Abstract: B. thuringiensis subsp.chinensis strain CT-43 highly produces thuringiensin (Thu). Meanwhile, other metabolizing product (ZZ) was synthesized, but was difficultly removed from Thu. In this study, various media were screened and the G medium was found to inhibite the ZZ synthesis. Based on the G medium, feed-batch fermentation and orthogonal arrays was combined to further inhibit ZZ synthesis and increase the yield of Thu.. The optimal fermentation process was that, After strain CT-43 was cultured in G medium for 20h, patching with 2.0 g/L sodium citrate and 16 g/L glucose and culturing for additional 32h, the peak area ratio of Thu could get 74.84 in the primary extract, which was 2.5 times for 29.85% of LB fermentation. These onditions could create a favorable fermention process to produce more pure Thu and it will enefit for further research. Lastly, HPLC-MS was used to verify the produced Thu. Thu became he major element during the feed-batch fermentation. No ZZ was detected and Thu was not modified after above fermentation process.

The new DNA extraction method we established was based on the traditional alkaline lysis method. We changed the volume of neutralization solution and introduced an isopropanol precipitated step. The result showed that DNA extracted by this method can amplify target genes fragments which were short than 600 bp. Only 1% expected material contained in other samples can generate the specific amplified. The whole process can be finished in 21 minutes, and the applicability of NaOH-based method is extended.

Abstract object To prepare the rapid diagnostic kit of H5 subtype avian influenza virus (AIV-H5) and set up AIV-H5 rapid diagnostic technology. Methods Balb/c mice were immunized by purified antigen, to produce two good matching monoclonal antibodies (McAb) against AIV-H5. It’s to develop the rapid diagnostic kit using gold immunochromatography, and evaluate its capability. Result The kit has high sensitivity, specificity and good stability. Detecting the man-made infected chicken tissues , the kit’s detecting result was as same as embryo culture methods. Contrasting the results of filed experiments with fluorescent RT-PCR, the coincidence rate was 100%. Conclusion The rapid diagnostic kit of AIV-H5 is a pecical , sensitive and rapid production for AIV-H5 detection.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation in many organisms, as a new generation of molecular markers, it has been widely used in genetics and breeding research of poultry due to the characteristics of high density, duble-allele and easy to automate detecting. In this paper, some new progresses on poultry SNP discovery, genotyping, polymorphisms characteristic and the application in genetics and breeding research were reviewed, moreover, the prospect of SNP in the research of poultry was also briefly discussed.

Abstract：To investigate the gene expression and to separate clones which are important genes related to fiber development,a cDNA library of Ramie（Boehmeria nivea(L.)Gaud）bark was constructed. Some quality analysis,such as the titer and insert cDNA length of cDNA library,indicated that the library was eligible.The sequencing of cDNA clones and the analysis of expressed sequence tags(EST) were reported,274 valid ESTs were generated from 384 cDNA clones.Result showed that EST sequences assembled out 217 TUTs and endued 20.8% redundancy. 91 ESTs with known or putative function and 126 novel ESTs were identified by Blast searches against the NCBI non-redundant databases.These ESTs were classified into 9 different categories,the cell wall related ESTs were 5.5%.The expression levels in different development, different tissues of different genotype was detected by semi-quantitativeRT-PCR with 18S rRNA as internal control. The results indicated the expression of ADF and Tubulin beta genes could be found in these tissues,.The hightest expression level were appeared in bark of cover-line stage. The expression level of ADF and Tubulin beta genes is particular to tissues and development stages.

Abstract: In order to clone the full-length of gene related to egg laying performance and further study the molecular mechanisms for the process of egg laying in the Zi geese, the experiment was conducted to constructed the full-length cDNA library of the ovary in the Zi geese by SMART (Switching Mechanism at 5’ end of RNA Transcript) using λTriplEx2 as vector. The results showed that the titration of the cDNA library we constructed was 3.1×106 pfu/mL, the rate of recombination was 94 %, and the fragment length of inserted DNA ranged mainly from 0.5 to 2 kb. 120 clones were chosen at random for sequencing from the cDNA library, and after BLASTn analysis of cDNAs,there possible function were predict. The results show that cDNA library we constructed can provide a base for further study on the full length cloning of important genes of egg laying.

GL2 gene plays key roles in trichome development of Arabidopsis. A putative GL2 promoter was isolated from genomic DNA of Brassica napus by genomic walking method. Bioinformatics analysis indicated GL2 promoter has low homology between Arabidopsis and B. napus. To study the function of this promoter, a GP1-GUS construct was made and introduced into Arabidopsis by Agrobacterium-mediated transformation method. Ten positive transformants were obtained. Segregation analysis in T2 generation revealed that each of six lines segregated as 3:1 for kanamycin-resistance, suggesting T-DNA insertion in a single copy. Histochemical staining analysis showed that the GUS gene mainly expressed in young tissues of Arabidopsis, such as cotyledons, trichomes of true leaves and roots, which are basically consistent with the expression style of Arabidopsis.GL2 promoter. However, compared with Arabidopsis GL2 promoter expressed in the whole processes of trichome development, B. napus GP1 only expressed in the early stage after the trichome initiation of Arabidopsis.

To define whether the expressed ompA of Aeromonas hydrophila (Ah) AS1.927 strain has antigenicity and determine the relative percentage survival (RPS) for the mice, the 1020 bp fragment encoding ompA was cloned from the Ah AS1.927 genome with a pair of primers designed according to the sequence of omp in GenBank (AF146597)., After digesting the fragment with Sal I ＋Xhol I, the gene was inserted into pET-30a(+) vector. The recombinant pET-30a-ompA plasmid was transformed into the expressing strain BL21 and induced expression by IPTG. The recombinant protein ompA was analyzed by SDS-PAGE and Western blot, and results showed that the ompA fusion protein was about 40.7kD and could react with sera from mice infected with A. hydrophila. BALB/c mice were orally immunized at 1st, 2nd, 3rd, 20th, 21th, and 22th day with the engineering bacteria E.coli [pET-30a-ompA]. The serum IgG was detected by ELISA at 7days post-vaccination. On the 14th day after last immunization, the mice were challenged with 3.3 × 105 cfu of live Ah AS1.927 (100LD50). The results showed that mice vaccinated with E.coli [pET-30a-ompA] showed significantly increased antigen-specific IgG level in the serum (P<0.05) and SIgA level in the intestinal mucosa compared with the control and 75% relative percent survivals (RPS). This indicated that oral immunization of E.coli [pET-30a-ompA] could induce an immune response protective upon challenge with live Ah AS1.927 in BALB/c mice. The present study will provide technical support for developing of efficient gene engineering vaccine against A. hydrophila.

Zearalenone (ZEN) was the most widely distributed Fusarium toxins all over the world. ZEN could lead to a series of estrogenic effects symptoms such as infertility and abortion , damage the internal organs, and even promote the cancer cells to grow.. Over the past decade, the researches on ZEN degradation had made some progress and a gene zhd101 encoded lactose hydrolase that possessed the capability to transform ZEN into non-toxic products(including plant cells) had been reported in Japan. In our research, a DNA fragment (ZEN-jjm ) was obtained by coloning from Gliocladium roseum, and there were 9 different bases in this sequence contrasting to the gene zhd101. Subsequently, ZEN-jjm was expressed through prokaryotic host, and the results indicated that the expressed protein could degrade ZEN significantly.

Hf2 gene(1527bp) encoding flavonoid 3’,5’-hydroxylase (F3’5’H) and dihydroflavonol 4-reductase (dfr) (1212 bp) gene were isolated from Petunia hybrida and Ipomoea nil petals respectively. Hf2 and anti sense dfr genes were subcloned into pBRLys vector together generating pUbi-Hf2-dfr vector which contained Ubiquitin(Ubi) promoter, and then the pUbi-Hf2-dfr was transferred into Agrobacterium tumefaciens strain EHA105. Result of PCR amplification with transformant colony showed that the expression vector pUbi-Hf2-dfr was successfully transferred into Agrobacterium. Hf2 and anti sense dfr genes were transformed into callus of Narcissus tazetta var. chinensis mediated by Agrobacterium. Extracted the genomic DNA of 130 regenerated plants with CTAB method, and then performed PCR with specific primers and electrophoresis results showed that there were 3 regenerated plants with the intergration of Hpt and blue genes. PCR-Southern blotting indicated the same result.

Plant resistance genes play an important role in genetic resistance mechanism of plants. Based on the disease resistance gene analogs Pb-Zs3 which was cloned from Zaosu pear in our previous study, a full-length cDNA sequence was acquired with the method of homology-based cloning, and this gene was named Vnp1(GenBank accession No. FJ763188). Bioinformatic Analysis revealed that Vnp1 was a novel gene, and its cDNA was 1200 base pairs. The complete open reading frame (ORF) is 921bp in length, encoding 306-residue protein with a calculated molecular mass of 34. 6 kD. Meanwhile, two domains which related to disease resistance NB-ARC and disease resistance protein were also detected in its encoding protein. What’s more, a sequence of apple EST project induced by Venturia inaequalis showed 71% identity with Vnp1 by homology comparison. Semi-Quantitative RT-PCR revealed that the transcript of Vnp1 was significantly enhanced in the leaves of pear under the inducing of Venturia nashicola. This demonstrated that Vnp1 was correlated with the resistance to pear scab.

An antimicrobial protein, with an apparent molecular mass of 12 kD, was isolated from the seeds of Ginkgo biloba. The isolation procedure entailed extraction, ammonium sulfate precipitation, cation exchange chromatography on CM Sephorose F.F, gel filtration chromatography on Superdex 75. MALDI-TOF-MS PMF identification showed higher homology with a novel antifungal protein Ginkbilobin-2. We have designed degenerate primers to clone a fragment of the antimicrobial protein gene, according to the ESI-MS/MS sequence results of internal peptide. The amplified fragment also showed 99% similarity with the Ginkbilobin-2 gene. Further, the homological primers were designed for cloning the full length cDNA of the antimicrobial protein by RT-PCR. The sequence has been deposited in the GenBank (Accession No.FJ865399). In addition, we have constructed the prokaryotic expression vector pGEX-GK2-1 which was used to express the antimicrobial protein in vitro. Results of SDS-PAGE and Western blot showed that the recombinant protein with the calculated molecular mass of 38kD was highly expressed in Escherichia coli BL21(DE3). Our studies lay a foundation for the further research of the antimicrobial mechanism and obtaining the transgenic disease-resistant germplasm resources.

LJAMP2, a member of nonspecific lipid transfer protein, isolated from motherwort seeds, displays antimicrobial activity against fungi. To obtain enough LJAMP2 with activity for performing function analysis in vitro, the LJAMP2 gene was cloned into the pPIC9K vector and transformed into Pichia pastoris using electrotransformation. A transformant with a high copy number of the gene integrated into the chromosome was obtained by YPD plates which contains 1.75 mg/mL G418. The active LJAMP2 was secreted into the culture medium under the induction of 1% methanol and purified more than 90% purity from culture medium by desalting chromatography, CM FF cation exchange chromatography, and reversed-phase HPLC. SDS-PAGE results showed that there was a faint target band in Mut+ recombinant culture supernatant after induction 24 hours culture and the yield of LJAMP2 got to the top after 168 hours culture. The antifungal activity of recombinant LJAMP2 was studied by plate inhibition analysis, which revealed that the recombinant LJAMP2 exhibited activities against Bipolaris maydis and Aspergillus niger. Together, these observations indicate that the recombinant LJAMP2 is an active antifungal protein that can be successfully produced in the yeast, and therefore may be a potential antifungal candidate for practical use.

Abstract: Objective: To construct the recombinant plasmid pMAL-c4x -GnRH6 with gene fragment of the GnRH hexamer ,and express recombinant GnRH6 fusion protein in Escherichia coli TB1, and identification the fusion protein. Methods: The gene fragment of the GnRH hexamer t was connected to the pMAL-c4x plasmid, then recombinant pMAL-c4x -GnRH6 was induced in E.coli TB1under IPTG. SDS-PAGE, Western blot and animal experimentation was used to identify the expression product. Result: The molecular weight of fusion protein MBP-GnRH6 was about 50kD. And the reactivity of fusion protein and GnRH-antibody was better. Active immunization against MBP-GnRH6 resulted in the atrophy and degeneration of testis in male mouse. Conclusion: The recombinant plasmid pMAL-c4x -GnRH6 and engineering bacteria were successfully constructed and the recombinant MBP-GnRH6 fusion protein was verified with better reactionogenicity and immunogenicity, which can be used to immunocastrate in male mice.

Apple (Malus domestica Borkh) is a typical Rosaceae species that exhibits gametophytic self-incompatibility (GSI). Identifying the S-genotype of apple cultivars has the practical significance for selecting appropriate pollinated varieties and incompatibility parents for hybridization breeding. In this investigation S-allele-specific PCR was conducted using genomic DNA extracted from 46 apple cultivars as templates and S-genotypes of these cultivars were identified. Furthermore, two new genes named S53- and S54-RNase respectively were cloned from apple cultivars 'Darling' and 'Jinshayilamu'. They were deposited in GenBank under the accession numbers of FJ602074 and FJ602075 respectively.

Abstract: Cultivars identification is an important method to resolve seedling confused. SRAP were used to detect the genetic diversity of 12 Changlin series superior clones. 15 primers pairs were screened out and 423 SRAP sites were Amplified from these clones, of which 394 markers were polymorphic loci and 50 markers were unique fragments. There were 13 primers pairs which could identify all clones respectively. In order to improve the identification speed, 24 loci were selected to establish the DNA finger print of the 12 clones. Genetic distance and cluster analysis were done, and the results showed that there was a great genetic diversity among the clones. All of these provide the rationale for camellia oleifera breeding.

Abstract:The regulatory networks of the related genes at sow pregnancy stage was constructed by other in our laboratory, and some new and valuable candidate genes associated with sow pregnancy screened by bioinformatics analysis. A expressed sequence tag(EST) on the top of the networks was studied in the research. Primers were designed according to the sequence of the EST, the total RNA was extracted from sow ovaries，cDNA complete sequence cloned using RT-PCR and RACE-PCT was 596bp, the sequence was submitted and the accession number(FJ36413) was got from the GenBank database. The complete sequence included untranslated region( located in 1-67 and 422-596) and coding region(located in 65-421) coding a polypeptide of 117 amino acids. The predicted nucleotide sequence similarity between pig and human, mouse, and macaca mulatta were83%, 70%, 83%, amino acid sequence were 74%, 55%, 74%, respectively. The relative molecular weight of predicted DPPA5 was 13.60KD , and its isoelectric point was 5.24. The hydrophobic region located in 10-19, 24-42, 47-77, 84-90, and 101-118. The analysis result indicated that DPPA5 was an extrinsic protein.

abstract： The study was to investigate the effect of EGF on Bovine preadipocytes which were isolated from bovine perirenal adipose. Effect of doses 10 ng/ml、50 ng/ml、100 ng/ml、200 ng/ml EGF on proliferation and differentiation of preadipocytes were analyzed by MTT colorimetric and incorporation of oil red O. and mRNA expression levels of LPL,PPARγ,A-FABP genes,which were adipocyte differentiated marker genes were determined by semi-quantitative RT-PCR. The preadipocyte proliferation was stimulated significantly by EGF of 10ng/ml (P<0.01). Treated with different doses EGF after induced differentiation could improve adipocyte differentiation and promote the expression of the differentiation marker genes LPL 、PPARγand A-FABP at 8th day .EGF could improve the proliferation and differentiation of bovine preadipocyte.

Abstract: iSingle strand conformation polymorphism (PCR-SSCP) and direct sequencing methods were used to analyze the polymorphism at 3′-UTR of HSP70 gene in bovine.The PCR-SSCP products showed that there was polymorphism and could be divided into three genotypes: AA, AB, BB among 673 Luxi and Holstein cattle. The direct sequencing of products showed that there was a new SNP in the 3′-UTR of the hsp70 gene.In this study the cows with BB genotype showed higher milk fat percentage, milk proteinum percentage and 305 days milk yeld than that of AA genotype,and cows with BB genotype showed lower blood potassium and milk production decrease ratio (p<0.05). Cows with BB genotype have higher heat tolera nce than AB and AA. Luxi cattle have higher heat tolerance than Holstein. B allele frequency in Luxi cattle is much higher than that in Holstein which also suggest BB genotype have higher heat tolerance than AB and AA.This mutation site can be used as molecular genetic markers to assist the selection for anti-heat stress cows.

The objective of this study was to investigate the difference of ruminal cellulytic bacteria quantities in lactating dairy cows under heat stress using real-time PCR. Forty eight cows were assigned to mid-stage or late-stage, high producing or medium producing, primiparous or multiparous groups with a factorial design. Rumen fluid was sampled 5h in the morning postfeeding via oral cavity with stomach-tube when the THI of barn was continuously high than 72 for one week. The real-time PCR was employed with the specific primers targeting 16s rDNA genes of the fermentation bacteria to test the abundance of six cellulytic bacteria. The quantities of Ruminococcus flavefaciens, Prevotella ruminicola and Genus-level ruminicola was 13.9%, 5.3% and 3.7% higher for late-stage than mid-stage cow, respectively. The quantities of Prevotella ruminicola and Ruminococcus flavefaciens was 14.4% and 12.6% higher for high producing cow than medium producing cow, respectively. The quantities of Ruminococcus flavefaciens, Prevotella ruminicola, Fibrobacter succinogene and Ruminococcus albus was 12.6%, 3.2%, 2.4% and 2.1% higher for multiparuos than primiparous cow, respectively. In summary, there is a variance for Ruminococcus flavefaciens and Prevotella ruminicola in lactating dairy cows under heat stress.

In the McCoy's 5a serum-free culture system, the combined effects of bovine follicular fluid(BFF) with gonadotrophins (FSH and LH) on in vitro growth, development, suvival and secretion of estradiol (E2) of preantral follicles during culture were studied. The results showed that adding individually different doses of BFF to the culture medium did not influence obviously on the development of preantral follicles, but it could promote the secretion of estradiol. Under the condition of addition with appropriate proportion of BFF in presence 10ng/ml LH and 50ng/ml FSH, the development and survival rates of bovine preantral follicles in each control groups were higher than the 0% BFF group. Especially for 10% BFF group, the development（78.52%）of preantral follicles after 6 days culture was higher strikingly than contrast group(49.28%) (p<0.05) and preantral follicles diameter increased to 33.7+4.8 μm by 12 days of culture, which was significantly higher than the other treatment group (p <0.01). In the meantime, antrum formation occurred in the 9th day of culture. Secretion of E2 for each BFF group became more higher in the presence of gonadotrophins(FSH and LH). The level of E2 in the 10% BFF group arrived the highest value(48.24pg/mL) in the sixth day of culture. All these reaveled that 10% BFF combined with a certain proportion of gonadotropin can stimulate strongly preantral follicles in vitro growth, development and survival and induced follicular cavity formation.

Single nucleotide polymorphisms (SNPs) of exons 2、3 of IGF-1 gene were detected in Bian chicken and two reference chicken populations (Jinghai yellow chicken and Youxi chicken). There were no polymorphisms detected in the amplified region of primer P1 in three chicken Breeds, which may indicate that the sequence of exon2 of IGF-1 gene were rather conserved. While the region amplified by primer P2 displayed polymorphisms. Three genotypes( AA、AB and BB) were all detected in three chicken breeds. The allele A was the predominance allele in Bian chicken and Jinghai yellow chicken and that was allele B in Youxi ckicken. Sequencing revealed two mutations (93A→G and 148G→A ) of IGF-1 gene in the genotype AA in comparison to the genotype BB. The former mutation did not cause any amino acid change, and the latter mutation resulted in an amino acid change of Glu→Lys. Compared with the sequence of IGF-1 of chicken in Genbank, we found the mutations were at the point 62bp and 117bp of Exon3 of IGF-1 respectively.

In this study, the effect of oleic oil on cytoactive, triglycerides (TG) accumulation, lipids drop and mRNA expression of tumor necrosis factor α（TNFα） and peroxisome proliferator-activated receptor α（PPARα） was measured in primary goose hepatocytes. Results were as follows: 1.5 mmol/L oleic oil had an inhibiting effect on the cell viability; 1.5 mmol/L oleic oil could induce the goose hepatocytic steatosis and the increase of TG content and lipids drop. 0.5，1.0 and 1.5 mmol/L oleic oil all could increase the mRNA level of PPARα; 0.5 and 1.0 mmol/L oleic oil could increase the mRNA level of TNFα, but 1.5 mmol/Loleic oil had no evident effect, demonstrating that the expression of PPARα and TNFα in mRNA level is important for the balance of lipid metabolism in goose hepatocyte.

Abstract: The liver proteome of Pseudosciaena crocea was analyzed through two-dimensional gel electrophoresis (2-DE) after acute low temperature stress at 8.5 ℃. Protein spots obtained from low temperature stress group were 1593± 41, and control group 1620± 37 by means of PDQuest analysis. The expressions of 16 spots expressed differentially of low temperature stress group varied significantly after challenge. 4 spots from 16 were picked out to analyze through MALDI-TOF-MS or LC-MS/MS, then searched in database. The result demonstrated that the expression of N-ethylmaleimide-sensitive fusion protein (NSF), cytokeratin-18 (CK-18) and 2-cys peroxiredoxins (2-Cys Prxs) were down-regulated remarkably, however, myosin heavy chain (MHC) up-regulated remarkably. It was concluded that the dynamic equilibrium in vivo environment of Pseudosciaena crocea was broken by low temperature stress, and various physiological reactions were induced and even death if the change was out of control.

AIM To study the distribution of I integron and resistance phenotype in Enterococcus isolates from different animals. Methods According to NCCLS (2007), we test 140 isolates resistant phenotype of different animal-derived using of the concentration dilution, sensitive tablet (PA) and the VITEK-AMS sensitivity test. We test the distribution of I integron by using I integrase gene PCR, and clone and sequence the representative isolates of integrase gene. Results I integrase gene positive rate was 52.14% of all and 59.52%, 52.63%, 46.67% and 46.67% of the pigs, chickens, cattle and sheep-derived. There are not significant difference (p<0.05) in Enterococcus faecalis and Enterococcus faecium. The resistant rate to amoxicillin, ampicillin, cefazolin, ciprofloxacin, clindamycin, erythromycin, gentamicin, furadantin, penicillin, rifampin, tetracycline and vancomycin were 62.14%, 60.00%, 50.71%, 77.86%, 17.14%, 69.29%, 76.43%, 20.00%, 68.57%, 80.71%, 51.43%, 0.00%. Integron-positive isolates to amoxicillin, ampicillin, gentamicin resistance compared to integron-negative isolates showed highly significant (p<0.01), to cefazolin, ciprofloxacin, clindamycin, Furadantin, rifampicin, tetracycline showed not significant (>0.05), to erythromycin and penicillin is between of the highly significant and significant (0.05>p>0.01). Conclution There are a large number of multi-drug resistant enterococci and I integron of the different animals. Suggesting that livestock production should be strengthened to monitor the resistance of enterococci to block the spread of drug-resistant strains in animals and human beings. It is indicted that I integron is very important in the dissemination of multi-drug resistant enterococci.

The transformation system by electroporation of the biocontrol bacteria ANTI-8098A (Bacillus cereus) was optimized in this paper. The results showed that the system was best for the electroporation when the concentration of compentent cell was 1.14×108 CFU/mL and the content of plasmid was 118 ng. The proportions of the green cells decreased when the strains of ANT8098A:pCM20 was cultured in the non-selective medium. And the growth speed of gfp-tagged strains was almost the same to the wild type strain ANTI-8098A. The best culture conditions of wild type strain and gfp-tagged one including the culture temperature of 35 ℃ and rotation speed of 250 r/min were almost the same. Both strains ANTI8098A and ANTI8098A: pCM20 showed the same in vitro inhibition of Ralstonia solanacearum from different crops.

In this paper, intestinal microbiota of ICR mice fed with Cry1C protein in acute oral toxicity test were analyzed, furthermore, edible safety of insect-resistant protein was evaluated deeply. DGGE maps by UPGAMA cluster analysis indicate that the intestinal flora in the mice of this study show significant difference; the mouse in the control group have unique and stable intestinal flora in this test period; the intestinal flora of the mouse in the test group have significant changes before and after gavage, however, the diversity in test group decreases gradually, and restore the structure of pre-administered after the peak. Therefore, Cry1C protein is safe for mice from the point of intestinal microbiota.

Abstract： The sequences of the HA and NA gene of the new pandemic A/H1N1 2009 influenza virus were obtained from GenBank. After multi-sequence alignment, two sets of specific PCR primers and TaqMan probes were designed. Two fluorescent probes were labeled with FAM and CY5 as reporter, respectively, and BHQ (black hole quencher) as quencher. A multiplex real-time RT-PCR assay was established to detect this new influenza A/H1N1 virus. The amplification curves of HA and NA gene of positive controls both exhibited standard S shape, suggesting a good amplification efficiency and linear relationship. Several other sub-types of influenza A virus samples including traditional H1N1, H3N2, H5N1, H6N2 and H9N2 were used as negative controls to validate this assay, for which no positive amplification signal was detected. The results indicated that our newly established assay had high specificity. In sensitivity test, 10 copies of plasmid DNA template can be detected, which nearly reach the detection limit. The whole multiplex real-time RT-PCR reaction could be finished in 90 min. 243 human clinical samples and 1351 swine clinical samples were examined with this assay. 7 human clinical samples were positive and all swine clinical samples were negative, which were consistent with the results from the test by using the commercial kits purchased from China academy of inspection and quarantine. Our study indicates that this newly established multiplex real-time RT-PCR assay is rapid, reliable and sensitive and it is suitable for application in new influenza A/H1N1 virus screening in laboratory.

Bioinformatics analysis revealed that a functionally unknown sequence, named as Ttx31, from the ?-amylase/pullulanase of Thermoanaerobacter thermohydrosulfuricusits could encode a novel carbohydrate binding module (CBM), but its’ binding specificity remains unknown. The sequence of Ttx31 was obtained by colony PCR from Thermoanaerobacter thermohydrosulfuricus and was cloned into the expression vector pET22b (+) to construct a recombinant plasmid named as pEX31. Sequence analysis revealed that pEX31 contained the correct Ttx31. After transforming pEX31 into Escheriachia coli Tuner, the recombinant protein, TtX31, was induced by IPTG and purified. The interaction between TtX31 and sugars was investigated by SDS-PAGE and non-denaturing affinity gel electrophoresis. Data obtained here showed that TtX31 displayed binding specificity for dextrin other than soluble starch, pullulan, and amylopectin. This finding indicates that TtX31 could be a functional novel CBM with dextrin binding activity.

The gla gene of Thermomyces lanuginosus was cloned by PCR. Sequencing analysis revealed that gla has an open reading frame of 1854bp, which encodes a putative polypeptide of 617 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 64 kDa. The glucoamylase belongs to the glycoside hydrolase family 15. The gla sequence without signal sequences was obtained and cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. The recombinant enzymatic activity reached 11.6 U/mL after fermatation by Erlenmeyer flask. The recombinant glucoamylase was purified by using ammonium sulfate fraction、DEAE-sepharose fast flow chromatography. A molecular mass of the purified enzyme is 67 kDa determined by SDS-PAGE and a little bigger than the deduced molecular mass, possibly related to the glycosylation site. The recombinant glucoamylase exhibited optimum catalytic activity at pH5.0 and 60 ℃ respectively. It was thermostable at 60 ℃ and remained 54% of its original activity after 20 min at 70 ℃.

Abstract：The mature peptide coding sequence of xylanase gene xynB was amplified by RT-PCR from Aspergillus niger GIM3.452 total RNA extracts. The result suggested that the mature peptide sequence of xynB is consisted of 567 bp,and encodes 188 amino acids. Then,the mature peptide sequence and signal peptide sequence of pig parotid secretory protein gene were splicing by overlap extension PCR (SOE-PCR). PSxynB was subcloned into the eukaryotic expressing plasmid vector pcDNA6/His A and transformed into host E. coli strain DH5a for identification. The recombinant plasmid pcDNA-PSxynB was identified by PCR, enzyme digestion and DNA sequencing. The result showed that the recombinant plasmid of pcDNA-PSxynB was constructed correctly. Meanwhile,the PK15 cells were transfected with pcDNA-PSxynB by cationic liposome，and the mRNA of the target gene was determined by RT-PCR. The maximum yield of the recombinant xylanase in cell culture medium was 35IU/ml.