS, IHHN and TS in shrimp.