Fas-associated death domain protein (FADD) functions as an apoptotic adapter in mammals, recruiting caspases for death-inducing signaling complexes, while in lower animals, it interacts with IMD and DREDD to initiate antimicrobial responses. In this study, we examined the T. molitor FADD sequence (TmFADD) using molecular informatics methods to understand its involvement in the host's immune response against microorganisms. Knocking down TmFADD transcripts resulted in increased susceptibility of T. molitor larvae to E. coli, underscoring the significance of FADD in insect defense mechanisms and providing valuable insights into insect immunity.

새롭게 부상하는 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) 9 유전자 편집 기술은 장기 이식(organ transplantation)과 같은 생의학 연구(biomedical research)와 동물 산업에 대한 전통적인 접근 방식을 빠르게 변화시키고 있다. 돼지 생식 및 호흡기 증후군 바이러스(porcine reproductive and respiratory syndrome virus; PRRSV)와 전염성 위장염 바이러스 (transmissible gastroenteritis coronavirus; TGEV)는 돼지 산업에 막대한 경제적 손실을 초래하는 치명적인 바이러스이다. 바이러스의 숙주 수용체 단백질 CD163과 pAPN에 대한 이중 유전자 녹아웃(double knock-out; DKO) 돼지는 PRRSV와 TEGV에 내성을 나타내었으며, 정상(wild-type; WT) 돼지와 비교할 때 성장과 생식 특성의 차이가 없었다. 이러한 결과는 경제 동물 돼지에 CRISPR-Cas9 매개 유전자 편집 기술을 적용하여 바이러스 저항성 유전자 변형에 의한 품종 개량이 달성될 수 있다는 것을 보여주며, 질병 저항성 돼지 생산을 위한 육종 시작점을 제공한다. 종간 배반포 보완(interspecies blastocyst complementation)은 이종 만능 줄기세포 유도체(xenogenic pluripotent stem cell derivatives)의 장기 특이적 생산(organ-specific enrichment)을 가능하게 한다. CRISPR-Cas9 매개 접합자 유전자 편집(CRISPR-Cas9-mediated zygote gene editing)을 이용하여 췌장 생성(pancreatogenesis), 신장 생성(nephrogenesis), 간 생성(hepatogenesis) 및 혈관 생성(vasculogenesis)이 불가능 생쥐 숙주를 만들었으며, 이러한 숙주와 배반포 보완 플랫폼을 결합하여 키메라를 만들었다. 또한 돼지와 소 같은 유제류(ungulate)의 섬유아세포(fibroblasts)를 이용하여 CRISPR-Cas9 매개 유전자 편집과 체세포 핵 치환(somatic cell nuclear transfer) 과정을 거쳐 복제 배아(genome-edited cloned embryos) 를 생산하였다. 복제 배아의 1차 배양 섬유아세포(primary cultured fibroblasts)를 재복제하여 배반포 보완을 위한 숙주 배아로 이용하였다. CRISPR-Cas9 유전자 편집 기술과 종간 배반포 보완 플랫폼 전략의 조합은 유전자 변형 돼지를 생산하는 데 유용하다. 본 논문에서는 CRISPR/Cas9 유전자 편집 기술과 배반포 보완 플랫폼, 질병 저항성(disease resistance) 돼지, 이종장기이식(xenotransplantation) 목적의 키메라 생산을 소개하고자 한다.

A γ-aminobutyric acid (GABA) producing microorganism was isolated from Sun-Tae Jeotgal, a Korean traditional fermented seafood. Two thousand presumptive lactic acid bacteria (LAB) isolates were screened for GABA production by thin layer chromatography. One isolate, T118, produced GABA profusely, and identified as Lactobacillus brevis. Growth of Lb. brevis T118 was examined during 120 h cultivation in MRS broth under different conditions. Lb. brevis T118 grew well at 30-37℃, initial pH of 4-7, and up to 5% NaCl (w/v). A gene, gadB, encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was cloned and gadC located immediately upstream of gadB, indicating gadCB operon structure. The operon structure was confirmed by reverse transcription (RT)-PCR. gadB was overexpressed in Escherichia coli BL21 (DE3) and recombinant GAD was purified. The size of recombinant GAD was 54.4 kDa by SDS-PAGE, which matched well with the calculated size from the nucleotide sequence.

본 연구는 돼지 농가의 생산성 향상 및 수익성 증대를 위한 분자 육종 기술에 적용할 유전자 마커를 개발하기 위해 수행 되었다. 분석 결과에 따르면, 돼지의 간 조직을 이용하여 RNA-Sequencing을 수행한 결과 ECI2 유전자의 SNP를 발견 하였고 발견된 SNP chr 7:g.2302809는 c.608로 608번째 C가 G로 변환되어 Threonine에서 Serine으로 아미노산이 치환되는 단일염기다형성임을 확인하였다. Berkshire 돼지 430으로 ECI2 유전자형과 육질 형질과의 연관성 분석 결과 도 체중, 적색도, 육즙 손실, 수분 함량 및 pH24hr에서 유의성을 확인할 수 있었다. 그중 GG 유전자형은 pH24hr에서 다른 유전자형에 비해 수치가 증가시키는 경향을 확인하였다. 성별에 따른 유전자형과 육질 형질과의 연관성은 거세돈에서 GG 유전자형이 육즙 손실의 감소와 pH24hr에서 유의성이 확인되었고, 암퇘지의 GG 유전자형도 수분함량이 증가되었다. 따라서 ECI2 유전자의 GG 유전자형을 가진 돼지가 육질이 더 좋은 것으로 판단된다. 이러한 결과를 바탕으로 ECI2의 GG 유전자형을 고정시킨다면 육질이 우수한 돼지고기 생산이 가능할 것이다. 또한 ECI2 유전자를 이용하여 품종개량 및 조기 선발 기술에 바이오마커로 활용한다면 농가의 경쟁력 강화 효과에 도움이 될 것으로 사료된다.

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 sweetpotato whitelfy Bemisia tabaci distribute worldwide and infests more than 500 species of plants. To determine nutritional stress of whiteflies at molecular level, we identified a full cDNA of glucose regulated protein 78 (grp78) which is known to be respond to nutritional restriction in vertebrate species. GRP78 of B. tabaci was highly conserved motifs of the HSP70 family and the C-terminal motif of KDEL characteristic of endoplasmic reticulum-specific HSPs. Real-time RT-PCR analysis showed that the grp78 level was not changed by thermal stress treatment from 4°C to 40°C for 1 h. However, the grp78 level was proportionally increased to the ingestion of a sucrose solution ranging in concentrations from 0% to 30% in a Parafilm feeding chamber. In addition, the grp78 levels were various by the ingestion of leaves of 10 different plants for 24 h; its level was higher with eggplant and pepper but lower with rice and apple. Our study suggests that the grp78 may have a role for cellular chaperones in relation to nutritional uptake of B. tabaci.

Cotton aphid infests more than 700 plants and a major pest of various horticultural crops worldwide. The glucose regulated protein 78 (GRP78) is a member of heat shock protein 70. Its expression is associated with the nutritional changes as well as environmental stresses. The full sequences of grp78 cDNA of Aphis gossypii was determined. It had conserved motifs of hsp genes and terminated in KDEL which is common to GRP78. Quantitative realtime PCR showed that its level was changed during development and also upregulated by starvation. However, its level was not much changed by heat stress. The level of grp78 can be use to understand nutritional physiology on insects.

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.

Immune defense is indispensible for insect survival. However, uncontrolled and excessive immune responses would be highly detrimental and energy-consuming processes. An insect cytokine, plasmatocyte-spreading peptide (PSP), induces hemocyte-spreading behavior as well as activating phenoloxidase (PO) in the beet armyworm, Spodoptera exigua. A hemocyte transcriptome of S. exigua contains a partial sequence of a putative PSP-binding protein (SePSP-BP). SePSP-BP was expressed in all developmental stages especially in hemocytes and fat body. A quantitative RT-PCR showed that the bacterial infection significantly up-regulated the expression level of SePSP-BP. A double-stranded RNA specific to SePSP-BP (dsRNASePSP-BP) was injected and suppressed SePSP-BP expression even in response to bacterial challenge. The larvae treated with dsRNASePSP-BPsuffered high mortality to infection of nonpathogenic bacteria and prolonged high PO activity after the immune challenge. These results suggest that SePSP-BP may play a role in suppressing immune responses as a negative controller

The Diadegma fenestrale is known as parasitoid on potato tuber moth, Phthorimaea operculella and diamondback moth, Plutella xylostella. The Diadegma genus is reported to have symbiotic virus, ichnovirus, D. fenestrale Ichnovirus (DfIV) was identified from this species which is a first report. DfIV showed typical ichnovirus shape with two membranes surrounding the virus capsid. To identify DfIV genes, whole genome sequencing based on GS-FLX was conducted using purified total DfIV genomic DNA extracted from D. fenestrale calyx. About sixty ORFs were analyzed and several typical ichnovirus gene families were detected such as cys-motif, repeat element, vinnexin and vankyrin. Present study was focused on the gene expression patterns in two different lepidopteran hosts.

This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (～35 kDa), dimer (～70 kDa), trimer (～105 kDa) and hexamer (～210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (～300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher’s study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (β- MeOH), anionic detergent (SDS) and heat (95℃) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) of the species collected from seven Korean localities. A total of 17 haplotypes (CACOI01 ~ CACOI17), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 16 sequence types (ITS2CA01 ~ ITS2CA16), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in COI gene sequence. Phylogenetically, the COI gene provided two haplotype groups with a high nodal support (≥ 87%), whereas ITS2 provided one sequence type group with a high nodal support (≥ 92%). The result of COI gene may suggest the presence of historical biogeographic barriers that bolster genetic subdivision in the species. Different grouping pattern between COI gene and ITS2 sequences were interpreted in terms of recent dispersal, reflected in the ITS2 sequence. Finally, finding of unique haplotypes and sequence types only from Beakryeng-Islet population was interpreted as an intact remnant of ancient polymorphism. As more samples are analyzed using further hyper-variable marker, further fruitful inference on the geographic contour of the species might be available.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from seven Korean localities. A total of 18 haplotypes (BARCA01 ~ BARCA18), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 17 sequence types (ITS2CA01 ~ ITS2CA17), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in mitochondrial gene sequence. Phylogenetically, the mitochondrial DNA has shown several haplotypes formed independent groups with substantially high node support (≥ 90%), whereas no such grouping was evidenced for ITS2, indicating different behaviors of the two molecules. Such difference may reflect a diverse dynamics of the species such as biogeographic history, mating behaviors, and also possibly different mode of inheritance of the two molecules, but requires further scrutinized examination of the dataset. In terms of population genetic perspective, overall no population subdivision was detected from both molecules, except for locality 7 (Eocheong islet) from mitochondrial DNA. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

The seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae), known also as the seven-spot ladybird, is natural enemy for aphids and has a broad ecological range, living almost anywhere there are aphids for it to eat. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from nine Korean localities. A total of 21 haplotypes (CSCOI01 ~ CSCOI21), with the maximum sequence divergence of 4.56% (30 bp) were obtained from COI gene sequence (from 78 individuals), whereas 65 sequence types (CSITS201 ~ CSITS265), with the maximum sequence divergence of 2.06% (11 positions) were obtained from ITS2 (from 79 individuals), indicating substantially larger sequence divergence in mitochondrial gene sequence. Both COI gene and ITS2 shows the distribution pattern that only a few haplotypes or sequence types are widely distributed, whereas majority of them are highly restricted in one geographic location, even represented as a single individual. Unlikely the ITS2 sequence types the mitochondrial COI haplotypes evidenced the presence of two main phylogenetic groups, reciprocally monophyletic to each other. Geographically, these two groups are spread in all localities surveyed. Considering both COI gene and ITS2 sequence together, current our data may suggest the presence of ancestral polymorphism, rather than on-going speciation, but more scrutinized analysis will be performed soon. Due partially by the presence of both COI groups in all surveyed localities, the genetic diversity estimates of all localities are similar from the perspective of COI gene, but ITS data showed extremely lower genetic diversity of one islet locality, Anmyeon-do (locality 2; 0.002530 vs. 0.008054 ~ 0.012060). Analysis of gene flew estimates between localities indicates that most populations are highly interconnected to each other. However, one islet locality, Anmyeon-do (locality) has shown statistically significant distance from the remaining localities on the basis of only ITS2 data (FST = 0.19 ~ 0.34), requiring scrutinized phylogeographic inference on this population with expanded sampling. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.