The Japanese oak silkmoth, Antheraea yamamai Guérin-Méneville 1861 (Lepidoptera: Saturniidae), is one of the important natural resources possessing industrial value for silk fiber production. In this study, ten microsatellite markers and two mitochondrial DNA (mtDNA) gene sequences (COI and ND4) were used to investigate the genetic variation and geographic structure of A. yamamai populations in South Korea. Two mtDNA gene sequences revealed very low total genetic variation and resultant low geographic variation, validating to use further variable molecular markers. Population-based FIS, FST, RST, and global Mantel test consistently support that A. yamamai populations are overall well interconnected with a relatively high gene flow. Nevertheless, STRUCTURE analysis using microsatellite data and mtDNA sequences coincidently indicate the presence of two genetic pools in many populations.

한국 및 그 인근에 서식하는 매미나방의 개체군 구조 분석을 수행하였다. 한국 및 러시아의 33개 지역 중 샘플이 채집된 32개 지역 960 개체를 대상으로 COI gene 서열을 분석하였으며, 이 중 서열이 확보된 906개체(n=20~30)를 이용하여 개체군 구조를 분석하였다. 그 결과, 전체 개체에 대한 haplotype diversity는 0.6774±0.0144으로 나타났는데, 33번 지역(러시아)이 0.3034 ± 0.1041로 가장 낮았고, 25번 지역이 0.8128±0.0648로 가장 높았다. 전체 개체에 대한 nucleotide diversity는 0.016701±0.012517로 나타났는데, 2번 지역이 0.005875±0.006619로 가장 낮았고, 25번 지역이 0.027003±0.018376으로 가장 높았다. Fst pairwise distance를 이용한 개체군 구조 분석 결과, 러시아를 비롯한 한국 내 분포하는 매미나방은 2개의 계통이 존재하는 것으로 나타났으며, 이에 대한 F-statistic value는 계통 간(Fct)에는 0.27066 (P=0.00000), 각 계통 내의 개체군 간(Fsc)에는 0.01174 (P=0.00489), 전체 개체군 간(Fst)에는 0.27922 (P=0.00000)으로 나타나 각 계통 간의 유전적 격리가 있음을 확인할 수 있었다. Median joining network에서도 한국의 남부지역에서만 나타나는 haplotype이 확인되었다. 이로 볼 때, 한국 내로의 매미나방 개체군의 형성은 크게 2가지 경로로 이루어진 것으로 사료되었다. mismatch analysis를 통해 각 계통에 대한 demographic history를 추론하였다. 그 결과, 러시아를 비롯한 한국 중부 지역의 개체군은 약 35,000년 전(34,782 (18826~54652) generation time)에 sudden expansion이 있었던 것으로 나타났으며, 지리적 거리와 유전적 거리의 상관관계를 나타내는 mentel's test에서도 지리적 거리가 증가함에 따라 유전적 거리도 증가하는 것으로 나타났다. 남부 지역 개체군은 약 48,000년 전(47,826 (35,478~66,652) generation time)에 확산이 일어난 것으로 나타났다. 다만, 남부지역 개체군의 경우, mentel's test에서 지리적 거리와 유전적 거리의 상관관계가 반비례하는 것으로 나타났으며, 남부지역에만 나타나는 haplotype은 NCBI GenBank Blast search 결과, 한국 내에만 분포하는 유전자형으로 나타나 유전적 병목현상이 발생했을 가능성이 있을 것으로 추론되었다. 그러나 이에 대한 분석은 추후 Mitochondrial control region이나 Microsatellite loci의 분석을 통해 확증할 예정이다. 결과적으로 한국 및 인근 지역의 매미나방 개체군 구조 분석을 통해 지역 개체군간의 관계를 밝힘과 분산에 대한 가설을 제공함으로서 유사한 양상을 나타내는 곤충 종에 대한 한국 내에서의 개체군 형성 모델을 제시할 수 있을 것으로 사료된다.

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.

The morecular cloning, gene structure, expression and enzyme activity of a serine-like proteas frome Laccotrephes Japonensis were examined. In this study, RT-PCR was used to amplify cDNA fragments for serine-like proteases from total RNA the hole body of Laccotrephes japonensis. The flanking sequences of the 5'- and 3'- end of the this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained an 963bp ORF encoding 321 amino acids. The deduced amino acid sequence of this protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin precuror LlsgP4, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine-like protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant JG showed activity in the protease enzyme assay using gelatin as a substrate.

The subfamily Hoplolaiminae included economically important plant parasitic nematodes and consisted of more than 400 species, all having the diagnostic characters of a strongly annulated cuticle and a large stylet. Among the Hoplolaiminae genera, the genus Hoplolaimus species include species such as H. columbus, and H. galeatus that cause serious damage to crops and turf grass in the Southeastern United States. Traditional identification of species has been approached by interspecific variation of phenotypic traits that rely on morphological and morphometric characters. However, these taxonomic criteria are sometimes not practical because of their limited ability to discriminate species among closely related groups due to overlapping of important taxonomic characters. The exact species identification is needed to control target nematode and also quarantine. Therefore, genetic studies for development of molecular diagnostics, population biology, and disease management are required. In recent years, many molecular diagnostic methods have been used for the identification of plant parasitic nematodes. Advanced molecular techniques have been used that test traditional identification methods. In our studies, Hoplolaimus species showed that high genetic divergence in rDNA sequence is combined with low morphological diversity. Based on genetic information, we developed multiplex PCR for H. columbus, H. galeatus, and H. magnistylus and successfully amplified mixed populations. In molecular phylogeny, the subfamily Hoplolaiminae is an important out‐group of the Heteroderidae, a notorious plant parasite nematode group. Molecular phylogeny of the Hoplolaiminae will help us understand of pathways of pathogenesis. In our phylogenetic analysis using D2 and D3 expansion segments of 28S gene, the molecular data supported morphological based taxonomic schemes. To reconstruct more reliable phylogenetic analysis, correct assignment of each nucleotide within multiple sequence alignment is an important step. Sequence alignments based on secondary structure have been proposed as new alternative methods to obtain this goal. We predicted the secondary structure of D2 and D3 domain using computational predictions method such as the minimization energy method and comparative sequence analysis (co‐variation). Predicted secondary structure included 18 species with two outgroup species, Globodera rostochiensis, Rotylenchulus reniformis. Consensus secondary structure was obtained from closely related and distantly related species. Phylogenetically informative characters were distributed in the stem region (86.7%). These results support the effectiveness of stem and loop regions for phylogenetic analysis of the Hoplolaiminae.

Inhibitor <SUB>K</SUB>B (I<SUB>K</SUB>B)-like gene has been found ill the genome of Cotesia plutellae bracovirus (CpBV), which is the obligatory symbiont of an endoparsitoid wasp, C. plutellae. The open reading frame of CpBV-I<SUB>K</SUB>B was 417 bp and encoded 138 amino acids. Four ankyrin repeat domains were found in CpBV-I<SUB>K</SUB>B, which shared high homology with other known polydnavirus I<SUB>K</SUB>Bs. Considering a presumptive cellular I<SUB>K</SUB>B based on Drosophila Cactus, CpBV-I<SUB>K</SUB>B exhibited a truncated structure with deletion of signal-receiving domains, which suggested its irreversible inhibitory role in NF<SUB>K</SUB>B signal transduction pathway of the parasitized host in response to the wasp parasitization. CpBV-I<SUB>K</SUB>B was expressed only in the parasitized diamondback moth, Plutella xylostella. Its expression was estimated by quantitative RT-PCR during parasitization period, showing a constitutive expression pattern from the first day of parasitization. An indirect functional analysis of CpBV-I<SUB>K</SUB>B was conducted and suggested a hypothesis of host antivirus inhibition.

야생주 핵다각체병 바이러스의 낮은 병원성에 대해 살충제로서의 파밤나방 핵다각체병 바이러스(Spodoptera exigua nucleopolyhedrovirus: SeNPV)의 병원성 향상을 꾀함과 동시에 재조합 바이러스가 다각체 내에 매립됨으로써 살충제로의 적용시 야외에서의 안정성을 확보할 수 있는 p10 유전자의 프로모터를 이용한 새로운 발현벡터를 개발하기 위하여 국내분리주 SeNPV의 p10 유전자 구조를 분석하였다. 그 결과, SeNPV p10 유전자의 프로모터와 구조유전자 부위를 포함한 545염기서열을 결정하여 기존에 보고된 SeNPV p10 유전자(Zuidema et al., 1993)와 비교한 결과 구조유전자 부위에서는 100%의 상동성을 보였으나 5` 및 3` flanking region의 4개의 염기서열에서 차이를 보였다. Southern hybridization에 의하여 SeNPV전체 genomic DNA상에서 p10 유전자는 Sph I 2.4Kb와 Cla I 4.0Kb 단편내에 각각 존재함을 확인하였으며, p10 유전자를 포함하는 이들 단편을 각각 클로닝하여 제한효소 지도를 작성한다.

Genomes of clusters of related eukaryotes are now being sequenced at an increasing rate. In this paper, we developed an accurate, low-cost method for annotation of gene prediction and exon-intron structure. The gene prediction was adapted for delta 1-pyrroline-5-carboxylate-synthetase (p5cs) gene from China wild-type of the halophytic Leymus chinensis (Trin.), naturally adapted to highly-alkali soils. Due to complex adaptive mechanisms in halophytes, more attentions are being paid on the regulatory elements of stress adaptation in halophytes. P5CS encodes delta 1-pyrroline-5-carboxylate-synthetase, a key regulatory enzyme involved in the biosynthesis of proline, that has direct correlation with proline accumulation in vivo and positive relationship with stress tolerance. Using analysis of reverse transcription-polymerase chain reaction (RT-PCR) and PCR, and direct sequencing, 1076 base pairs (bp) of cDNA in length and 2396 bp of genomic DNA in length were obtained from direct sequencing results. Through gene prediction and exon-intron structure verification, the full-length of cDNA sequence was divided into eight parts, with seven parts of intron insertion. The average lengths of determinated coding regions and non-coding regions were 154.17 bp and 188.57 bp, respectively. Nearly all splice sites displayed GT as the donor sites at the 5' end of intron region, and 71.43% displayed AG as the acceptor sites at the 3' end of intron region. We conclude that this method is a cost-effective way for obtaining an experimentally verified genome annotation.

Microsomal Ω-3 fatty acid desaturase (FAD3) is an essential enzyme in the production of the n-3 polyunsaturated fatty acid α-linolenic acid during the seed developing stage. To understand the regulatory mechanism of the gene encoding the Ω-3 fatty acid desaturase, a genomic fragment corresponding to the previously isolated perilla seed PfFAD3 cDNA was amplified from perilla (Perilla frutescens Britt) by GenomeWalker PCR. Sequence analysis of the fragment provided with identification of a 1485-bp 5'-upstream region and a 241-bp intron in the open reading frame. To determine the tissue-specificity of the PfFAD3 gene expression, the 5'-upstream region was fused to the β-glucuronidase (GUS) gene and incorporated into Arabidopsis thaliana. Histochemical assay of the transgenic plants showed that GUS expression was restricted to seed and pollen, showing that PfFAD3 gene was exclusively expressed in those tissues.