One pair of primers was designed and synthesized according to duck IL-18 gene sequences published in GenBank．Duck IL-18 mature protein gene was amplified by RT-PCR from total RNA extracted directionally from ma duck splenocyte ,then was cloned and sequenced．The result suggested that the nucleotide sequence of duck IL-18 mature protein gene be consisted of 513 bp in length, encoding 170 amino acid residues．A prokaryotic expression plasmid of mDuIL-18 , pQE30-mDuIL18 , was obtained by subcloning the encoding region of the DuIL-18 mature peptide into pQE30. pQE30-mDuIL18 was transformed E.coli M15 ,and induced by IPTG. The expression of p IL-18 mature protein gene was identified by SDS-PAGE and Western-blotting. The results revealed it had a molecular weight of 19755,and could be specifically recognized by the rabbit sera to chicken IL-18. The expressed products exist at the form of inclusion body. After being degenerated and then renaturated, the activity of the inclusion bodies were detected by by MTT. In ducks injected intramuscularly with rduIL-18 protein (150 ng or 200 ng per duck, respectively) and AIV vaccine 2 weeks after immunization, the average titers of HI antibodies to AIV reached 7.5–7.7 log 2, while the average titers of HI antibody to AIV were 6.3–6.6 log 2 in ducks only vaccinated with AIV vaccine or with 100 ng rduIL-18 and AIV vaccine. The results clearly showed that 150 ng rduIL-18/duck strengthened in vivo immune responses induced by the inactivated oil emulsion AIV vaccine.Development of recombinant pQE30-mDuIL18 paved the way for future study of biological function of expressed product and development of recombinant fowlpox viruses(rFPV) coexpressing duck IL-18 and protective antigen gene.

Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the nucleus under the downstream of the ethylene gas signaling transduction. In this paper two oligonucleotide primers were designed from conserved domain of EIN3 and EIL gene family. The forward primer sequence of the primer was 5’ —ttg gag agg agg atg tgg ag—3’ and The reverse primer 5’ —ata ata gca agc cag gta gc—3’. PCR amplifications were performed on Cucumis sativus, Citrullus lanatus and Cucurbita maxima DNA template and produced three fragments of 725 bp Separately. The three sequences that highly matched with other 151 sequences of EIN3 gene were all EIN3 genes by using the program of BLASTn on NCBI GenBank database. The three 725 bp fragments of EIN3 gene in Cucumis sativus, Citrullus lanatus and Cucurbita maxima were deduced 241 amino acid sequences via using the program of ORF Finder (Open Reading Frame Finder). The three sequences had been submitted to the GenBank database and the accession number is AY973275、DQ023225、DQ￥023224 separately. By using the program of BLASTp on NCBI GenBank database, The results which were searched out in MMDB (Molecular Modelling Database) indicated that putative conserved domain 3D structure of 241 amino acid sequences in Cucumis sativus, Citrullus lanatus and Cucurbita maxima had the same 3D structure which are very similar to structure of the DNA-binding domain of ethylene-insensitive3-like3 of the 1WIJA in Arabidopsis thaliana. The results of phylogenetic analysis showed that the EIN3 family from the dicot of cucumber, watermelon, pumpkin, melon, mungbean, barrel medic, tobacco, tomato, Arabidopsis thaliana and the monocot of rice, phalaenopsis equestris had a closer phylogenetic affinity.

The efe gene encoding ethylene-forming enzyme was PCR amplified from Pseudomonas syringae pv. glycinea ICMP2189. The efe gene was cloned to expression vector pET28a, yielding recombinant plasmid pET28a-efe. Then plasmid pET28a-efe was introduced into Escherichia coli BL21(DE3). SDS-PAGE showed that ethylene-forming enzyme with molecular weight of 45 kDa was effectively expressed in E. coli. Gas chromatography analysis demonstrated that the E.coli strain carrying the efe gene could efficiently produce ethylene.

Inverted repeat plant expression vector pKcp was constructed with peanut stripe virus(PStV) coat protein gene, then transformed into Agrobacterium tumefaciens strain GV3101. Transgenic plants were obtained by transformation of Nicotiana benthamiana leaf discs by agrobacteirum and kanamycin screening. The transgenic plants were tested for its resistance by inoculation with PStV in 5~6 leaf stage. By symptom observation and ELISA detection, there was 87% transgenic plants immune to PStV in T0 , 60~100% immune to PStV in T1 different lines, most of T2 lines immune to PStV. siRNA was detected by Northern blot by PStV cp probe in T1 plants, different amount of siRNA was detected in transgenic plants, no siRNA was found in nontransgenic plants; siRNA amount was higher in resistant plants than susceptible plants in the leaves before inoculation; about same amount of siRNA of leaves were detected in different days post inoculation in the same resistant plant, different amount of siRNA was found in different resistant plants. In this experiment, dsRNA strategy were succeeded inducing high frequency of transgenic resistant tobacco plants to PStV.

Abstract: The fermentation conditions of mutant EGs2 (obtained from directed evolution for thermostable β-glucanase) for β-glucanase production were investigated with orthogonal experiment.Fermentation was conducted in 250 mL flask, each containing 30 mL of medium contained Whey 10 g•L-1,Yeast extract 5 g•L-1,NaCl 10 g•L-1,the temperature was 37 ℃.The optima culture conditions were as following: initial pH value 6.0, shaking speed 170 r•min-1,inoculation volume 1 %,inoculation time 12 h.β-glucanase production by mutant EGs2 was associated with cell growth and biomass,β-glucanase activity was increased significantly when cells entered growth phase.The bacterium began the stable phase after cultured for 14 h with the highest biomass 1.71 g•L -1 and β-glucanase activity 321.56 U•ml-1.When β-glucan was used as a substrate,the optimum temperature and pH were respectively 60 ℃ and 6.0.After 20 min incubation at 40 ℃,50 ℃,55 ℃,60 ℃,65 ℃,70 ℃, the residual activity remained at least 80%.After 48 h conservation at pH 5.0-9.0,the residual activity remained at least 90%.The enzyme was stable below 70 ℃ and at pH 5.0-9.0.

In order to provide a rapid, selectivity and very high sensitivity method for the determination of zearaleone(ZEN), a competitive indirect time-resolved fluoroimmunoassay(TRFIA) was used. An immunogen of ZEA-oxime-BSA conjugate coated onto the microtitre plate competes with ZEN standard controls or free ZEN in the sample for anti-ZEN antibody. A goat antirat IgG-Eu3+ conjugate was used to enable detection. The suitability of the assay for quantification of ZEN was also studied. Results showed the limit of detection of the assay to be 0.01μg/L(10ppt) for indirect competitive TRFIA formats. The assay range was 0.01-20 μg/L. The concentration of ZEN that is required for 20%, 50% and 80% binding inhibition (ED20, ED50, ED80) were 0.156±0.035 μg/L, 0.634±0.091 μg/L and 2.595±0.274 μg/L, respectively. Cross-reactivity of Zearalanol was 15.16%. The inter- and intra-assay variability of the ZEN-TRFIA were 7.2% and 14.6%, respectively. The mean recovery of ZEN from corn samples was 94.4%. The kit can be stored at 4℃over 6 months. It was shown that the newly developed TRFIA could be applied to detect the ZEN contamination in corn. The ZEN-TRFIA provides very high sensitivity and optimal range, it will be useful to screen ZEN contamination easily, simply and economically when the the number of sanples is large.

To investigate the effects of IGF-I, GH and CLA on the proliferation and differentiation of preadipoctyes obtained from different depot sites. Our present study separated and cultured the preadipocytes from rat epididymis and abdominal subcutaneous adipose tissues. After diversity dose of IGF-I, GH and CLA treatments, cell proliferation and the extent of differentiation were detected by MTT method and oil-red O pigmentation respectively. The results showed that every dosage of IGF-I and CLA chould significantly promote both the proliferation and the extent of differentiation of the preadipocyte from each depot site, however, no significant change was observed after GH treatment. For the better understanding of the mechanism of IGF-I and CLA on the xubcutaneous preadipocytes, we also examed the mRNA expression levels of PPARγ and C/EBPα by semi-quantitative RT-PC method. It was found that 100ng/ml IGF-I markedly up-regulated the PPARγ and C/EBPα mRNA expression levels, while 48ng/ml GH and 100μM CLA could slightly increase that of PPARγ only, but no change of C/EBPα mRNA expression. This study suggested that IGF-I may stimulate the differentiation of rat preadipocyte primarily by enhancing the transcriptional factors mRNA expression, while the effect of CLA was mediated probably by up-regulating PPARγ mRNA expression, as well as activating of PPARγ as a ligand.

To reveal the differentiation of phenotypic values and QTLs for grain quality traits in rice, we evaluated a doubled haploid (DH) population from a cross between two japonica cultivars for milling quality traits including brown rice (BR), milled rice (MR) and head milled rice (HR) and grain appearance traits including grain length (GL), grain breadth (GB), grain length- breadth ratio (LWR), and chalkiness (C) cultivated under upland and lowland environments. The analysis of variance (ANOVA) between upland and lowland environments for all the traits showed that HR, GL, GB and C were more easily influenced by environment than BR, MR and LWR. The values of BR, MR and HR of the lowland parent Yuefu were higher than that of the upland parent IRAT109 under lowland environment. But under upland environment, the BR and MR for the parents were both declined by 5.8% and 5.5% for IRAT109，and 11.7% and 11.5% for Yuefu respectively. The values of GL, GB and C were all reduced under upland condition than that under lowland condition for all the two parents while the value of LWR had the opposite tendency. Compared to lowland environment, some quality traits changed such as an increased HR, decreased C, and thinner grain shape when cultivated under upland environment. Using a complete genetic linkage map with 165 molecular markers covering 1535 cM, QTLs for these traits were mapped under upland and lowland environments. A total of eleven additive QTLs and ten pairs of epistatic QTLs associated with BR, MR, HR, GL, GB, LWR, and C were detected. It was found that epistasis was an important genetic component underlying BR, MR, HR and C. The result also showed that MR, GL, GB and LWR were less affected by upland environment. However, HR was affected greatly by upland environment. Four intervals on chromosomes 1, 3, 6 and 7 were found to contain multiple QTLs(co-localization). Eight common QTLs for these traits across different studies were also revealed. These co-localized QTLs and common QTLs will facilitate marker- assisted selection for grain quality traits in rice breeding.

Abstract:120 couples of laying pigeons of 720 days of age were randomly divided into four groups, each of which included three replicates of 20 doves. The doves were kept in individual cages and provided with purified diet ad libitum. The difference of control and treatment groups diet was the amount of olanzapine (the diet of control group contained 0 mg olanzapine /kg and the diet of treatment groups contained 0,0.75,1.25mg olanzapine /kg, respectively).The effects of Olanzapine on the expression of PRLR gene were investigated by means of RT-PCR. Supplementation of 1.25 mg/kg Olanzapine decreased the relative mRNA expression of PRLR to β-actin (PRLR/β-actin ratio) by 14.3%（P<0.05）.These results implicated that Olanzapine can significantly increase egg production by inhibiting the expression of PRLR gene from pituitary gland. These results also indicate that hypothalamic DA and 5-HT activities are the vital causes of development and maintenance of broodiness and pituitary PRL secretion.

Objective: In order to study on the growthy characters of co-cultures in porcine, we established the method for co-culture between preadipocytes from porcine firstly. Method: Differential attachment was used to purify preadipocytes and myogenic satellite cells, then both were mixtured and co-cultured in the ratios of 1:10 as preadipocytes and myogenic satellite cells for controls. Preadipocytes and myogenic satellite cells were identified respectively with Oil Red O and Desmin immunohislochemistry staining. Their morphological changes, MTT, growth curve all described the growthy regularity of co-cultures. Result: The myoblasts purity exceeded to 97% by Desmin immunohislochemistry staining test, and more than 60% of preadipocytes developed to differentiated adipose cells as defined by Oil Red O staining. Before confluence the micrography of typical co-cultures was fusiform shape by survey, but after confluence changed into irregular with coinciding and interlace with each other, the cells filled lipid appear later and fewer than control group. HE staining demonstrated polykaryocytes fuse into myotubes. MTT and growth curves for 8 days show the growthy rate of co-cultures is more than control groups, a rapid growth until 7 days after the delitescence of growth from 2 to 3 days like as the control groups, but keep on slow later growth to 9 days. Conclusion: The method for co-culture of preadipocytes with myogenic satellite cells in porcine is established premilinaryly, and the basic growthy characters of co-cultures are identified.

Genome RNA was extracted from bone marrow of pig (Duroc×Landrace× Yorkshire) and antimicrobial peptide PMAP-37 mRNA was amplified using RT-PCR. A DNA fragment about 282 bp in length was obtained and the PCR product was cloned into pGEM-T vector. The PMAP-37 gene was isolated and sequenced from the screened positive clones. Result of sequence analysis showed that this fragment was the partial sequence of PMAP-37 cDNA and coded 167 amino acid residues. The gene homology of the obtained fragment compared with that of reported PMAP-37 gene sequence in bone marrow of porcine was up to 98%. Based on the PMAP-37 gene clone, an optimal semi-quantitative RT-PCR method was successfully constructed. Using 18S rRNA as inner control, the difference of PMAP-37 gene expression at different avoirdupois of pig was researched, which increased from new born to 20 kg, decreased from 20 to 40 kg, increased from 40 to 60 kg and decreased again at the stage of 60 to 90 kg.

Abstract: mitochondrial is the only organelle containing extranuclear genetic material in mammal cell, which proper function required extensive nuclear-mitochondrial interactions. Mitochondrial DNA(mtDNA) encodes some kinds of subunits of respiratory chain enzymes, tRNAs and rRNAs; other hundreds of mitochondrial components are encoded in the nucleus. The heteroplasmic somatic nuclear transfer possibly causes mtDNA heteroplasmic phenomenon in reconstructed embryos. In the present study, applying the method of PCR-restriction fragment length polymorphism(PCR-RFLP),examination of the mtDNA origin and proportion in reconstructed embryos derived from goat fetal fibroblasts and enucleated sheep MⅡoocytes was performed. The result shows that, the proportion change of nuclear donor to recipient mtDNA is not big, the proportion of nuclear donor to recipient mtDNA in 1-cell,2-cell,4-cell and 8-cell reconstruction embryos is 2.5±0.8%; 2.8±0.6%; 1.9±0.4%; 1.7±0.6% separately and the difference is not significant (p> 0.05), however, in the heteroplasmic blastocysts, the proportion of nuclear donor mtDNA declined to 0.3±0.1%,and the difference is significant (p< 0.05).

Abstract: The spike protein gene of porcine epidemic diarrhea coronavirus (PEDV) DX strain was cloned and sequenced. The results showed that the nucleotide sequence of the gene was 4152 nt in length and encoding a protein of 1383 aa, the homology of amino acids between it and other PEDV strains exceeded 90%. The results of phylogenetic tree analysis showed all the reference PEDV isolates could be divided into 3 genetic groups and the isolates in the same group came from the same country. Bio-soft analysis showed that PEDV muting slowly, especially in Korea, DX strain and JS-2004-2 strain have a closed relationship.

Abstract: To study immunoprotection against Schistosoma japonicum(Sj) elicited by immunization of DNA vaccine co-encoding SjFerritin and GM-CSF. Methods SjFer were amplified by PCR from pGEX-5x-3/SjFer temple. GM-CSF were amplified by RT-PCR from totle mice RNA. Reconstructed plasmids were constructed by routine methods. Eighty-five female mice were randomly divided into 5 groups, 17 each .Group A, B and C are controls, immunized with monmal saline, pcDNA3.0 and GM-CSF/pc respectively, and group D, E were immunized with SjFer/pc and GM-CSF-SjFer/pc at weeks 0,2,4. Sera were collected before immunization and challenge infection . Serum lgG were detected by ELISA, Spleen lymphocyte cells of mice were collected before challenge infection.Proliferation activity of spleen lymphocytes was detected by MTT assay. Mice were infected with (40±1) Sj cercariae per mouse 2 weeks after final immunization. Forty-five days later, adult worms and eggs were counted. Results Level of IgG in sera increased after immunization. And proliferation activity of spleen lymphocytes were increased compared with group A, B and C. The worm reduction rate of groups D and E were 36.27 % and 39.22 %. The egg reduction rate of them were 36.10 % and 49.04 %, respectively. Inclusion GM-CSF increased immunoprotection against challenge infection of Sj.

To express the bovine interleukin-18(BoIL-18) protein, cDNA fragment encoding the mature BoIL-18 protein was subcloned into pGEX6p-1 and transformed into E.coli BL21(DE3) . The expressed BoIL-18 protein was purified by affinity Chromatography. MTT method ,ELISA method and VSV were used to assay the functional activity of BoIL-18 protein. Results: The recombinant BoIL-18 was highly expressed in E.coli and purified by affinity Chromatography method. The reBoIL-18 could enhance the proliferation of PBMC, induce IFN-γproducing in spleen lymphocyte. Conclusion: The purified reBoIL-18 has functional activity and can be applied for further studies.

EST-SSR marker is a new kind of SSR markers which is developed from the ESTs（expressed sequence tags）. This novel type molecular maker was part of expressed gene, therefore, when used in Brassica napus genetic analysis, they could reveal differences in related gene expression among varieties. In this study, a total of 21 EST-SSR markers were used to analyze the genetic diversity of 42 Brassica napus varieties. All the primer sets produced clear and strong amplifications, and 85 alleles were generated and examined. 49 alleles of all showed polymorphic among the 42 Brassica napus varieties, accounting for 57. 65%. The range of the 42 varieties’ genetic distance was 0.0087-0.1885 computed by NTSYS（software）. when GD on 0.1508, the varieties were divided into 5 groups, which indicated difference of these germplasm based on different cultural area approximately.

Abstract： Two primers are designed on the base of Ghrelin gene sequence in genbank in this study, and the polymorphism of Ghrelin gene in 140 Jinghai yellow chickens and 30 Suqin yellow chickens was analyzed by PCR-SSCP. The result indicates that there were no polymorphism detected by Primer 1,and there were polymorphisms detected by Primer 2. BB, BC and CC genotypes were detected in Jinghai yellow chicken ,but only BB and BC genotypes were detected in Suqin yellow chicken; the allele B was obviously higher than that of C allele in two chicken breeds. The frequency of BB genotype is 0.933 in Suqin yellow chicken and 0.657 in Jinghai yellow chicken. The sequencing results showed that there was one single nucleotide mutation : T→A at the point of 546bp of Ghrelin gene in chicken.

To investigate in vitro surviving and proliferating characteristics of mouse spermatogonia and its ability to endure freezing-thawing procedure by single cells or in their functional structure (seminiferous tubes or testes). Kun ming mouse testes of 7 day of age were collected to isolate the seminiferous epthelial cells (spermatogonia and Sertoli cells mainly) using one step of enzymatic digestion. After being plated D-MEM, D-MEM/F12 or KSOM (each contained 10% FBS) separately, the spermatogonial survival and proliferation in vitro was tracked systematically. The seminiferous epthelial cells were stored at -70℃ and liquid nitrogen, the seminiferous tubes and testes were stored at -70℃. The results indicated mouse spermatogonia could survive and proliferate in D-MEM, D-MEM/F12. Spermatogonia decreased gradually from 1d to 9d or 1d to 8d, increased rapidly from 9d to 13d or 8d to 12d in primary culture, while the increase slowed down after 17d or 16d. Mouse spermatogania could survive a short period but couldn’t proliferate in KSOM. No visible proliferation but differentiating characteristics was observed when mouse spermatogia cultured alone. The ideal percentage of viable cells (all above 85 percent) was got after the seminiferous epthelial cells, seminiferous tubes and testes stored at -70℃ for 1 or 2 weeks or the seminiferous epthelial cells stored in liquid nitrogen for 2 weeks, 1month and 3 months.

Female, 6-8w Balb/c mice were immunized with the purified recombinant fusion protein. After three immunizations, the spleen cells from Balb/c immunized were fused with mouse myeloma cells SP2/0. An indirect ELISA with purified VP1 protein as coated antigen was used to screen antibody-producing hybridomas. By 3 cycles of limited dilutions, Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. All MAbs reacted with MDCC-MSB1 cell infected with CAV by indirect immunofluorescence assay (IFA) and they recognized the VP1 protein expressed in Baculovirus by Western blot. This demonstrated that all MAbs have good specificity. The subtype of all MAbs is IgG1 and light chains of all MAbs are kappa. Taking advantage of VP1 fragments expressed, the epitopes corresponding to the MAbs are analyzed. The results showed that the monoclonal antibodies 1C5、2F3、4D2 and 4E8 recognized the same epitope, which was located in 218-274aa. Moreover, the two other epitopes were identified and they were located in 274-301, 324-369aa, respectively.

To obtain recombinant canine interleukin-2(cIL-2) with natural bioactivity expressed in Pichia pastoris, the cDNA of canine IL-2 was amplified by reverse transcription polymerase chain reaction(RT-PCR) from the total mRNA of the lymphocyte from canine blood, which stimulated with ConA for 20h. The amplified fragment was cloned into pMD18-T vector and confirmed it was identical to that published in GenBank. The fragment was inserted into expression vector pPICZα-A. After linearized by SacⅠ, the recombinant plasmid was transformed into X-33 strain. The recombinant strain was isolated and identified by PCR. After induced by methanol, the recombinant protein was examined by SDS-PAGE. The result of SDS-PAGE in concentrated fermentation supernant showed that the molecular weight of the recombinant protein was approximately 20kDa. The expressed protein was larger,which may be due to glycosylation of protein. The result of MTT assay proved the recombinant protein could induce canine T lymphocytes proliferation in vitro. It concluded that canine recombinant IL-2 expressed in Pichia pastoris had good bioactivity.

ABSTRACT：The NP gene segment deleted part of sequence，with a length of 1320bp，and coding for 440 amino acids，was amplified by PCR．The NP gene fragment was then inserted into the EcoR I site of pR vector to obtain recombinant plasmid pR-NP. The recombinant plasmid was used to transform E．coli E2, the host bacteria of T4 phage. and then infected with lysozyme-defective phage T4-Z1.The NP gene were then integrated into the SOC site of the genome of SOC-deleted T4 phage mutant by homologous recombination. The recombinant phage were screened by PCR and designated as T4-ZI-NP．The immunological test applying Western blot showed that SOC-NP fusion protein expressed by the phage T4-ZI-NP could react to AIV specifically. Recombinant bacteriophage T4 expressing the NP Gene of Avian Influenza Virus was constructed successfully.

Abstract: HA gene of H5N1 avian influenza virus was amplified from the plasmid pGEM–HA and inserted into baculovirus transfer plasmid pFastBacHT to construct recombinant transfer vector pFastBacHT-HA. The pFastBacHT-HA was transformed into E.coli DH10Bac competent cells , transposited with baculovirus shuttle vector (Bacmid) and constructed recombinant transposition rBacmid-HA. After the rBacmid-HA transfected into sf9 cells , the recombinant baculovirus was harvested. The expressed HA protein was analyzed by SDS-PAGE, Western-blot and hemadsorption assay. The specific protein band of 66kDa was identified, which can only react with chicken sera against H5 subtype AIV and not react with the sera against H7 and H9 subtype respectively. Hemadsorption assay showed that sf9 cells that infected rBacmid-HT baculoviws can absorb chicken red blood cells. These results indicated that the HA protein was accurately expressed in sf9 cells, displayed nice specificity to H5 subtype antisera and biologic activation.

Abstruct：Cells derived from porcin fetus expresse PDX-1, GLUT-2,PCNA, Glucagon, somatostatin，polypeptide.Nestin was weakly expressed.These cells have strong viability,It were subcloned over 58 passages during 13 months and identified as pancreatic stem cells by immunochemical method。 The cell can form three-diemention islet-like structure by induction in vitro.It stongly express insulin and ck-19 after forming ICCs.This result reveal that the cell is pancreatic stem cells and pancreatic stem cells do exist in pancreas,It maintains the steady of pancreas by neogenesis and differation. At present,our laboratory can isolate and expand this cell steadily.This study can provide reference for isolation and expansion of pancreatic stem cells.Also,it can provide new source for diabetes therapy.

The exon 2 of SLA-DQB and DRB genes in Sutai pigs were analyzed by PCR-RFLP method., the results showed that the exon 2 of SLA-DQB gene digested by restriction endonuclease RsaⅠcould be divided into three kinds of alleles , five kinds of genotypes, the exon 2 of SLA-DRB gene digested by restriction endonuclease RsaⅠcould be divided into three kinds of alleles , four kinds of genotypes. The results of Chi Square test indicated that the frequencies of patterns types of SLA-DQB and SLA-DRB genes digested by RsaⅠwere fit with Hardy-Weinberg equilibrium in Tibet pig populations(p>0.05). There were twelve combined patterns of SLA-DQB and DRB genes in Sutai pigs, the correlation between these combined patterns with the productive performances of Sutai pigs was analyzed, the results showed that productive performances (the total litter, number of viable pigs, litter weight at birth and number of ablactation pigs in the third litter ) of Sutai pigs with BBDFcombined pattern were significant higher than those with AADD, AADF, ABDD and CCDF combined pattern. As the productive performances of Sutai pigs, the combined pattern of BBDF was the most favorable.

Primordial germ cells (PGCs) were isolated from the gonads of chicken embryos at stage 28, PGCs were co-cultured with gonadal stroma cells. Identification of PGCs was carried out by periodic acid-Schiff (PAS) and alkaline phosphatase (AKP) staining. We constructed a lentiviral vector pLenti-CMV-eGFP and harvested the virus by cotransfecting 293FT cells with the vector and packaging plasmids. Lentiviruses after concentration were used to transfect chicken PGCs, the transfection efficiency of PGCs was up to 24.19%. The impacts of different volume of lentiviruses on transfection efficiencies were also compared, the results indicated that as the increase of lentiviruses, the transfection efficiency was significantly improved.

N-acyl-homoserine lactones were typical quorum sensing signal molecules and regulated the expression of many physiological characteristic in Gram-negative bacteria. But the AHL was smaller chemical molecules and the AHL concentration produced by the bacteria was very low, so it was very important to set up the methods of assay for the AHLs. In this paper, the biological methods were established of agar diffusion assays, thin-layer chromatography combination with biosensor analysis andβ-galactosidase activity to detect AHL based on the biosensor Chromobacterium violaceum CVO26 and Agrobacterium tumefaciens A136（pCF218/pCF372）.The powerful manners were developed to research the quorum sensing in Gram-negative bacteria.

Abstract：Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. Plant aquaporins play an important role in response to water balance, since their discovery, advancing knowledge of their structure led to an understanding of the basic features of the water transport mechanism. In the present study, the gene encoding the Solarium tuberosum L. aquaporin (StPIP1) cDNA was isolated from leaf of “GannongShu No.2” under PEG6000 stress by reverse transcription-polymerase chain reaction(RT-PCR ), the analysis of the sequence showed that StPIP1 cDNA was 867-bp in length , the protein encoded by 288 amino acids with a predicted molecular mass of 30. 9KD; StPIP1 exhibited six predicted transmembrane helices and possesses two sets of conserved NPA motifs as well as the highly conserved motifs on M1 and M4 as EXXXTXXF/L which may be involved in the recognition and transport of water molecules across the water channel, and possessing the MIP family signal consensus sequence SGXHXNPAVT and the higher plant PIP highly conservative sequence GGGANXXXXGY and TGI/TNPARSL/FGAAI/VI/VF/YN. As compared to PIP1 identified from other 14 plant species PIP1 subfamily by phylogenetic trees, StPIP1 has the highly sequence identity at the amino acids level (more than 92%), so StPIP1 should be a member of PIP1 subfamily. The protein 3D structure was predicted by homology comparatvie modeling in Swiss-Model, the results feedback from Swiss-model showed that the 3D structure of StPIP1was highly similitude with spinach(2B5F).

Abstract: Pig myoblasts were obtained by pronase digestion and identified by Desmin immunohistochemistry and the mRNA expression of MyoD and MyoG. The effects of leptin on the expression of thermogenesis-related genes PGC-1αand UCPs were determined by RT- PCR. It was showed that 30 nmol/L and 100nmol/L leptin promote the mRNA expression of PGC-1αin myoblasts. The effect of 100nmol/L Leptin treated for 24h was significantly higher than that of 30 nmol/L Leptin for 12h(p<0.05). Moreover, 30 nmol/L leptin markedly promoted the UCP2 mRNA expression of myoblasts, and it was higher when treated for 24h than that of 12h (p<0.05). The effect 100nmol/L leptin on UCP2 mRNA expression was significantly lower than that of 30 nmol/L Leptin in 12h and 24h, but markedly higher than that of control(p<0.05). Additionally, the mRNA expression of UCP3 was not significantly different when treated by 30 nmol/L and 100nmol/L leptin compared to control in myoblasts(p<0.05). It suggests that leptin stimulate energy consumption by promoting the mRNA expression of PGC-1αand UCP2 in myoblasts.

Two pairs primers were designed according to the already known granule-bound starch synthase (GBSS) gene sequence, one of them was used for pointing mutation. First by means of pointing mutation、RT-PCR、amalgamation PCR methods with reverse Transcription cDNA first chain as the template, the all cNDA (1818bp) fragment of maize GBSS gene was cloned from the total RNA of isolated from 15d maize Endosperm after pollination. The fragments have 99.72％ homeologous identical to the already reported sequence. Then the plant expressing vector pBI-Gt1-GBSS of containing NPT‖selective mark gene was constructed by using Endosperm-specific Promoter Gt1 as primer, NOS-ter as the teminater. It was looked forward to realizing the abundant expression of GBSS gene in maize endosperm in specific period.

Abstract: The human erythropoietin gene (hEPO), containing all exons and introns of the hEPO gene except intron 1, was cloned by using PCR technique and synthesized. The hEPO gene was subcloned into the pIRES2-AcGFP1 to construct the eukaryotic expression vector to co-expression hEPO and AcGFP1. The vector was transfected into Chinese hamster ovary cells (CHO) and goat fibroblast by lipidosome transfection. The results showed that CHO and goat fibroblasts appeared bright fluorescence 48h after transfection. Screened by G418, expression cells were obtained. This indicated that the eukaryotic expression vector of hEPO gene with reporter gene has been constructed and expressed in CHO cells and goat fibroblasts. The results paved the way for detecting whether hEPO gene had been transduced into cells, as well as screening positive cells.

Genome RNA was extracted from mesentery adipose of porcine (Duroc×Landrace×Yorkshire) and suppressor of cytokine signaling -3(SOCS3) mRNA was amplified using RT-PCR. A DNA fragment about 332 bp in length was obtained and the PCR product was cloned into pGEM-T vector. SOCS3 gene was isolated and sequenced from the positive clones screened. Sequence analysis suggested that this fragment corresponded to a portion of the sequence of SOCS3 cDNA, its homology with SOCS3 from GenBank was up to 100%. Based on the SOCS3 gene clone, an optimal semi-quantitative RT-PCR method was successfully constructed. Using 18s rRNA as inner control, the difference of SOCS3 gene expression at different stages of swine was studied. The results showed that the amount of SOCS3 gene expression increased from 1 kg to 90 kg. and the SOCS3 mRNA level at 90 kg significantly increased by 141% (p<0.05) as compared with 1 kg.

Oxygen tension and culture media (CM) as well as growth factors are the key factors to the development of embryos in vitro. Systematically study had been carried on effect of oxygen tension（7% O2和20% O2）, culture media (NCSU-23、 PZM-3、G3) and growth factors(EGF, bFGF）on in vitro development of porcine parthenogenetically activated(PA) embryos, including blastocyst formation rate and cell number per blastocyst. Compared with 20%O2, low oxygen atmosphere improved blastocyst rate and total cell number; PA embryos in PZM-3 and 7 %O2 had a greater cell number (P < 0.05, 51.0±4.0 vs. 38.8±2.2). Regardless of atmosphere, PZM-3 supported a higher development rate and quality of porcine PA embryos than NCSU23 respectively. 2-4 cell stage PA embryos cultured in NCSU-23 supplemented with 10ng/mL EGF or bFGF had a significantly greater developmental ability to blastocyst than the control （P < 0.05, 36.2±5.3％，44.3±5.8％ vs. 20.7±6.3％）, no difference at blastocyst cell number between them.

Abstract: Aim：Glucagon-like peptide-2 (GLP-2) plays an important role in the regulation of intestinal epithelium growth. To investigate the patterns of GLP-2 expression in duodenum, jejunum and ileum of weaning piglets, to explore whether the low intestinal epithelial restitution rate in weaning piglets could be reflected, to some extent, by the expression of proglucagon that encoding GLP-2. Method: Nine litters of newborn piglets were employed in the present experiment to determine the temporal and spatial pattern of proglucagon mRNA expression using semi-quantitative RT-PCR. Piglets were weaned at 35 days (d) of age. Six piglets were sacrificed respectively on 28, 35, 38, 42 and 45 d. Mucous membrane of duodenum, jejunum and ileum was taken and immediately frozen in liquid nitrogen. Results: The proglucagon mRNA expression exhibited significant spatial-specific pattern. Duodenum expressed little, if any, proglucagon mRNA, while jejunum and ileum expressed remarkably higher proglucagon mRNA. Furthermore, proglucagon mRNA expression in jejunum and ileum followed different temporal (age-related) patterns. One week (42d) and 10 days (45d) after weaning, jejunum proglucagon mRNA expression decreased significantly compared with the levels of pre-weaning (28 and 35d) and 36 h post-weaning. However, this down-regulation was not observed in ileum where proglucagon mRNA expression kept constant during the whole period of observation. Conclusion: The results indicated that the expression of GLP-2 mRNA is spatio-temporal specific in small intestine of weaning piglets.

One handred fifty-four bacteria were isolated from tobacco leaves on the medium with nicotine as unique carbon source. A high nicotine-degrading bacterium (L1) was obtained after screening. Based on morphology, physiological and chemical tests, 16S rDNA sequence and phylogenetic analysis, L1 was identified as Bacillus simplex. The concentration of nicotine for the optimal growth of L1 was 1.0g/L. The nicotine was degraded up to 75% by strain L1 under the optimized incubation conditions for 36 h monitored by high-performance liquid chromatography. There was no pigment observed at degrading process. This study demonstrates that Bacillus simplex L1 had strong ability to degrade nicotine, and the degrading mechanism may be different from Arthrobacter sp.

Inter-Simple Sequence Repeat ( ISSR) is a good molecular marker for revealing genetic diversity. In our study, the systems of DNA extracting and ISSR-PCR reaction were constructed using eggplant(Solanum melongena L) leaves. The results indicated the DNA quality was improved by modified CTAB protocol and applied to ISSR-PCR system. The amylase, polyphenol and pigment of the eggplant leaves were wiped off completely. Orthogonal design was used to optimize ISSR amplification system on eggplant in four factors(Taq, dNTP, primers, template) three levels respectively. Using eggplant genome DNA as template, Optimal PCR(20μL) mix for eggplant contained: 2. 0μL 10 ×stock buffer (25mM Mg2+), 375μmol/ L of each dNTP, 0.4μmol/ L primer , 1 unit of DNA polymerase and 4ng DNA template. The study construct a base for the research of genetic diversity of eggplant.

Abstract: To express the Staphylococcal α-Hemolysin gene in E. coli and study its primary biological characterristics, α-Hemolysin (α-HL) gene without signal peptide sequence was amplified from Staphylococcus aureus by PCR and inserted into pMD18-T vector. The cloned α-HL gene was then inserted into prokaryotic expression vector pET32a+ and transformed into E. coli BL21. The predicted protein was detected by SDS-PAGE after IPTG induction, which had molecular weight approximately 53 kD. Hemolytic experiment demonstrated the expressed protein can lyse mouse red cell and its hemolytic titer attained 2.26 ×104 HU/mg. Conclusively, the Staphylococcal α-HL gene was successfully cloned and expressed in E. coli. The successful expression of α-HL in E. coli BL21 constituted a solid foundation for further researches such as pathogenesis and immune mechanism and vaccine development.