GAL1 promoter is one of high-efficient inducible promoters of yeast (Saccharumyces cerevisiae), which is regulated by GAL4 and induced by galactose. GAL genotypes of yeast have significant effects on the expression levels of exogenous proteins controlled by GAL1 promoter, so the characteristics of recombinant GAL yeast strains need further study. In this study, the growth, fermentation performance and genetic stability of the recombinant GAL yeasts with different copies of GAL1 and GAL4 genes were evaluated. The results indicated that, the changed GAL genotypes were stable and recovery mutation didn't occurred during cultivation. The deficiency of GAL1 gene slightly affected the abilities of yeast to use glucose and the different cultivation modes had also impacts on galactose utilization for the recombinant yeasts. In yeast peptone galactose (YPG) and yeast peptone galactose glycerin (YPGL) medium, the depletion time of galactose was 20, 20, 20, 26, 20, 44, 44 and 68 h for MS-1, SG1, DG115 and DPG65, respectively. For GQ21, the galactose concentrations were always constant in medium, indicating that GAL1 genes' knockout. However, the heat-resistance temperature of yeast didn't change for the different GAL yeasts. This study will be helpful for large-scale and low-cost production of recombinant proteins using yeast expression system as bioreactor.

Invariant chain (Ii) can bind to major histocompatibility complex (MHC) classⅠorⅡmolecules, and plays an important assistant role in antigen presentation by MHC molecules. In order to explore what about the binding of Ii with MHC molecules, a Pull-down method for detection of association of grass carp(Ctenopharyngodon idellus) Ii with MHC molecules was reported in the present study. DNA segments of Ii (711 bp), MhcⅠα (540 bp) and MhcⅡβ (549 bp) in grass carp were cloned, and then respectively inserted into prokaryotic expression plasmid PET-22b (no His-tag) or PET-28a (carry with His-tag) to construct 3 recombinant plasmids of PET-22b-Ii, PET-28a-MhcⅠα and PET-28a-MhcⅡβ. The above 3 recombinant plasmids then were transfected into engineering bacteria (Escherichia coli) Rosetta (DE3), respectively. After an induction to express and renature the interest proteins and purification by Ni+-NTA affinity chromatography column, the purified expressed protein products were detected by polyacrylamide gel electrophoresis (PAGE). The results indicated that MHCⅠα and MHCⅡβ labeled with His-tag could well express and be purified each with a molecular weight of 24 kD. The result of Western blot showed that Ii also well expressed and had a molecular weight of about 30 kD. Finally, the recombinant plasmid PET-22b-Ii was cotransfected with PET-28a-MhcⅠα or PET-28a-MhcⅡβ into Rosetta (DE3), respectively, to construct 2 recombinant strains of Rosetta(DE3)(PET-28a-MhcⅠα+PET-22b-Ii) and Rosetta(DE3)(PET-28a-MhcⅡβ+PET-22b-Ii). Induced expression and PAGE detection showed that MHCⅠα or MHCⅡβ could bind Ii to form a complex of 54 kD, respectively, which could be dissociated into simple molecule of Ii (30 kD), MHCⅠα or MHCⅡβ (24 kD) after SDS treatment, respectively. Western blot result showed that the dissociated Ii could bind specific antibody and be coloured by electrogenerated chemiluminescence (ECL). In conclusion, the prokaryotic expressed grass carp Ii and MHC molecules had activity, and its Ii could bind with MHCⅡβ as well as MHCⅠα. This research provides basic data for further research on relation between Ii and MHC molecules in animals.

Aiming at enriching novel lepidoptera insecticidal genes, Bacillus thuringiensis strains BN23-5 was analyzed and identified by the PCR-restricted fragment length polymorphisms (PCR-RFLP) method, which indicated that crystal(cry) gene of cry2Ab-type gene was contained. According to the NCBI open cry2Ab-type gene encoding area, a pair of degenerate primer was designed. A full-length of 1 902 bp cry2Ab-type gene fragment, which was amplified by PCR with the degenerate primers using strain BN23-5 genome plasmid DNA as template, was inserted into the Escherichia coli expression vector pET-28a to obtain the recombinant plasmid pET-2Ab, and then transformed into E. coli BL21(DE3) cell. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) verified that the target gene could express as 65 kD protein in E. coli BL21(DE3) strain by isopropyl-β-d-thiogalactoside (IPTG) induced at low temperature. What's more, expressed product of cry2Ab32 existed in bacteria precipitation after ultrasonic broken but not in the supernatant expressed product. The encoded protein composed 634 amino acid residues and shared a 96.5% sequence homology with Cry2Ab1 with 21 amino acids difference, which was designated as cry2Ab32 (GenBank No. KJ710647) by International Nomenclature Commitee of B. thuringiensis endotoxin. Bioactivity of cry2Ab32 gene expressed product was tested against Plutella xylostella, Ostrinia fumacalis, Laphygma exigua and Helicoverpa armigera by feeding method. The bioassay results indicated that expressed product of cry2Ab32 had a high insecticidal activity against P. xylostella, O. fumacalis and H. armigera, with median lethal concentration (LC50) of 8.99 μg/mL (95% confidence: 5.82~14.91 μg/mL), 19.34 μg/mL (95% confidence: 9.64~28.31 μg/mL) and 21.24 μg/mL (95% confidence: 13.35~30.32 μg/mL), respectively. However, Cry2Ab32 protein had no insecticidal activity against L. exigua, but could obviously inhibit the growth of L. exigua. In view of few previous reports about the high insecticidal activity of cry2Ab-type gene against O. fumacalis pest, the result was a major breakthrough for its prevention and control. The discovery of new gene cry2Ab32 will provide more choices for important agricultural pests prevention, and supply materials for further study of Cry2Ab-type protein structure and insecticidal mechanism.

In order to study the quality condition of the spring wheat (Triticum aestivum) in Gansu Province and identify the corresponding quality-affecting factors, the present research, based on the determination of their high molecular weight glutenin subunits (HMW-GS), identified the 1BL/1RS translocation lines using molecular and biochemical detection technologies, as well as the genomic in situ hybridization (GISH) and analyzed gluten cooking features. The result showed that Ganchun 24, Ganchun 22, Wuchun 4 and Wuchun 121 were truly the translocated line and Ganchun 23, Ganchun 20, Yongliang 4 and Heshangtou were not. The result explained why the 4 varieties of translocated lines had high quality HMW-GS without high quality. Comparing the 3 detection technologies, it was found that acid poly acrylamide gel electrophoresis (A-PAGE) and GISH could produce more accurate results in determining 1BL/1RS than molecular detection technology. The gluten cooking features analysis showed that the varieties of 1BL/1RS translocated lines were inclined to swollen and collapse. Ganchun 24, Ganchun 22, Wuchun 4 of the 4 of 1BL/1RS translocated line varieties were middle gluten and had the fine subunit of (5+10), while Wuchun 121 was weak gluten variety and did not have the subunit (5+10). This indicated that the high-quality subunit (5+10) could compensate the negative effect on variety quality caused by 1BL/1RS translocation lines. The result could provide theoretical basis for accurate and effective determination of 1BL/1RS translocation lines, as well as for wheat quality breeding and food processing.

Chinese wheat mosaic disease, which is caused by Chinese wheat mosaic virus (CWMV), leads to severe yield and huge economic losses on wheat (Triticum aestivum). The purpose of this study was to develop serological methods for detecting CWMV using prepared monoclonal antibodies (MAbs) against CWMV, and to provide technical support for the diagnosis and scientific prevention and control of CWMV in fields. Using the purified CWMV virus particles as an immunogen, 4 hybridoma lines (5H5, 7C2, 9A7 and 12E5) secreting MAbs against CWMV were obtained via cell fusion, cell culture, antibody detection and cell cloning, and injected intraperitoneally into BALB/c mice (Mus musculus) to produce ascitic fluids. The titers of all 4 MAbs in ascites determined by an indirect-ELISA were up to 10-7. Isotypes and subclasses of all 4 MAbs belonged to IgG1, κ light chain. Western blot analysis indicated that all 4 MAbs could specifically react with the coat protein of CWMV. Using the MAbs, antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA) and dot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting CWMV were established, respectively. Specificity analysis of the ACP-ELISA and the dot-ELISA indicated that both 2 serological detection methods had positive reactions of detection with CWMV infected wheat plant tissue crude extracts and negative reactions of detection with wheat or barley (Hordeum vulgare) plant infected by Wheat yellow mosaic virus (WYMV) or Barley yellow mosaic virus (BaYMV) and healthy wheat plant. This result suggested that 2 serological detection methods could specifically detect CWMV in wheat plants. Sensitivity analysis showed that the sensitivity of the ACP-ELISA based on the MAbs (5H5 and 12E5) for detecting CWMV in infected wheat tissue could reach 1∶81 920(W/V, g/mL), while the detection sensitivity of ACP-ELISA based on the MAbs (7C2 and 9A7) was up to 1∶40 960 (W/V, g/mL). The sensitivity of the dot-ELISA based on the MAbs (5H5 and 12E5) for detecting CWMV in infected wheat tissue could reach 1∶2 560 (W/V, g/mL), while the detection sensitivity of the dot-ELISA based on the MAbs (7C2 and 9A7) was up to 1∶1 280 (W/V, g/mL). The field sample detection results suggested that 2 serological detection methods could accurately and reliably detect CWMV in field wheat samples, and furtherly confirmed that CWMV was prevalent in Jiangsu and Shandong Provinces. The anti-CWMV MAbs and the developed serological detection methods provide material and technology for diagnosis, forecast and scientific prevention and control of CWMV.

PCR amplification and direct sequencing were used for single nucleotide polymophisms (SNPs) searching from growth hormone secretagogue receptor gene (GHSR) of the 2 populations of Nile tilapia (Oreochromis niloticus) (i.e., Fast-growing population and Base population). Snapshot method was used for genotyping of SNPs, and the correlation between genotypes of SNPs and growth traits of tilapia were analyzed using general linear model (GLM) in the offsprings of the 2 populations. Five SNPs loci (S1 (A-409G), S2 (G-424T), S3 (T-553A), S4 (T-1114A) and S5 (A-1168C)) were found in GHSR gene, and they were located in 5′ flanking region. The results showed that individuals with genotype AA or AT at S4 site had significantly higher body length, body height, head length, caudal peduncle length, caudal peduncle depth, and larger body weight than the individuals with genotype TT (P＜0.05). The dominant genotype frequency of S4 site in the generation of Fast-growing population and Base population was AT (55%) and TT (75.94%), respectively. The 5 SNPs sites composed 7 diplotypes (D1~7). The body weight, body length, body height, head length and caudal peduncle depth of individuals with diplotypes D3 and D5 were significantly larger or higher than that of the individuals with diplotypes D4 and D7 (P＜0.05). The growth traits of individuals with diplotypes D1 (except for body width), D2 (except for body width and the shank length) and D6 (except for body height) had no significant difference compared with individuals with the other diplotypes. The dominant diplotypes in the generation of Fast-growing population and Base population were diplotypes D3 (46.25%) and D4 (35.44%), respectively. In conclusion, the genotypes AA and AT at S4 site, diplotypes D3 and D5 of GHSR gene were closely related to fast growth rate in Nile tilapia, which can be used as candidate molecular markers for tilapia breeding.

Maize (Zea mays) is one of the main food crops in the world. Through breeding to cultivate maize rich in β-carotene is one of the effective methods to solve the problem of lower β-carotene content in maize relatively. In order to screen out maize inbred lines and hybridized combinations of high agronomic traits combining ability and rich in β-carotene, 64 hybridized combinations which were planted at Yuanjiang and Ya'an were made according to the complete diallel cross method with total 8 inbred lines as parents which were high or low in β-carotene content, and 13 agronomic traits of the hybridized combinations were observed by combining ability analysis based on Griffing 3 method. The results showed that the general combining ability (GCA) of agronomic traits related to yield of inbred lines 9636, R08 and 698-3 performed higher and the effects of yield per plant and ear weight were the largest. The specific combining ability (SCA) of agronomic traits related to yield of hybridized combinations 9636×Huangzaosi, 698-3×Mo17, Dongdan54-3-1-1-1×R08 and 5311×698-3 performed better through SCA analysis. The reciprocal cross effect of agronomic traits related to yield of hybridized combinations 9636×48-2, 9636×Dongdan54-3-1-1-1, Dongdan54-3-1-1-1×Mo17 and Dongdan54-3-1-1-1×Huangzaosi performed higher through reciprocal cross analysis. Combined with the results of β-carotene content combining ability analysis, it is finally supposed that both β-carotene content and agronomic traits GCA of the inbred line 9636 are higher, and β-carotene content and agronomic traits of its hybridized combination 9636×Huangzaosi are better at the two environments. In agronomic trait aspect, the research provides a theoretical reference for maize quality breeding rich in β-carotene.

This experiment was conducted to reveal the effect of the polymorphism of exon 1 and 10 of follicile stimulating hormone receptor gene (FSHR) on the reproductive traits of Qianbei Ma goat (Ovis Linnaeus). The expression of FSHR gene in hypothalamus, pituitary, uterus, fallopian tubes and ovaries was studied using high prolificacy goat breed (Qianbei Ma goat and Guizhou White goat) and low prolificacy goat breed (Guizhou Black goat) as a control. The single nucleotide polymorphism (SNP) of FSHR gene in exon 1 and 10 was detected by direct sequencing technology of PCR products and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and different expression in Guizhou native goat 5 tissues was detected by quantitative Real-time PCR (qRT-PCR). The result showed that 3 SNPs locus (1-C126T,10-C1246A and 10-A1412C) were deteced in exon1 and 10 in Qianbei Ma goat, and 10-A1412C was a sense mutation and devided into 3 genotypes: CC, AC and AA, and the genotypes frequency were 0.472 7, 0.309 0 and 0.218 3, respectively, the populations was in high polymorphism situation. The relationships between reproductive trait of litter size of Qianbei Ma goats and genotype CC and AC were significantly higher than those with genotype AA (P＜0.05). qRT-PCR result showed that the expression level of FSHR was high in hypothalamic but low in ovarian ducts, and with no significant difference in hypothalamus, pituitary, uterus, fallopian tubes and ovaries in 3 breeds goats (P＞0.05). This study indicates that FSHR gene may be a potential major gene or link to major gene effecting goat litter sizes, and a candidate molecular marker for reproductive traits selection in Qianbei Ma goats.

Listeria monocytogenes is an important food-borne pathogen due to its widespread distribution. Consumption of food contaminated with this pathogen can lead to listeriosis. This condition has high rates of morbidity and mortality (25%~30% over all). The organism can endure adverse conditions such as freezing, drying, mild heat and anaerobic conditions. It can also grow at temperatures used in refrigeration (-1.5~4 oC). Due to these characteristics, L. monocytogenes is a great concern for food safety. In this study, the expression timing of Listeria virulence genes in the viral growth cycle was studied to understand the basis for food poisoning caused by pathogens and the effectiveness of food safety control mechanisms. The growth curves for the pathogen were measured at OD600, and 27 of the Lm 319 virulence genes were detected by PCR. Quantitative Real-time PCR (qRT-PCR) was used to analyze 12 batches of 10 virulence genes. The growth curve showed that the first 2 hours of L. monocytogenes Lm319 growth was the lag phase and 2~8 h was the exponential growth phase. During 8~18 h, cell growth entered stationary phase, followed by the declining phase after 18 h. PCR results for 27 virulence genes of L. monocytogenes Lm319 showed that they explicitly contained 23 virulence genes. Quantification by qRT-PCR indicated that 10 virulence genes had similar expression patterns in a 24 h period, with peak expression levels at about 16~18 h in late stationary phase, which thereafter declined but increased again in the death phase. At the same time, it was also found that the expression levels of the 10 virulence genes were significantly different, with invasion associated protein gene (iap), fibronectin-binding protein gene (fbp), hexose phosphate transporter gene (hpt) and host factor for RNA phage Qβ replicase gene (hfq) having the highest expression levels, actin polymerizing protein gene (actA) and hemolysin gene (hlyA) having the lowest expression levels. This study demonstrated that the major virulence genes of L. monocytogenes Lm 319 peaked in expression during the late-stationary phase of cell growth and maintained the high expression levels through the death phase. This conclusion provides a theoretical basis for L. monocytogenes detection and prevention.

In the face of the inefficiency of bioconversion of agricultural biomass waste in low temperature environment in the winter, this study was aimed at enrichment culture and community structure analysis of a cold-adapted cellulose degrading microflora A25-3, which was isolated from the soil in alpine region. The microflora A25-3 could grow rapidly by enrichment culture at 15 ℃, with 10% cow dung and 10% LB sodium salt, and the weight loss ratio of cow dung was 48.28% in 72 h. The carboxymethyl cellulose (CMCase) activity of the supernatant of microflora culture at 15 ℃, with a peak of 42.37 U in 120 h was higher than that at 25 ℃. Total microbial DNA was directly extracted from the microflora cultured at different temperatures of 15 and 25 ℃. The clone library of 16S rRNA genes was amplified using PCR with universal bacterial primer sets. Each unique restriction fragment polymorphism pattern, which was created with 2 restriction endonucleases (HinfⅠand Csp6Ⅰ), was designated as an operational taxonomic unit (OTU). Amplified DNA was used for diversity analysis. The microflora phylogenetic trees of bacterial clone library at different temperatures of 15 and 25 ℃ was constructed. Community structure analysis revealed that the genera Brevundimonas, Pseudomonas, Chryseobacterium, Acinetobacte, Sphingobacterium and Bacillus were dominant in the clone library from microflora cultured at 25 ℃, while the genera Acinetobacte, Psychrobacillus and Bacillus were dominant at 15 ℃. The results suggested that the dominant population of cold-adapted cellulose degrading bacteria important species, especially Acinetobacte, provided the effect of increasing the CMCase activity at 15 ℃. The result will provide a scientific basis for the next study to separate active bacteria strains from the bacterial community and to optimize cellulase synthesis conditions.

DNA barcoding technique, taking a standardized DNA region as a tag, has been proved to be a new accurate approach in species identification and phylogenetic analysis of creatures. As the largest family of Eulamellibranchia, Unionidae plays a key role in freshwater ecosystems. We sequenced partial fragments of 2 mitochondrial genes (cytochrome c oxidase 1 (CO1) and NADH dehydrogenase subunit 1 (ND1)) in 19 species from China, and analyzed their relationships together with the published data of 10 American unionids from NCBI. The research based on the amplification and sequencing of 2 mitochondrial genes CO1 and ND1 in 19 species of mussels from China, and combined with the sequence information of 10 American unionids in the NCBI database. By using DNA barcoding technology, the research analyzed intraspecific distances and interspecific distances for unionid species, and phylogenetic tree of these species was also constructed. For most of the species, the relationship between species based on the genetic distance were consistent with that by traditional taxonomies according to morphological characteristics. However, as for Solenaia, 2 species (Solenaia triangularis vs Solenaia rivularis) showed little molecular differentiation. The intraspecific genetic distances were much less than interspecific genetic distances for both CO1 and ND1 sequences in the 19 unionids species. These results suggested that both CO1 and ND1 should be useful DNA barcoding for identification and phylogenetic analysis of mussel species. This research provides important references for identifing species through DNA barcoding technology, studying the biomass and conservation strategies of freshwater Unionids.

Multigene family of uridine dipfosphate glycosyltransferases (UGTs), comprising the greatest number of glycosyltransferases, involve in various biological processes, such as regulation of plant growth and development, and response to abiotic stresses. In the present study, one UGT gene, named as GhUGT85O1(GenBank No. KP300000) in cotton (Gossypium hirsutum) was identified. The full length of its open reading frame (ORF) and predicted peptide chain was 1 422 bp and 473 amino acids, respectively. The C-terminal contained a plant specific putative secondary plant glycosyltransferase (PSPG) motif found in UGTs family. Quantitative Real-time PCR (qRT-PCR) result indicated that GhUGT85O1 expressed extensively in the mature and old leaves, and was induced by abscisic acid (ABA), jasmonic acid (JA) and polyethylene glycol (PEG) in cotton. To illustrate its biological function, 35S::GhUGT85O1 vector was constructed and transformed into Arabidopsis thaliana. The over expression of GhUGT85O1 in transgenic Arabidopsis thaliana showed significantly early flowering (P＜0.01) and accelerated senescence phenotype compared with that in wild type. The chlorophyll content in transgenic Arabidopsis thaliana was significantly lower than that in wild type at senescence stage(P＜0.01). Meanwhile, the expression levels of AtORE1, senescence-associated gene12 (AtSAG12), AtSAG13, WRKY DNA-binding protein 6 gene (AtWRKY6), NAC transcription factor (AtNAP) and pathogenesis-related protein 1 gene (AtPR1) of senescence associated genes were higher than that in wild type(P＜0.05 or P＜0.01). Promoter deletion analysis result located the essential region of GhUGT85O1 promoter on -1 077~-1 363 bp, which contained 3 cis-acting elements of anaerobic response element(ARE), WRKY transcription factors binding site(W box) and TGA element (TGA). The result preliminarily suggested the involvement of GhUGT85O1 in response to abiotic stresses, regulation of both flowering time and senescence. It will not only establish a solid foundation for further function identification of GhUGT85O1 in cotton, but also provide gene resources for breeding short season cotton.

In order to obtain high quality genomic DNA of rumen microbes in goats(Capra hircus) and analyze the microbial diversity in the rumen, 6 kinds of DNA extraction methods, including bead mill+Tris-Phenol method, bead mill+Sodium dodecyl sulfate (SDS) method, bead mill+SDS/Guanidine thiocyanate (GITC) method, bead mill+SDS/NH4Ac method, bead mill+SDS/GITC/NH4Ac method and SDS/GITC methods, were used to extract microbial genomic DNA of goats in the present research. These extraction methods were compared through measuring DNA concentration and purity, polymerase chain reaction(PCR), denaturing gradient gel electrophoresis(DGGE) technique to find the suitable method for extracting microbial genomic DNA in the rumen of goats. Results showed that microbial genomic DNA extracted by using bead mill plus sodium dodecyl sulfate/guandine thiocyanate/ammonium acetate method had better purity and the concentration of it was 96.4 ng/μL. Meanwhile, ruminal bacterial and methanogenic 16S rDNA was amplified simultaneously, and the highest 16S rDNA diversity indexes of ruminal bacteria and methanogen were found when using this method. Therefore, bead mill plus sodium dodecyl sulfate/guandine thiocyanate/ammonium acetate method is more suitable for the subsequent molecular biology research of ruminal microflora.

In order to improve the accuracy of denaturing gradient gel electrophoresis (DGGE) in the analysis of rumen bacteria community diversity, the best range of DGGE denaturing gradient, PCR amplification procedure and treatments methods of PCR products had been optimized in this study. Three denaturing gradients, 35%~70%, 40%~60% and 55%~65%, were designed for DGGE, and normal PCR and reconditioning PCR were used to amplify rumen bacteria 16S rDNA, respectively. After that, the PCR products were purified via denatured polyacrylamide gel electrophoresis (d-PAGE) and mung bean nuclease respectively, as well as cluster and some bands sequences analysis. The results showed that the DNA bands in gel were well-distributed at 40%~60% denaturing gradients. Reconditioning PCR plus d-PAGE purification could minimize single-stranded DNA (ssDNA) to a certain extent. According to cluster and sequence analysis, different treatments method could result in different bands distribution in DGGE profiles, and the single strand could not completely represented unique bacteria species. In conclusion, the better denaturing gradient for rumen bacteria community DGGE was 40%~60%, and reconditioning PCR plus d-PAGE purification could be used to purify samples before DGGE analysis to obtain the better profile. The study provides basic references to DGGE in the analyzing of rumen bacteria community diversity.

Capsular polysaccharide of Haemophilus parasuis (HPs) is known as one of crucial virulence factors in pathogenicity. Wza gene plays a vital role in transporting the capsular polysaccharide from inside to the surface of the cell. In this study, an Wza-deficient mutant of HPs (ZJ1208-ΔWza) was successfully screened from Kan-TSA following natural transformation of Haemophilus parasuis with pUC18-LR-Kan. According to preliminary research on biological characteristics, ZJ1208-ΔWza mutant and ZJ1208 wild strain had no visual difference in colonial morphology and capsular after 12 h culture in vitro. ZJ1208-ΔWza mutant reached the maximum OD600 at 1.246, while wild strain reached 2.196, nearly 0.95 higher than ZJ1208-ΔWza mutant. The logarithm of viable count was 9.7634 for ZJ1208 wild strain after 8 h culturing, and 9.322 2 for ZJ1208-ΔWza mutant. With regard to mycelial morphology, the long-chain or long rod of ZJ1208-ΔWza could be observed in 6 h culture in vitro, whereas wild strain formed shorter cells. Moreover, ZJ1208-ΔWza mutant had higher ratio of sedimentation, and was more sensitive to porcine alveolar macrophage (PAM) phagocytosis, and was less virulent in mice toxicity assay than ZJ1208 wild strain. This study showed that Wza deficiency had a multiple affects on biological characteristic of haemophilus parasuis and also lay a foundation for furtherly researching the roles of Wza gene in formation of capsular polysacchaaride around HPs.

This study aimed at establishing a TaqMan quantitative Real-time RT-PCR (qRT-PCR) assay for detecting Apple stem grooving virus (ASGV). A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ASGV coat protein (CP) gene. The recombinant plasmid of ASGV-cp was constructed as positive standard to generate standard curve, and the specificity, sensitivity and reproducibility of this method were evaluated. The results showed that the correlation coefficient (R2) of standard curve was 0.999 and the amplification efficiency (E) was 96.8%. There was no crossing reaction with Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), indicating a strong specificity. The sensitivity of this method was 10 copies/μL which was 1 000 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 1%. This method could be used to detect ASGV rapidly in practical samples with strong specificity, high sensitivity and reliable reproducibility.