• Proceedings of the Korean Society of Crop Science Conference
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• 2019.09a
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• pp.165-165
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• 2019

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.419-424
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• 1998

The present study was carried out to examine the efficiency of cloning of transgenic embryos by nuclear transfer(NT) using gene-injected rabbit embryos. The rabbit embryos at pronuclear stage were microinjected with methallothionein-human growth hormone(MT-hGH) gene and cultured to 8- and 16-cell in TCM-199 containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% $CO_2$incubator. The recipient oocytes were collected from the oviducts 14~16 h after hCG injection. The oocytes were enucleated and activated with 5$\mu$M ionomycin and 2mM 6-dimethylaminopurine. Blastomeres form gene-injected embryos were transferred into the enucleated oocytes by micromanipulation. The nuclear transplant oocytes were electrofused and co-cultured with rabbit oviductal cells. Following 120 h of culture, blastocysts were prepared for gene analysis by polymerase chain reaction(PCR). In previous experiment, the rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased while developing to blastocyst(25%)(Kang et al., 1998). The fusion rate of gene-injected blastomeres was significantly(P＜0.05) lower than non-injected blastomeres(66% vs 80%). However, the NT embryos that were derived from gene-injected donor embryos did not differ from control embryos in development to the blastocyst stage(39% vs 31%). Of the 43 NT blastocysts developed from the gene-injected donor embryos, twelve(28%) were positive for the injected DNA. The results indicate that NT with gene-injected embryos can be successfully used for cloning and multiplication of transgenic embryos, furthermore applicable to improvement of transgenic animal production.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.311-317
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• 2006

Using PCR amplification, we cloned a cellulase gene (ce/H) from the Bacillus subtilis AH18 which has plant growth-promoting activity and antagonistic ability against pepper blight caused by Phytophthora capsici. The 1.6 kb PCR fragment contained the full sequence of the cellulase gene and the 1,582 bp gene deduced a 508 amino acid sequence. Similarity search in protein database revealed that the cellulase of B. subtilis AH18 was more than 98% homologous in the amino acid sequence to those of several major Bacillus spp. The ce/H was expressed in E. coli under an IPTG inducible lac promoter on the vector, had apparent molecular weight of about 55 kDa upon CMC-SDS-PAGE analysis. Partially purified cellulase had not only cellulolytic activity toward carboxymethyl-cellulose (CMC) but also insoluble cellulose, such as Avicel and filter paper (Whatman No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. The optimum pH and temperature of the ce/H coded cellulase were determined to be pH 5.0 and $50^{\circ}C$. The enzyme activity was activated by $AgNO_3$ or $CoCl_2$. However its activity was Inhibited by $HgC1_2$. The enzyme activity was activated by hydroxy urea or sodium azide and inhibited by CDTA or EDTA. The results indicate that the cellulase gene, ce/H is an antifungal mechanism of B. subtilis AH18 against phytophthora blight disease in red-pepper.

• Polymer Science and Technology
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• v.23 no.3
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• pp.289-295
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• 2012

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.2
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• pp.113-120
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• 2009

Embryonic lethal abnormal visual (elav) is a lethal gene in Drosophila inducing the abnormal development and function of nervous system. We cloned a Bm-elav gene by bioinformatics and biological experiment, based on sequence of ELAV protein and dbEST of Bombyx mori. The full-length of Bm-elav cDNA is 1498 bp, contains a 906 bp open read frame (ORF) encoding a precursor of 301 amino acid residues with a calculated molecular weight of 34 kDa and pI of 8.99. Bm-ELAV protein precursor contains three RNA recognition motifs (RRM) in $24{\sim}91$, $110{\sim}177$ and $222{\sim}295$ bit amino acid residues respectively, and belongs to RNA-binding protein family. Bm-ELAV shared varying positives, ranging from 56% to 60% (Identities from 41% to 45%), with RRM from other species of Xenopus tropicalis, Apis mellifera, Tribolium castaneum, Branchiostoma belcheri and Drosophila. Gene localization indicated that Bm-elav is a single-copy gene, gene mapping within 12-chromosome from 7916.68 knt to 7918.16 knt region of nscaf2993. Spatiotemporal expressions pattern analysis revealed that Bm-elav expressed higher in most tested tissues and developmental stages in whole generation, such as silk gland, fat body, midgut, hemopoietic organ and ovary, but almost no expression in terminated diapause eggs. This suggested that the expression of Bm-elav in early developmental embryonic stages might induce abnormal development like in Drosophila. Cloning of the Bm-elav gene enables us to test its potential role in controlling pests by transferring the gene into field lepidopteran insects in the future.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1673-1679
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• 2008

In order to study the biological function of gapdh gene in yak, and prove whether the gapdh gene was a useful intra-reference gene that can be given an important role in molecular biology research of yak, the cDNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase from yak was cloned by the RT-PCR method using gene specific PCR primers. The sequence results indicated that the cloned cDNA fragment (1,008 bp) contained a 1,002 bp open reading frame, encoding 333 amino acids (AAs) with a molecular mass of 35.753 kDa. The deduced amino acids sequence showed a high level of sequence identity to Bos Taurus (99.70%), Xenopus laevis (94.29%), Homo sapiens (97.01%), Mus musculus (97.90%) and Sus scrofa (98.20%). The expression of yak's gapdh gene in heart, spleen, kidney and brain tissues was also detected; the results showed that the gapdh gene was expressed in all these tissues. Further analysis of yak GAPDH amino acid sequence implied that it contained a complete glyceraldehyde-3-phosphate dehydrogenase active site (ASCTTNCL) which ranged from 148 to 155 amino acid residues. It also contained two conserved domains, a NAD binding domain in its N-terminal and a complete catalytic domain of sugar transport in its C-terminal. The phylogenetic analysis showed that yak and Bos taurus were the closest species. The prediction of secondary structures indicated that GAPDH of yak had a similar secondary structure to other isolated GAPDH. The results of this study suggested that the gapdh gene of yak was similar to other species and could be used as the intra-reference to analyze the expression of other genes in yak.

• Korean Journal Plant Pathology
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• v.10 no.3
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• pp.157-162
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• 1994

Erwinia rhapontici causes soft-rot disease in a number of plants such as onion, garlic and hyacinth. There has been no report that E. rhapontici produces pectate lyase. Pel gene was cloned from genomic DNA of the parasitic soft-rot E. rhapontici polymerase chain reaction by using synthetic oligonulceotide primers designed from the pel 1 to E. carotovora. The recombinant plasmid pJE101 containing pectate lyase gene, when introduced into E. coli DH5$\alpha$, produced pectate lyase an macerated hyacinth tissue.

• Proceedings of the Ginseng society Conference
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• 2004.12a
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• pp.50-52
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• 2004

Introduce of gene connected with disease and transformation system of ginseng, Squalene systhase(PSS) gene cloned from and disease resistant gene were carried out for expression and transformation of plant using Agrobacterium. PSS of 35S-35S-AMV-PSS-Tnos, has been constructed which were mobilized into Agrobacterium tumefaciens strain MP 90 disarmed Ti-plasmid. PSS gene were introduced into the binary vector pRD 400. The transgenic ginseg plants were propagated using repetitive secondary embryogenesis and introduced NPTII and PSS genes of the transgenic ginseng were successfully indentified by the PCR and survival test on the medium.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.619-624
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• 1992

Tn5 lac 삽입으로 채소입고병원균에 길항력이 약화된 T-67 및 고추역병균과 참깨역병균에 길항력이 약화된 T-81의 Tn5 lac 유전자 일부와 오른쪽 말단에 있는 길항관련 유전자의 flanking sequence가 cloning된 pAG67 및 pAG81 clone을 선발하였고, pAG67 및 pAG81 clone된 길항관련 유전자의 flanking sequence를 야생 길항균 Pseudomonas maltophilia B-14의 DNA를 probe로 사용하여 Southern hybridization으로 확인하였으며, 제한효소 지도를 작성하여 8Kb 및 4Kb 크기의 flanking sequence가 cloning되었음을 확인하였다. pAG6 및 pAG81의 flanking sequence를 EcoRi-BglII와 EcoRI-MpaI으로 분리하여 유전자 은행으로부터 길항관련 유전자가 cloning된 cosmid clone 7개주를 선발하였다.

• Genomics & Informatics
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• v.4 no.2
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• pp.80-86
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• 2006

It is clear that the construction of large insert DNA libraries is important for map-based gene cloning, the assembly of physical maps, and simple screening for specific genomic sequences. The bacterial artificial chromosome (BAC) system is likely to be an important tool for map-based cloning of genes since BAC libraries can be constructed simply and analyzed more efficiently than yeast artificial chromosome (YAC) libraries. BACs have significantly expanded the size of fragments from eukaryotic genomes that can be cloned in Escherichia coli as plasmid molecules. To facilitate the isolation of molecular-biologically important genes in Ashbya gossypii, we constructed Ashbya chromosome-specific BAC libraries using pBeloBAC11 and pBACwich vectors with an average insert size of 100 kb, which is equivalent to 19.8X genomic coverage. pBACwich was developed to streamline map-based cloning by providing a tool to integrate large DNA fragments into specific sites in chromosomes. These chromosome-specific libraries have provided a useful tool for the further characterization of the Ashbya genome including positional cloning and genome sequencing.