최근 Flammulina 종들에 대한 유전체 염기서열 분석 결과가 보고되었고, 그로 인해 다양한 유전자 정보가 밝혀지고 있다. 본 연구에서는 Flammulina elastica 전체 유전체 서열의 laccase 유전자를 동정하고 구조적 특징 분석을 수행하고자 하였다. 유전체 분석 및 생물정보분석을 통하여 F. elastica 유전체 내 3개의 laccase 유전자(Felac1, Fe-lac2, Fe-lac3)를 확인하였고, 이들 유전자 내에는 10개의 히스티딘 잔기와 1개의 시스테인 잔기를 가지는 구리 이온 결합 영역과 4개의 시스테인 잔기를 가지는 이황화결합 형성 부위가 존재하는 것을 확인하였다. 1,548~1,602 bp의 laccase 유전자에 대한 전장 cDNA 염기 서열 분석을 통하여 12~16개의 인트론이 존재하는 것을 확인되었으며, N-말단으로부터 17~22 bp의 사이에 신호 펩타이드가 존재하는 것이 확인되었다. 본 연구를 통하여 F. elastica의 laccase 유전자를 최초로 동정하여 구조적 특징을 분석하였고, 이러한 결과는 F. elastica의 바이오매스 분해에 대한 이해를 돕는데 활용될 것으로 사료된다.

본 연구는 옥수수 재배 시 환경에 영향을 미치는 노균병 저항성과 관련된 유전자 후보군을 탐색해서 노균병으로 인한 토양오염과 옥수수 생산량 감소를 해결하기 위하여 노균병 저항성 품종을 효율적으로 발굴하기 위한 연구이다. 옥수수의 6번 염색체의 152,892,333과 154,335,437 사이에 있는 노균병 저항성 유전자를 탐색하였으며 이 부분에 존재할 것으로 예상되는 전사체에서 38개의 프라이머 세트를 디자인하여 이 중 16개의 예측 전사체를 가려 내었다. 또한 RT-PCR을 수행하여 감염된 Ki11의 발현이 높은 7개의 전사체로 5개의 품종에 대하여 건강한 샘플과 감염된 샘플을 검정하였고 최종 5개의 후보 유전자군[알려지지 않은 미확인 유전자 2개, OFP transcription factor, bZIP transcription factor, pentatricopeptide repeat (Ppr)]이 발견 되었다. 본 연구의 결과로 추가적인 실험 설계를 통해 5개의 후보 유전자군에 대한 재검정을 통하여 확실한 노균병 저항성 유전자를 발굴하고 이를 노균병 저항성 품종 개발 및 방재에 이용할 수 있을 것으로 사료된다.

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges. fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylationsites(Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1(Asn-454), fvLac-2(Asn- 437andAsn-455), fvLac-3(Asn-111andAsn-237), and fvLac4 (Asn-402andAsn-457). In addition, the first 19–20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction(RT-PCR) analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes.

Recently, Bacillus thuringiensis (Bt) cry genes encoding insecticidal Cry proteins have been widely applied for the construction of transgenic crops resistant to insect pests. This study aimed to construct novel mutant cry1Ac genes for genetically modified crops with enhanced insecticidal activities. Using multi-site directed mutagenesis, 34 mutant cry1Ac genes were synthesized and converted at 24 amino acid residues, located on domain I (8 residues) and domain II (16 residues). These mutant genes were expressed as a fusion protein with polyhedrin using the baculovirus expression system. The expressed proteins were occluded into polyhedra and activated stably to 65 kDa by trypsin. Among these, Mut-N04, N06, and N16 showed high levels of insecticidal activites against larvae of Plutella xylostella, Spodoptera exigua, and Ostrinia furnacalis. Mut-N16, which showed the highest insecticidal activity, is expected to be a desirable cry gene for introduction into transgenic crops. This study could provide useful means to construct mutant cry genes with improved insecticidal activities and expanded host spectrum for transgenic crops.

Crystals of proteinaceous insecticidal proteins, Cry proteins, produced by Bacill us thuringiensis (Bt) have been generally used used to control insect pests. In this st udy, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Plutella xylostella, Spodopt era exigua and Ostrinia furnacalis were identified. To construct novel cry genes wi th enhanced insecticidal activity, we randomly mutated these 24 amino acid sequen ces by in vitro muti site-directed mutagenesis, resulting in totally 34 mutant cry gen es. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded in to polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activit ies of these mutant Cry proteins against to larvae of P. xylostella, S. exigua, and O. furnacalis were assayed, they showed higher or similar insecticidal activity compar ed to those of Cry1Ac and Cry1C. Especially, among them Mutant-N16 showed th e highest insecticidal activity against to both of P. xylostella, S. exigua and Ostrinia furnacalis. Therefore, Mutant-N16 is estimated to have the potential for the efficac ious bioagent.

Crystals of proteinaceous insecticidal proteins, Cry proteins, produced by Bacillus thuringiensis (Bt) have been generally used to control insect pests. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. To construct novel cry genes with enhanced insecticidal activity, we randomly mutated these 23 amino acid sequences by in vitro muti site-directed mutagenesis, resulting in totally 24 mutant cry genes. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella and S. exigua were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, among them Mutant-N16 showed the highest insecticidal activity against to both of P. xylostella and S. exigua. Therefore, Mutant-N16 is considered to have the potential for the efficacious biological insecticide.

Varieties of Bacillus thuringiensis (Bt) crystal proteins, Cry proteins, have so far been found as one of the most successful biological control agents which are safe to natural environments for a long time. Recently, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. To construct novel cry genes with improved insecticidal activity, we randomly mutated these 23 amino acid sequences by in vitro muti site-directed mutagenesis, resulting in totally 24 mutant cry genes. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. In the further study, we plan to investigate their insecticidal activity against Plutella xylostella, S. exigua and O. furnacalis larvae.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.

In order to unravel unidentified genes from human salivary gland, a cDNA library of human submandibular gland was constructed in the Uni‐ZAP XR vector by use of mRNA from human submandibular gland and ZAP‐cDNA® Gigapack® III Gold Cloning Kit. cDNA of salivary gland was subtracted with cDNA of immortalized human keratinocyte cell line, Rhim Human Epithelial Keratinocyte cell line. The phage cDNA library was converted into a pBluescript phagemid cDNA library, which was subsequently plated on LB plates with ampicillin, IPTG, and X‐gal, and white colonies were selected for sequencing. Among 200 clones analyzed, four clones containing C77‐091, C75‐014, C76‐022, and C76‐012 designated orphan genes that are intensely expressed in the interlobular ductal and serous acinar cells of human submandibular gland. Particularly C77‐091 gene expresses 46 amino acids peptide (pI=9.45). C75‐014 and C76‐022 genes were characterized as those expressing excretory basic proteins primarily consist of alanine, proline, and leucine residues, mimicking a basic proline‐rich protein (bPRP) showing helical structures and having multiple consensus sequences of phosphorylation sites. The strong expression of C76‐012 mRNA in the nuclei of salivary ductal and acinar cells suggests a role of C76‐012 gene as a DNA binding RNA/protein. These data suggest that the identification of four orphan genes from the human salivary glands may add further understanding of greater role of salivary proteins providing innate immunity by protecting and stabilizing the mucosal epithelium in the maintaining homeostasis of oral mucosa.

In order to obtain novel genes related to the human craniofacial development, molecular cloning and sequencing, and in situ hybridization using craniofacial tissue sections were performed and followed by protein structure simulation. Totally 231 clones were obtained from the subtracted craniofacial tissue cDNA library of human embryo. Random cloning using the non-redundant clones from the craniofacial tissue of human embryo was done and obtained 398 clones from the premade human chondrocyte cDNA library. Their partial sequence data showed that 214 clones of subtracted cDNA library of craniofacial tissue were still non-redundant in Genebank search. And 20 clones among 498 clones of premade chondrocyte cDNA library were known to be undefined genes. Through in situ hybridization screening in the craniofacial tissue sections of 10 weeks old human embryo 36 clones were found to be positive in specific tissues. Depending on the cell types of sirnilar developmental origin, the positive reactions could be divided into five groups. Among the 20 clones of undefined genes from human chondrocyte cDNA library, 7 clones showed characteristic positive reaction in human cartilage tissue by in situ hybridization. From the simulated protein structure, motif analysis and in situ hybridization studies for the 7 undefined clones, Ch89, Ch96, Ch129, Ch285 clones may function in the outer space of the cell constituting a part of matrix protein complex, and Ch276 as a transmembrane protein which might partic ipate in matrix calcification around chondrocytes. Ch153 is a kind of antirnicrobial protein also acting as an inflammation mediator, and Ch334 clone is a zinc finger protein, of which expression increases in human adult tissues We presume these novel genes from human chondrocytes may provide a new path of chondrocyte development and functions of human craniofacial tissues

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.