Xenorhabdus와 Photorhabdus 속은 각각 곤충병원성 선충인 Steinernema와 Heterorhabditis에 공생하는 공생세 균이다. 감염성 선충의 유충은 공생세균을 표적 곤충의 혈강에 전달하고, 여기서 세균이 증식하여 숙주 선충의 발달을 돕는다. 이러한 선충과 세균 복합체의 성공적 공생관계는 세균의 이차대사산물을 통한 숙주의 면역억제 에 달려져 있다고 알려져 있다. 본 연구에서는 서로 다른 살충력을 보이는 6종의 Xenorhabdus를 확보하고 이러한 차이가 세균의 성장속도와 NRPS (Non ribosomal peptide synthease)에 의해 생성되는 세균의 이차대사산물 발현에 서 기원한다는 것을 확인하였다. 서로 다른 균주들은 콩명나방 (Tenebrio molitor)에 대한 살충력에 차이를 가지고 있었다. 이러한 세균들은 TSB 배지에서는 세균 성장 속도에 차이가 존재하지 않았지만 콩명나방 혈강 내에서는 세균의 성장 속도에 차이가 존재하는 것으로 나타났다. 또한 각 세균의 이차대사산물 추출물을 통한 곤충의 면역 억제 실험 결과 PLA2 활성 억제, 세포독성 능력들이 살충력과 상관관계가 있는 것으로 나타났다. 이러한 이차대 사산물의 경우 많은 물질이NRPS (Non ribosomal peptide synthease)에 의해 생성되므로 각 세균 별 NRPS의 유전자 발현을 보았을 때 흥미롭게도 살충력이 더 높은 스트레인의 세균이 일부 NRPS 유전자의 발현이 더 높은 것으로 나타났다. NRPS에 의해 합성되는 물질을 포함한 세균의 이차대사산물의 차이를 서로 비교하기 위하여 이차대사 산물 추출액을 GC-MS/MS를 이용하여 분석하였다. 본 연구를 통해 곤충병원세균에 살충력의 기원이 NRPS를 통해 합성되는 이차대사산물에 있다는 것을 확인하였으며 이를 이용한 다양한 NRPS 유래 물질 연구는 신규 살충 물질 개발에 들어가는 비용과 시간을 획기적으로 줄일 수 있을 것으로 기대된다.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. Success parasitism of the nematode-bacterial complex depends on the host immunosuppression by the bacteria via their secondary metabolites. Lrp (Leucine-responsive regulatory protein) is a global transcriptional factor of the bacteria and play a crucial role in the parasitism. However, its regulatory targets to suppress the insect immunity were not clearly determined. This study investigated the regulatory target genes and subsequent secondary metabolites by Lrp in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage in a target insect, Spodoptera exigua. Among eight non-ribosomal peptide synthetase (NRPS1-NRPS8) genes, six gene (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites along with alteration of the NRPS expression levels. The immunosuppressive activities of X. hominickii depended on the Lrp expression level. The metabolites produced by Lrp expression possessed the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (= cPF) was produced under Lrp control and identified to inhibit phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays crucial role in insect immunosuppression by modulating NRPS expressions.

BALB/c mice were vaccinated with Brucella (B.) abortus recombinant protein L27 (50S ribosomal protein L27) cloned into a pMal vector system. L27 was induced, purified and injected intraperitoneally (IP). Mice were vaccinated on 0-, 15- and 35-day. Serum cytokines were evaluated on 36- and 49-day from first vaccination. Mice were intraperitoneally infected with 5×104 CFU of virulent B. abortus 544 on day-50 and sacrificed after two weeks from infection. Bacterial burden from the spleen was quantified and showed a 0.7- and 0.9-log reduction in vaccinated mice in comparison to PBS and MBP (maltose binding protein) groups respectively. Cytokines in the serum demonstrated increased interferon-gamma (IFN-γ) and other pro-inflammatory cytokines such as macrophage chemoattractant protein-1 (MCP-1) and interleukin 6 (IL-6). On the other hand, interleukin 10 (IL-10) was attenuated in the sera of vaccinated mice. This cytokine profile is indicative of a cell-mediated type of immune response which is favorable for the eradication of intracellular infections. The current study showed the potential of another B. abortus ribosomal protein in inducing protective immunity against B. abortus infection.

NRPS (non-ribosomal pepetide synthetase) plays an important role in the biosynthesis of secondary metabolites, which includes economically important compounds such as antibiotics, antitumor agents, immune-suppressants, siderophores, toxins, etc. In most cases, however, their functions and biosynthesizing pathways have been still remained riddles because of their cryptic expression and recalcitrant genetics. In our previous genome analyses of Cordyceps bassiana C101, we predicted the sequences of secondary metabolite biosynthesizing gene clusters, which contain non-ribosomal pepetide synthetases whose functions are already known such as beauvericin, bassianolide, bassiatin, tenellin, beauverolides, and oosporein. The authors focused on nrps6 that has not been functionally studied. The single-gene deletion and overexpression mutants were established via Agrobacterium-mediated transformations. PCR, quantitative real-time PCR, and southern blot analyses were carried out to confirm the correct single-gene integrated homologous recombination. In further study, comparative metabolic profiling between wild-type and mutant strains will be followed by their detailed chemical structure analysis through NMR spectroscopy.

A karyotype analysis of five wild rose species (Rosa helenae, R. mulliganii, R. multiflora, R. rubus, and R. solieana) was performed using bicolor fluorescence in situ hybridization (FISH). All five species were found to be diploid (2n = 2x = 14). The chromosome length in the metaphase stage ranged from 1.29 to 2.05 μm in R. helenae, 3.32 to 6.82 μm in R. mulliganii, 1.58 to 2.24 μm in R. multiflora, 2.05 to 3.46 μm in R. rubus, and 1.62 to 2.46 μm in R. solieana. The chromosomes were either metacentric or submetacentric, with no subtelocentric or telocentric chromosomes being observed. Each p air of 5 S and 4 5S rDNA sites was detected at the proximal region of the long arm and the terminal region of the short arm of chromosome #7, respectively.

본 연구는 RPS3 유전자의 SNP를 탐색하고 버크셔 돼지 433두에서 RPS3 유전자 SNP와 육질형질과 의 연관성을 규명하였다. SNP 탐색을 위해 버크셔 돼지의 간 조직을 사용하여 RNA-Seq을 수행한 결 과, RPS3의 염색체 451번째 G 서열이 C로 변환되어 arginine에서 serine으로 아미노산이 치환되는 non-synonymous SNP를 확인하였다. 동일한 조건에서 사육된 버크셔 돼지 433두에서 RPS3 SNP의 유전자형을 분석한 결과 major allele은 G이며 minor allele은 C로 확인되었다. RPS3의 유전자형과 육 질형질과의 연관성 분석 결과 공우성 모델에서 등지방두께(Backfat thickness), 가열감량(Cooking loss), 적색도(CIE a), 사후 45분 후 삼겹살과 등심 pH(pH45minB and pH45minL), 지방(Fat)과 단백질 (Protein) 함량 형질에서 유의성을 가졌다. 성별에 따른 RPS3 SNP 유전자형의 육질분석 결과 거세돈 은 단백질 함량에서만 유일하게 유의성이 나타난 반면 암퇘지는 등지방두께를 포함한 7가지의 육질 형 질에서 유의성을 나타나 거세돈 보다 육질 형질과의 연관성이 높은 것으로 확인되었다. 육질형질과 RPS3 SNP를 비교하였을 때, G allele을 가진 유전자형이 C allele에 비해 사후 pH45minB, 사후 pH45minL, 단백질 함량을 증가시키고, 등지방두께, 가열감량, 지방 함량을 감소시키는 것으로 확인하였 다. 따라서 G allele을 가진 돼지가 육질이 더 좋은 것으로 판단된다. 본 연구의 결과를 통해 RPS3의 SNP를 육질이 우수한 돼지고기를 생산하기 위한 분자유전 육종에서 육질 연관 biomaker로 응용 할 수 있을 것으로 사료된다.

The genetic relationships among five genera, seven species of Theaceae were examined through a random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS). In RAPD analysis, five of 15 arbitrary primers showed polymorphic bands, which were able to classify different genera and species of Theaceae. The genetic variations of Theaceae were from 0.031 to 0.484. In ITS analysis, the ITS sequences were analyzed using BLAST and showed high identities with sequences of Theaceae, seven species published in NCBI GenBank database, which ranged from 98 to 100%. Sequence alignment of seven species showed 34.9% identities for ITS 1 region and 43.7% for ITS 2 region. Pairwise sequence divergences among seven species ranged from 0 to 0.330%. In phylogenetic tree, they were divided into three groups. In conclusion, the molecular data generated in the present investigation will help to understand the genetic relationships of Theaceae and also might be useful for further studies in intra-species, inter-species, and molecular evolution researches.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is widely utilized for greenhouse pollination in South Korea. Understanding the magnitude of genetic diversity and geographic relationships is of fundamental importance for long term preservation and utilization. As a first step, we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to the “DNA barcode” region and the complete internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA from 88 individuals collected in nine South Korean localities. The complete ITS2 sequences were longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated 112-bp long repeats. The 658-bp long mtDNA sequences provided only six haplotypes with a maximum sequence divergence of 0.61% (4 bp), whereas the ITS sequences provided 84 sequence types with a maximum sequence divergence of 1.02% (21 sites). The combination of the current COI data with those of published data suggest that the B. ignitus in South Korea and China are genetically a large group, but those in Japan can be roughly separated into another group. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population were found to exist among the South Korean populations of B. ignitus, which suggests panmixia. This finding is consistent with our understanding of the dispersal capability of the species.

This work was performed to confirm the molecular discrimination through the nrITS1 sequences among 3 taxa of Scirpus L. sensu lato (s.l.) species. S. planiculmis represented only 2 base sequence variations with S. maritimus in spite that they showed different morphological features. The nucleotide sequences of the ITS1 region from S. planiculmis were shown to have 99.1% homology with S. maritimus and 60.4% homology with S. triqueter. Although the morphology of S. planiculmis is similar with S. triqueter, molecular basis of the size and sequences on ITS1 region were shown to have distinctive differences. For divergency investigation on same sites and metapopulation, sequencing was conducted on ITS1 region with partial 5.8S and 18S regions. All plants of each species collected at the same site had identical band size pattern and sequences. Intraspecific molecular divergency was not identified in spite that these species live in different wetland sites. The ITS1 sequences described here provided a powerful genetic tool for phylogenetic studies which was difficult by morphological identification as high rate of morphological plasticity

The pear psylla, Cacopsylla pyricola Foerster (Homoptera: Psyllidae), is a serious insect pest of commercial pear crops. The species, which resides on pear trees throughout its life cycle, is rapidly spreading in some regions of the world. Given the life cycle, it is unclear how such a rapid spread has been facilitated. Presently, the population genetic structure of the species including genetic diversity and gene flow was studied to understand the nature of dispersal and field ecology of the species. Pear psylla was collected from several pear orchards in Korea. The 658-bp region of mitochondrial COI gene and the 716-bp long complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA were sequenced. Unlikely other previously studied insect pests, the COI-based genetic diversity of the pear psylla was extremely low (maximum sequence divergence of 0.15%). This finding allowed us to conclude that the species may have been introduced in Korea relatively recently, possibly with the phenomenon of genetic bottlenecks. ITS2 sequence-based analyses of phylogeny, population differentiation, gene flow, and hierarchical population structure all concordantly suggested that the pear psylla populations in Korea are neither genetically isolated nor hampered for gene flow. These genetic data are concordant with the dispersal of an overwintering winterform morph outside the non-pear habitat in the fall and the possibility of subsequently longer distant dispersal.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.61% (4 bp), the ITS sequences provided 84 sequence types with the maximum sequence divergence of 1.02% (21 sites), confirming better applicability of the ITS sequences to the study of intraspecific variation. The complete ITS2 sequences of B. ignitus were shown to be longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated repeats. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/ population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability of the species

Taxonomic resolution of the Nosema/Vairimorpah clade has been augmented with DNA sequences of the small subunit (SSU) and large subunit (LSU) ribosomal RNA (rRNA) and the arrangement of SSU and LSU. Based on the two characteristics, the clade is largely divided into two, i.e. 'true' Nosema sub-group and 'non-true' Nosema sub-group within the clade. Our study shows that a novel Nosema species isolated from Pieris rapae has mixed characteristics of the 'true' and non 'true' Nosema sub-group based on the topology of SSU and LSU sequences, and rRNA of the isolate is normally organized. Additionally, the length of ITS can be a diagnostic tool to distinguish 'true' Nosema from non 'true' Nosema in the Nosema/Vairimorpha clade based on its nucleotide length as reported before. To our knowledge, this is the first report of recombination event in the Nosema/Vairimorpha clade.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) with the individuals collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.456% (3 bp, from 91 individuals), but the ITS sequences provided 33 sequence types with the maximum sequence divergence of 0.78% (16 bp, from 35 individuals), confirming better applicability of the ITS sequences to the study of intraspecific variation. The ITS2 sequences of B. ignitus were shown to be the longest among known insects, ranging in size from 2,034 bp ~ 2,045 bp and harbored two duplicated repeats, indicating unusual structure of B. ignitus ITS2 sequences compared with other insect ITS sequences. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability.

There has been a substantial controversy on the phylogenetic relationships among butterfly families and several competing phylogenetic hypothesis have been suggested. Among them the relationships of (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae) has been further widely accepted. In this study, we sequenced EF1-α, COI, and 16S rRNA from 62 species belonging to four true butterfly families, Papilionoidea. Phylogenetic analyses using BI, ML, and MP showed that the traditionally recognizable families were strongly supported as monophyletic groups, with the exception of Nymphalidae, wherein the singly included species of Danainae was placed as basal lineage of the Nymphalidae + Lycaenidae group. Phylogenetic relationships among families supported the sister group relationship of Nymphalidae and Lycaenidae strongly by all analyses and placed Papilionidae as the most basal lineage of the Papilionoidea. On the other hand, the relationships of Nymphalidae and Lycaenidae group to Pieridae were either unresolved, revealing trichotomy, or the relationships of (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae) as previously supported by several morphological and molecular works supported. Detailed within-family relationships among some genera also are shown in the presentation.

The bumblebees, Bombus species are valuable natural resources being utilized for greenhouse pollination. Low level of genetic variation of Bombus species has been reported previously. In this study, we sequenced complete internal transcribed spacer 2 (ITS2) of the nuclear rDNA from 100 individuals of B. ardens collected from seven localities in Korean peninsula. The ITS2 sequence of B. ardens is longest, ranging in size from 1,940 bp-1,954 bp among known in insects, which ranges approximately from 241 bp-1,728 bp. The ITS2 sequences have ~51% of C+C content and contain each two 27 bp repeats, 20 bp repeats, 33 bp repeats, and 34 bp repeats at the beginning. Such repeats were not found in other insects. Uncorrected pairwise distance among 96 sequences that were obtained from 100 individuals revealed a maximum sequence divergence of 1.03%. Genetic diversity (π) of each population ranged from 0.007801 to 0.009627, and the lowest diversity was obtained from islet population of Ulleungdo, indicating possibly small, isolation of the population. Significant level of genetic distance was only found when Ulleungdo population was compared to two other mainland populations. Except for this, overall, a very high rate of per generation migration ratio (Nm=7.1-infinite) and a very low level of genetic fixation (FST=0-0.06546) were detected between pairs of localities. Analysis of hierarchical relationships among localities consistently revealed no statistically significant structure among populations. Taken these together, the B. ardens populations on the Korean peninsula are panmictic this is consistent with our understanding of the dispersal capability.

Halacarid mites inhabit mainly in the bottom of the sea and their body lengths are approximately 0.5 mm. Few biological information about halacarids have been obtained. The phylogenetic study of halacarid mites has not been progressed and the phylogenetic relationship within the family Halacaridae is still unknown. Particularly there is no molecular study to infer phylogenetic relationship within the family. In the annual meeting of Acarological Society of Japan 2007, we developed the method for molecular analysis of 28S nuclear ribosomal gene in haracarid mites. Subsequently, we have further progressed the technique of the molecular analysis to infer the phylogenetic relationship of haracarids. The aim of the present study is to elucidate the phylogenetic position of the subfamily Rhombognathinae on the bases of molecular data. Halacarid specimens which were obtained from seaweeds and sandy segment at Japanese seacoasts, consist of eight genera in five subfamilies: Halacarinae (Halacarus sp., Agauopsis spp., Thalassarachna spp.), Halixodinae (Badyagaue sp., Agaue sp.), Copidgnathinae (Copidognathus spp.), Actacarinae (Actacarus spp.) and Rhombognathinae (Rhombognathus spp.). The template DNA was extracted and 28S nuclear ribosomal gene of the DNA was amplified by PCR reactions. And we determined approximately 400 base pair length of the gene. To construct phylogenetic trees by neighbor-joining method we calculated genetic distances of each operational taxonomic unit considering 1) transversions and 2) transitions and transversions based on Tamura - Nei model by using MEGA ver. 4 (Tamura et al. 2007). As a result, we got two trees both showing two main clusters. One of them includes only one subfamily Rhombognathinae and another consist of Halacarinae, Copidgnathnae, Actacarinae and Halixodinae. Rhombognathinae was greatly differentiated from the other subfamilies. Thus, Rhombognathinae possibly is a distinct taxon phylogenetically and this result supports the monophyletic nature obtained previously by morphological analysis.

지렁이과 (family Megascolecidae) 왕지렁이속 (genus Amynthas) 그룹을 rDNA ITS 유전자를 이용하여 계통 유연관계를 알아보고자 하였다. 2과 10속에서 26종의 DNA 염기서열을 이용하여 종간 계통적 유연관계를 MP (Maximum Parsimony), NJ (Neighbor Joining), QP (Quartet Puzzling)로 계통도를 작성하였다. 염기서열이 확보된 Megascolecidae (지렁이과)의 국내외

국내에서 재배하여 생산되고 있는 상황버섯의 일종인 PMO-P4균주에 대한 ITS 영역의 염기서열 분석을 실시하였으며 목질 진흙버섯으로 잘 알려져 있는 P. linteus와 함께 RFLP분석을 통하여 상호 비교한 결과 PMO-P4균주는 P. baumii로 판명되었다. 이 결과를 토대로 이미 보고 되어 있는 Phellinus속 균주들과의 종간 ITS 영역의 상동성을 비교한 결과 48.6%-72.2%였으며 본 연구에서 비교한 종들 가운데서는 P. linteus와 상동성이 가장 높았으며 P. gilvus와 상동성이 가장 낮았다.