Abstract: The insulin-like growth factors (IGFs) play a central role in cellar growth, division and differentiation, their binding proteins (IGFBPs) with various posttranslational modification are important for metabolism and distribution of IGFs, but their biological activity is independent from IGFs. In this study, one SNP mutation of G/A was rapidly screened by PCR with direct sequencing based on gene-pool in position +534 of IGFBP5 coding sequence, 300 F2 pigs of Jinhua×Pietrain crossbred was genotyped by PCR-RFLP. The results showed that three genotypes (GG, GA and AA) were found, the frequency of genotype GG, AG and AA were 0.064, 0.589 and 0.348 respectively, and the allele frequency of G and A was 0.358 and 0.642 respectively. The significant effect of various genotypes was found on weaning weight and water holding capacity by using the general linear model (p<0.05).

CAST gene plays an important role in muscular protein degradation, muscle cell proliferation and post-slaughter calpain activation. Study on interaction effects of polymorphisms of CAST gene and nutrition levels has an essential role in the regulation of pig carcass quality. Interaction effects of polymorphisms of CAST gene at HinfⅠ, MspⅠand RsaⅠsites and three nutrition levels on meat carcass traits were studied among four pig population including Duorc, Daweizi, Durocⅹ(LandraceⅹLarge White) and Larege White ⅹDaweizi. The results showed that the three RFLP markers of gene CAST were all polymorphic in the four pig populations. These RFLPs are bi-allelic, with alleles A and B at HinfI, alleles C and D at MspI, and alleles E and F at RsaI. Genotype AA was significantly associated with reduced cooking percentage and drop loss in the high nutrition group, and with augmented drop loss in the moderate nutrition group (P<0.05). Genotype AB was associated with significantly decreased carcass length and loin eye area, and with increased drop loss in the high nutrition group, and with significantly decreased dressing percentage, back-fat thickness, and cooking percentage in the moderate nutrition group. Genotype CD showed significant interaction with high nutrition level on all traits. Genotype CC significantly increased drop loss (P<0.05). The performance of pigs of genotype EF overcame the average by 10% in some traits, when fed with high nutrition diets (P<0.05). The interaction between genotype EE and moderate nutrition level was significant on back-fat thickness.

The 667 bp control region of mitochondrial DNA from 8 domestic duck breeds and Anas zonorhynchto were sequenced. The genetic polymorphism and the origin of the 8 domestic ducks were analyzed. The result showed that content of nucleotide A, G, C, T were25.5%,15.3%,33.4%and 25.8% respectively. The mutation types were transition and transversion in this region. 20 haplotypes were found in this study, in which Hap2(A7) was major haplotype in 72 domestic ducks. The haplotype diversity(Hd) and average nucleotide diversity(Pi) were 0.67136 and 0.19％, respectively. Hd and Pi of Youxian Sheldrake was the highest in the 8 domestic duck breeds. Kimura 2 parameter genetic distance between the breeds was 0.00056-0.00414. A phylogenetic analysis of the 38 haplotypes identified that the 8 domestic ducks were only origin from Anas platyrhynchos.

Abstract: 268 samples from 7 sheep populations including Gansu Tibetan sheep, Gannan Tibetan sheep, Qinghai Tibetan sheep, Hu sheep, Small- tailed Han sheep, Tan sheep and Minxian Black Fur sheep are involved to detect the alleles in 15 microsatellites DNA loci. The analyses of the variations in alleles, populations’ heterozygosity, the ratio of Hardy-Weinberg, genetic distance, phylogenetic tree and genetic structure are performed. As the result shows, total 187 alleles are discovered in 15 microsatellites DNA site. And 43 population-loci deviated from the Hardy-Weinberg equilibrium. The quantity measure of the deviation in Hu sheep is the largest in our investigated populations. The Populations heterozygosity analysis shows: in concerned 7 populations, Qinghai Tibetan sheep has rich genetic diversity, while Hu sheep and Minxian Black Fur sheep are lower than others. The genetic distance and phylogenetic tree results shows: the Hu sheep, Tan sheep and the Small-Tailed Han sheep's relationship are closer than other populations involved, which have the same ancestor. The genetic structure shows that the these 7 populations can divide into three genetic clusters.

Abstract: Traditional method of screening homozygous transgenic plants is several generations self-pollenation, this is a time consuming work and low efficiency. We created a new method by flanking sequences amplification of T-DNA insertion site to screen homozygous plants. Firstly, we using modified adaptor ligation PCR method amplified flanking sequences of T-DNA in tomato transgenic plants. Then flanking sequences were comfirmed by PCR analysis in transgenic plants. BLAST results in SGN database showed that T-DNA integration site lost 40 base pair. Finally, we screened out homozygous lines in T1 transgenic generation using flanking sequence primers and border sequence primers, and verified the results in T2 generation.

The anther proteins in uninucleate-stage and binucleate-stage from wheat male sterile line induced by CHA SQ-1 were separated by two-dimensional electrophoresis with immobilized pH(4~7) gradients as the first dimension and SDS-PAGE as the second. After Coomassie Blue G-250 staining and analysis by PDQuest 2DE software, over 500 protein spots were detected reproducibly, and these protein spots mainly focused on pI5~ 6, molecular weight ranging from 20.0kD to 40.0kD. 43 in uninucleate-stage and 45 in binucleate-stage differentially expressed protein spots were detected by PDQuest 2DE software, Molecular weight and pI of these spots were calculated. Among these, in uninucleate-stage, 11 protein spots were absent in the protein map of treatment anther but present in that of check, 7 present in that of check but absent in that of treatment, 14 and 11 up-regulated significantly in that of treatment in comparison with that of check; in binucleate-stage, 13 protein spots were absent in the protein map of treatment anther but present in that of check, 7 present in that of check but absent in that of treatment, 12 down-regulated and 13up-regulated significantly in that of treatment. The protein spots A-Z3（26.8/5.7）in uninucleate-stage and C-Z5（26.8/5.7）in binucleate-stage were absent in the protein map of treatment anther. The spot A-L10(26.8/5.9) of treatment anther in uninucleate-stage was down-regulated but spot C-Z6（26.9/5.9）absent in binucleate-stage. These differential proteins may participate in pollen development process directly or indirectly. It can be deduced that the differentially expressed protein spots might relate to male sterility induced by CHA-SQ-1.

The Xyn2 gene, which encodes the main Trichoderma reesei Rut C-30 endo-β-1, 4-xylanase was successfully cloned into the pPICZαA vector and expressed in Pichia pastoris. The desired P. pastoris strains produced β-xylanase under the control of the methanol inducible alcohol oxidase 1 (AOX1) promoter, and the secreted recombinant Xyn2 was estimated by SDS-PAGE as 21 kDa. The activity of the recombinant Xyn2 was highest at 60℃ which was 5℃ higher than native xylanse. In addition, the recombinant Xyn2 was active over a broad range of pH 3.0-8.0 with maximal activity at pH6.0. The enzyme was quite stable at 50℃ and retained more than 95% of its activity after 30 mins incubation at this temperature. These properties should make the enzyme an attractive candidate in various industrial applications.

Stenotrophomonas maltophilia YHJ-1, isolated from the poultry factory, shows the high feather keratin degradation activity as well as many antibiotics resistances including kanamycin. In this study, a transposon plasmid pRH30 was developed using chloramphenicol gene to replace kanamycin gene with transposon plasmid pRL27 containing both hyperactive Tn5 transposase and kanamycin resistance gene and pMCm, carrying chloramphenicol cassette. Finally, an insertion library containing 1246 mutants was produced with pRH30 by biparental conjugation between donor E. coli UQ3201/pRH30 and recipient strain YHJ-1. The results of insertion mutants, subjected to detection of chloramphenicol resistance and PCR sequencing analysis, indicated that the library was reliable. The fact that 3 mutants were unable to degrade feather suggested that transposon mutagenesis with pRH30 was a highly efficient method for facilitating the isolation of genes and analyzing the molecular mechanism on feather-degrading of S. maltophili YHJ-1.

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant eukaryotic expression plasmid pVAX1-F, and subcloned into the plasmid pTR-C3d3 containing three copies of C3d. The F-C3d3 gene was identified by sequencing and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX1-F-C3d3 was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207(pVAX1-F-C3d3). One-day-old SPF chickens were immunized orally with SL7207(pVAX1-F-C3d3) at the dosage of 1×109 CFU and boosted two weeks later with the same dose. The serum antibodies responses specific to NDV were observed in chickens immunized with SL7207(pVAX1-F-C3d3) on day 14 and 28, and their levels were significantly higher than that of SL7207(pVAX1-F) (P < 0.05). Intestinal mucosal immune response were observed in chickens immunized with recombinant bacteria on day 28, and their levels were significantly higher than that of negative and positive control (P < 0.05), but there was not significance between the groups SL7207(pVAX1-F-C3d3) and SL7207(pVAX1-F). These results indicated that three copies C3d could enhance humoral immune response against NDV induced by attenuated Salmonella typhimurium harbouring DNA vaccine. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.

The eight full-length genes of H9N2 subtype avian influenza virus A／Chicken／Zhejiang／HJ／2007(H9N2) were amplified by reverse transcription-polymerase chain reaction (RT-PCR),and then cloned to pMD18-T vector and sequenced. The sequenced result revealed that the hemagglutinin（HA）gene’s deduced amino acid sequence at the cleavage site of the HA protein was -PSKSSR*G-, which was match to the characteristic of low pathogentic avian influenza（LPAI）, which belong to the A/Quail/Hong Kong/G1/97 (H9N2) sublineage, not the same as most of the viruses belong to the A/Chicken/Beijing/1/94 (H9N2) sublineage.The neuraminidase (NA) gene had deletions at 63, 64 and 65 amino acids, which derived from the A/Chicken/Beijing/1/94(H9N2)[A/Duck/HongKong/Y280/97(H9N2)]sublineage.The nucleoprotein （NP） was similar with some H5N1 subtype influenza viruses which also isolated in Zhejiang Province, they all had near phylogenetic relationship. The matrix （M）protein was at one sublineage with A/Hong Kong/1073/99(H9N2) and A/Hong Kong/1074/99(H9N2), those two viruses infected two children in Hong Kong at 1999. The nonstructural （NS） protein was at one sublineage with H9N2 subtype swine influenza viruses isolated during the past few years, at one sublineage with A/Swine/Hong Kong/9/98(H9N2). However, the polymerase protein: the PB1 gene had 15 nucleotides（caatccgactttact）deletions from 33 to 47, which was first report had deletion 5 amino acids from four to eight in ORF. The PA gene and the PB1gene had familiar phylogenetic, which were near to the H5N1 subtype influenza viruses. But the PB2 gene didn’t show any immediate ancestor with the different avian influenza viruses used in the paper. Therefore, A／Chicken／Zhejiang／HJ／2007(H9N2) is product of natural reassortment from different influenza viruses sublineages.

Adipose triglyceride lipase (ATGL) was recently identified and described as an important factor in fat metabolism. In this study, we construct specific small interfering RNA (siRNA) expressing vectors of ATGL gene. Porcine ATGL-specific small interferencing RNA (siRNA) was designed and inserted into pSilencer 4.1-CMV neo vector after annealing. The constructed recombinant was analyzed and identified by PCR, enzyme digested and sequencing. The results showed that the porcine ATGL-specific siRNA recombinant expressing vectors was constructed successfully. The results will benefit for further study on the function of porcine ATGL gene in fat metabolism using RNAi technology .

Abstract:Suppression subtractive hybridization method was employed to construct a cDNA library expressed in mammary gland of early and peak lactation stages of the Xinong Saanen goat. Osteopontin was one of the positive clones . The CDs of goat osteopontin gene was cloned from the goat mammary gland by RT-PCR , real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analysis the change of OPN mRNA level between early and peak lactation stages,The sequence was predicted and registered in GenBank EU295699. Osteopontin gene with an ORF of 834 nucleotides encoded a polypeptide of 277 amino acids with a putative signal peptide of 16 amino acids. The nucleotide sequence homology of osteopontin of Xinong Saanen goat was found to be 98%, 95% ,71% and 70% compared with that of sheep(AF_152416), bovine (NM_174187), human(DQ_846871) and mouse (NM_009263), while the amino acid sequence homology to be 98%, 91%,55% and 54%, respectively.The mRNA level of OPN gene in mammary gland of early lactation stages was 5.03 folds higher than that in peak stage normalized by housekeeping gene β-actin .Goat osteopontin had two additional potential binding motif s compared to humans’ and mouses’.

CELⅠ is a key enzyme in TILLING(Targeting Induced Local Lesions In Genomes)technology, and it can cleave heteroduplex DNAs with high specificity at sites of base mismatch and DNA distortion. Here we extracted CELⅠ from celery(Apium graveolens), and made detection by SDS-PAGE, which showed the main band at 43kD. Then used heteroduplex DNAs as the substracts, investigated the activity of CELⅠ. The results indicated that CELⅠ can cleave heteroduplex DNAs of different materials with high specificity at sites of base mismatch. Through the extraction of CELⅠ, activity investigation, and application for detection of gene polymorphism in rice, we will provide a simple and practicable method for detection of gene polymorphism.

The Bt gene expression cassettes of GM cotton 33B and GK-12 were sequenced and analyzed. Differences were found both in the Bt gene and the junction region of Bt gene/terminators except the junction region of CaMV 35S promoter/Bt gene. In order to detect the two transgenic cotton lines, three specific primers, MG-P1, MG-P2 and MG-P3, were designed and the duplex PCR detection method was established based on the differences. Forty transgenic cotton samples were tested by this duplex PCR method, thirty-two samples were identified to contain 33B Bt gene structure, six samples were identified to have GK-12 Bt gene structure, and two samples were identified to have both 33B and GK-12 Bt gene structures.

Despite the increasing importance of the pig as a large animal model, little is known about porcine amniotic fluid–derived stem (AFS) cells. AFS cells were isolated from 30-day porcine fetuses amniotic fluid. Plasmid containing EGFP gene was transfected into AFS cells by lipofection. The positive AFS cells were abtained. AFS cells were induced to differentiate into cell types representing each embryonic germ layer, including cells of neuronal, adipogenic, osteogenic, myogenic and endothelialand lineages. Expanded AFS cells and differentiated cells were banked or harvested for analysis using reverse transcription–polymerase chain reaction (RT-PCR).Cultured porcine AFS cells widely expressed β-Actin, NogoA, DCX, CyclinD2, CD133, Hes1, Oct4, Desmin, CD-90, Nanog and Sox2. AFS cells were differentiated into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+、NSE+ and MAP2+), adipocyte (LPL+ and PPARγ-D+), osteoblast (Osteonectin+ and Osteocalcin+), myocyte (myf-6+ and myoD+) and endothelium (CD31+, CD34+, CD144+ and eNOS+). This study shows that porcine AFS cells are broadly multipotent, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of disease and injury.

The objective of the present study was to investigate the effects of NO donor nitroprusside sodium (SNP) (1mM) on mouse oocytes spontaneous maturation and to explore the mechanisms by which NO influenced oocyte spontaneous maturation. Oocytes maturation was evaluated through in vitro cell culture and intracellular cGMP levels were determined by Radioimmunoassay (RIA). The results showed that NO donor SNP (1mM) could delay the occurrence of GVBD and inhibit the first polar body (PB1) extrusion of cumulus-oocyte complexes (COCs). The data of RIA demonstrated that NO donor SNP significantly increased the cGMP levels in COCs and that the stimulative effect of SNP on cGMP level could be eliminated by soluble guanylate cyclase (sGC) inhibitor ODQ (10μM). ODQ could also reverse the inhibitory effects of SNP on COCs spontaneous maturation. However, the suppressive effects of SNP on COCs spontaneous maturation were not abolished by PKG inhibitor KT5823 (1μM). These results indicated that NO elicited its effects on spontaneous maturation through activating sGC and elevating the intracellular cGMP levels and suggested that PKG signal pathway was not involved in NO-mediated mouse COCs spontaneous maturation.

Based on the cyclophosphamide (CP) induced immunosuppressed mice model, we investigated the effect of the enriched-selenium polysaccharide produced by Z0206 on the immunoloregulation in mice. The mice were divided into 4 groups, control groupⅠ, CP group Ⅱ, enriched-selenium polysaccharide group Ⅲ and prevetion of enriched-selenium polysaccharide group Ⅳ. They were given different doseages of normal saline or the enriched-selenium polysaccharide Z0206 by gavage respectively. Compared to the control group, immunosuppression appeared in the CP group. Results showed that compared with the CP group, the enriched-selenium polysacchride Z0206 could remission the immunosuppression, enhanced the spleen and thymus index(p<0.05), regulated T cell subsets(p>0.05), enhanced the NK cell activities(p<0.05), raised the plapue forming cell assay and hemolysin level in mice (p<0.05). However, compared with the control group, the spleen and thymus index, T cell subsets, NK activity, and humoral immunity increased significantly(p<0.05). Conclusion: the enriched-selenium have obvious improvement and arrest of immunosuppression induced by CP in mice.

The antigene FaEtr1 and FaErs1 were introduced into leaves of stawberrywith Agrobacterium tumefaciens, and regenerated plants were obtained by kanamycin selection. 15 plants were confirmed by PCR and Southern blot analysis that the antigene FaEtr1 and FaErs1 gene were transferred and integrated into the genome of strawberry. Northern blot analysis indicated that FaEtr1 and FaErs1 mRNA abundance were decresased in antisense strawberry plants.

Four cultivars of pepper（Capsicum annuum L.）were successfully transformed with the CBF-4 gene by Agrobacterium mediated transformation. This study established a highly efficient regeneration system of pepper . The main factors influencing regeneration and transformation rate were discussed and the best conduction of regeneration and transformation were determined. The positive regeneraion was obtained.60 Km-resistant regenerated plants were obtained, 46 of which were positive by the analysis of PCR and RT-PCR.The rate of transgene is 76.67%.The morphological indexes of transgenetic plants showed that it was compact plant type with thicker, darker green and larger area leaves compared with the non-transgenetic plants. The enzyme activities of SOD, POD and MDA in transgenetic plants are higher than that of the non-transgenetic plant (control group) after the cold resistant treatment.

Late embryogenesis abundant (LEA) protein gene is an important gene in plant stress response. In this paper, a full-length cDNA sequence of sugarcane Lea gene was obtained from sugarcane leaf full-length cDNA library through sequencing and validated by the corresponding bioinformatics analysis , termed Sc-Lea . The full-length of Sc-Lea is 921 bp, with a 495 bp open reading frame(ORF), with a 64 bp 5’ untranslated region(UTR) and 362 bp 3’ UTR . At the 3’ end UTR, it has a clear polyA structure, the sequence around the start codon is CCCCAGCCATGGC,and the base of -3、+4 is G and C respectively which is line with the rules of Kozak. Then, the prokaryotic expression vector, which contained the ORF of sugarcane Lea gene, was constructed and the targeted protein at the molecular weight 17.9kD coding by sugarcane Lea gene was induced by IPTG. Moreover, in order to explore the expression characters of sugarcane Lea gene under the stress of SA, H2O2, PEG and NaCl, the Real-time qPCR approach was applied. The results showed that the expression of the sugarcane Lea gene was induced both by PEG and NaCl, while inhibited by H2O2. Also, the expression of this gene could be influenced by SA. It could be to some extent inferred that the sugarcane Lea gene obtained in this study plays an important role in the sugarcane drought-tolerant and salt-tolerant mechanism.

In order to analyse gene expression pattern in the sugarcane leaves and set the basis for the cloning of some important functional gene, a full-length cDNA library was constructed by the method of Oligo-capping, taken functional leaves from three development stage of sugarcane as plant material. After identification, the titer of the library was 1.8×105pfu/mL, the recombinant rate was 89%, the average size of inserted fragment was about1.0kb. In total, 228 efficient EST(expressed sequence tags) of 5’ end were obtained from this cDNA library. bioinformatics analysis showed these ESTs were assembled into 154 TUTs(tentative unique transcript, TUT),The homologous analysis of the 154 TUTs on NCBI showed that these 64 TUTs (including 94 ESTs) were determined to be the tags of gene with putative function, these genes were found to be involved in the biological process including cell development, signal transport ,protein synthesis, transcription, stress tolerance response, energy metabolism and so on.

Abstract: Comparing with their corresponding wild allele genes, a single nucleotide mutation had been taken place in high pigment genes hp1 and hp2, respectively, which was very difficult to be discriminated by normal PCR technique. In this experiment, the relationship between specific PCR and mismatched bases as well as their positions were studied. Results showed that the primer with double-base mismatch TA-TG can distinguish mutation location of hp1 from its wild allele location at annealing temperature 49.3 +0.1 ℃. A primer with T-C mismatch can distinguish mutation location of hp2 from its wild allele location at annealing temperature 53.5+0.1 ℃. The postion of mismatched bases at 3` end of primers had little effect on specificity of PCR.

The genetic diversity of 51 parental lines used for hybrid production and breeding in Brassica napus L. was studied by SSR and SRAP markers, and the results of these two maker systems were compared. One hundred and ninety-four polymorphic fragments were detected using 45 SSR primer pairs, with an average number of polymorphic fragments per primer pair of 4.3. Whereas 197 polymorphic fragments were detected using 25 SRAP primer pairs, with an average number of polymorphic fragments per primer pair of 7.9. The 51 parental lines could be divided into five groups based on either SSR or SRAP markers by UPGMA cluster analysis. The main maintainer lines and restorer lines of Polima CMS were classified into the same group but different sub-groups. The groups classified by SRAP markers were more in accord with pedigree information than SSR markers.The SRAP markers were more effective in genetic analysis of closely related lines. The genetic distance calculated based on SRAP markers was larger than that on SSR markers. There is difference between the genetic diversity evaluated by SRAP and SSR markers, even though the same number of polymorphic fragments was used for evaluation. This difference may be caused by different numbers of variation alleles of the same gene detected by different primer sequences.

Acid polyacrylamide gel electrophoresis (A-PAGE) was employed to detect the gliadin genetic diversity among fifty-four wild accessions of Elymus sibiricus L. collected from Qinghai-Tibetan plateau. The follow results were obtained: (1) A total of 42 bands were detected in all accessions, of which 92.86% were polymorphic. The average number of Shannon index to four electrophoretic zones (α, β, γ, ω) was 0.4627. The Nei’s genetic similarity coefficient of the tested accessions ranged from 0.2424 to 0.9767, and the average Nei’s coefficient was 0.5822. These results suggested that there was a rich genetic polymorphism among the tested wild resources of E. sibirucus. (2) 54 wild accessions can be clustered into 4 groups at GS = 0.562 level on dendrogram. The principal coordinates (PCA) reflected almost the same relationships among the studied materials as showed in cluster analysis. Moreover, the accessions from the same origin frequently clustered into one group. The findings implied that a correlation among gliadin patterns, geographical and ecological environment. (3) Genetic differentiation of between and within five eco-geographical groups of E. sibiricus is estimated by Shannon’s diversity index, which shown that 68.17% genetic variance existed within group, and 31.83% genetic variance was among groups. (4) The unweighted pairwise groups method using arithmetic average (UPGMA) cluster analysis based on Nei's unbiased measures of genetic identity was assayed for five geographical groups of E. sibiricus, which indicated that there was a significantly positive correlation between genetic differentiation and geographical habits among the five groups.

Effects of EGCG, Cd2+ and EGCG+Cd2+ on the growth of PC-3 cells were investigated by MTT assay. Morphological changes of PC-3 cells were obtained with a inverted microscope. Apoptosis of PC-3 cells were measured according to Cell Apoptosis Rhodamine 123 detection Kit. Cell membrane fluidity was detected with Bruker ESR A300 spectrometer. EGCG and Cd2+ were screened for growth inhibition of PC-3 cells. And they were found to bring the changes of PC-3 cells morphologically. Apoptosis of PC-3 cells treated with 80 µmol/L EGCG were not induced. Meantime, cell necrosis was found by 20 µmol/L Cd2+. But apoptosis of PC-3 cells was induced by 80 µmol/L EGCG in the presence of 20 µmol/L Cd2+. Also, the membrane fluidity of PC-3 cells was decreased by EGCG and EGCG+Cd2+.

We identified for cry7Ab gene by PCR –RFLP from Bacillus thuringiensis HQ40.Then we designed a pair of special primer for full length DNA according to the published sequence of cry7A genes, successfully cloned it into Bt-E.coli shuttle vector pSTK and and transformed into acrystalliferous strain HD73cry-.The Bt engineering bacteria was named HD7Ab4.The gene had been registered in GenBank with accession number EU380678 and designated as the novel gene cry7Ab4 by International Nomenclature Committee of Bt.The SDS-PAGE showed that the cry7Ab gene could be expressed as 130 kD peptide.In addition, rhombohedral crystals was observed.Subsequently,we purified crystal proteins from Bt engineering bacteria HD7Ab4 and wild-type HQ40 respectively,we used the trypsin-activation of pro -endotoxins to do toxicity assay against a series of Orthopteran、 Lepidoptera、Coleoptera.The bioassay results indicated that the cry7Ab4 toxic protein had distinctly insecticidal activity against Colaphellus bowringi Baly.However,it exhibited little toxicity towards Oriental migratory locust ,Plutella xylostella、Spodoptera exigua、Ostrinia furnacalis、Pyrrhalta aenescens、Leptinotarsa decemlineate.

Purification and characterization of an antilisteria bacteriocin produced by Enterococcus faecium M-2, isolated from Inner Mongolia traditional cheese, were studied. Enteriocin M-2 was purified by 80% ammonium sulphate precipitation, followed by SP-Sepharose FF cation exchange chromatography. And the specific activity of purified bacteriocin was 411.204AU/mg. The inhibitory activity was heat-stable, and insensitive to pH and surfactants. However, it was sensitive to proteolytic enzymes. In addition, the bacteriocin had a broad inhibitory spectrum. It not only had a strong effect on gram-positive, such as Listeria bacteria, Staphylococcus aureus, Bacillus, and etc, but also can inhibited several gram-negative bacteria. These results showed that the bacteriocin had a potential application as food additives and bio-preservatives in food industry.

In this study, a high-frequency transformation system has been established by Agrobacterium tumefaciens soaked seeds transformation technology, Chinese zoysia germinating seed were used as transformation receptor. External factors that affect the efficiency of genetic transformation were studied and optimized, on the base of NPTⅡ. The major results as followed: seed was germinated for 7 days, then was infected with Agrobacterium which OD600 was 0.9 with 100μmol.L-1 AS for 48 hours after pumping 5min+30s vacuum treatment. Then co-cultivation for 3 days, selected the transferred plant by 75mg.L-1 kanamycin. It was originally proved by the PCR and GUS examination that NPT-Ⅱand GUS gene had been transferred into the genome. The positive transformation rate was 55.21% by the analysis of PCR.

Five thousand and twenty five ESTs of Juglans regia from the database of dbEST were screened using SSR-Hunter software, and 540 SSRs in 485 ESTs, accounting for 10.75% of ESTs were mined out. The SSR-ESTs occupied 9.7% of the total number of ESTs. Forty three primer pairs were designed for polymorphism analysis in 6 genotypes of J. regia, 1 genotype of J. nigra and 1 genotype of J. mandshurica. 40 primer pairs showed the amplification, accounting for 93% of designed primers, and 35 primer pairs showed polymorphisms, accounting for 87.5% of primers available. In the polymorphic 35 primer pairs, 27 primers showed polymorphisms in J. regia, the other 8 primers did not display polymorphism in J. regia, but showed polymorphisms among J. regia, J. nigra and J. mandshurica. Cluster analysis of the 8 genotypes was performed by UPGMA method, the result accords with the conventional taxonomy.

Liver tissue was taken from Wanxi White Geese and Landaise Geese at 90days of age. The number of each breed is 20. The total RNA was extracted from liver to measure the abundance of the growth hormone receptor（GHR）, insulin like growth factor-1（IGF-1）and insulin like growth factor-1 receptor（IGF-1R）by Semi-quantitative RT-PCR using β-actin as an internal standard. Results show that the liver weight and liver index of Wanxi White Geese were significantly higher than in that of Landaise Geese（p<0.05）. IGF-1 mRNA expression was higher in Wanxi White Geese than that in Landaise Geese （p<0.01）. Significant positive correlation was found between body weight and liver weight（r=0.35, p<0.05）. The liver index with the expression of IGF-1mRNA in liver also had significant positive correlation（r=0.67, p<0.01）. The results suggest that, at 90 days of age, (1) The growth of liver was better in Wanxi White Geese than that in Landaise Geese; (2) the higher of liver wight of Wanxi White Geese could be the results of higher expression of IGF-1 mRNA in liver .

Using the genomic DNA extracted from Yane geese, the orthogonal design was used to optimize ISSR-PCR amplification system in five levels of five factors(Taq DNA polymerase, Mg2+,DNA template, dNTPs and primer)， respectively. Based on the preliminary experiment, the density within factors of ISSR-PCR was further optimized. The purpose of this experiment was to determine the reaction condition of ISSR-PCR fleetly and accurately, which was basis for the study on genetic diversity, map construction and gene localization of Yane geese. The results showed that a better amplification of ISSR was obtained with the reaction system containing 0.20umol/L primer, 0.28mmol/L dNTPs, 40ng templet DNA of Yane geese, 1.5mmol/L Mg2+,1.0U Taq DNA polymerase in the total volume of 25uL. Based on the orthogonal design experiment, the prime reaction condition of ISSR-PCR can be established through the density within factors of ISSR-PCR further optimized.

Based on the conserved sequence of Perilla’s MYC mRNA gene from GenBank, the conserved fragment of MYC like anthocyanin transcriptional activator gene was amplified by using RT–PCR from the RNA of purple potato coat. Then the cDNA 3-end sequence was amplified by using 3’-Race technique from the RNA of purple potato coat. Sequence analysis show that the nucleotide sequence of this gene is 1106bp, containing a complete open reading frame and encoding 326aa, named stmyc. The deduced protein of stmyc gene shares 46.98％identity with MYC-GP protein. stmyc gene encodes a typical bHLH protein domain that is more important for MYC-like proteins. The results of RT-PCR expression analysis show that stmyc expressed in the leaves, stems, tuber skins, tuber fleshes and roots of purple potato and the leaves, tuber skins, tuber fleshes of white potato with the different expression levels. It is suggested that the stmyc is constitutive expressed in potato.