The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.

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.

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.

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.

Bacillus thuringiensis 1-3 (Bt 1-3), isolated from Korean soil sample, showed high insecticidal activity against Plutella xylostella. Recently, we improved plasmid capture system donor-s (PCS-S) by inserting attB sites including lacZ between transposable elements (designated as pTroy), to reduce background and construct E. coli-Bt shuttle vector. Through in vitro transposition with total plasmid DNA of Bt 1-3, at least 6 different size plasmids of Bt 1-3 were cloned. Among them, 47 clones which have approximately 10 kb plasmid in size were sequenced and 5 contigs were assembled. These contigs showed partial similarity with two known plasmids, pGI3 or pBMB175, separately. These cloned plasmids will acquire erythromycin resistance by BP recombination reaction with pDonrattPEm vector. After transformation into Bt cells, final erythromycin resistant Bt cell might contain novel E. coli-Bt shuttle vector. This scheme proposes that pTroy and pDonr-attPEm system can easily construct new shuttle vector by in vitro transposition, BP reaction, and erythromycin selection with any Bt plasmids.

A strain of Bacillus thuringiensis, named Bt 1-3, was isolated from Korean soil sample and it showed high insecticidal activity against Plutella xylostella. Bt 1-3 was deterimined to belong to ssp. aizawai (H7) by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins. PCR analysis with specific cry gene primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2Ab genes. In addition, this isolate showed high uptake rate of foreign plasmid by electroporation. Based on these characteristics of Bt 1-3, we tried to construct a spore-free Bt 1-3 mutant by knock-out sigG gene, which is known as a key transcription factor during sporulation. First, we constructed a basal vector, named pDST, consisting of erythromycin resistant gene (EmR), partial polyhedrin gene and temperature sensitive origin of replication gene (Orits). Subsequently, according to the chromosomal DNA sequence of Bt subsp. konkukian 97-27, we amplifed upstream and downstream regions of Bt 1-3 sigG, and cloned into pDST (pDST-G). So far, several EmR colonies were obtained by electroporating into the wildtype Bt 1-3 and crossover by homologous recombination is going on.

The signing and implementation of RCEP mark the birth of a highly promising free trade zone. This agreement not only promotes the liberalization of regional trade and investment, creating an integrated regional market but also drives regional economic growth, supporting the stability and prosperity of the East Asian economic sphere. Simultaneously, it contributes to global economic recovery.The implementation of RCEP brings new development opportunities to China’s manufacturing industry but also imposes higher standards, stricter rules, and intensified competition. Chinese manufacturing will face more challenges. This article analyzes the opportunities and challenges brought by RCEP to China and proposes policy suggestions to promote the development of China’s manufacturing industry, including strengthening cooperation with RCEP countries, enhancing technological innovation capabilities, and prioritizing the development of the workforce.

The RCEP rules are the basic guarantee for the settlement of international trade competition disputes. Only by establishing fair competition in relevant markets can the objectives of the Free trade Agreement be fully realized. Therefore, we should study the RCEP rules and summarize the shortcomings of the RCEP rules. In view of these deficiencies, a scientific and reasonable solution to the RCEP international competition dispute settlement rules will be found to solve the practical difficulties existing in the relevant rules of international competition dispute settlement, so as to promote the better realization of the objectives and purposes of international competition dispute settlement, and ultimately provide a fair market environment for further promoting the development of foreign trade of all countries.

This study investigated in vitro antioxidant, anti-inflammatory and cytotoxicity on human lung epithelial A549 cells of different solvent extracts from Jerusalem artichoke (Helianthus tuberosus L.) tuber. The EtOH extract contained amounts of phenolics (22.20 tannic acid equivalent ㎎/ɡ) and exhibited the highest antioxidant activity and anti-inflammatory activity. Several methods were employed for measure the antioxidant activity: 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (IC50 = 206.79 ㎍/㎖), reducing power activity (21.26 ascorbic acid equivalent ㎎/ɡ) and total antioxidant activity (19.05 ascorbic acid equivalent ㎎/ɡ). Meantime, the EtOH extract inhibited the NO production completely with a concentration of 800 ㎍/㎖. Besides, the H2O extract exhibited more potent effect on human lung epithelial A549 cells. This study suggested that Jerusalem artichoke tuber had antioxidant, anti-inflammatory and cytotoxicity on human lung epithelial A549 cells.