Abstract:[Objective] To exploit resources of purple sulfur bacteria and their photosynthetic genes. [Methods] A purple sulfur bacterium strain 283-1 of okenone-containing, halophilic, high sulfide tolerance was isolated by agar dilution method in Pfennig medium from photolithoautotrophic enrichments of Dongfeng saltern, Qingdao, China. [Results] Cells of strain 283-1 were Gram–negative, halophile, straight or slightly curved rods, motile by monopolar single flagella, no gas vacuoles, carotenoid of okenone series and bacteriochlorophylla as photosynthetic pigment, purple red. It could photolithoautotrophically grow under anoxic condition in the light with sulfide as electron donor, sulfur globules accu-mulate as intermediate oxidation product and stored in the form of highly refractile globules inside the cells. The strain 283-1 belonged to Gammaproteobacteria, Chromatiales, Chroamtiaceae, genus of Marichromatium. Phylogenetic analysis based on 16S rRNA gene sequence also confirmed that strain 283-1 belonged to Marichromatium genus. However, the physiological characteristics of strain 283-1 were significantly different from four species of the genus Marichromatium. NaCl requirement range from 1% to 15%, good growth was observed at 7.5mmol· L-1NaS·9H2O, 45℃, 5000lux and pH9.0, a number of organic substances of C3 and C4 of TCA cycles and gluconate could be photoassililated in the presence of sulfide, no growth factors were required. [Conclusion] On the basis of 16S rRNA gene sequence analysis and its mor-phological and physiological characteristics, strain 283-1 is a new isolate of Marichromatium genus, named as Marichromatium sp. 283-1.

Abstract:[Objective] To identify Lactobacillus sp. strain S1 from the intestine of piglet, and compare the genomic difference between this strain and Lactobacillus sobrius 001T. [Methods] Analysis of 16S rRNA gene sequence and species-specific polymerase chain reaction assay were used to identify Lactobacillus sp. strain S1. Representational difference analysis (RDA) was used to compare these two strains. [Results] The 16S rRNA gene sequence analysis of strain S1 showed that its closest known species in the GenBank database was L. sobrius. Denaturing gradient gel electrophoresis analysis showed that the predominant bands in profiles from bacteria in the jejunum and ileum of piglets had the identical migrating position with strain S1. Cloning and sequencing of 16S rRNA gene analysis revealed that this band matched clone (Clone S) was also related closely to L. sobrius. Phylogenetic analysis of 16S rRNA gene showed that homology between strain S1 and Clone S was 99.8%, and that between strain S1 and L. sobrius was 99.6%. Both strains could be detected in 1.2% agarose gel by L. sobrius-specific PCR assay. Recently, RDA has been adapted to study the genomic diversity of bacterial strains. This analysis showed that there was no genomic difference between the two strains. [Conclusion] Piglet-derived strain S1 belonged to L. sobrius. Piglet-derived L. sobrius 001T and L. sobrius S1 were the similar strain.

Abstract:[Objective] To investigate the effect of the well-defined RNA elements (VR, RCS2, CS2, CS1 and SL) within the 3′untranslated region (UTR) on dengue virus (DEN) translation. [Methods] We constructed a virus induced reporter gene (VIRG) by inserting the firefly luciferase (LUC) gene between 5′- and 3′-UTRs of DEN2-43 genome. Subsequently, a series of modified VIRGs consisting of different RNA elements in the 3′UTR were constructed. A 3′UTR-deficient VIRG was also constructed. The translational efficiency of all VIRGs was then analyzed by LUC detection, real time RT-PCR and Western blot assays. [Results] The translation of 3′UTR-deficient VIRG was abolished. The translational efficiency of VIRG with RNA element VR was comparable with that of VIRG with unmodified 3′UTR. The translational efficiency of VIRG with RNA elements RCS2 or CS2 was significantly higher while the translational efficiency of VIRG with RNA elements CS1 or SL was substantially lower than that of VIRG with RNA element VR. [Conclusion] These results sug-gested that 3′UTR was indispensable for DEN translation, and some RNA elements within 3′UTR might either up-regulate or down- regulate translation.

Abstract:[Objective] Endophytic fungi can produce beneficial active components during symbiosis with host plants. We isolated a taxol-producing endophytic fungus strain from Podocrapus. [Methods] The anti-tumor activity of the endophytic funguswas detected by Methyl Thiazolyl Tetrazolium (MTT) method with Vero cells. The production of taxol by one fungus was confirmed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). This strain was classified by morphology together with similarity of internal transcribed spacer (ITS) sequence by Clustal W method. The deduced apoptosis of taxol produced from the strain was detected by fluorescent staining method with Vero cells. [Results] A total of 155 endophytic fungi were isolated from the tissue of Podocrapus. The result showed that 28 strains inhibited the growth of Vero cell (inhibitory ratio ≥ 10%), and 7 strains had high activity (inhibitory ratio ≥70%). The taxol-producing ability of strain A2 was confirmed by TLC and HPLC. Therefore, we recognized strain A2 as an endophytic fungus capable of producing taxol from Podocrapus-1 and named it EPTP-1. Its output of taxol was 0.56 mg/L when growing in liquid potato dextrose medium. EPTP-1 was classified as Aspergillus fumigates. Taxol extracted from strain EPTP-1 resulted in significant apoptosis of Vero cells at concentration of 5.553 mg/L for 24h. The activity of anti-Vero growth by extracts from strain EPTP-1 was similar to that of the purchased standard taxol (P>0.05). [Conclusion] The identified endophytic fungus, strain EPTP-1, can be a candidate for taxol production.

Abstract:[Objective] We used submerged fermentation to cultivate a strain of the entomopathogenic fungus Beauveria tenella isolated from the infected larvae of Dentrolimus tabulaeformis in Pinus tabulaeformis forest in Chengde of Hebei Province in China [Methods] We used ethyl acetate to extract antagonistic components from the fermentation broth and used silica gel column chromatography and GC/MS to separate and identify the components. [Results] Six compounds were obtained by silica gel column chromatography. The sixth compound had higher activity to kill the larvae of Den-trolimus tabulaeformis with a corrected mortality rate of 80%. Seventeen compounds were separated and identified by GC/MS in the 6th group, of which 3compounds were more than 10%, 2-Piperidinone(14.02%), 2-coumaranone(47.10%), and Pyrrolo[1,2-a]Pyrazine-1,4-dione, hexahydro (21.05%). [Conclusion] 2-Piperidinone and 2-coumaranone had insec-ticidal activity (corrected mortality rate reached 83.32% and 91.61% respectively) and were the most important toxic sub-stances to control pests.

Abstract:[Objective] Bacillus sphaericus is unable to use hexose and pentoses as the sole carbon source, due to the lack of key enzymes in Embden-Meyerhof-Parnas pathway (EMP), Hexose Monophophate Pathway (HMP) and Entner-Doudoroff (ED) pathway, such as phosphofructokinase (PFK). Based on the genome sequence annotation results of B. sphaericus C3-41, the phosphofructokinase gene pfk was verified with a single copy on chromosome, the aim of this research is to analysis the EMP pathway in B. sphaericus further, and confirm the function of phosphofructokinase. [Methods] The methods of southern-blot of pfk gene among different B. sphaericus strains, pfk ORF cloning from C3-41 and expressing in Escherichia coli, the corresponding sequence analysis and anlignment were used. [Results] The pfk ORF of B. sphaericus was composed of 960 bp nucleitides encoding a protein about 42 kDa, and the PFK sequence analysis showed it had the conservative amino acids-binding sites and an ATP-binding domain. The expression of pfk in recombinant E. coli strain could complement the PFK activity of a pfk mutated E. coli strain DF1020. [Conclusion] The expressed PFK had the conventional phosphofructokinase activity, and settled the foundation for the further research of catabolism of B. sphaericus.

Abstract:[Aim] Frataxin protein is a component of Fe-S clusters and closely related to metabolism of mitochondria. We identified an integrity mitochondrial protein frataxin gene (Nbfra), analyzed its phylogenetic relationship, and confirmed the transcriptase activity of Nbfra in N. bombycis. [Methods] We analyzed the sequence of the second structure, gene location in genome and construction of NJ phylogenetic tree through various bioinformatics software. We constructed recombinant vector pGEX-4T-1-Nbfra, expressed the 36.5kDa recombinant protein in E. coli BL21 (DE3), and then used the protein as antigen to produce its polyantibody in mice. [Results] Nbfra was lack of targeting signal into mitochondria and part of alpha helices in functional domain, and had a synteny character between N. bombycis and E. cuniculi. Phylogenetic trees of Nbfra suggested that the evolutionary position of microsporidia was closely related to that of higher eukaryote, rather than that of other protozoa. The result of western blot suggested the expression and transcription of Nbfra gene in N.bombycis. [Conclusions] Our results offered the new evidence to analysis the conservation of Nbfra and evolutionary position of N.bombycis, and would support the hypothesis of mitosome in microsporidia.

Abstract:[Objective] We investigated the intestinal microbial diversity in the larval gut of Hepialus gonggaensis, an economically important insect. [Methods] We used morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis method, and the molecular method of PCR-DGGE (denaturing gradient gel electrophoresis) analysis based on the sequence of 16S rRNA V3 region gene. [Results] By the traditional isolation method, 8 genera of bacteria were identified from 11 isolated bacterial populations. The dominant bacteria in intestine belonged to enterobacter. By 16S rRNA V3 region gene DGGE method, eleven distinct bands were obtained from 16S rDNA amplificons. The bands were purified, sequenced. The sequences aligned with GenBank database and showed that they were belonged to 8 different genera of bacteria. Phylogenetic analysis showed that the sequences of bacteria belonged to the Proteobacteria and Firmicutes. The most dominant bacteria group was Carnobacterium in the gut and Bacillus followed by it. The different patterns were observed in different instars larvae guts from DGGE profiles, which might be related to their physiological development stages. [Conclusion] 8 genera were obtained from intestine of H. gonggaensis by traditional culturing method and 16S rDNA analysis method respectively, but the two groups were not exactly same, and the dominant group was different also. This suggested that a combination of molecular and traditional culturing methods can be used to analyze and monitor the diversity of intestinal microflora effectively, and that will give us more information of microorganism diversity.

Abstract:[Objective] To identify and colonize an antagonistic bacterium, Lu10-1, isolated from the healthy mulberry.[Methods] Strain Lu10-1 was identified based on the analysis of its 16S rRNA gene sequence homology, the physiological and biochemical characteristics, and the recA gene sequence comparison. A spontaneous Lu10-1 mutant tolerant to rifampicin and ampicillin were isolated by gradually increasing the concentration of the two antibiotics. The mutants were used to assess the ability of Lu10-1 to colonize mulberry by different inoculation approaches, including stem and leaf acupuncturing, seed soaking, root soaking and leaf daubing. [Results] Lu10-1 belonged to Burkholderia. In the phylogenetic tree, Lu10-1 was the closest relative to B. cepacia (X80284) with more than 98% sequences similarity. The 16S rDNA sequences of Lu10-1 have been registered at GenBank database under the accession number EF546394. Moreover, our results also indicated that the population of strain Lu10-1 living in the mulberry tissues decreased as a whole after the treatment of seed soaking . The bacterial density inside the mulberry seedling tissues decreased to a steady level 20 days after germination. The population of strain Lu10-1 in mulberry leaves and stems after the treatment of root soaking increased first and then decreased. [Conclusion] The strain Lu10-1 fell into Burkholderia cepacia genomovarⅠas a single species. Furthermore, the strain Lu10-1 could colonize and transmit in mulberry, while its resistance to plant pathogen was not changed during the process of colonization compared to the original strains. Taken together, we suggest that Burkholderia. cepacia Lu10-1 will play an important role in the biological control of mulberry disease.

Abstract:[Objective] To screen antagonistic bacteria from rhizosphere of tobacco against Phytophthora parasitica var. nicotianae (Breda de Hann) Tucker and to analyze its antagonistic activity. [Methods] The antagonistic bacteria were screened by dual culture with P. parasitica var. nicotianae (Breda de Hann) Tucker on low iron (2μmol/L FeCl3) Sucrose-L-Asparagine (SA) plate. The chrome azurol S (CAS) assay was used to detect the siderophore production and its affinity to ferric ion. The strain was identified by using morphological, biochemical and physiological characteristics, 16S rRNA sequence homology, phylogenetics and specific species molecular analysis. The siderophore was isolated by column chromatography on Amberlite XAD-2 and analyzed by spectrophotometer. Antagonistic activity of the siderophore was confirmed by incubation of P. parasitica var. nicotianae (Breda de Hann) Tucker in liquid SA medium with different concentration of siderophore and FeCl3. [Results] We obtained an antagonistic bacterium G-229-21T against P. parasitica var. nicotianae (Breda de Hann) Tucker. The strain produced high-affinity siderophore under low iron conditions and was identified as Pseudomonas mediterranea. This strain produced carboxylate-type siderophore against P. parasitica var. nicotianae (Breda de Hann) Tucker. The suppression ratio was up to 92.3% under low iron conditions (0.16 mmol/L~10 mmol/L FeCl3). However, it was only 2.0% under Fe-replete conditions (100 mmol/L FeCl3). [Conclusion] P. mediterranea G-229-21T could produce high-affinity carboxylate-type siderophore against P. parasitica var. nicotianae (Breda de Hann) Tucker under low iron conditions.

Abstract:[Objective] The hemagglutinin-neuraminidase (HN) and fusion protein of Newcastle disease virus (NDV) plays a crucial role in the process of budding and infection. To understand the exact contribution of the HN gene to NDV pathogenecity, a reverse genetics system was developed using the lentogenic NDV LaSota strain . [Methods] the HN genes of an avirulent recombinant NDV strain (rLaSota) was replaced by an HN gene from Mukteswar mesogenic NDV strain by reverse genetics. Furthermore, the F gene of rL-MuHN was replaced with that of Mukteswar strain, resulting the double genes replaced chmeric virus, rL-MuFHN. [Results] Although the rescued chimeric virus (rL-MuHN) did not show significant increase in ability of hemadsorption, the intracerebral pathogenicity index test (ICPI=0) in chickens and mean death time for eggs (MDT≥90 h). rL-MuHN kept the low pathogenicity similar to its parent rLaSota strain. Compared to single gene replaced rL-MuHN, rL-MuFHN induced stronger cell fusion and showed a mild increase in ICPI (from 0 to 0.59) and no significant change in MDT (≥90 h). rL-MuFHN showed much lower pathogenicity than that of Mukteswar (ICPI.=1.32 and MDT=46,respectively). A HN gene exchange alone within the context of the NDV rLaSota backbone failed to increase virus virulence from unvelogenic to mesogenic pathotype. [Conclusion] These results indicated that the virulence of NDV is determined multigenically. The heterotypic HN and F pairs were not equally effective in virus pathogenicity. The HN gene derivated from mesogenic strain dose not alter the lentogenic property of NDV LaSota strain. NDV can be manipulated by gene replacement in the future for use as a vaccine candidate.

Abstract:[Objective and Method]: For rapid identification of Nesterenkonia species, one set of genus-specific primers was designed and synthesized for polymerase chain reaction based on the 16S rRNA gene sequences. [Results and Conclusion]: The genus specificity of these primers was validated with reference strains as well as with wild-type isolates. Partial sequencing results of 16S rRNA gene of the wild-type isolates confirmed that they are members of the genus Nesterenkonia.

Abstract:[Objective] To discriminate the strains of Bacillus subtilis group including B. subtilis, B. amyloliquefaciens, B. licheniformis, and B. pumilus, a rapid and accurate distinguishing method is essential for the identification of the target strains to ensure the quality and safety of microbial fertilizers. [Methods] By analyzing unique nucleotide sequences of the rpoA, gyrA and 16S rDNA genes, 4 pairs of species-specific primers were optimized and the multiplex PCR was de-veloped to discriminate and identify B. subtilis, B. amyloliquefaciens, B. licheniformis and B. pumilus. [Results] Thirty-three reference strains belonging to three genera of Bacillus, Paenibacillus and Brevibacillus were tested and the anticipated results appeared except for four species with cross amplification results with the primers of B. pumilus. How-ever, the four species can be easily discriminated by morphology characters. In addition, the multiplex-PCR results of 23 strains of B. subtilis group isolated from MF products were identical with the biochemical assay. [Conclusion] The newly constructed multiplex-PCR assay is species-specific and effective. This method can be used to detect and identify the strains of B.subtilis group from microbial fertilizers products.

Abstract:[Objective] To make a simpler and highly efficient replica plating tool to avoid the limitations of the traditional method. [Methods] A number of bamboo-toothpicks was sheaved to form a bundle with diameter close to the plate diameter. The sheaved bamboo-toothpicks were used for replica-plating. [Results] This replica plating tool was successfully used for the screening of Saccahromyces cerevisiae transformants. [Conclusion] Compared with the traditional tool, this tool was simpler and highly efficient.

Abstract:[Objective] To efficiently construct resistance gene-free Bacillius thuringiensis engineered strain that can stably express heterologous gene. [Methods] We amplified the trigger factor gene located in chromosome of XBU001 strain as homologous arms and constructed an integrative plasmid pKTF12 on the basis of plasmid pKSV7, a temperature sensitive plasmid. We also constructed a recombinant strain KCTF12 containing cry1Ac gene in its chromosome via the integrative plasmid pKTF12. [Results] Site-specific integration of cry1Ac into XBU001 chromosome did not affect its normal growth. The cry1Ac gene could stably express and form bipyramid crystals in KCTF12. When compared with HTX42 harboring a high-copy number plasmid, the recombinant strain KCTF12 has the merit of advanced sporulation and an increase in spore number. [Conclusion] The Site-specific integration proved to be a good approach to construct resistance gene-free Bacillius thuringiensis engineered strain that can stably express the heterologous gene.

Abstract:[Objective] We studied the differences in gene sequence of cellobiohydrolaseⅠgene (cbh1) from Penicillium decumbens 114-2 and its derepressed mutant JU-A10. [Methods] We cloned cbh1 and its full-length cDNA from Penicillium decumbens 114-2 by the modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and RT-PCR. [Results] The total length of cbh1 was 1500 bp. It contained 2 introns and encoded 453 amino acids (GenBank Accession No.EF397602). The upstream sequence (1.9 kb) of cbh1 gene was also cloned and sequenced. It contained two putative binding sites for the carbon catabolite repressor CREⅠand two putative binding sites for cellulases transcriptional regulator ACEⅠ. [Conclusion] The derepressed strain JU-A10 was a multiple mutant of the wild strain114-2. The mutant produced several times more cellobiohydrolase activity per ml of culture medium when compared with 114-2. The cbh1 gene sequence of the mutant was the same with the wild strain. While four single base mutations were detected on the upstream sequences (1.9 kb) of cbh1 gene.The result suggests that the evidently enhanced cellobiohydrolase activity of the mutant is not due to cbh1 protein-coded sequence . The true reason maybe refer to single base mutations of the upstream sequence that effect the transcription regulation of mutant JU-A10. As a result, the secretion of CBHⅠincreased.

Abstract:[Objective] To analyze the coordination function of cry2A sporulation-dependent promoter and the enhanced expression of molecular chaperone ORF1-ORF2 to crystal protein Cry11Aa. [Methods] We constructed three recombinant plasmids pHcy1, pHcy2 and pHcy4 containing cry11Aa gene. pHcy1 carried cry11Aa own promoter and p19 gene, and pHcy2 carried cry2A sporulation-dependent promoter and orf1-orf2 gene. pHcy4 inserted cry2A promoter and orf1-orf2 gene upstream pHcy1 plasmid. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of Bacillus thuringiensis sub sp. israelensis. We performed SDS-PAGE to analyze Cry11Aa protein expression in the recombinant Bt strains and carried out the mosquitocidal bioassay. [Results] SDS-PAGE showed that Cry11Aa protein was detected in 4Q7(pHcy1) and 4Q7(pHcy4), but not in 4Q7(pHcy2). The cry11Aa gene could not be regulated under cry2A promoter. Cry11Aa protein had a 1.25 fold expression amount in the equal volume culture of 4Q7(pHcy4) to that of 4Q7(pHcy1), which indicated that molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent. Both 4Q7(pHcy1) and 4Q7(pHcy4) formed Cry11Aa crystals in a similar size and shape during sporulation under the transmission electron microscope. Their LC50s against 3rd-instar Culex quinquefasciatus were 59.33 ng/mL and 66.21 ng/mL respectively. [Conclusion] Whether crystal protein from B. thuringiensis could successfully express might relate to the type of the used promoter and their coordination. Molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent with an unknown mechanism, but did not have an effect on high mosquitocidal toxicity of Cry11Aa protein. This research might play an important role to search the best collocation between ICP promoter or chaperone gene and ICP gene and to construct high-toxic Bacillus thuringiensis engineering strain by chaperone gene.

Abstract:[Objective] Xenorhabdus nematophila is an insect pathogen bacterium symbiotically associated with entomopathogenic nematode. The bacteria produce a number of toxins to overcome immune response of insect hosts and kill their hosts. We purified a novel haemocoel insecticidal protein from X. nematophila HB310, cloned and analysed gene sequence of this novel protein. [Methods] We isolated and purified the insecticidal protein by methods of salting out and native-PAGE from the intracellular proteins of X. nematophila HB310. We tested the virulence of the protein by direct injection into fifth-instar Galleria mellonella larvae. The protein was identified by western blotting. The gene of insecticidal protein was cloned by PCR and analyzed in GenBank. [Results] We purified a novel haemocoel insecticidal protein that was named as Tp40. The injectable hemocoelic potency (LD50) of Tp40 was 68.54ng/larva against fifth-instar G. mellonella larvae. The SDS-PAGE spectrum of Tp40 only showed a single ~ 42kDa band. Western blotting with an antibody that was highly specific to the known Txp40 indicated that Tp40 was homologous to the known Txp40 and only existed inside cells. The nucleotide sequences of tp40 gene have been deposited in GenBank (accession number GenBank: EU095326). The size of the open reading frame of tp40 was 1107bp, encoding a peptide of 368 amino acid residues, with a theoretical molecular weight 41.5 kDa and an isoelectricpoint (IP) 8.66. The Tp40 shared 85%-99% homology of nucleotide sequences and 70%-99% amino acids with those of 13 group strains. [Conclusion] Tp40 is high injectable virulent for G. mellonella larvae and its gene/protein sequence is highly conserved, which play a key role during the bacterium-nematode killing host insects process.

Abstract:[Objective] We characterized strain Rpp39 isolated from the soil of Sichuan, China. A type of cry2Aa gene was cloned and expressed in E. coli. [Methods] We characterized strain Rpp39 by scanning electron microscope, PCR-RFLP identification and SDS-PAGE analysis. We cloned cry2Aa gene by PCR clone. We constructed the recombinant plasmid pET-2Aa, and transformed E.coli.BL21(DE3) to express by IPTG. We determined the toxicity of expressed products to the larvae of Plutella xylostella and Chilo supperssalis through bioassay. [Results] The strain Rpp39 contained three types parasporal crystals, diamond, squareness and round, observed under the scanning electron microscope. SDS-PAGE analysis showed that two kinds of molecular mass of insecticidal crystal proteins, one was about 130kD and the other 60 kDa, were expressed in isolate Rpp39. The strain contained cry1Aa, cry1Ab, cry1Ac, cry1Ia and cry2Aa gene, analyzed by the PCR-RFLP method. One cry2Aa-type gene of Rpp39 was cloned and designated as cry2Aa12 by Bacillus thuringiensis Insecticidal Crystal Proteins Nomenclature Committee. Sequence analysis revealed that this gene contained an open reading frame of 1902 nucleotides encoding a protein of 634 amino acids. Compared with Cry2Aa1 protein, Cry2Aa12 protein has shown as high as 99.7% amino acid homology. We amplified the full open reading frame sequence of the cry2Aa12 gene by use of a pair of PCR primers P3/P4 designed according to its DNA sequence, and the PCR product was inserted into the Nde I / BamH I site of E. coli expression vector pET-30a to obtain the recombinant plasmid pET-2Aa. The result of SDS-PAGE proved that Cry2Aa12 could be expressed as 65 kDa protein in E. coli BL21(DE3) strain induced by IPTG. We found that Cry2Aa12 was highly toxic to the larvae of Plutella xylostella and Chilo supperssalis through bioassay, with LC50 as 5.4 mg/mL and 22.3 mg/mL, respectively. [Conclusion] Strain Rpp39 and cry2Aa12 gene cloned from strain Rpp39 were came from Sichuan ecological condition in China. They could be affluent in the resources of strain and gene. It has important significance to accumulate the resource.

Abstract:[Objective] To study the effect of Bt9875 crystal protein treated with proteinase K on human cancer cells, HL-60. [Methods] We used the methods of Thiazolyl Blue Tetrazolium Bromide, fluorescence microscopy examination, agarose gel electrophores and flow cytometry to detect the growth inhibition rate and apoptosis characters of the HL-60 cells that were treated with different concentration of Bt9875 crystal protein. [Results] Bt9875 crystal protein inhibited the growth of HL-60 cells evidently in a dose-dependent manner, with minimal effects on normal human peripheral blood mononuclear cells (PBMCs). The nuclei of HL-60 cells showed the characteristics of apoptosis. The analysis by flow cytometry indicated that the apoptosis rate of HL-60 cells was 52% after treatment with Bt9875 crystal protein (100 mg/mL). DNA analyzed by agarose gel electrophoresis showed "ladder" pattern. [Conclusion] Bt9875 crystal protein could inhibit the growth of HL-60 and induced its apoptosis, which provided a foundation for use of Bt9875 crystal protein to cure the acute myeloid leukemia.

Abstract:Microorganisms are an important component of polar ecosystems. Based on the unique features of geographical location, climate and environment of polar regions, the novelty and biodiversity of polar microorganisms have attracted an increasing attention for their significant values in both scientific research and resources exploitation. Bioprospecting and natural products research of polar microorganisms have become a hot field of microbiology. In this review, following the introduction of bioprospecting and culture collection of polar microorganisms in recent years, the research and development of microbial bioactive compounds aimed for pharmaceuticals and biological agriculture chemicals are summarized. In addition, factors that currently limit the research progress are discussed. It is believed that the resources exploitation of polar microorganisms can provide an opportunity for discovery of new natural medicines. Compared to human pharmaceuticals, the research and development of biological agriculture chemicals has more possibilities to achieve a breakthrough in a short period of time.

Abstract:The single-copied gyrB gene, encoding the subunit B of gyrase and distributing universally in all bacteria, has the average substitution rate of 0.7%~0.8% per million years. It’s proved that this region could be used to discriminate and identify the closely related species of different bacteria such as Pseudomonoas, Bacillus, Vibrio, Enterobacteriaceae, Mycobacteria, Aeromonas, Lactic acid bacteria et.al. It also can be used of quantitative or restriction fragment analysis of bacteria with the aid of species-specific primers or combined with DGGE(denaturing gradient gel electrophoresis). It really brings new promise for the identifying closest isolates or fingerprinting population due to overcoming the shortage of undistinguishing them acutely with the non-protein-encoding genes such as 16S rDNA or ITS(internal transcribed spacer) DNA. It’s an important new molecular marker for researches of closely related species and becoming an attractive topic in current microbial research world.