Abstract:Lipoxygenases (linoleate: oxygen oxidoreductase, EC 1.13.11.12; LOXs) are encoded by a multi-gene family in plants. The LOXs are monomeric non-heme, non-sulfur iron dioxygenases, which catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids containing a cis, cis-1, 4-pentadiene moiety. The LOX isoforms are distinguished by differences in optimum pH of the reaction, pI, substrate and product specificity, spatial and temporal expression, and subcellular localization. The function of various LOXs in plants has been suggested. Some of the physiological processes in which lipoxygenases have been implicated include wounding, pathogen attack, seed germination, fruit ripening, plant senescence, and synthesis of Abscisic acid (ABA) and Jasmonic acid (JA). During normal vegetative and reproductive growth, lipoxygenases have also been suggested to act as vegetative storage proteins, participate in transference of lipoid, and response to nutrient stress and source/sink relationships. Significant progress in understanding LOX families will be beneficial to the application of the LOX in crop breeding, research on new-type phytoalexin and food industry.

Abstract:In recent years, the potential value of nonstructural protein (NSP) 2C was well documented for distinguishing foot-and-mouth disease virus (FMDV) in infected animals and vaccinated animals. In order to develop a more sensitive approach to detect natural infected FMDV while there is no interact with vaccinated FMDV, we incorporated a major epitope region of 2C with whole 3AB coding region within NSP and expressed in Escherichia coli. We got a 47.6 kD fusion protein named 2C￠3AB. The product showed a specific reactivity with FMDV from serum of infected animal by using Western blotting analysis. This suggests that this protein could be applied to distinguish infected FMDV and vaccinated FMDV. We further compared 2C￠3AB protein with 3ABC fusion protein, another available protein used for detecting infected FMDV, using indirect ELISA assay. The results showed that 2C￠3AB-ELISA had higher sensitivity than that of 3ABC-ELISA for distinguishing infected FMDV and vaccinated FMDV of sera from epidemic region. Therefore, this recombinant protein 2C￠3AB is a good candidate protein to develop more sensitive method to differentiate infected FMDV and vaccinated FMDV from vaccinated animals. This finding will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.

Abstract:To explore the effect of chronic high dose of insulin on lipolysis in porcine adipocytes and the underlying molecular regulation mechanisms, we cultured primary porcine adipocytes and incubated them with different concentrations of insulin (0, 200, 400, 800, 1600 nmol/L) for 24~96 h in the absence or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the glycerol release into the culture media as an indicator of the lipolysis, and observed the lipid accumulation morphology by phase-contrast microscopy. Further, we analyzed the gene expressions of perilipin A and peroxisome proliferator-activated receptor-gamma2 (PPARg2) with semi-quantitative RT-PCR and Western blotting, respectively. The results showed that chronic high dose of insulin stimulated lipolysis in differentiated porcine adipocytes in a dose- and time-dependent manner, and significantly attenuated the lipolytic response to isoprenaline. Meanwhile, the protein and mRNA expressions of PPARγ2 and perilipin A were significantly reduced. In addition, both PKA and ERK inhibitors significantly suppressed insulin-stimulated lipolysis, however, only ERK inhibitor reversed the insulin-induced down-regulation of perilipin A. These findings imply that chronic high dose of insulin stimulates lipolysis in porcine adipocytes by repressing perilipin A, which is involved in ERK pathway.

Abstract:The cDNA of cattle Ghrelin gene was amplified from abomasum fundic gland mRNA of Qinchuan Cattle by RT-PCR. PCR product was cloned into the T vector pGEM-T to construct pGh-T1 for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a(+) and transformed into host Escherichia coli strain BL2l (DE3) for expression. The expression of pGh-32 mature Ghrelin protein was induced by IPTG and was identified by SDS-PAGE. The expression product was observed with soluble protein and inclusion body. Western-blotting showed that the recombinant protein was recognized by his-antibody specifically. The protein was purified by Ni-NTA column and was used to inject rabbits to obtain polyclona antibody. ELISA result showed that the antibody titer was 1:12 800. The immunohistochemistry test between the hypothalamus arcuate nucleus and the antibody showed that fusion protein had biological activity. This will provide a basis for further study on the biological function of Ghrelin protein to growth and development and fat deposition of cattle.

Abstract:Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. The objective of this study was to evaluate the potential of attenuated Salmonella enterica serovar Choleraesuis C500 strain with a Dasd mutant as an effective live vaccine vector by the Asd+ balanced-lethal host-vector system. Here, we compared the characteristics of S. enterica serovar Choleraesuis DasdC500 strain with the parent C500 strain, including phenotype, growth rate, virulence, safety, and expression for heterologous antigen. The mean generation times of ΔasdC500 mutant, the vector control DasdC500(pYA3493), and the parent avirulent C500 vaccine strain in Luria broth were 30.7, 28.1, and 27.9 min, respectively. The fermentation patterns of theses three strains on different carbohydrates, and the levels of production of H2S, were similar. The O and H antigens of DasdC500 mutant, DasdC500(pYA3493) and DasdC500(pYA-F1P2) were 6,7:C:1,5, identical to the parent strain C500. By the method of Reed and Muench, groups of mice were challenged by the intraperitoneal route with different amounts of DasdC500(pYA3493) or the parent C500 strain, and the virulence of DasdC500(pYA3493) with LD50 of 1.1×107 CFU was a little lower than C500 with LD50 of 4.4×106 CFU. All piglets inoculated with DasdC500(pYA3493) or C500 survived, and no signs of disease were observed during the entire experimental period. No major differences were found in these two groups. In addition, the recombinant pYA-F1P2 plasmid was very stable in the recombinant DasdC500(pYA-F1P2) strain, which expressed secretorily a large amount of the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen (rF1P2) of Bordetella bronchiseptica. In this study, we have shown that the DasdC500 mutant had a series of biological characteristics silimar to the parent vaccine strain C500. Furthermore, the strain could express secretorily a large amount of heterologous antigen. It is likely that this Salmonella expression and delivery system could be easily adapted to develop multivalent recombinant Salmonella vaccines against infectious agents using the Asd+ balanced-lethal host-vector system.

Abstract:The aims of this research were to construct prokaryotic expression vector containing fusion gene of Cholecystokinin 39 (CCK39) of pig and Urease subunit B (UreB) of coliform bacteria, and then to express the fusion protein in reconbinant Escherichia coli BL21(DE3). The CCK39 gene was amplified by RT-PCR from the extracted total RNA of pig’s duodenum, and the UreB gene was then amplified by PCR from the extracted plasmid DNA of bacillus of coliform bacteria from pig’s intestinal content. Then the CCK39 and the UreB were inserted into the prokaryotic expression vector pET43a(+) to construct a recombinant fusion expression vector pET43a(+)/CCK39/UreB and then, the recombinant vector was identified by PCR, endonuclease digestion and sequence analysis. It was identifier that the gene fragment of CCK39 at length of 117 bp and UreB at length of 324 bp were amplified and cloned into the vector pET43a(+) successfully. The recombinant vector was transformed into Escherichia coli BL21(DE3) and induced the expression of CCK39/UreB fusion protein with a molecular mass of approximately 80 kD by using isopropylthio-b-D-galactoside (IPTG) as inducer. The fusion protein was mostly located in the cytoplasm and it was soluble. The soluble protein was collected and purified by Ni2+-NTA column chromatograph and then reached a purity of more than 95%. It was proved by western blotting that the fusion protein could react with rabbit anti-CCK8 antiserum and rabbit anti-UreB antiserum. Therefore, the expressed fusion protein has good antigenicity. This work established a good foundation for further study on the production of anti-CCK/Urease vaccines.

Abstract:K88ac-LTB gene derived from pQE30-K88ac-LTB was cloned into the expression vector pLA and then the recombinant vector was transformed into the competent cells Lactobacillus casei 525. The recombinant bacteria were grown at 37°C, in MRS broth. Western blotting analysis with rabbit-anti-K88ac-LTB polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 71.2 kD. The K88ac-LTB fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy and flow cytometric analysis. In addition, the survival of recombinant Lactobacillus casei 525 was studied in imitative gastrointestinal environments such as artificial gastro fluid (pH 1.5–5.5), artificial intestinal fluid, bile(0.3–3.0 g/L).The results indicated that the recombinant strain survived well in artificial gastric fluids at pH 2.5–4.5 in 5 h. The recombinant Lactobacillus casei 525 could slowly grow in the artificial intestinal fluid for different time, and could survive in 0.3%bile.

Abstract:Ferulic acid esterase (FAE) was used to hydrolyze feruloyl ester linkages between hemicellulose and lignin in natural lignocellulose, and the possibility of FAE accelerating cellulase to hydrolyze steam-exploded rice straw by breaking covalent linkages was studied. When the dosage of FAE was 240 mu/g substrate, the cellulose conversion rate and the weight-loss rate of insoluble substrate at 72 h were respectively 32.00% and 32.77%, more than without using FAE; Cellulose conversion rate and the weight-loss rate of insoluble substrate were respectively 29.85% and 32.48% with FAE (300 mu/g substrate) processing time of 120 min. By comparison of the accessibility and FT-IR spectra of lignocellulosic material treated by different enzyme methods, it indicated that FAE hydrolyzed some ester bonds within it, and improved the accessibility by over 50%. It is concluded that FAE and cellulase have great synergistic effect, and FAE can help cellulase hydrolyze natural lignocellulose and enhance hydrolytic efficiency.

Abstract:We used four strains (Y1, Y2, M1 and M2) to screen out the high lipid production strains. We first adopted cell morphology and cytochemical methods (Sudan III dyeing technique) to observe intracellular characteristics. Observation results indicated that M2 strain had the potential lipid accumulation capacity. To prove this, lipid content of these strains was determined by soxhlet extraction. One strain (M2) was found to produce lipids up to 53.09%. In order to increase the production of oleaginous microorganism, the effects of hydrolysate concentration, nitrogen source, pH, fermentation temperature and time on cell growth and lipid accumulation were studied. The optimal fermentation conditions were obtained as follows: corn starch byproduct hydrolysate concentration at 10°Bx as carbon source; NaNO3 as nitrogen source at 0.2%; initial pH of 6.0; temperature at 28oC, cultivated for 6 d. Under these conditions, M2 strain accumulated lipids up to 75.21% on a cellular biomass basis with biomass yield of 30.40 g/L, and the corresponding lipid production reached 22.86 g/L. GC analysis demonstrated that the fatty acid composition of the lipid was similar to that of vegetable oil, which mainly contained 16-and 18-carbon fatty acids. Thus, microbial lipid is a promising material for biodiesel production, and its unsaturated fatty acid content reached around 68%. These unsaturated fatty acids show great potential applications in food, medicine and cosmetics industries.

Abstract:The effects of heavy metal cadmium (Cd), alone and in combination with zinc (Zn), on the root growth and activity of antioxidant enzymes superoxide dismutase(SOD) and peroxidase(POD) in Cucumis sativus L. hairy roots were studied. The purpose was to study the possibilities on using C. sativus hairy roots for phytoremediation of cadmium contamination. The results showed that less than 10 mg/L Cd enhanced the growth of C. sativus hairy roots and increased root diameter only in 5–15 days of root culture. At Cd concentrations above 15 mg/L hairy root growth was gradually inhibited with increasing Cd concentration. The roots formed were shorter with smaller lateral roots. Among all the Cd concentrations tested, except with 10 mg/L Cd, the soluble protein contents in the C. sativus hairy roots cultured with the other Cd concentrations decreased, but the POD and SOD activities increased gradually with time during the culture process. Further tests were conducted using a control culture containing 25 mg/L Zn alone. The addition of 1mg/L Cd to the 25 mg/L Zn culture stimulated the growth of C. sativus hairy roots after 7–15 days of growth, compared with the control. At all other Cd concentrations the growth of C. sativus hairy roots was inhibited compared to the control. Growth inhibition increased with increasing Cd concentration, and the hairy roots formed fewer, shorter and smaller lateral roots, the tips of which became swollen. After 5 days culture with different concentrations of Cd + 25 mg/L Zn, the root biomass and the activity of POD and SOD were lower than in C. sativus hairy roots cultured without the addition of Zn. However, the soluble protein content was significantly higher when the culture contained 25 mg/L Zn. Our results suggested that C. sativus hairy roots have higher tolerance to heavy metal Cd but higher concentration of Cd inhibited the growth. Cd in combination with Zn would result in more serious Cd-induced growth inhibition.

Abstract:Acidithiobacillus ferrooxidans is able to synthesize intra-cellular electron-dense magnetite, which formed by BCM method in Acidithiobacillus ferrooxidans. The whole genome of the type strain Acidithiobacillus ferrooxidans ATCC 23270 was analyzed by bioinformatics and some homolog genes of functional ones in magnetotactic bacteria were available. This study analyzed the different concentration of Fe2+ stress response of mpsA, magA, thy and mamB gene by using real-time PCR analysis. Temporal genes expression profiles were examined in cells subjected to different concentration of FeSO4·7H2O stress, they reached to high expression under 150~200 mmol/L FeSO4·7H2O stress. With this new method study, it is possible that we could do deeper research to generate a comprehensive description of the mechanism that how Acidithiobacillus ferrooxidans synthesize the magnetic particles.

Abstract:In order to effectively increase capacity of Cu2+ absorption by Penicillium from Cu2+-containing aqueous solution and to study the mechanisms of absorption, effects of eight pre-treatment methods on Cu2+ absorption of Penicillium janthinellum strain GXCR were compared. The results showed that the efficiency of Cu2+ absorption obviously increased through pre-treatment by homogenization, homogenization-basification (NaOH), oven dry (80oC), homogenization-salinification (NaCl), homogenization- detergent and homogenization-polarization (C2H6SO), but significantly decreased after acidification pretreatment with H2SO4. In comparison with the previous reports, the pretreatment in a homogenization-NaOH way could more efficiently enhance the Cu2+ absorption capacity of this fungus. Homogenization-basification (0.5 mol/L NaOH) increased Cu2+ biosorption by 47.95%. The Cu2+ absorption of the mycelia treated by homogenization-basification followed Langmuir isotherm equation, suggesting a surface absorption process. After four cycles of absorption-desorption, mycelia pretreated by homogenization-alkalization still had 70.82% of Cu2+ biosorption efficiency. Infrared reflectance analysis indicated that alkalization treatment made marked effects on molecular groups of C–H, C=O, and C=O in COOH on the mycelial surfaces, and -OH was a key Cu2+-binding group. It is therefore suggested that the Cu2+ absorption by the GXCR is likely to be a chemical absorption process through Cu2+ binding with -OH group on the mycelia.

Abstract:We assessed the effects of trxS gene on changes of proteinase activity, contents of different protein fractions and SDS-PAGE profiles in germinating seeds of contrasting transgenic and nontransgenic barley variety. Proteinase activity was enhanced by 70.28% in transgenic than nontransgenic barley seeds, whereas contents of albumin, globulin, hordein and glutelin in transgenic seeds were 3.68%, 23.52%, 31.37%, and 21.04%, lower than those in nontransgenic seeds. Degradation rates of hordein and glutelin in transgenic seeds were faster than those in nontransgenic seedlings as indicated by the SDS-PAGE profiles. Our data imply that the transformation of trxS gene could promote the degradation of protein, providing theoretic basis for the use of trxS gene and barley quality breeding.

Abstract:In this report, we utilized N-Acetylmuraminidase (AcmA) to develop a whole-cell catalyst of endo-beta-1, 3-1, 4- glucanase in Lactococcus lactis. The PCR-amplified full-length acmA gene from L. lactis MB191 was fused with the green fluorescent gene (gfp), followed by ligating the chimeric acmA-gfp into the Escherichia coli-L. lactis shuttle expression vector pMG36k, yielding the recombinant plasmid pMB137. SDS-PAGE analysis showed that the constitutive expression of AcmA-GFP fusion protein in the L. lactis AS1.2829 construct harboring pMB137 (named MB137), with the predicted Mr of 74 kD. Western blotting, GFP specific fluorescence intensity assays and flow cytometry analysis confirmed that AcmA-GFP was immobilized on the outer membrane, which constituted approx. 35% of the total intracellular fusion protein. Furthermore, acmA was fused with a PCR-amplified encoding fragment of the endo-beta-1, 3-1, 4-glucanase gene (gls) from Bacillus sublitis BF7658, resulting in the recombinant plasmid pMB138. By transferring pMB138 into L. lactis AS1.2829, the derived L. lactis MB138 expressing the AcmA-GLS fusion enzyme exhibited a distinct whole-cell glucanase activity (by 12 U/mL) compared to the control strain, indicating AcmA had served as a functional anchoring motif to immobilize the heterologous enzyme on the cell surface of L. lactis.

Abstract:It is of theoretical and practical significance to understand the mechanism of enzyme adaptation to acidic and alkaline environments and classification of them based on sequence information. In present work, the amino acid composition of 105 acidic and 111 alkaline enzyme sequences was systematically analyzed. Acidic enzymes contained significantly more Trp, Tyr, Thr and Ser, whereas less Glu, Lys, Met and Arg. On the other hand, alkaline enzymes have slightly more Trp, Ala and Cys, whereas less Lys, Arg and Glu. The amount of Ala, Glu, Leu, Asn, Arg, Ser and Thr in acidic and alkaline enzymes varied largely. Hence, a weighted amino acid composition method was developed for the discrimination of acidic and alkaline enzymes. Using the back-check and the 5-fold cross validation methods, the overall accuracy could reach 86.1% and 83.3%, respectively. A new method to classify acidic and alkaline enzymes based on their sequences was established.

Abstract:Amphibian skin antimicrobial peptides exhibit a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacterium and cytotoxic activity responsible for inhibiting the growth of cancer cells. In this present study, six cDNAs encoding antimicrobial peptide precursors were cloned from the skin of Chinese brown frog, Rana chensinensis by RT-PCR and 3￠-RACE procedure and identified as preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, preprobrevinin-1CEa, preprobrevinin-1CEb, and preprochensinin-1, respectively. The nucleotide sequences of cDNA encoding 59-65 amino acid composed of 289-315 bp. Preprotemporin-1CEa, preprotemporin-1CEb and preprotemporin-1CEc are members of temporin family, which usually are short, hydrophobic, and C-terminally a-amidated antimicrobial peptides. Preprobrevinin-1CEa and preprobrevinin-1CEb were identified as the members of the brevinin-1 family of antimicrobial peptides since both peptides contain “RANA box” that it’s responsible for forming Cys-bridged cyclic heptapeptides at the C-terminal region of peptide. The nucleotide acid sequence and the deduced amino acid Sequence of preprochensinin-1 were not found to be identity with any known amphibian skin defensive peptides, so, preprochensinin-1 was identified as a novel peptide precursor. Four of bioactive peptides: temporin-1Cea, temporin-1Ceb, brevinin-1CEa and chensinin-1 were synthesized to investigate their antimicrobial, anticancer and haemolysis activities. The results showed that all of the synthesized antimicrobial peptides in this study inhibited the growth of the Gram-positive bacterium, and exhibited the anticancer activity against the growth of MCF-7 cells and HeLa cells. Analysis of the R. chensinensis bioactive peptides and their gene expression will be beneficial for preservation of this species.

Abstract:We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC?-TOPO? by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC?-TOPO? vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine? 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in a-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-Ig G4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.

Abstract:To study the genetic basis of spontaneous transformation of mesenchymal stem cells (MSCs) and clinical application value MSCs were isolated by combining utilization of density gradient centrifugation and adherence screening. After cell homogeneity analysis by flow cytometry, spontaneous transformation MSCs were isolated after six-month in vitro expansion. Then cell total RNA was obtained with Trizol reagent and studied for gene expression profile. Differentially expressed genes between normal MSCs and spontaneous transformation MSCs in cDNA microarray were determined by real-time RT-PCR for validation of the microarray data. Forty-four genes were differentially expressed after spontaneous transformation of MSCs, among which 21 were up-regulated and 23 down-regulated. The result of real-time RT-PCR was in accordance with that of the cDNA microarray. Several genes in SHH, Notch, TGFβ/BMPs signal pathway play an important role in spontaneous transformation of MSCs.

Abstract:This study is aimed to design a chemically-defined serum free medium (CDSFM) to support in vitro culture of marrow-derived mesenchymal stem cells (BM-MSCs). BM-MSCs were isolated from the femoral bones of one month old New Zealand Rabbits with density gradient centrifugation. We compared the proliferation capability, cell cycle, colony-forming efficiency, osteogenic and adipogenic differentiation capabilities of BM-MSCs cultured in CDSFM with those cultured in serum-containing medium (SCM). After 10 days culture, BM-MSCs were expanded by 50 folds in CDSFM, while only 40 folds in SCM. EGF, bFGF and hy-drocortisone were the most important additives and significantly stimulated BM-MSCs proliferation. The percentage of cells at G0-G1 cell cycle was 80.31% ± 0.58% after CDSFM culture, with no significant difference (P>0.05) compared to 75.24% ± 4.05% for SCM culture. However, the cloning efficiency of BM-MSCs cultured in CDSFM was significantly lower than that in SCM (P<0.01). The expanded BM-MSCs in CDSFM preserved differentiation potentials into mesenchymal lineages in vitro, including adipocytes and osteoblasts. We have designed a chemically-defined serum free medium that could support in vitro proliferation and maintain the properties of BM-MSCs as stem cells, which could be applied to cell-based therapy and biomedical research.

Abstract:To improve the efficiency of targeted gene replacement (TGR), a dual screen (DS) system with gusA gene as negative selective marker (GUS-DS) was developed in Magnaporthe oryzae. First, we tested the endogenous β-glucuronidase (GUS) activities of 78 fungal strains. All tested strains were GUS-, only with 3 exceptions. Whereas, after the gusA being introduced in, M. oryzae, Fusarium oxysporum and Colletotrichum lagenarium acquired high GUS activities. The gusA is thus usable as a selective maker in most fungal species. With gusA as the negative marker, HPH gene as the positive marker, and the peroxisomal targeting signal receptor genes MGPEX5 and MGPEX7 as 2 instances of target genes, we established the GUS-DS system. After transformation, we collected the transformants from hygromycin B screen media and then tested the GUS activities of them. The GUS– ones were selected as potential mutants and checked in succession by PCR and Southern blotting to identify the true mutants and calculate the efficiency of GUS-DS. As a result, GUS-DS improved the screen efficiency for Δmgpex5 from 65.8% to 90.6%, and for Δmgpex7 from 31.2% to 82.8%. In addition, we established a multiple PCR (M-PCR) method for mutant confirmation. By amplifying the different regions at the targeted locus, M-PCR differentiated the wild type, the ectopic transformants and the mutants effectively and rapidly, and had the same reliability as Southern blotting. In conclusion, GUS-DS and M-PCR are useful tools to improve the efficiency of TGR and would be helpful for fungal genomics.

Abstract:We designed the specific primers and TaqMan probes targeting cytochrome b genes of mitochondrial DNA from bovine, goat and sheep. We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a multiplex fluorescent real-time PCR method to detect bovine, goat and sheep derived materials, simultaneously. We finished the detection tests of 17 kinds of animal DNA and 200 DNA samples from different sources with the developed method and the National Standard GB/T 20190Y-2006 routine PCR method. The coincidence rate of these two methods was 100%. Without electrophoresis or restriction digestion, the developed method could reduce the test time to one third as routine PCR and identify three kinds of animal derived materials including bovine, goat and sheep in one reaction. The developed method was approximately 10 times more sensitive than routine PCR, and was applicable to identifications of bovine, goat and sheep derived materials in feed stuff, meat, milk, pelt and grease, etc. The study showed that the developed real-time PCR method is a rapid, sensitive and efficacious detection assay for bovine, goat and sheep derived materials in animal products.

Abstract:Lysostaphin, a specific endopeptidase enzyme derived from Staphylococcus aureus, is a bactericidal agent against Staphylococcus and difficult to be drug-resistant. This study established monoclonal antibody affinity chromatography to obtain lysostaphin of high purity for drug-use standard. The purified Lysostaphin was of > 95% purity and its recovery rate more than 90%. Moreover, the affinity column kept its efficiency of purification invariable after more than 30 times repeat. Also, the dye release assay validated that the purified lysostaphin had significant bactericidal activity. This method was simple and of high efficacy for the lysostaphin purification and showed its potency in commercial production.