Bacillus thuringiensis 1-3 (Bt 1-3) which was isolated from a Korean soil sample showed high insecticidal activity against Aedes aegypti as well as Plutella xylostella. The isolate was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with cry gene specific primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A gene, differing from spp. aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D. We modified the plasmid capture system (PCS) to clone plasmid from Bt 1-3 through in vitro transposition. Fifty-three clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified according to 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 20 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.

Baculovirus chitinase gene (ChiA) is a late gene and is essential for liquefying host insect at the late stage of infection for its hydrolyzing chitin function. In previous report, baculovirus ChiA can offer many interseting new opportunities for pest control. Recently, a putative chitinase gene (ChiA) was identified in the Spodopter litura nucleopolyhedorvirus (SlMNPV-K1) genome. The open reading frame (ORF) contains 1,692 nucelotides (nt) and encodes a protein of 563 amino acids (aa) with a predicted molecular weight of 62.62 kDa. To conform the insecticidal activity of ChiA from SlMNPV-K1, we constructed a baculovirus transfer vector, pBac-SlChiA, and this transfer vector was co-transfected with the bApGOZA DNA into sf9 cell to generate corresponding recombinant viru which designed Ap-SlChiA. Western blot analysis indicate that SlMNPV-K1 ChiA was successfully expressed. We found the chitinase activity of recombinant virus was enhanced 53% than wide type AcMNPV by chitinase assay, and the recombinant virus showed higher evidently insecticidal activity against 3rd instar larvae of Spodotera exigua than wide type AcMNPV (4.5 time). These results suggested that the chitinase gene from SlMNPV-K1 could be successfully applied to improve pathogenicity of bauclovirus

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and comprises 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we 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, 56 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells to verify viral replication. Interestingly, both lef-1 and p48 knockout mutants showed normal viral replication in infected cells, which are reported to essential for viral replication. These results suggest that these single ORF-truncated mutants are useful for elucidation of viral replication cascade.

Through an application of plasmid capture system (PCS) to Bacillus thuringiensis plasmid DNAs, we acquired 21 polymorphic clones of putative genomic DNA of bacteriophage. The genome size of phage 1-3 (PhBT1-3) was determined to be 46,517 base pairs (bp) with 35.43% G + C content and 83% coding region. Sixty-five putative open reading frames (ORFs) with more than 50 codons were found in the new phage genome. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Morphological characterization and infectivity assay demonstrated that PhBT1-3 belongs to the family Siphoviridae and it showed infectivity to three B. thuringiensis type strains, galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phages in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and the similarity of those putative amino acids was more than 70%. Furthermore, we verified the existence of various shaped phages from the supernatants of 10 B. thuringiensis type cultures. In conclusion, we characterized a putative genome of phage, PhBT1-3 from B. thuringiensis 1-3, and confirmed the distribution of phages in the group of 67 B. thuringiensis type strains.

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.

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.

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.

Background : This study is designed to select the best shading materials for the 4-year old ginseng K-1 variety by examining the light volume, photosynthesis and growth characteristics of ginseng. Methods and Results : The ginseng seedling was transplanted in the 7 lines x 9 rows (63 plants)/ 1.65 ㎡. The shading materials were installed in the form of rear line with 170 ㎝ high for front pole and 100 ㎝ high for rear pole. The installation of shading materials were installed in combination of polyethylene, blue polyethylene sheet + polyethylene, polyethylene + polyethylene, polyethylene + aluminium screen 70%, polyethylene + aluminium screen 100%, aluminium shade plate to inspect the photosynthesis and ginseng's growth characteristics. The photosynthetic rate was measured on the middle of small leaf using the photosynthetic tester (L1-6400, Li-COR) a t 10 : 00 – 14 : 00 on a clear day with the light volume of 100, 200, 400, 600, 800, 1,000, 1,400, 1,800 μmol s-1m-2.For the ginseng K-1, which is 4-years old, the aerial parts growth (such as survival rate, height of plant, length of stem, diameter of steam and length of leaf) and underground part (length, diameter and weight of root) were examined in 2016. As for the PAR (photosynthetically active radiation), the aluminum shade plate showed the best performance while the polyethylene + polyethylene showed the least. As for the photosynthetic rate, the following order was shown in the decreasing order: blue polyethylene sheet + polyethylene > polyethylene > aluminium shade plate ≒ polyethylene + aluminium screen 100%, > polyethylene + polyethylene > polyethylene + aluminium screen 70%. As for the survival rate for 4-year old ginseng, the polyethylene + aluminium screen 70% showed the highest survival rate with 91.0%. As for the growth of aerial part of ginseng, the polyethylene and polyethylene + polyethylene showed the best growth. As for the underground part of ginseng, the polyethylene, blue polyethylene sheet + polyethylene, and polyethylene + polyethylene showed the best growth in the length of roots and length of main root while polyethylene and blue polyethylene sheet + polyethylene showed the best growth in the weight of roots. Conclusion : In conclusion, considering the characteristics of photosynthesis and the underground parts of ginseng, it was found that the best shading materials for K-1 4-year old ginseng is blue polyethylene sheet + polyethylene.

Background : The study is designed to select the organic fertilizer as an additional fertilizer suitable for improving the yield of ginseng cultivar K-1 at low productive land when it is cultivated as 6-year old red ginseng. Methods and Results : This study was conducted in 2016 at a farm in Paju cultivating 6-year old ginseng root. As for the management of the cultivation land, in 2010 and 2011, Sudangrass was cultivated in order to make the organic matter content and chemical composition suitable for ginseng cultivation, and more than 15 tillage works were carried out a year. The transplantation was carried out at the planting density of 63 weeks / 1.62 ㎡ in April 2012. On May 2, the amount of 15 ℓ / 1.62 ㎡ of N 1.5 ㎏/1,000 ㎡ was supplied to the non-treatment, liquid fertilizer of fermented rice bran, liquid fertilizer of fermented soybean, seaweed and liquid yeast. N 1.5 ㎏/1,000 ㎡ was put into the soil for the fermented soybean and leaf mold. The photosynthetic rate was measured on the middle part of the small leaf by using a photosynthetic analyzer (LI-6400, Li-COR) at 10 : 00 - 14 : 00 on a clear day under PAR (photosynthetically active radiation) of 100, 200, 400, 600, 800, 1,000, 1,400, 1,800 μmol s-1m-2. The growth features of ginseng (including the length of stem, length of leaf, width of leaf, length of root and yield, etc.) as well as leaf discoloration, and root rot, etc were examined. The statistical analysis was performed 3 times in the randomized block design. For the rate of photosynthesis, the liquid fertilizer of fermented rice bran > liquid fertilizer of fermented soybean ≒ untreated ≒ fermented soybean > seaweed > liquid yeast > leaf mold was higher in that order. The survival rate ranged from 42.9% to 51.9%. As for the above-ground growth characteristics, the liquid fertilizer of fermented rice bran, liquid fertilizer of fermented soybean, seaweed and liquid yeast showed higher performance than the non-treated while the fermented soybean and leaf mold showed similar performance to the non-treatment. As for the underground growth, the liquid fertilizer of fermented rice-bran, liquid fertilizer of fermented soybean, fermented soybean, or leaf mold showed the same performance as non-treatment while the fermented soybean or leaf mold showed shorter length than non-treatment. As for the yield, the liquid fertilizer of fermented rice-bran, or liquid fertilizer of fermented soybean showed better performance than non-treatment. Conclusion : In conclusion, it was found that liquid fertilizer of fermented rice bran and liquid fertilizer of fermented soybean are the suitable organic fertilizers which can be used in ginseng cultivation for improving the quantitative yield of 6-year-old ginseng in low-producing area.

Background : Ginseng rain cover farming is expanding around Jinan county and Jangsu country of North Jeolla Province. Some farmers doing ginseng rain cover farming have suffered from difficulties due to hot weather damages. However, it is a situation that the study on mitigation techniques for high temperature damage do not exist with ginseng rain cover farming. Methods and Results : The test covering work was firstly done on April 28th for heat block film+90% black light blocking net, blue double sided film, and PE film+75% black light blocking net and when it comes to second treatment, 30% and 40% shading were implemented for heat block film group and blue double sided film group respectively and 75% black light blocking net was installed on PE film+75% shading group. When it comes to micro-climate measurement in rain cover facility, temperature, humidity and light intensity were measured during the growing period of ginseng. The results are as follows. Regarding the light transmittance (per PAR, 10 am in clear day) in facility with 1st covering, light block film covered group (LBF), blue double sided film group (BDF) and PE film group have 12.9±1.8%, has 11.6±1.0% and 27.1±1.1% respectively and after 2nd covering, in LBF groups, 30% blocking, 40% blcoking and no blocking have 10.6±1.3%, 8.2±0.9% and 12.9±1.8% and in BDF groups, 30% blocking, 40% blcoking and no blocking have 9.4±0.8%, 7.9±0.7% and 11.6±1.0 respectively and PE film group has 10.6±0.7%. Relative humidity also showed the same trend as temperature. The average monthly amount of light and maximum light intensity were lower in 30% and 40% light blocking groups of LBF and BDF and a little higher in no light blocking group compared to PE film group. The degree of high temperature damage was 1 in no LBF of BDF, but no LBF of LBF was so bad like 3. However, there was no high temperature damage in the test groups of blocking films or BDF with 30% and 40% light blocked light screens. Regarding root weight, all secondly treated groups of LBF group and BDF group were lighter compared to 4.36g of PE film group and especially, prism sheetof no light blocking group has 2.5g and BDF of of no light blocking group has 3.21g. 30 % and 40% light blocking groups of LBF group and BDF group were light with 3.20~4.07g. Conclusion : Regarding the analysis result on micro-climate in facility with different cover materials for 2 years old ginseng in ginseng rain cover farming of Gyeonggi Province, the covering method suitable for high temperature damage mitigation in ginseng rain cover farming was that 1st covering was done by PE film+75% black light blocking net and then 75% black light blocking net is additionally covered at a time when outside temperatures reaches 30℃.

Background : The research is designed to investigate the optimal cultivation technology and the growth of above-ground and below-ground sections as well as the photosynthetic characteristics for new ginseng variety “K-1” by differentiating the planting density under the conditions of transplanting and direct seedling. Methods and Results : The K-1 variety and hybrid variety (Jakyungjong) were selected for the research and the ginseng varieties were transplanted and directly sown in Yeoncheon area in 2013. The transplanting was made in the form of 5 lines × 9 rows (45 plants), 6 lines x 9 rows (54 plants), 7 lines × 9 rows (63 plants) and 8 lines × 9 rows (72 plants) in each lot (1.65㎡) while the direct seedling for testing was conducted three times in randomly blocked design in the form of 11 lines × 14 rows (154 plants), 12 lines × 14 rows (168 plants), 13 lines × 14 rows (182 plants). Various measures were collected from the 4-year transplanted ginseng and 3-year direct seedling ginseng in 2015 to find out the growth features and photosynthesis of above-ground section (rate of germination, leaf length, leaf width, stem length and leaf area index (LAI)) and the below-ground section (length, diameter, weight and class of roots). Conclusion : After the planting of the ginseng, the germination rate of K-1 for the transplanting was 85.1 ~ 92.0% across different plantation densities while that for the direct seedling was 67.7% ~ 77.9% across plantation densities, thus showing no significant difference between the two planting methods. LAI was higher for the higher planation density for both transplanting and direct seedling. As for the photosynthesis speed, the form of 6 lines × 9 rows showed the higher speed in transplanting while the form of 12 lines x 14 rows showed the higher speed in direct seedling. The photosynthesis of K-1 was higher than that of Jakyungjong. In the 4-year ginseng cultivated under the transplanting, diameter of roots, number of branch roots and weight of raw ginseng were the highest in the plantation density of 5 lines × 9 rows. The distribution of root weight was high with 23.3% and 20.0% for the 51~70g group and the 71g or above group, respectively, for the 4 year transplanted plants in the form of 5 lines × 9 rows. The growth for above-ground and below-ground sections for K-1 was better than that for Jakyungjong. As a result, it was found that the proper plantation density for the 4-year root in the transplanted K-1 was 5 lines × 9 rows considering the growth of the above-ground section, quantity and distribution of root weight.

‘Namcheondeulkkae’ was developed as a new vegetable perilla variety using the pure line selection from Milyang local strains at the National Yeongnam Agricultural Experiment Station (NYAES), Rural Development Administration in 1998. The advanced and regio