Autoinducer 2 (AI-2), used to communicate among bacterial species, regulates numerous physiological functions of bacteria. In this study, we studied the effect of AI-2 on adherence and invasion of Riemerella antatipestifer (RA) strain CH3 to Vero cells and transcriptional levels of virulence-related and metabolism-related genes were investigated.To verify whether the adherence and invasion of CH3 was affected by AI-2, we added different concentrations of AI-2 to the cocultures of Vero cells and CH3 and then calculated adherence percentages and invasion percentages of tested groups. We further added AI-2 (184.0 micromol/L) to the tryptone soya broth culture of CH3 and then detected the effect of transcriptional levels of related genes of CH3 using real-time PCR.The adherence of CH3 to Vero cells was decreased most to 62% with 18.4 micromol/L AI-2 and the invasion of CH3 to Vero cells was increased most to 194% with 184.0 micromol/L AI-2. The result of real-time PCR shows that AI-2 increased transcriptional levels of some virulence-related genes and decreased transcriptional levels of some metabolism-related genes.These results suggest that AI-2 affected adherence and invasion of CH3 to Vero cells. Moreover, AI-2 could regulate some genes of CH3 to modulate particular physiological behaviors.

We selected reference genes for virulence gene expression of Vibrio parahaemolyticus under different environmental conditions.Using qRT-PCR, we evaluated the expression stability of four housekeeping genes (GAPDH, pvuA, pvsA and rpoS) of V. parahaemolyticus cultivated separately in seawater, filtered seawater, shrimp and Tryptone Soya Broth.The result shows that all the candidate reference genes could be amplified specifically in qRT-PCR reaction. The expression stability of the four reference genes ranked pvuA (2.906) > pvsA (3.197) > GAPDH (3.746) > rpoS (6.512). Further analysis with geNorm program reveals that the highest stability was observed in pvuA and pvsA. The geometric average score of the two genes was considered as the most appropriate reference gene for normalizing the expression of virulence genes of V. parahaemolyticus.The pvuA and pvsA genes could be used as reference genes to study the virulence gene expression of V. parahaemolyticus.

To prepare anti-fps mono-specific serum, and detect the fps antigen in tumors induced by acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.Two part of v-fps gene was amplified by RT-PCR using the Fu-J viral RNA as the template. Mono-specific serum was prepared by immuning Kunming white mouse with both two recombinant infusion proteins expressed by the prokaryotic expression system. Indirect immunofluorescent assay was used to detect fps antigen in tumor tissue suspension cells and CEF infected by sarcoma supernatant. Immunohistochemical method was used to detect fps antigen in tumor tissue.The mouse mono-specific serum was specific as it had no cross reaction with classical ALV-J strains. The result reveals that the tumor tissue suspension cells, the CEF infected by sarcoma supernatant, and the slice immunohistochemistry of the sarcoma showed positive results.The anti-fps mono-specific serum was prepared, and the detection method was established, which laid the foundation for the study of viral biological characteristics and mechanism of tumourgenesis of acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.

With the application of high-throughput sequencing methods, more and more sRNAs are required to be verified. In this study, we developed the digoxigenin-labeled Northern blot method for detection of Yersinia pestis sRNA.Total RNAs extracted from Yersinia pestis grown under low-iron conditions were loaded onto 10% denaturing urea polyacrylamide gel (dPAGE), electrophoresed and transferred to nylon membranes. Northern blots were fixed to the membrane by UV cross-linking and subjected to hybridization with 3' -end digoxigenin-labeled oligonucleotides RNA probe for RyhB1 and RyhB2 overnight and followed by washing, blocking, immunological detection and finally exposed to film.Exposure time of digoxigenin-labelled Northern blot was 20s-3 min. The detection sensitivity of RyhB1 and RyhB2 was 0.005 microg and 0.05 microg, respectively. RyhB1 and RyhB2 probe specificity was high and no cross reaction with each other was found. Positively charged or neutral nylon membranes are applicable to the hybridization reaction. Hybridization for RNA probe can proceed within 42 to 65 degrees C with reduced non-specific probe interactions by increasing the temperature while the hybridization temperature for DNA oligonucleotide probes seemed to be determined empirically.A digoxigenin-labeled Northern blot has been developed for detection of Yersinia pestis sRNA, which was characterized by good specificity, high sensitivity, longer shelf life and short exposure times and provided an power tool for validation of bacterial sRNAs and function studies.

To obtain mice and rabbit polyclonal antibody of serotype I Marek's disease virus (MDV) sorf2 protein with higher titer and to identify the specificity.Using serotype I MDV GX0101 as template, we amplified sorf2 gene and then cloned it into pET-28a (+) and pET-32a (+) respectively. The recombinant plasmid pET-28a-sorf2 and pET-32a-sorf2 was separately transformed into Escherichia coli BL21 (Rosetta) competent cell which was induced with isopropylthio-beta-D-galactoside (IPTG). After purification, immuned 6-8 Balb/c mice and adult New Zealand white rabbit with the purified fusion protein and the anti-sorf2 polyclonal antibody were prepared. The specificity of the serum was detected by Western blot and the indirect immunofluorescence assay (IFA) method.Serotype I MDV sorf2 protein was expressed successfully in the recombinant E coli. Mice and rabbit anti-sorf2 polyclonal antibody with higher titer could react with sorf2 protein specifically.The prepared anti-sorf2 polyclonal antibody could identify MDV sorf2 gene deletion strain specifically. In addition, it could be used for differential of MDV vaccine poison HVT and serotype I MDV, which was useful for the separation and identification of clinical MDV.

To construct the virB1-89K gene knockout mutant and its complementary strain of Streptococcus suis serotype 2 (SS2) highly virulent strain 05ZYH33 and evaluate the role of virB1-89K in the pathogenesis of SS2.The virB1-89K gene was knocked out by homologous recombination, then multiple-PCR and sequence analysis were used to identify the knockout strain deltavirB1-89K. The virB1-89K gene and its upstream promoter were cloned into the E. coli-S. suis shuttle vector pSET1, and the recombinant plasmid was electrotransformed into the deltavirB1-89K mutant to generate the complementary strain CvirB1-89K. The effects of virB1-89K deletion on the basic biological characteristics and virulence of SS2 were then determined in this study.The isogenic mutant deltavirB1-89K and its complementary strain CvirB1-89K were successfully constructed. No significant differences in biological characteristics were found among the three strains. However, the virulence of the deltavirB1-89K mutant was reduced to 30% of the wild-type level and functional complementation of virB1-89K restored its pathogenicity.The virB1-89K gene plays an important role in the pathogenesis of S. suis 2 infection.

This study aimed to investigate the diversity of endophytic bacteria isolated from rice seeds, and screen indole acetic acid secrecting srtains.Conventional culture-dependent methods were used to isolate the endopytic bacteria from rice seeds. Phylogenetic analysis was done based on partial 16s rRNA gene sequences. The ability to indole acetic acid secretion of tested strains was analyzed qualitatively and quantitatively by colorimetry.In total 66 isolates were identified as belonging to 26 species of 15 genera of 5 phyla. Of them 26 strains were chosen to test indole acetic acid secretion. Four isolates had more ability of indole acetic acid secretion; they belonged to the genera of Staphylococcus, Rhizobium, Microbacterium and Methylobacterium.The endophytic bacteria in rice seeds are diverse. Some of them could produce indole acetic acid.

In this study, we constructed a recombinant Escherichia coli (E. coli) strain to produce hemolytic phospholipase C and optimized the fermentation conditions.We screened a high phospholipase C activity strain, Pseudomonas aeruginosa (P. aeruginosa) 41, through yolk borax plate method, and cloned the hemolytic phospholipase C gene (plcH) from it. The plcH was inserted into pET-28a (+) and then obtained the recombinant expression plasmid (pET28a-plcH). We selected the correct recombinant plasmid and transformed it into E. coli BL21 (DE3). Furthermore, we determined the PLC activity and hemolytic activity in positive transformants on yolk borax plate and columbia blood agar plate. Finally, we optimized the fermentation conditions.We successfully constructed a recombinant E. coli strain (E. coli BL21 (DE3)/pET28a-plcH) that showed significant phospholipase C activity. Moreover, hemolytic phospholipase C of the recombinant strain showed strong hemolytic activity. The enzyme activity of phospholipase C was 722.9 +/- 0.47 U/mL with 5% of inoculation amount, 200 r/min for 4 hours at temperature of 37, induced by 0.9 mmol/L IPTG for 14 hours.We constructed a recombinant E. coli strain with high hemolytic phospholipase C activity under optimized fermentation conditions. It is the first time in domestic to successfully clone and express phospholipase C gene from P. aeruginosa in E. coli. These research results are helpful to advance the industrialization and application of phospholipase C.

By analyzing the function and mechanism of nitric oxide in initiating producing lignin peroxidases by phanerochaete chrysosporium, we studied the regulation mechanism triggering the secondary metabolism of white-rot fungi.Mutant (pcR5305) and wild-type (pc530) strains of phanerochaete chrysosporium were respectively cultured under both the conditions of nitrogen limitation and nitrogen sufficiency. To compare their lignin peroxidases (LiP)-production and nitric oxide(NO)-production kinetics and their different influences on producing LiP after the NO donor Sodium Nitroprusside (SNP) and scavenger cPTIO were respectively added to the nitrogen limitation or sufficiency culture medium to show the function and mechanism of nitric oxide in initiating production of lignin peroxidases by white-rot fungi.Both strains produced nitric oxide (NO) under the two opposite nutritional conditions, but the levels of NO produced were related with the type of strain and the nutritional conditions. Strain pc530 produced NO requiring nutrition depletion and producing of NO was strongly delayed and reduced when it was cultured under nitrogen sufficiency condition. On the contrary, pcR5305 did not require nitrogen depletion to trigger and the levels of NO were higher than that of pc530. The results indicate that LiP content had positive correlation with NO value except the occurrence time of LiP peak value was later than that of NO. The ability of producing LiP was promoted after the NO donor SNP added, but SNP affected more on pc530 than pcR5305 in promoting producing LiP. 15mM cPTIO would greatly repress producing LiP, but could not completely restrain the synthesis of LiP for both strains.By producing NO, Phanerochaete chrysosporium triggers LiP synthesis. However, the evidences do not indicate that NO participates or effect directly in LiP synthesis. It is more likely that NO is reacting as an upstream signal molecule. Besides NO, there are other signal molecules that have a positive effect on NO levels also involving in the regulation producing LiP. The mechanism of the resistance to nutritional repression of pcR5305 in synthesizing lignin degrading peroxidases may be the answer to the different NO production mechanism of pcR5305 from pc530.

To identify the transcriptional regulation of exosporium basal layer structural gene exsB in Bacillus thuringiensis.We analyzed exsB and its promoter sequence in Bacillus cereus group by sequence alignment. We performed beta-galactosidase assay of exsB-lacZ gene fusion to analyze transcriptional activation of exsB promoter; we used Electrophoretic mobility shift assays to detect binding of GerE and exsB promoter.exsB was the high similarity in Bacillus cereus group strains. Beta-galactosidase assay showed that exsB promoter had the strong transcriptional activation on the late sporulation phase. Deletion of gerE or sigK gene decreased the activation of exsB promoter. Electrophoretic mobility shift assays showed that GerE could bind with exsB promoter.The exsB gene is regulated by SigmaK and GerE on the late sporulation phase.