Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.

Recently, the genome of Spodoptera litura granulovirus (SlGV) which encodes 133 putative open reading frames (ORFs) was completely sequenced. In this study, to screen novel insecticidal genes of SlGV, we first constructed an advanced plasmid capture system, pPCS-TPI, which contains not only pUC19 ori and ampicillin resistance gene but also Autographa californica nucleopolyhedrovirus (AcMNPV) ORF603 and ORF1629 homologous region between Tn7L and Tn7R. In order to introduce genomic segments of SlGV into the genome of AcMNPV, genomic DNA of SlGV was digested with EcoRI and self-ligated. These self-ligated segments were in vitro transposed with the pPCS-TPI donor by the help of TnsABC* transposase. By this, 10 EcoRI-digested genomic segments of the SlGV were cloned, and these clones were co-transfected with the bApGOZA DNA into sf9 cells to generate corresponding recombinant virus, respectively. The resulting recombinant viruses harboring genomic segments of the SlGV could be used to investigate the insecticidal activity and/or other functions originated from the introduced genomic segments of the SlGV.

The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), a large circular double-stranded DNA virus whose genome encodes at least 155 open reading frames (ORFs), is highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. Although many genes of AcMNPV have been identified, the genome-wide study related to viral replication has not been well announced. In this study, to elucidate DNA replication cascade of AcMNPV, we firstly developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, ORF knock-out mutants were generated by random insertion into bAc-MK genome. These mutants will be suffered DNA microarray to elucidate AcMNPV replication cascade.

Plasmid capture systems (PCS) facilitate cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in invertebrate pathology, and we are extending its application to include vertebrate research.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, and mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with Polh gene at the N-terminus including an adaptor and enterokinase (EK) site between Polh and EGFP was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells according to three steps; cell harvest, sonication and EK digestion. Through the final enterokinase digestion, EGFP was presented mainly in the supernatant (93.3%) and the supernatant also showed a pure EGFP band. These results suggest that the combined procedure of Polh fusion expression and enterokinase digestion can used for the rapid and easy purification of other proteins.

A survey was conducted in the northern conifer forests of Korea from February to May in 2009 for the Monochamus species vectored Bursaphelenchus species. Dead pine trees infested with bark beetle larvaes were collected from Pinus densiflora and P. koraiensis located in the following target areas: Pocheon, Yeongi, Jincheon, Chuncheon, and Bonghwa. A cerambycid beetle, M. saltuarius was only found from sampled log piles in target areas. Bursaphelenchus species carried by M. saltuarius was only isolated in Pocheon, Gyeonggi province. The nematodes recovered from 17 (71%) of 24 beetles from P. koraiensis and 12 (75%) of 16 beetles from P. densiflora. The average number of nematode per adult was 1,974 and 11,823 from P. koraiensis and P. densiflora, respectively. The nematode was also recovered from the inner bark of both Pinus species infested with M. saltuarius larvae. Bursaphelenchus species identification was performed by morphological characters and molecular analysis with ITS-RFLP patterns and sequences of ITS and D2D3 region. Both morphological and molecular characters match well with the original description of the European type of B. mucronatus. This is the first report of M. saltuarius species as a vector of the European type of B. mucronatus on both P. densiflora and P. koraiensis in Korea.

Basic creep in concrete at an early age plays an important role in the mechancal properties of young concrete and many researches on this field have been carried out. According to recent some studies, however, it is the conclusion that for some cases the basic creep measured from the sealed concrete specimen shows inaccurate results. More specifically, for high-strength concrete (HSC), the incorrectness is more apparent due to autogenous shrinkage occurred at an early age. This paper shows the results obtained from experimental study to evaluate the effect of autogenous shrinkage on basic creep. In this study, four different mixture proportions of which primany test variable is water-cement ratio (w/c) were placed and autogenous shrinkage and basic creep tests on the specimen were performed with age and various loading levels. From this research, it was found that the differences between apparent creep and real creep were remarkable in HSC at an early age. Therefore, it is recommended to modify existing basic creep model equation by considering autogenous shrinkage.

From 2002 to 2007, two different systems (shrubs and grasses) were established to raise appropriate ruminants for the purpose of improving biological diversity and fragile ecological environment in the karst-areas through grazing. The objective was to find out a novel way to promote the application of ecological restoration and reconstruction, and the agricultural economy could develop in sustainable way in the karst areas in Guangxi and similar areas in Southwest China.

To explore the relationship of species diversity and aboveground productivity in grazing ecosystem is very important to manage grassland. We used the four years' data to check this relationship and to look how abiotic factor affect species diversity and aboveground productivity. We found a good linear relationship between species diversity and aboveground productivity in all previous grazing sites, while no any relationship was found in the no grazing site. From our results, we concluded that drought affects aboveground productivity more than grazing, while heavy grazing affects species diversity more than drought in Inner Mongolian steppe.

Whole plant com fertilized with or without nitrogenous fertilizer during its growth was harvested at milk matured stage for silages. The lactic acid bacteria inoculants Lactobacillus＞6×10¹? cfu/g, Pediococcus＞2×10¹? cfu/g) was used at 0.01 g/㎏ of fresh matter. Cellulase (CF) (concluding cellulase, acremmoocium and trichioderma) was used at 0.033 g/㎏ of fresh matter. And the mixture of lactobacillus and enzyme (LF+CF) were used respectively 0.01 g/㎏ and 0.033 g/㎏ of fresh matter. After 45 days, when the silages had been made, opened to air and sampling for the analysis of nitrate contents and nitrite contents at 0, 1, 3, 6 and 10 days. The results showed that, the nitrate content of fertilized com silages was higher (p＜0.05) than that of unfertilized com silages significantly at the beginning of opening the silages. As the day's extension, the nitrate content of all silages trended to decline. For the fertilized com silages, the nitrate content of untreated silages higher (p＜0.05) than that of treated silages and declined sharply throughout the process. This phenomenon appeared from 3 day point to 10 day point for treated silages. For unfertilized com silages, there was no significant difference between untreated silages and treated silages at 0 day point. The nitrate content of untreated silages sharply declined from 0 day to 10 day point, which of treated silages began at 3 day point. From above, we can conclude that nitrogenous fertilizers can affect the nitrate content of com silages, and adding LAB and enzyme can retard the nitrate transforming into nitrite.

In this paper, the effect of different land-use types on soil organic carbon (SOC) and Particulate Organic Carbon(POC) was studied by analyzing the soils in the meadow and conversing land of grassland to cropland in ecotones of agriculture and animal husbandry. It is shown t㏊t, compared with meadow, rotations of P/O and P/Cwill reduce SOC in 0-30㎝ soil layers by 47.54%-65.11% and 34.16-64.26% and POC by 16.90%-24.48% and 11.94%-20.29%, respectively. And SOC in meadow is more t㏊n in conversing land of grassland to cropland (P＜0.05). Conversing grassland to cropland in ecotones of agriculture and animal husbandry will decrease the contents of SOC and POC.

Biolog ECO plates (31 different carbon sources, Biolog Inc., Hayward, USA) were used to discriminate between rhizosphere soil samples from 4- and 9-year alfalfa stands each with three replication. The growth curves for different groups of carbon sources were nearly sigmoidal, but the maximum rate of utilization was faster for amino acids, carbohydrates and polymers t㏊n for amides, miscellaneous and carboxylic acids, and carbon sources utilization efficiency were all higher in 9-year t㏊n in 4-year alfalfa stand.

Storage and disposal of digested effiuent of manure (DEM) produced by biogas plant is a challenge to beef producers because of the mitigation of environmental pollution. The DEM contains essential plant nutrients and to use DEM as a soil amendment for crop production is a practical method to solve the disposal problem. In 2 years of cropping with dwarf napiergrass as a perennial summer grass and Italian ryegrass as an annual winter grass, DEM revealed to be so effective fertilizer as chemicals to increase dry matter yield and forage quality, especially crude protein (CP) content and in vitro dry matter digestibility (IVDMD). The efficient use of DEM can be an agronomic-and economic-viable management practice for sustainable crop production in Miyazaki, southern Kyushu of Japan.

Forage production and water use efficiency (WUE) of Lucerne were investigated in three varieties at different water availabilities. Forage production decreased with the severity of soil water availability. At 50% field water capacity (FWC), forage production dropped but in two varieties, Algonquin and Longdong, was still high and from 75%FWC to 50%FWC, forage production in Longdong decreased at the least rate. The greatest leaf WUE was observed in Longdong at all soil water availabilities. From 75%FWC to 50%FWC, it increased in Longdong and Xinjiangdaye, but decreased in Algonquin. With the severity of water deficit, δ¹³C value increased in all three varieties. At the same water availability, the greatest value was observed in Longdong. It suggested that moderate water stress can improve WUE in Lucerne. Longdong is to some extent more efficient in water use and may be more drought-tolerant with more steady production at moderate water deficit.

Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki K1 using PCS which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pK1S-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pK1S-1, ORF2 (MobK1) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8-25.2%) with the Rep protein coded by RCR plasmids, however. The putative double-strand origin of replication (dso) and single-strand origin of replication (sso) of pK1S-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pK1S-1, seven subclones were contructed in the B. t huringiensis ori-negative pHT1K vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (Rep1K), dso and ORF4, exhibited replication ability. These findings identified pK1S-1 as a new RCR group VII plasmid, and determined its replication region.

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. This strain exhibited dual insecticidal activity against Aedes aegypti as well as Plutella xylostella. Recently, we improved the donor-s of plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy), to construct E.coli-Bt shuttle vector. Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their range of sizes were approximately 10 kb. Based on the sequence analysis, they were classified in 4 groups showing similarity with 4 known plasmids, pGI1, pGI2, pGI3 and pBMB175, respectively. One of pGI3-like clones was fully sequenced and its open reading frames were analyzed. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily construct novel shuttle vectors with any Bt plasmids and this combined procedure can introduce foreign gengs into various circular DNA molecular.

Plasmid capture system (PCS) was developed for easy cloning and manipulation of circular double-stranded DNA from various sources. Recently, we improved PCS system (named PCS-LZ) to clone relatively large-sized DNA molecules (30-150 kb). PCS-LZ donor consists of a Mini-F replicon and a kanamycin resistance marker between Tn7L and Tn7R regions. Both replicon and marker gene of PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition and the transposed DNAs can replicate in E. coli cells by transformation. White/blue screening by LacZ expression is also available to avoid backgrounds. Up to now, we acquired various circular DNA clones from four sources such as plasmids of B. thuringiensis, bacteriophage genome isolated from B. thuringiensis, genome segments of Cotesia glomerata bracovirus, and polymorphic genomes of Autographa californica nucleopolyhedrovirus. Among them, interestingly, the genome clones of bacteriphage (Ph1-3) were screened from the PCS transposition with plasmids of B. thuringiensis 1-3 strain. The genome of Ph1-3 was fully sequenced (46517 bp) and open reading frames were analyzed. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Ph1-3 showed infectivity to B. thuringiensis type strains such as subsp. galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phage in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and their sequence similarity was more than 70%. Conclusively, we expect this PCS-LZ system would be a powerful tool for genomic analysis and mutagenesis study at the area of invertebrate pathology and further its application will be enlarged to the vertebrate pathology area.

We've collected litter-dwelling predatory arthropods (spiders and carabid beetles) by using pitfall traps at four different fire intensity treatment areas (control, ground fire, canopy fire, and canopy fire with plantation) at four different regional areas in 2005. We analyzed the arthropod community structures with PC-Ord for the difference of arthropod community among the various fire intensity treatments and control. Our objective was to verify if there was any difference between fired areas and non fired area (control) and between canopy fire with plantation and canopy fire without plantation. From our arthropod community structures, we found there was distinct community composition difference between fired areas and non-fired area as well as between control and canopy fire treatment with plantation or non-plantation. However, we are not sure yet that there was any distinct difference between control and ground fire treatments. Our results from the multivariate analysis, Non-parametric Multidimensional Scaling ordination, could be relatively conclude that the main difference of arthropod community between fired areas and non-fired areal and canopy fire with plantation or non-plantation was caused from the difference of arthropod habitat pattern such as litter depth, litter volumem, CWD cover or volume, and et. al. When we compared to control and other treatments, we could also conclude that the canopy fire treatment with non-plantation was relatively closed to control than the canopy fire treatment with plantation.