미토콘드리아 시토크롬 c 산화효소 1 (COX1) 유전자 염기서열(658 bp)을 사용하여, 콩 포장에서 채집된 어리팥나방(Matsumuraeses falcana)과 팥나방(Matsumuraeses phaseoli)의 종을 실험실 집단의 종들과 비교하여 동정하였다. COX1 염기서열 분석에서, 어리팥나방 47개체 로부터 10개의 하플로타입이 발견되었고, 종내 유전적 거리는 0.15~0.46%이었다. 이중 하프로타입 A형이 약 70%로 우점형이었다. 팥나방의 30개체로부터는 모두 동일한 하나의 서열만이 확인되었고, 어리팥나방과의 종간 유전적 거리는 4.11~4.61%이었다. 두 종의 COX1 염기서열을 번역한 아미노산 서열은 모두 동일하여 동의적 염기서열 변이(동의치환, 同義置換, synonymous substitution)를 확인할 수 있었다. 포장 조사에 서 두 종의 유충이 콩의 잎과 꼬투리를 가해하였고, 한 포장에서 동시에 발생하였다. 전체 포장에서 어리팥나방의 평균 밀도는 팥나방보다 약 1.5 배 높았다. 이 결과는 콩이 두 종의 동일 기주임을 명백하게 제시하였다. 별도로 이 속의 유충 기생파리로서 Elodia flavipalpis (파리목: 기생파리 과)가 발견되었고, COX1 서열로 동정되었다.

보구치는 난류성으로 전 세계적으로 널리 분포하며 해양 저층에 주로 서식하는 어종이다. 광양만에 서식하는 보구치의 미토콘드리아 DNA에서 cytochrome c oxidase subunit I (COI) 유전자를 발굴하고 해양 어류종에서의 계통유전학적인 위치를 분석하였다. 발굴된 미토콘드리아 DNA 내 605 bp COI 시컨스의 다중배열 결과 광양만 보구치들 에서는 높은 염기서열 상동성을 확인하였다 (98~100%). 하지만 광양만 내해와 외해의 어획지점에 따라 염기서 열 변이가 다르게 나타나는 것을 확인하였다. 외해지점의 보구치들에서 COI 내 염기서열 변이가 높게 나타났다 (43.2~70.3%). 나아가 13종 어류의 COI 계통유전학적 분석결과 광양만 보구치는 타이완에서 보고된 보구치와 하나의 계통군 (clade)으로 묶이고 진화적 거리는 0.036으 로 나타났다. 또한 민어 (M. miiuy)와 대두이석태 (Pennahia Macrocephalus)에 속한 어종과 진화적 거리가 가까운 것으로 나타났다 (0.041~0.048). 본 연구의 결과는 국내산 보구치의 분자 계통유전학적 정보를 제공함으로 연안환경에 따른 어류자원 모니터링 및 종다양성 관리에 주요한 유전 적 자료로 활용될 것이다.

2019년에 서남해안 지역인 고창군의 옥수수 밭 주변에서 성페로몬을 이용하여 열대거세미나방(Spodoptera frugiperda) 성충을 효과적으로 모니터 링하는 방법을 조사하였다. 총 함량이 300 또는 1000 ㎍인 2종류 성분 조성의 성페로몬 미끼[(100%) (Z)-9-tetradecenyl acetate and (2%) (Z)-7-dodecenyl acetate]를 설치한 깔대기형 트랩과 델타형 트랩 중에서 열대거세미나방은 300 ㎍ 미끼의 깔대기형 트랩에서 8월 6일에 처음 잡혔고 가장 많이 포획되었다. 또한 깔대기형 트랩 모두에서 비표적 종인 뒷흰가는줄무늬밤나방(Mythimna loreyi)이 많이 포획되었다. 총 함량이 1000 ㎍인 위의 2종류 성분 조성과 4종류 성분 조성의 미끼[(100%) (Z)-9-tetradecenyl acetate, (8%) (Z)-11-hexadecenyl acetate, (2%) (Z)-7-dodecenyl acetate, and (1%) (Z)-9-dodecenyl acetate]를 설치한 날개형 트랩에서 열대거세미나방은 비슷한 수준의 낮은 포획수를 보였으나 뒷흰가는줄무늬밤나방은 4종류 성분 조성의 미끼에서 훨씬 더 많이 포획되었다. 성페로몬 트랩에 포획된 열대거세미나방 70마리의 미토콘드리아 시토크롬 옥시다제 1(CO1)의 부분 염기서열(1,004 bp)을 이용하여 계통수를 분석한 결과, 두 개의 종내 변이군으로 나눠졌으며 66마리가 CO1-RS로, 나머지 4마리는 CO1-CS로 분지되었다. 또한 두 개의 CO1 변이군과 기주식물계통(벼, 옥수수)에서 일관되게 차이가 있는 총 12개의 CO1 단일염기다형성(SNP)이 확인되었으며, 전체 73마리 중 4마리만 CO1-CS 그룹(옥수수계통 포함)과 동일한 패턴을 보였으며 나머지 69마리는 CO1-RS그룹(벼계통 포함)과 같았다.

We investigated the effects of two Brucella proteins expressed in a pMAL expression system, RocF and EF-Ts, as subunit vaccines on immune modulation and protective efficacy using a mouse model. Mice vaccinated with MBP-RocF and MBP-EF-Ts displayed increased production of TNF, IFN-, MCP-1, IL-10 and IL-6, and TNF and MCP-1, respectively. Furthermore, mice vaccinated with MBP-EF-Ts showed decreased induction of IFN- and Th2-related cytokines, IL-10 and IL-6. Higher proportions of CD4+ and CD8+ T cells were observed in the blood of mice vaccinated with MBP-RocF than in the PBS-vaccinated group, although the increases were not significant. Furthermore, significantly reduced Brucella proliferation in the spleens of the MBP-RocF and MBP-EF-Ts groups were observed, but inflammation of these organs was not attenuated. Overall, these results indicate that RocF and EF-Ts could be potential subunit vaccine candidates against animal brucellosis.

풀무치의 전국적인 발생현황 및 밀도조사의 결과, 한국에서는 전라남도 해남군 산이면과 전라남도 무안군 망운면 간척지에서 2015년 이후 지속적으로 높은 밀도의 발생이 관찰되었다. 우리는 두 지점에서 발생하는 풀무치의 기원을 알아내기 위하여 NADH dehydrogenase subunit (NAD) 2, NAD4 와 NAD5의 염기서열을 분석하였다. 그 결과 해남풀무치의 경우는 중국동북부의 Liaoning성 과 Heilongjiang성 개체군과 기 원이 비슷하고, 무안풀무치의 경우는 일본풀무치와 기원이 비슷하다는 결론에 도달했다. 이전의 전 세계적인 풀무치의 진화에 관한 연구에서 한 국의 풀무치가 포함이 되지 않아서 한반도 풀무치의 기원은 알 수 없었다. 본 연구의 결과는 중국북동부 지방에서 8만 년 전에 분리된 풀무치 중 일부가 한반도로 이동을 하여 해남 지역에 정착을 하고 일부는 러시아 사할린과 일본 홋카이도섬을 거쳐서 무안으로 이동하였을 가능성을 보여주 고 있다. 하지만, 한반도로 내려온 풀무치가 해남과 무안계통으로 분리된 후 일본으로 이동하였을 가능성도 배제할 수 없다.

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.

Synthesis of Li+-selective 14-crown ether (CE) having rigid and bulky subunits was reported. CE-poly(vinyl alcohol) (PVA) dope solutions were electrospun. CEs were immobilized on PVA matrix via acid-catalyzed acetalization using novel aerosol method. Structures of new compounds and their immobilization to PVA were confirmed and characterized. Adsorption experiments show superior lithium capacity and selectivity among previously reported solid-supported CEs. Dihydroxy-dibenzo-14-crown-4 ether-PVA nanofiber membrane showed superior performance. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2015R1A2A1A15055407) and Ministry of Education (2009-0093816).

Recently, RNA interference (RNAi) technology has been emerged as a potent tool for pest control strategy. Based on the previous studies on RNAi via leaf disc-mediated systemic delivery of dsRNA and in planta expression of hairpin RNA by agroinfiltration, the coatomer subunit alpha (COPA) gene has been found to be a crucial target for RNAi against Tetranychus urticae. In current study, transgenic plants of Arabidopsis thaliana expressing COPA hairpin RNA were generated by the floral dip method. Putative transgenic plants were screened by PCR and positive transformants were subjected to bioassay using age-synchronized and host-adapted T. urticae. T. urticae feeding on plants expressing dsRNA/siRNA showed more than 80% mortality as compared to the mites feeding on control plants at 6 days post-infestation. Our data shows that in planta expression of hairpin gene such as COPA may serve as an effective way for the control of this important pest in ornamental and economically important plants.

Na+/K+-ATPase is a membrane protein and plays a key role in osmotic regulation in living organisms. In the present study, a cDNA sequence encoding the Na+/K+-ATPase alpha subunit from the monogonont rotifer, Brachionus koreanus was cloned by rapid amplification of cDNA ends technique. To investigate the role of this enzyme in osmotic stress, enzymatic activities of Na+/K+-ATPase were measured after exposure to different salinities for 48 h. The full-length Bk Na+/K+-ATPase cDNA was 3069 bp-long, encoding a 1022-amino acid polypeptide. Bk Na+/K+- ATPase possesses eight membrane spanning regions and five conserved domains. Phylogenetic analysis showed that Bk Na+/K+-ATPase had high identity with those of other species, and was closely clustered with other Brachionus sp. These findings indicate that this protein was conserved both structurally and functionally. B. koreanus Na+/K+-ATPase activity was stimulated in both hyposaline (6 psu) and hypersaline (32 psu) conditions, suggesting that this protein may play a role in osmoregulation. This study would provide better understanding of the physiology of B. koreanus and this enzyme may be useful as a molecular marker for evaluation of osmotic stress in aquatic environment.

Synthesis of Li+-selective 14-crown ether (CE) having rigid and bulky subunits was reported. CE-poly(vinyl alcohol) (PVA) dope solutions were electrospun. CEs were immobilized on PVA matrix via acid-catalyzed acetalization using novel aerosol method. Structures of new compounds and their immobilization to PVA were confirmed and characterized. Adsorption experiments show superior lithium capacity and selectivity among previously reported solid-supported CEs. Dihydroxy-dibenzo-14-crown-4 ether-PVA nanofiber membrane showed superior performance. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2015R1A2A1A15055407) and by the Ministry of Education (No. 2009-0093816).

Double-stranded RNA(dsRNA) had been used to specitically suppress target gene expression at post-tanscription level. Injection of dsRNA to hemocoel is the most efficient to knockdown target mRNA. However, some insects have shown to be susceptible to feeding dsRNA. Spodoptera exigua was susceptible to dsRNA at oral treatment. Especially dsRNA specific to β-integrin was potent to survival of S.exigua larvae. This study advanced our dsRNA application technology by generating recombinant E.coli expressing dsRNA specific the β-integrin. A recombinant vector L4440 was constructed with a partial β-integrin gene under T7 RNA polymerase promoter. The recombinant vector was used to transform HT115 competent cells of E.coli. The transformed E.coli expressed the dsRNA. The production of dsRNA was proportional to the bacterial number. By feeding the recombinant E.coli, S.exigua underwent significant mortality. By adding E.coli expressing Cry1Ca Bt toxin to E.coli expressing dsRNA, S.exigua exhibited highly enhanced mortality. This study suggests a possibility to use a recombinant E.coli expressing dsRNA to control S.exigua.

The cotton aphid, Aphis gossypii (Glover), is one of the main pests in various vegetable crops due to insecticide resistance in Korea. Some insect pests noticed neonicotinoid insecticide resistance such as Nilaparvata lugens, Bemisia tabaci and Myzus persicae etc. and the major player which contributed for developing the resistance was over-expression of P450, particularly CPY6 family. However, A. gossypii was a unique case that they developed non-P450 dependent resistant mechanism. Previously we reported that two point mutations (RtoT in nicotinic acetylcholine receptor, nAChR, beta 1 subunit and RtoT with LtoS in a transcript variant) contribute to develop the imidacolprid resistance in A. gossypii. Moreover we surveyed the mutation(s) in various local field populations. Based on the 3D modeling, we hypothesize that RtoT mutation can reduce the imidacloprid sensitivity. A stretch of 33 amino acid was deleted in the N-terminal region of original transcript of nAChR beta 1 subunit that contained RtoT with LtoS mutations in resistant strain. Among the two transcripts, only original transcript differently expressed between imidacloprid susceptible and resistant strain (resistant ratio = 3,800). Six alpha subunit (1∼5, 7) transcript levels were not significantly different between two strains. Therefore mutation and down-regulation of nAChR beta 1 subunit is also associated with imidacloprid resistance in the A. gossypii.

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2,517 bp) of a integrin subunit β1 (HaITGβ1) was cloned from the oriental tobacco budworm, Helicoverpa assulta. Phylogenetic analysis showed that HaITGβ1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (61%) to β1 of other Noctuidae such as Spodoptera exigua and S. litura. Structural analysis of the HaITGβ1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that HaITGβ1 was expressed in all developmental stages and all tested tissues of H. assulta. Injection of double-stranded HaITGβ1 RNA (dsHaITGβ1) into third instar of H. assulta suppressed HaITGβ1 expression and resulted in significant delay from last larval stage to pupal stage. The dsHaITGβ1 injection significantly impaired nodule formation of H. assulta in response to bacterial challenge and hemocyte adherence. These results suggest that HaITGβ1 plays crucial roles in cellular immune responses as well as development in H. assulta.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pests in various vegetable crops. In Korea, some field populations of A. gossypii especially in greenhouse showed high resistance against neonicotinoids. The imidaclopridresistant strain (IR) selected from one of the greenhouse strains was found to be about 3,800 folds more resistant to imidacloprid, compared to the susceptible strain (S), as judged by LC50 values. To identify differentially expressed genes in IR, an isogenic strain, reverse susceptible strain (IRS) was generated from IR and comparative transcriptome analyses based on GS-FLX were conducted using total RNAs extracted from both IR and IRS. Also we confirmed protein expression patterns by 2DE and detoxification enzyme over-expression by synergist test. However there was no significant variation among IR, IRS and S. Comparison of the nucleotide sequence of seven nicotinic acetylcholine receptor (nAChR) subunit (alpha 1-5,7 and beta 1) genes from S and IR strain revealed a point mutation causing an arginine to threonine substitution (R81T) in the loop D region of the nAChR beta 1 subunit of the IR. These mechanisms were also reported in M. persicae and this amino acid change confers a vertebrate-like character to the insect nAChR and results in reduced sensitivity to neonicotinoids. Moreover an extra point mutation, L80S (leucine to serine substitution) was also detected nearby R81T mutation in nAChR beta 1 subunit variant. These mutations can be an additive factor in imidacloprid resistance in A. gossypii. This is the first report of imidacloprid resistance mechanism in A.gossypii. Further, this would be helpful in managing A. gossypii resistant populations in field.

콩과(Fabaceae) 작물 해충인 팥나방(Matsumuraeses phaseoli)과 어리팥나방(M. falcana) (나비목: 잎말이나방과)은 형태적으로 매우 유사하여 종 구별이 힘든 것으로 알려져 있다. 본 연구에서는 PCR-SSP(PCR with Sequence Specific Primers) 방법으로 두 종을 빠르고 정확하게 구별할 수 있는 판별법을 찾고자 두 종의 미토콘드리아 시토크롬 옥시다제 I(mtCOI) DNA 부분영역(439 bp)의 염기서열을 해독하였다. 그리고 다른 나방 종의 mtCOI 염기서열과 함께 나열하여 비교한 후 팥나방과 어리팥나방에서 종 특이적으로 차이가 나는 단일 뉴클레오티드를 프라이머의 3ʹ말단으로 하는 염기서열 특이 프라이머 조합을 만들었다. PCR 산물들을 전기영동 한 결과, 어리팥나방은 245 bp, 팥나방은 409 bp와 245 bp의 특이적 밴드 패턴을 보여 두 종을 구별할 수 있었다.

사체의 사후경과시간을 추정하는데 사체에 출현하는 검정파리과 곤충을 이용할 때 파리 종의 정확한 동정이 요구된다. 최근 미토콘드리아DNA(mtDNA)의 염기서열이 종의 동정에 많이 이용되고 있으며, COI-II 유전자 부위는 상대적으로 염기서열 변화가 많기 때문에 종간의 분류를 위한 마커로 적합하다고 알려져 있다. 본 연구에는 부산에서 채집된 검정파리과에 속하는 파리들의 mtCOI의 염기서열을 분석하고, GenBank에 등록된 종들과 비교하였다. COI 염기서열의 한 부분을 증폭하여, 염기서열들을 398 bp 크기로 정렬하였다. 전체 34종의 계통수에서 Lucilia 와 Calliphora 속 사이는 확연한 계통학적 분리가 나타났지만, 동일 속내 일부 종 사이에서 계통학적 거리가 나타나지 않았다. 계통수에서 C. stygia 와 C. albifrontails, C. augur 와 C. dubia, L. cuprina와 L. sericata 및 L. caesar 와 L. illustris 사이에서는 혼합된 집락이 나타났다. 전제 34종 가운데 표본이 1개체뿐인 종을 제외한 16종에서 종내 염기서열 변이도를 조사한 결과 ~ 0.044까지의 종내 염기서열변이도를 나타내었으며, 종 내의 염기변이결과로 각 종에 따라 1 ~ 17개의 haplotype 이 관찰되었다. 동일 종 내에서 다양한 haplotype이 보임으로서 종의 동정에 이용될 수 있는 염기서열의 정보가 매우 제한적임이 시사되었다. 다양한 지역에서 다수의 개체를 이용한 연구를 통하여 각 종들에 대한 종내 변이의 범위를 확인하는 연구가 필요할 것으로 사료된다.

콩은 식물성 단백질 및 지방의 주요 공급원이고 콩 종실에는 기능성 성분이 많이 함유되어져 있어 소비 가 점차 증가하고 있지만 Lipoxygenase, Kunitz Trypsin inhibitor, 7S α′-subunit 단백질과 같은 성 분들이 존재하는데 이는 품질과 영양가치를 떨어뜨리고 섭취시 알러지를 일으키기도 한다. 콩에서 유전적 으로 이러한 성분이 결핍되어져 있는 유전자형의 선발은 품질이 우수한 콩 육종의 기초단계이다. “개척2 호”와 PI506876의 교배로부터 434개의 F2 종자를 얻어 F2 종자의 일부를 사용하여 SDS-PAGE로 각각 의 종자를 분석한 결과 Lipoxygenase와 Kunitz Trypsin inhibitor 및 7S의 α′-subunit 단백질이 모두 결핍되어져 있는 lx1lx1lx2lx2lx3lx3titicgy1cgy1 유전자형을 가진 종자를 선발하여 F2 식물체로 길러 성 숙 후 F3 종자를 수확하였다. F3 종자로부터 Lipoxygenase, Kunitz Trypsin inhibitor, 7S α′-subunit 단백질이 모두 결핍되어져 있음을 재확인하였으며 선발된 종자는 고품질 콩 품종 육성에 유용하게 활용될 것으로 기대된다.

Glycoprotein hormones have a common α-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the α-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the NH2-terminus of the α-subunit to the COOH-terminus of the β-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form Δ96, Lys was substituted at position 95 to form Δ95, His was inserted at position 93 to form Δ93 and Tyr was substituted at position 87 to form Δ87. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and Δ96, Δ95, and Δ93 mutants were efficiently secreted into the medium but the Δ87 mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the Δ87 mutant. However, the Δ87 mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The Δ95 and Δ93 mutants were completely inactive in both the LH- and FSH-like activity assays. The Δ96 mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the Δ96 mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the α-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the α-subunit is very important for the secretion and functioning of this hormone.