Abstract:Abstract: Polyketides represent a large group of natural products with diverse structures and biological activities, which could be divided into two structural types: aromatic polyketides and complex polyketides. Cyclases are key enzymes involved in the early tailoring reactions after polyketide chain synthesis; they act in concert with other early tailoring enzymes to control the folding and cyclization pattern of aromatic polyketides. Therefore, they are the key determinants of the final polyketide scaffold. Here, we classified cyclases involved in aromatic polyketide biosynthesis based on their sequences and structures. The reactions catalyzed by these cyclases and the possible catalytic mechanisms were described. We also described the research progress on the characterization of cyclases in jadomycin B biosynthesis.

Abstract:Abstract：[Objective] In order to investigate bacteria diversity in a hot gas spring soil of MaNasi county, Xinjiang. [Methods] Total DNA were directly extracted from the soil of a hot gas spring from Xinjiang.16S rDNA were amplified directly from purified DNA by PCR with universally bacteria-specific rDNA primers and cloned. Positive clones were identified by restriction fragment length polymorphism(RFLP), and unique rDNA types clones were sequenced, analysed and then constructed phylogenetic tree. [Results] Twenty-nine operational taxonomic units (OTU) from 170 positive clones were found belonging to 6 phyla: Firmicutes, Proteobacteria, Acidobacteria, Bacteroidetes, Planctomycetes, Actinobacteria. Firmicutes (71%) was the absolutely dominant components of the soil bacterial community. 14 sequences (about 1.5 kb) from 29 OTUs showed less affiliation with known taxa(<97% sequence similarity).[Conclusion] Soil bacterial diversity is low in the hot gas spring soil, but exit a large number of new unknown taxon in this environment.

Abstract:Abstract: [Objective] We surveyed the composition and diversity of archaea in Xinjiang Shawan cold spring sediment. [Methods] We studied the archaeal diversity in the cold spring sediment by direct extracting environmental DNA with liquid nitrogen grinding method and constructing clone libraries of 16S rRNA gene amplified with archaeal-speci?c primers. Amplified 16S rRNA gene fragments were analyzed by Restriction Fragment Length Polymorphism (RFLP), and the unique RFLP patterns were selected for sequencing, alignment and constructing 16S rRNA gene phylogenetic tree. [Results] A total of 121 positive clones were randomly screened from the library and 22 Operational Taxonomic Units (OTUs) were determined. BLAST analysis indicated that all OTUs were affiliated with the phylum Crenarchaeota. Phylogenetic analysis classified them into two subgroups of Soil-Freshwater-subsurface group, and Marine group I and represented 50% of total clones, respectively. Of them, clones with the potential to assimilate nitrate accounted for 40% of the total archaeal clones. In addition, 40% of clones were related to the cold-loving Crenarchaeota. [Conclusion] These results suggested that the spring sediment harbors a low diversity of archaea, however, a large fraction of Crenarchaeota indigenous species might exist and well adapted to the cold and oligotraphic environment.

Abstract:Abstract: 【Objective】To obtain new strains that can dissolving phosphate, we screened and identified a new strain Z32 capabile of phosphate dissolving from soil samples. We studied the colonization and phosphate-dissolving characteristics of Z32 in soil.【Methods】Morphological, growth on various media, and ITS rDNA sequences homology analysis were performed to identify the strain Z32. We conducted a soil experiment in pot to investigate the colonization and the capbility of the different forms of inorganic phosphorus transformation of the strain Z32.【Results】The strain Z32 was identified as Penicillium aculeatum. Z32 dissolved all Ca3(PO4)2 in solid culture for 4 days and decreased the pH from 7.0 to 1.5 in potato liquid culture for 18 hours. Z32 presented high colonization ability in soil and the optimum colonization temperature was 20℃. The number of Z32 increased 9.83 times in 21 days and kept alive in 49 days at the optimum temperature. Z32 promoted the transformation of insoluble phosphate like Ca8H2(PO4)6?5H2O, AlPO4 and FePO4 to soluble CaHPO4 and the CaHPO4 content increased by 58.83% in soil compared to CK at the 21st d at 20℃. Conclusion】 A new strain Z32 with high performance of phosphate dissolving had colonized well in soil. Z32 showed high phosphate-dissolving ability for different kinds of inorganic phosphorus in soil. At the same time, Z32 inhibited the transformation of CaHPO4 to insoluble phosphate.

Abstract:Abstract: [Objective] To isolate and characterize aerobic bacteria to degrade effectively chlorobenzene (CB). [Methods] We used enrichment culture to isolate and characterized bacterial strain through the observation of morphological and biochemical characters and analysis of 16S rRNA gene sequences. We determined the concentration of CB, other chlorobenzenes and released Cl-, densities of strain cells and activities of catechol dioxygenase in crude extracts from cells in pure culture liquid. [Results] We isolated a bacterium able to effectively degrade CB and assigned as strain CB001.Phylogenetic analysis based on 16S rRNA gene showed the similarity of 98.5% between strain CB001 and Acinetobacter calcoaceticus. In pure culture with CB as the sole carbon and energy source, 98.2% of CB at initial concentration of 50 mg/L was degraded; the molar ratio of the amount of net released Cl- to the amount of CB degraded by strain CB001 ranged from 1:1.85 to 1:1.39; the average activity of catechol 1,2-dioxygenase in crude extracts was 0.538 U/mg protein. The addition of glucose increased significantly the density of cells and the concentration of net released Cl-, but decreased obviously the ability of per cell to degrade CB. The capacity of strain CB001 to degrade CB was inhibited in di-chlorobenzenes and tri-chlorobenzenes coexisting culture system. The order in which strain CB001 readily degraded di-chlorobenzenes was 1,3-dichlorobenzene >1,2-dichlorobenzene >1,4-dichlorobenzene. [Conclusion] The results showed that strain CB001 belonged to the genus Acinetobacter, which showed capacity to degrade CB, di-chlorobenzenes. Strain CB001 possibly degraded CB through the pathway of meta ring cleavage. The addition of CB in the culture liquid increased significantly the ability of strain CB001 to degrade CB and the activity of catechol 1,2-dioxygenase.

Abstract:Abstract: [Objective] In this study, we generated knockout strains of rpn4, rpn7, and rpn10 genes respectively in Neurospora crassa, and studied their phenotypes to uncover their functions in the 26 S protesome pathway. [Methods]We used gene-replacement strategy to make knockout strains. After electrotransformation, the resulted transformants were examined by PCR. To get homokaryotic mutants, the heterokaryotic strains were crossed with the wild-type strain. The ascospores were germinated after heat shock, and the resulting strains were examined by PCR to confirm the homokaryotic strains. [Results] Homokaryotic rpn4 and rpn10 knockout strains, and heterokaryotic rpn7KO strains were obtained. [Conclusion] Compared to the wild-type strain, rpn7KO strains exhibited severe defects in hyphal growth and conidial formation; rpn4KO strain had defect in hyphal growth too, but displayed normal conidiation; the growth and developmental phenotypes of rpn10KO strain were slightly affected. Taken together, these data demonstrated that these three genes were important for the growth and developmental phenotypes of Neurospora crassa.

Abstract:Abstract: [Objective] We cloned a novel cry9Ea gene encoding a Cry9Ea protein with a high insecticidal activity against Trichoplusia ni. [Methods] We identified a cry9-type gene from Bt strain (Bt4) by PCR-RFLP. The full length cry9Ea gene was amplified by PCR with a pair of primers F9EA/R9EA. [Results] We cloned the complete cry9Ea7 gene into pET21b. The SDS-PAGE results showed that the 130 kDa Cry9Ea7 protein was expressed in E. coli BL21(DE3). We also constructed an engineering strain BioHD9Ea7 by transforming a shuttle vector pSXY422b containing the cry9Ea7 gene into Bt acrystalliferous mutant HD73-(cry-). The bioassay results indicated that the Cry9Ea7 protein was highly toxic against Trichoplusia ni neonates, and the LC50 value was 0.044 μg/mL. However, the Cry9Ea7 protein showed no activity against Spodoptera exigua and Helicoverpa armigera neonates. [Conclusion] The novel cry9Ea7 gene encoding Cry9Ea7 is highly toxic against Trichoplusia ni neonates.

Abstract:Abstract: [Objective] The purposes of this paper were to evaluate the adhesion ability of bifidobacteria in vitro and analysis its surface properties. [Methods] We selected Caco-2 cell as the adhesion model to determine the adhesion ability of 7 bifidobacterial strains in vitro. The autoaggregation ability and surface hydrophobicity were also assayed; moreover, the substance on the surface of bifidobacteria related to adhesion was characterized by treatments of different enzymes and chemicals, and surface proteins of bifidobacteria were analyzed by electrophoresis. [Results] The strain with high surface hydrophobicity and strong autoaggregation ability also showed higher adhesion ability than the strain with low surface hydrophobicity and weak autoaggergation. The correlation among the three properties was obvious. On the other hand, the adhesion of bifidobacteria was sensitive to proteinase and metaperiodate, and the adhesion ability of bifidobacteria decreased significantly after the surface protein extracted with LiCl. The result of SDS-PAGE showed that many proteins with different molecule weights were extracted from bifidobacteria by LiCl. [Conclusion] It could be concluded that bifidobacteria adhering to Caco-2 cell was strain-specific; surface hydrophobicity and autoaggregation ability of bifidobacteria exhibited positive correlation to the adhesion ability. On the other hand, glycoprotein might exist on the surface of bifidobacterial cells which could mediate the adhesion ability of bifodobacteria.

Abstract:Abstract: [Objective] To construct a prokaryotic protein expression vector of green fluorescent protein (GFP) with the non-sporulation promoter of cry3a gene of Bacillus thuringiensis (Bt), and to obtain recombined engineering bacteria with labled gfp gene and non-sporulation promoter of cry3a gene the construction of insecticidal engineering strains. [Methods] The promoter of gene cry3a and gfp gene was connected by SOE-PCR, The recombined gene pro3a-gfp was inserted into shuttle vector pHT304 at sites BamHⅠand SphⅠ Then plasmid pHT3AG was introduced into Brevibacillus brevis CQUBb and Bacillus thuringiensis CQUBt isolated from Apriona gemari larvae intestines. The expression of the recombinants was analyzed with fluorescent microscope, SDS-PAGE, and then the stability of engineering strains was tested under the conditions with or without antibiotics. [Results] Two engineering strains labeling with GFP were obtained. They expressed green fluorescent protein from the vegetative growth period and about 29kDa green fluorescent protein band was detected by SDS-PAGE. Heterogenous plasmid replicated and expressed stably in engineering strains and did not lead to significant adverse effects on these two host strains. After 30 generations, 95% and 67% of clones was still bearing recombined plasmids respectively. Compared to CQUBt, CQUBb had higher efficiency of electroporation, and the CQUBb-pHT3AG had longer GFP expression time, expressed more protein than CQUBt-pHT3AG, and the stability of CQUBb-pHT3AG was higher than CQUBt-pHT3AG. [Conclusions] We successfully obtained two transgenic insecticidal engineering strains.

Abstract:Abstract: [Objective] To investigate the structural distribution responsible for protein piezophilicity is of great significanceimportant for understanding the stability of piezophilic protein and would help to develop a practical strategy for designing piezophilic proteins. [Methods] We introduced a systematic comparative analysis of 43 pairs of piezophilic and non-piezophilic proteins. Three kinds of residue structural states such as external, intermediate and internal were considered for analyzing the structural patterns of single amino acids and amino acids in different groups. [Results] The statistical tests revealed that higher frequency in external state of Cys, Asp, Asn and Lys at the expense of Pro and Arg, higher frequency in intermediate state of Ile and Met at the expense of Trp, higher frequency in internal state of Cys and Ile at the expense of Ala, higher frequency in external and internal state of non-barophilic amino acids groups, lower frequency in external state of neutral and large amino acids groups could be critical factors related with protein piezophilicity. [Conclusion] The external state was the domain that had the most differences between piezophilic and non-piezophilic proteins, and it should be a hot spot for designing and engineering new piezophilic proteins. At the same time, the pressure asymmetry index should be revised based on more datasets.

Abstract:Abstract:[Objective]Bovine prochymosin is expressed by the nisin controlled gene expression system in Lactococcus lactis.[Methods]we amplified bovine prochymosin gene from pS19-PPC vector by PCR,and ligated the gene with expression vector pNZ8148.Ligated products were electrotransformated into Lactococcus lactis NZ9000.Then we identified transformants by restriction,PCR and sequencing, and molecular weight and immune characteristics of expression product by SDS-PAGE and Western blot after inducting using nisin.Eventually we tested milk-clotting activity after purification.[Results]Recombinant bovine prochymosin was not significant difference in molecular weight,immune characteristics, bioactivity and sensitivity of inhibitors comparison to nature bovine prochymosin.The milk-clotting activity of recombinant bovine prochymosin was 2×103 international milk-clotting unit per millilitre.[Conclusion]We expressed recombinant bovine prochymosin of milk-clotting activity in Lactococcus lactis. This study suggested potential application in cheese manufacture as a new method,which could regard Lactococcus lactis as starter of milk and host of expression bovine prochymosin.

Abstract:Abstract: [Objective] We screened a new lipase gene to asymmetricly hydrolyze the rac- ketoprofen Chloroethyl ester, to construct a secretive expression strain and to improve its enantioselectivity.[Method] Strain NK13 was screened for asymmetricly hydrolyzing the rac-ketoprofen Chloroethyl ester. We prepared NK13 chromosomal DNA library to screen the lipase gene. An inducible secretion vector (pHY300–plk- sacR-gene) was developed based on the sequence that was ligated by the lipase gene and the regulatory region and the signal sequence (sacR) of the SacB gene. Transforming Bacillus subtilis WB600 with this vector, we attained a new recombinant strain. The expression of the lipase gene was analyzed by SDS-PAGE. The lipase was purified through native PAGE. The ability of enantioselected Ketoprofen Chloroethyl ester of the recombinant was identified by thin-layer chromatography (TLC) and HPLC. [Results] A 633bp lipase gene with enantioselectivity was attained (GeneBank accession number: EU381317). SDS-PAGE analysis showed that the expression of the inserted lipase gene could be induced by addition of sucrose into the medium. The results of TLC and HPLC showed that the highest enantiomeric excess of (S)-Ketoprofen was 93.64% at 36 h, and the conversion rate was about 45% of the Bacillus subtilis recombinant strain by adding Tween-80. The enantiomeric excess of (S)-Ketoprofen of was 16 times of that of NK13. For the purifed lipase the highest enantiomeric excess was 60.22% at 40 h and the conversion rate was about 30%, which was same to the recombinant strain without Tween-80. [Conclusion] The lipase screened from NK13 had high ability to hydrolyze the rac-ketoprofen Chloroethyl ester to (S)-Ketoprofen. The 633 bp lipase gene of NK13 was induced to secretively express. By adding Tween-80, the enantioselectivity of the lipase was improved.

Abstract:Abstract: [Objective] Bacterial blight and blast are the most severe rice diseases. Xa21 confers resistance to bacterial blight, while Pi-d2 confers resistance to rice blast. Both Xa21 and Pi-d2 encode receptor-like kinase proteins. The aim of this study was to express kinase domain of XA21 and PI-D2 proteins in Pichia pastoris yeast system. [Methods] We amplified the coding regions for the kinase domains of Xa21 and Pi-d2 and constructed recombinant plasmids pPICZαA-Xa21K and pPICZαA-Pi-d2K, respectively. Restriction enzyme digestion and sequence verified plasmids were linearized and transformed into yeast strains. We compared the expression of recombinant proteins in 3 Pichia pastoris strains (KM71,GS115 and X33), with various methanol concentration (1%, 2% and 3%), pH (pH5, pH6, pH7 and pH8) and induction time (24 h, 48 h and 72 h). [Results] The recombinant kinase domain of XA21 and PI-D2 proteins were expressed in yeast, however, they were detected only in pellet of yeast cells but not in the supernatant of medium. This indicated that the recombinant proteins were not secretive. Comparison results revealed that Pichia pastoris strains (KM71 and X33), 2% methanol, pH5 and 48 h or longer induction time were optimal for the expression of the two rice kinase domain proteins. Finally, we purified soluble recombinant proteins and detected them by SDS-PAGE. [Conclusion] We obtained purified domain of XA21 and PI-D2 proteins from Pichia pastoris expression strain, which will facilitate the investigations of their biochemical properties.

Abstract:Abstract: [Objective] We isolated and characterized potassium-bearing mineral-solubilizing bacteria from the surfaces of weathered feldspar as well as soil samples. [Methods] We isolated cultivable mineral-solubilizing bacteria by plating and screening from the surfaces of different level of weathered feldspar as well as soil samples. We characterized bacteria regarding their ability of releasing Si from feldspar samples and further classified by amplified rDNA restriction analyses (ARDRA). Twelve typical bacterial strains were identified by 16S rDNA sequence analysis. [Results] Thirty-five Si-releasing bacteria were isolated from the surfaces of weathered feldspar and soil samples and classified by ARDRA in 11 different operational taxonomic units (OTU) at the similarity level of 60%, belonging to 5 major phylogenetic groups, 6 families, and 7 genera (Pantoea, Pseudomonas, Serratia, Agrobacterium, Sphingobacterium, Exiguobacterium, and Microbacterium). Pantoea, Pseudomonas and Serratia were the dominant groups. Inoculation with the isolates was found to increase the Si in the solution by 101~206% compared to the control. [Conclusions] Different mineral- solubilizing bacteria inhabited in different weathered K-feldspar surfaces and soils in the potassium mine. γ-Proteobacteria might play an important role in the process of K-feldspar weathering.

Abstract:Abstract: [Objective] Salmonella isolates isolated from retail meats in Shaanxi Province in 2007-2008 were characterized to determine their serotypes and genotypes. [Methods] Serotyping of 359 Salmonella was performed using slide agglutination method using Salmonella hyperimmune sera, whereas genomic DNA profiles were obtained using pulse field gel electrophoresis (PFGE) with XbaI. The data were analyzed and a dendrogram was generated using the BioNumerics Software. [Results] A total of 24 serotypes were identified among the 359 Salmonella isolates. The most common serotypes were Enteritidis, Typhimurium, Shubra, Indiana and Derby. Other serotypes were also identified, including Saintpaul, Rideau, Tennessee, Thompson, Galiema, Kallo, Ⅲa, Rissen and Brancaster. Enteritidis and Rideau were most common in chicken and beef, respectively, whereas Derby was most common in in pork and lamb. Seven PFGE clusters were observed among the 359 isolates with 88% similarity. Salmonella isolates with the same serotypes were assigned to the same clusters with only a few exceptions. [Conclusion] Salmonella present in retail meats were phenotypically and genotypically diverse. A database on serotyping and molecular subtyping of Salmonella will enable us better understand the epidemiology of foodborne salmonellosis and identify their source more rapidly and accurately.

Abstract:Abstract：[Objective] The rabbit’s intestinal tract model was constructed to analyze the different expression proteins of Bacillus anthracis in vitro and in vivo. [Methods] We used the rabbit’s intestinal tract model to culture the Bacillus anthracis, using different methods to extract the extracellular proteins, cell wall proteins and whole cellular proteins from the in vivo and in vitro samples. Those proteins were separated by two－dimensional gel electrophoresis and analyzed by mass spectrum. [Results] We had been distinguished about 144 differential expression protein spots between in vivo and in vitro samples, and 124 spots were identified, including 19 supernatant proteins, 29 cell wall proteins and 78 whole cellular proteins. [Conclusion] Those proteins which related to the anabolic metabolism were down-regulated in vivo, including threonyl－tRNA synthetase, prolyl-tRNA synthetase, long-chain-fatty-acid-CoA ligase. There were also many up-regulated proteins in vivo, such as DnaK, SodA and S-layer proteins.

Abstract:Abstract: [Objective]The objective is to improve the affinity of antibody against parathion with an aim to enhance the sensitivity of ELISA assay. [Methods] We recombined anti-parathion single-chain variable fragment gene (scfv) and the core streptavidin gene(sa) to produce an anti-parathion single-chain variable fragment-core of streptavidin-biotin fusion gene (scfv-sa), and inserted the fusion gene (scfv-sa) into the vector pET28a (+) in E.coli BL21 for transformation by prokaryotic expression. The expression was analyzed with SDS-PAGE and Western blot. the affinity of the recombinant protein was measured by ELISA after purified by metal affinity chromatography using Ni-NTA. [Results] The recombinant gene could express a fusion protein about 46 kDa, which formed a tetravalent domain, tetravalent antibody. ELISA results showed that the tetravalent antibody could bound to parathion specifically with the Affinity constant of 4.25 × 107 L / mol and titer of over 1:1×106. [Conclusion] The recombining anti-parathion tetravalent antibody could react with parathion specifically with significantly more binding sites with the monoclonal antibody, based on which the detection sensitivity of ELISA would be improved.

Abstract:Abstract：[Objective] The autolysis rate of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. To establish a rapid and sensitive method to detect autolysis rate of lactic acid bacteria, A Flow Cytometric Method (FCM) can be employed. [Methods] Cell suspensions were dyed 30 min by PI-PBS (20 mmol/L) in 4℃ dark environment. A 630 nm long pass filter was used to collect the red fluorescence (FL3 488 nm), and 1×105 cells were collected each sample. Data were analyzed by CellQuest online analysis system. Test time depended on different concentrations of cell suspensions, were about 40 s to 100 s. FCM enumerations were accurate down to a concentration of 104 cells/mL. [Results] Correlation test results reported that there were no significant difference between FCM test and other normal methods for the determination of Autolysis rate of lactic acid bacteria. [Conclusion] FCM by use of fluorescent probes propidium iodide (PI) is a good analytical method that allows the rapid and sensitive measurement of viability of lactic acid bacteria. This FCM method provides tools to assess the viability of different lactic acid bacteria in fermentation starters and probiotic products.

Abstract:Abstract: [Objective] Concentration of endomycorrhizae spores in Jatropha rhizosphere were detected er to obtain the distribution characteristics of endomycorrhizae, for effective technical support in seedling, cultivation and afforesttation of Jatropha. [Methods] Concentration of endomycorrhizae spores in Jatropha rhizosphere were studied by random sampling method in Renhe borough of Panzhihua City. Then 20 samples of soil and root mixture from 20 sites were collected to measure the concentration of endomycorrhizae under macroscope. We analyzed soil moisture and organic matter on the basis of The Forestry Industry Standard issued in 1999. [Results] Rhizosphere soil samples from 1025m to 1500m altitude in natural Jatropha contained abundant Arbuscular mycorrhizas (AM) fungi spore. The highest number of AM fungi spore was 236/g dry soil, the lowest was 9/g dry soil, the average was 80/g dry soil. Soil moisture samples correlated positively with the number of AM fungi spores. Similarly, organic matter of soil correlated positively with the number of AM fungi spores. [Conclusion] AM fungi spores could be detected in the rhizosphere soil of natural Jatropha, with high concentration and asymmetrical distribution. The spore concentration was inversely correlated with the altitude whereas positively correlated with moisture and organic matter in the soil.

Abstract:Abstract：【Objective】This study was to clarify pathogens and its sources of the disease called rotting edges syndrome at the auricularia stage in the larval culture of Apostichopus japonicus, and further to find out effective medicines for this disease.【Methods】Etiological analysis was performed on larvae with typical rotting edges syndrome from larvae culture farm. Suspicious pathogen was used for artificial infection test, and then identified through morphological, physiological/biochemical tests, and 16S rDNA sequence analysis. Bacterial analysis on the culture system of the factory, including water sources, feeds，rearing water, bottom ordure and water from cultivation pond for parent sea cucumber, was carried out. Finally, drug-sensitive tests were performed on the pathogens.【Results】A common dominant bacterium strain was isolated from all ill larvae included in the study. Artificial infection test showed it was the causative pathogen associated with the disease, and the artificially infected sea cucumbers had the same syndromes to the naturally ill ones. The bacterium was identified as V. lentus. Bacterial quantity of rearing water system were at a high range (2.8 × 102–8.8 × 107 cells/mL). The sources of the pathogen were complicated, since pathogens were discovered in the rearing water, bottom ordure and water from cultivation pond for parent sea cucumber. However, the density of causative bacteria was the highest in the bottom ordure, middle in the rearing water, and lowest in the water from cultivation pond for parent sea cucumber. Fifteen antibiotics could inhibit growth of the pathogens.【Conclusion】The possible pathogen for rotting edges syndrome was V. lentus. The bottom ordure, rearing water and parent sea cucumber may be the main infective origin of the pathogen. Fifteen antibiotics including neomycin, could be applied for disease prevention and treatment of Apostichopus japonicus.