Abstract:Abstract: Microbial secondary metabolites have a wide range of biological activities due to their structural diversity and have been proved a major source for drug lead compounds. However, the traditional method of a single culture restricts the metabolic pathways of microorganisms and as a result many metabolites cannot be formed. Recently, it has attracted much attention to use various techniques to activate those metabolic pathways restricted by the traditional method to get metabolic products with rich variety of structures. “One strain many compounds” (OSMAC) is a simple and effective approach for activating metabolic pathways and has been successfully applied. This review summarizes the common strategies of the OSMAC approach (altering cultivation parameters, co-cultivation, addition of enzyme inhibitors, etc）and the recent advances of OSMAC combined with genomics scanning. This review also introduces the research of our studies using the OSMAC approach on a fungus Spicaria elegans KLA03 which yielded a series of cytochalasins.

Abstract:Abstract: [Objective] We isolated and characterized phosphor-dissolving endophytic bacteria from two commonly cultivated crops. [Methods] Phosphor-dissolving endophytic bacteria were isolated by plating and screening from interior tissues of rape and maize plants on NBRIP medium with tricalcium phosphate as sole phosphate source. Bacteria were characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction---indoleacetic acid, siderophore and 1-aminocyclopropane-1-carboxylic acid deaminase production, and further classified by restriction analysis of 16S rDNA. Eleven typical strains were identified by 16S rDNA sequence analysis. [Results] Thirty-two phosphor-dissolving endophytic bacteria were isolated from maize and rape plants and classified by restriction analysis of 16S rDNA in 8 different taxonomic groups at the similarity level of 76%. All the isolates could release phosphor from tricalcium phosphate and decrease the pH of the medium. The maximum phosphor content (537.6 mg/L) in the solution was obtained with strain M1L5. Thirteen isolates isolated from rape produced indoleacetic acid and siderophore, 68.4% and 63.2% of the strains isolated from maize produced indoleacetic acid and siderophore, respectively. 63.2% of the strains isolated from maize were able to grow on 1-aminocyclopropane-1-carboxylic acid as the sole nitrogen source. The eleven strains belonged to five different genera including Pantoea、Pseudomonas、Burkholderia、Acinetobacter and Ralstonia. [Conclusions] Phosphor-dissolving endophytic bacteria isolated from rape and maize plants have abundant characteristics relative to promoting plant growth and genetic diversity.

Abstract:Abstract: 【Objective】To better elucidate the functions of RpfFxoo, RpfCxoo and RpfGxoo, 3 proteins of diffusible signal factor (DSF)-dependent cell–cell signaling system in regulation of virulence expression of Xanthomonas oryzae pv. oryzae (Xoo). 【Method】 △rpfxoo, the gene deletion mutants were generated from PXO99A, the wild-type strain of Xoo via marker-exchanging and DSF biosynthesis and extracellular polysaccharide production and virulence to rice of the mutants were assayed.【Result】rpfFxoo, rpfCxoo and rpfGxoo were cloned from the genomic DNA of PXO99A and the relative single or double mutants were constructed. Compared to PXO99A, DSF production was deficient in △rpfFxoo, △rpfFCxoo and △rpfFGxoo, while DSF was overproduced in △rpfCxoo and reduced in △rpfGxoo. DSF production of △rpfFxcc, △rpfCxcc and △rpfGxcc, the mutants of X. campestris pv. campestris can be restored as XC1, the wild-type strain by in trans complementation of rpfFxoo, rpfCxoo and rpfGxoo. All the mutants except △rpfFxoo were remarkably deficient in production of extracellular polysaccharide. All the mutants significantly exhibited the reduced bacterial virulence to rice.【Conclusion】 DSF signaling proteins RpfFxoo、RpfCxoo and RpfGxoo might function in regulation of DSF biogenesis and EPS production and bacterial virulence.

Abstract:Abstact: [Objective] We isolate the gene involved in osmotolerance of Beauveria bassiana for investigation the adaptation mechanism of the fungus to adversity. [Methods] T-DNA tagging and genomic DNA walking were carried out by using Y-shaped adaptor dependent extension (YADE) method. RT-PCR was used to analyze the transcription profile of Bbmpd. [Results] A gene (Bbmpd) encoding mannitol 1-phosphate dehydrogenase in B. bassiana was identified. The transcription of Bbmpd was induced by osmostress (0.8 mol/L NaCl) in wild type strain, while significantly decreased in Bbhog1 mutant, indicating that the transcription of Bbmpd was regulated by Hog1 pathway in B. bassiana. Bbmpd-disrupted mutants showed sensitive to osmostress on Czapek’s medium containing 0.8 mol/L NaCl. However, the growth and sporulation of the Bbmpd-disrupted mutants in vitro were not significantly different from that of wild type strain. [Conclusion] Mannitol 1-phosphate dehydrogenase gene was isolated by T-DNA tagging, and was found to be involved in the osmostress of B. bassiana. The transcription of Bbmpd was induced by osmostress and regulated by Hog1 pathway.

Abstract:Abstract:【Objective】The performance of Lactobacillus salivarius BBE09-18, L. fermentum BBE09-29 and L. casei Zhang to remove the cyanobacterial toxin microcystin-LR ( MC-LR) were determined in this study, and the key factors related to the removal efficiency were investigated to analyze the specific mechanism and provide a novel way to deal with the MC-LR contaminated foods. 【Methods】 Investigate the microcystin-removing capability of cells in different physiological status, e.g. viable and nonviable cells, and compare their removing effects with different biomass, toxin concentration and glucose supplement.【Results】Among these strains, L. casei Zhang removed the microcystin-LR most effectively. After a removal treatment with L. casei Zhang for 24 h (109 CFU/mL, pH 7, 37 ?C), the MC-LR concentration deduced from 150 μg/L to 85.5 μg/L. Results also showed that the viable cells were more effective for MC-LR removal than the nonviable ones. Moreover, the removal performance can be improved significantly when glucose was added, and the maximum removal of 92% was observed for L. casei Zhang ( toxin initial concentration 1800 μg/L, 1010 CFU/mL, pH 7.0, 37℃, 24 h) with 5% glucose supplement.【Conclusion】All of the three LAB used in this study can remove MC-LR. It can be confirmed that some factors, such as biomass, toxin concentration and energy supplement of cells play important roles on the removal efficiency. These results suggest further that the mechanism of MC-LR removal in LAB may be related to their metabolic activity.

Abstract:Abstract: [Objective] We amplified and overexpressed the FHL activator (fhlA) in E. aerogenes ATCC13408 to enhance hydrogen production. [Methods] By using universal primers and genome walking, we cloned the full open reading frame (ORF) of fhlA gene. We inserted it into the glutathion S-transferase (GST) fusion expression vector pGEX-4T-2-Cat, and transformed the recombinant plasmid into E. aerogenes ATCC13408 via electroporation for expression. Then we measured the hydrogen production of the recombinant strain in a batch culture. [Results] We found that the ORF of fhlA was 2073 base pair in length, potential to encode a 690 amino acid peptide (GenBank accession GU188474). The FhlA protein from E. aerogenes ATCC13408 shared high amino acid identities with those from other bacterial species. By using SDS-PAGE and Western blot analysis, we confirmed that the fhlA gene had successfully expressed in the strain. The hydrogen yield of the recombinant strain was increased from 1.23 to 1.48 mol H2/mol glucose. [Conclusion] Enhancement of hydrogen productivity was attained under anaerobic conditions with the recombinant strain.

Abstract:Abstract: [Objective] We identified the interaction between toxin Slr0664 and antitoxin Slr1114, encoded by ssr1114/slr0664 system in the chromosome of cyanobacteria Synechocystis sp. PCC6803. [Methods] We constructed a recombinant plasmid in which only H6-Ssr1114 was induced to express, and another plasmid in which both H6-Ssr1114 and Slr0664 was co-expressed in E. coli Bl21(DE3). After induction, we used affinity capture technique to purify H6-Ssr1114 and copurified H6-Ssr1114 and Slr0664 under different conditions. We confirmed the co-purified H6-Ssr1114 and Slr0664 by using mass spectrographic analysis. [Results] When induced to express, Slr0664 showed cell toxicity leading to cell growth suppression or death. However, cells could grow normally if both H6-Ssr1114 and Slr0664 were induced to co-express. We could purify both H6-Ssr1114 and Slr0664 by His-Bind under native conditions, but only H6-Ssr1114 could be purified under denature conditions. The results of mass spectrometric analysis showed that the copurified proteins were H6-Ssr1114 and Slr0664. [Conclusion] The antitoxin Slr1114 and toxin Slr0664 in ssr1114/slr0664 TA system was interacted with each other.

Abstract:Abstract: [Objective] To heterogeneously express the chitinase gene of Bacillus licheniformis strain MY75 in E. coli, and to characterize the recombinant chitinase ChiMY. [Methods] The extracelluar crude protein from B. licheniformis MY75 was analyzed by zymogram analysis. The partial amino acid sequence of the protein owned chitinase activity was given by time-of-flight mass spectrometry (TOF-MS). Then the corresponding chitinase gene chiMY was cloned and heterogeneously expressed in E. coli. The optimum temperature and pH of the ChiMY, and the effect of various metal ions on chitinase activity were studied. The antifungal activity and the synergistic effect on insecticidal activity were demonstrated by bioassays. [Results] A 55 kDa extracelluar protein produced by B. licheniformis MY75 exhibited chitinase activity in zymogram analysis. The chiMY gene was 1797 bp long and encoded a 599 amino acid protein. The molecular weight of the recombinant protein ChiMY over-expressed in E. coli was 67 kDa. The amino acid sequence of the 55 kDa extracelluar protein was proved identical to the 67 kDa ChiMY by the TOF-MS. The optimum temperature and pH were 50 °C and 7.0, respectively. The enzyme activity was improved by Li+, Na+ and Mg2+ and inhibited by Mn2+, Cr3+, Zn2+, and Ag+. Cu2+ and Fe3+ can inactivate the enzyme. The bioassays demonstrated the heterogeneous expressed ChiMY could inhibit the sporangia germination of G. saubinetii and A. niger. and reduce the LC50 (50 % lethal concentration) of the crystal protein of Bacillus thuringiensis against S. exigua by approximately 27 %. [Conclusions] The B. licheniformis MY75 could produce a 55 kDa chitinase. The corresponding chitinase gene was over-expressed in E. coli. The molecular weight of heterogeneous expressed ChiMY showed significant different to the wild-type chitinase protein. This implicated the the protein processing of chitinase in the B. licheniformis MY75. The ChiMY also owned the antifungal activity and could improve the insecticidal activity of the Bacillus thuringiensis crystal protein against to S. exigua. This is the first report about the chitinase from B. licheniformis in China.

Abstract:Abstract: [ Objective ] To produce Proteus vulgaris lipase (PVL) in large quantities, we cloned and expressed the lipase gene in Escherichia coli. [ Methods ] We cloned PVL gene by PCR method and then inserted the open reading frame of PVL gene into pET-DsbA and pMBP-P vectors. PVL gene was expressed in E. coli with the introduction of isopropyl β-D-1-thiogalactopyranoside (IPTG). We also studied the optimal culture conditions, including the concentrations of glucose, IPTG and ampicillin, induction temperature, and pH value of the medium. The characteristics of recombinant lipase were examined after affinitive purification by His-chelating affinity chromatography. [ Results ] The open reading frame of PVL gene consisted of 864 base pairs, encoding a protein of 287 amino acids. The sequence was deposited to GenBank with the accession number FJ643627. The gene was expressed in E. coli and active lipase was obtained from E. coli BL21 cells by the induction of IPTG, and the lipase production reached 192.2 U/mL in BL21[pET-PVL] after culture for 15 h at 15 ℃. The maximum production was obtained by culturing BL21 cells in LB medium (pH8.5) with 15 mg/mL glucose and 200 μg/mL ampicillin, as well as adding 100 μg/mL IPTG at OD600 of 1.2. A single protein band of 31 kDa was displayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after affinitive purification. The properties of lipase expressed in E. coli were similar to the native one，which could hydrolyze all three esters of triglyceride. [ Conclusion ] We have succeeded in over-expressing the lipase gene from P. vulgaris in E. coli, and this research has laid a foundation for improvement and industrial application of this lipase.

Abstract:Abstract: [Objective] An integrative vector of Saccharomyces cerevisiae for xylulokinase gene expression was constructed to overexpress xylulokinase activity. [Methods] On the basis of plasmid p406ADH1, 4 components were integrated, which were KanR gene as G418 resistant marker, ADH1 terminator fragment, xylulokinase gene from Saccharomyces cerevisiae W5 and 18S rDNA sequence for homologous recombination. After enzyme digestion and ligation, high copy recombinant expression vector pCXS-RKTr was constructed. pCXS-RKTr was linearized and transferred into Saccharomyces cerevisiae W5 , then xylulokinase activity was detected to determine the expression of pCXS-RKTr. [Results] Xylulokinase gene located on pCXS-RKTr was highly expressed in W5. The xylulokinase activity was 2.87 times of the original strain. [Conclusion] An integrative vector of industry strain Saccharomyces cerevisiae is successfully constructed and xylulokinase gene of Saccharomyces cerevisiae itself was over expressed by this vector. This intergrative vector can efficiently raise the xylulokinase activity of Saccharomyces cerevisiae. This system laid a foundation for the construction of gene engineering Saccharomyces cerevisiae strain which can ferment xylose to ethanol.

Abstract:Abstract:[Objective] To study the relation between mRNA expression of △5-desaturase gene and arachidonic acid production. [Methods] Using fluorescence quantitative PCR, we determined the mRNA expression levels of △5-desaturase gene of five Mortierlla isabellina strains cultured for the same time and strain YZ-124 cultured for different time. Simultaneously, we measured the contents of arachidonic acid by gas chromatography. [Results] The results showed that △5-desaturase gene mRNA expression level of distinct strains was different. Expression level of the original strain As3.3410 was the lowest, whereas that of the mutant strain YZ-124 was the highest. The mRNA expression of △5-desaturases gene decreased gradually with fermentation time. [Conclusion] According to arachidonic acid yield, mRNA expression quantity of △5-desaturase gene presented a positive correlation with arachidonic acid content in cultured oil.

Abstract:Abstract: [Objective] To investigate the effect of putative lipoate-protein ligase (LPL) on the virulence of Streptococcus pneumoniae. [Methods] lpl gene deficient strain was constructed by LFH-PCR and identified by PCR and sequencing. The cell adherence assay and mice challenge assay were used to observe the differences between wild strain and the mutant in the pathopoiesis of Streptococcus pneumoniae. [Results] Mice virulence experiments showed that the median lethal time of wide type and the lpl mutant are both 12h, no statistics difference ;The ability of adherence of the mutant was greater than the wild strain( P < 0.01); The capsule stain in-vivo showed that the wild strain and the mutant both had the capsule. [Conclusion] lpl gene inhibits the adherence to host, but no affect on the ability to infection mice by intraperitoneal injection.

Abstract:Abstract：[Objective]To illuminate the impact of humic acid (HA) on soil ammonia oxidizing archaea and then reveal the effect of HA on soil nitrogen cycle. [Methods]Two humic acids (cHA and bHA) were added into the soil amended with urea. Community changes of ammonia oxidizing archaea (AOA) and total archaea were studied with terminal restricted fragment length polymorphism (T-RFLP) and real time PCR in the microcosm experiment. [Results]We found that the AOA population size increased significantly and AOA community changed greatly in the urea only treatment. However, HA could inhibit the increase of AOA population, moreover, HA could buffer the change in AOA community showed by canonical correspondence analysis (CCA）result. On the other hand, the total archaeal population decreased significantly in the urea only treatment, but stabilized in the urea with HA treatments, which indicated HA could eliminate the toxicity of urea to total archaea. CCA results showed that incubation time was the most important factor for the total archaeal community, and partial CCA (pCCA, when time as a covariable) result demonstrated that cHA was the most important environmental variable for total archaeal community. [Conclusion]These results showed that HA diminished ammonia loss by inhibiting the increase of AOA competing with plant for ammonia, thus HA can increase the urea efficiency.

Abstract:Abstract: [Objective] Salmonella isolates from retail food were examined for antimicrobial susceptibility and further characterized to better understand the development and dissemination of antimicrobial resistance among foodborne Salmonella in China. [Methods] Antimicrobial susceptibility of 359 Salmonella isolates was determined by using agar dilution methods recommended by the Clinical and Laboratory Standards Institute. Antimicrobial resistance integrons and resistance genes were identified using PCR. Mutations in gyrase and Topoisomerase genes related to fluoroquinolones resistance were also determined using PCR and gene sequencing analysis. [Results] Among the 359 Salmonellae isolates, 67% were resistant to Sulfamethoxazole, followed by resistant to trimethoprim/Sulfamethoxazole (58%), tetracycline (56%), kanamycin (37%), nalidixic acid (35%), ampicillin (33%), amoxicillin/clavulanic acid (32%), streptomycin (29%), chloramphenicol and gentamicin (26%), ciprofloxacin (21%), ceftriaxone (16%), cefoxitin (9%) and cefoperazone (8%). Among the 284 resistant isolates, 79% were resistant to at least one antimicrobial, 25.9% to 10 or more than 10 antimicrobials, and 2.5% to 14 antibiotic agents. Integrons were detected in some of sulfamethoxazole-ressitant Salmonella, and the most common integron was 1.4 kb, Antimicrobial resistance genes carried by integrons included aadA1, aadA2, aadA5, tetR, blaPSE-1, blaDHA-1, blaVEB-1, dhfrⅠ, dhfrⅤ, dhfrⅦ and dhfr17. The blaTEM gene was also detected in 51.6% of 62 ceftriaxone and / or cefoperazone resistant isolates, and blaCMY-2 was detected in 56.5% of the isolates. 13.6% of the Salmonella isolates carried Salmonella Gene Island. Sixty-eight point mutations were detected in gyrA, parC and parE of 35 fluoroquinolone-resistant Salmonella isolates. The common mutations in gyrA gene were Ser83Phe, Ser83Tyr, Asp87Gly and Asp87Asn, whereas ser80Arg was detected in parC. Mutations including Lys441Ile, Lys428Gln, Asp494Asn, Lys428Gln and Gly442Ser were detected in parE, which was first reported in Salmonella. [Conclusion] Antimicrobial resistance of Salmonella recovered from food in Shaanxi province was common. Several genetic elements including integron, Salmonella Gene Island, β-lactamase genes and mutations in gyrase and topoisomerase genes played an important role in antimicrobial resistance of Salmonella.

Abstract:Abstract: [Objective] Bezafibrate is one of the most frequently detected pharmaceuticals at relatively high concentration in surface water and even in drinking water. Biodegradation is an important way to solve the problem. This study aimed to isolate, identify and characterize a bezafibrate-degrading bacterium. [Methods] Strain B31 capable of degrading bezafibrate by cometabolism was isolated from activated sludge of sewage treatment plant in Shanghai, China , and identified based on its morphology, physiology and phylogenetic analysis of 16S rRNA sequence. To evaluate the ability of degradation, the concentration of bezafibrate was detected by high performance liquid chromatography. [Results] Strain B31 was identified to be closely related to Pseudomonas putida. The optimum condition of degrading bezafibrate was at 30 ℃, pH 7. After 5 days, Strain B31 could degrade 30 mg/L bezafibrate by 48% in liquid mineral salt medium with 1% methanol as primary substrate. And the rate of degradation could enhance to 61%, 72.6%, 76.67%, when 5g/L glucose, peptone and yeast extract as primary substrate, respectively. [Conclusion] The strain has the potential for bezafibrate biodegradation.

Abstract:Abstract: [Objective] In order to improve the rate of the heterotrophic nitrification, we screened and identified a high-efficient heterotrophic nitrifier, as well as studied its nitrification characteristics and nitrification conditions.[Methods] We obtained activated sludge samples from sewage and chemical fertilizer factories and farmland. We then utilized sodium citrate and ammonium chloride as carbon and nitrogen source. We used methods including domestication, gradient dilution of domestication liquid, isolation from streaking plate and color indicator as rapid nitrification detection. Finally a high-efficient heterotrophic nitrifier was obtained. We identified this strain according to its physiological, biochemical properties and the sequence analysis of 16S rDNA. After inoculating the strain into artificial ammonia-nitrogen wastewater, changes of nitrogen compounds were measured in order to understand the nitrification characteristics. Nitrification condition was also optimized by changing the carbon source, dissolved oxygen, C/N ratio, temperature and pH of the medium. [Results] The heterotrophic nitrifier was a gram-negative bacilli. It neither fermented glucose, nor produced indole. Oxidase and catalase tests were positive. It could produce alkali if organic salt was provided. The strain shared 99.7% sequence identity of its 16S rDNA with ES-SDK-3 of Alcaligenes sp. In the artificial wastewater with 182.30 mg/L ammonia nitrogen as initial concentration, the removal efficiency by the strain was 99.8% after 30h cultivation. The average nitrogen removal rate was 9.61mg-N/L/h in its exponential phase. It produced almost no NO2―-N and NO3―-N in the entire nitrification process. The optimal carbon source is sodium citrate. Higher dissolved oxygen and C/N ratio favor its nitrification. When temperature is ranged from 30℃ to 35℃ and pH is ranged from 5.0 to 9.0, it can completely remove ammonia nitrogen. [Conclusions] The strain was identified as Alcaligenes genus, and named as Alcaligenes sp. HN-S. Our research confirmed that the Alcaligenes sp. HN-S had significant advantages over heterotrophic nitrifiers that were screened previously with aspect of ammonia nitrogen removal rate. The research of its nitrification condition definitely provided necessary theory support for a new biology process to remove nitrogen with high efficiency.

Abstract:Abstract: [Objective] We constructed recombinant adenovirus rAd-Ag85B expressing antigen 85B of Mycobacterium bovis and analyzed cellular immune responses after mice immunization. [Methods] We amplified the fbpB gene from genomic DNA of BCG by PCR and cloned into pMD18-T vector for sequence analysis. Then we constructed pDC516-Ag85B and co-transfected with plasmid pBHGfrtΔE1,3FLP into Ad293 cells by lipofectamineTM2000. Then we identified the recombinant Ad-Ag85B virus by RT-PCR, Western blot assay and TEM observation after negative staining. BALB/c mice were subcutaneously immunized with the dose of 108 TCID50 rAd-Ag85B or rAd. Two weeks after immunization, CD69 expression, T lymphocyte proliferation and ELISPOT were detected in spleen cells of immunized mice. [Results] We obtained the correct recombinant adenovirus rAd-Ag85B expressed Ag85B antigen. The level of both splenic CD69+CD4+T and CD69+CD8+T cells of rAd-Ag85B group were significantly higher than those of rAd control. The SI index of rAd-Ag85B was much higher than rAd. In ELISPOT assay more IFN-γ specific secreting cell spots other than IL-4 were detected in rAd-Ag85B group. [Conclusion] The recombinant adenovirus rAd-Ag85B can induce specific Th1 cell immune responses in mice.

Abstract:Abstract:[Objective] To study the influence of native bacterial magnetic particles on mouse immune response. [Methods] Ovalbumin was used as an antigen, mixed with complete Freund’s adjuvant, bacterial magnetic particles (BMP) and phosphate buffer solution, to immunize BALB/C mouseAfter 14 days later, we detected the titers of the anti-ovalbumin (IgG) and subtype (IgG1, IgG2a), the proliferation ability of T lymphocyte, and the expression of IL-2, IL-4,IL-10 and IFN-γ. [Results] Compared to the negative control group, the BMP group had the similar level in the titer of specific antibody, the proliferation ability of T lymphocyte and the expression of cytokines (IL-2, IL-4, IL-10 and IFN-γ). [Conclusion] Native BMPs has no significantly influence on mouse immune response.

Abstract:Abstract：[Objective] To construct the in-frame deletion gene mutants in streptococcus equi subsp. zooepidemicusby (S. zooepidemicus) by pJR700 plasmid, a thermosensitive delivery vector system. [Method] With PCR we amplified a 4017-bp DNA fragment of streptococcal hemoprotein receptor gene from chromatosome DNA. This DNA fragment was subcloned into the pJR700 plasmid to create pXL28. Using pXL28 as the template we got the DNA fragment deleted 1831 bp in streptococcal hemoprotein receptor gene by inverse PCR amplification and ligated the in-frame deletion DNA fragment by T4DNase to create pXL30. Then pXL30 was transformed into S. Zooepidemicus by electroperation. The kanamycin(kan)-resistant clones were selected at 37°C in the presence of kan for three rounds. To induce excision of the plasmid vector sequence, the culture was shifted to 30°C and grown without antibiotics for four rounds. Colonies with kan sensitivity, which indicated that the plasmid had been excised, were selected by plating on THY agar at 37°C. S. Zooepidemicus mutants were identified through PCR with primers homologous to the flanking regions and the streptonigrin sensitive test. [Result] The in-frame deletion streptococcal hemoprotein receptor mutants were successfully built in S. Zooepidemicus. [Conclusion] The thermosensitive delivery vector system of pJR700 could be utilized to construct the in-frame deletion gene mutant strains of S. Zooepidemicus.

Abstract:Abstract: [Objective] To investigate the midgut bacterial community of sensitive and resistant Helicoverpa armigera for the emergence of resistance to Bacillus thuringiensis. [Methods] Two culture-independent techniques (16S rDNA library and DGGE) were introduced. The genomic DNA of midgut bacteria was extracted. The full length gene of 16S rDNA was amplified by PCR and then cloned. The 16S rDNA library was analyzed with Alu I and Sac I. The V3 region of 16S rDNA was amplified by PCR. The intrapopulation variation and variation between resistant and sensitive Helicoverpa armigera were assayed by DGGE. [Results] Analysis of 16S rDNA library showed similar patterns of midgut bacterial structure and diversity in terms of the dominant bacteria, but the inferior bacteria were significant different. The resistant population harbored abundant phylotypes belonging to uncultured bacterium (56.4%), Enterococcus gallinarum (17.0%) and Enterococcus casseliflavus (17.0%). For the sensitive population, the dominant bacteria were uncultured bacterium (60.2%), Enterococcus casseliflavus (14.7%) and Enterococcus gallinarum (19.3%). The divergence of midgut bacteria community between resistant and sensitive populations detected by 16S rDNA library analysis was verified by PCR with specific primers, and the result showed that all those inferior bacteria presented in both varieties. DGGE profile revealed that the similarity of the two varieties was 92.3%. [Conclusion] The midgut bacterial community of resistant and sensitive H. armigera was similar, which had no direct effect on Bt-resistance of H. armigera.