리스테리아 모노사이토젠스 감염에 대한 선천면역에서 SIRT6의 역할

오준택 전북대학교 일반대학원 2021 국내석사

Listeria monocytogenes is a foodborne pathogenic gram positive bacterium. When L. monocytogenes invade the body, the bacteria grow in liver or spleen mainly. After bacterial infection, myeloid cells such as macrophages, dendritic cells and neutrophils which contribute to innate immune responses infiltrate into infection site and eliminate bacteria. Sirtuin 6 (SIRT6) which belongs to Sirtuin family of NAD+-dependent deacetylase regulates glucose metabolism, fat metabolism and embryonic stem cell differentiation. SIRT6 also affect pro-inflammatory and anti-inflammatory responses but it doesn’t demonstrate clearly whether SIRT6 is involved in innate immune responses against gram positive bacteria. Thus, experiment was designed to elucidate role of SIRT6 in innate immune responses during infection. First, SIRT6 wild type mice (WT) and knockout (KO) mice were infected with L. monocytogenes to check the effect of SIRT6 in susceptibility. Additionally, SIRT6 WT and KO mice were challenged with 5×105 CFU of L. monocytogenes and sacrificed to collect serum, liver and spleen at indicated day. The liver and spleen were homogenized to assess colony forming units (CFU) and RNA expression level of cytokine. In addition, the liver was treated with paraffin for tissue staining. The spleen and serum were used for assessment of secretion of cytokine. After SIRT6 WT and KO mice were infected with L. monocytogenes, survival rate of KO mice was higher than WT. Deletion of SIRT6 in mice caused decreased production of inflammatory cytokine such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and bacterial colony forming units of KO mice were lower than WT mice in liver or spleen. In histological evaluation, lesions of SIRT6 KO mice were relatively mild compared with WT mice. Altogether, this study suggests possibility that SIRT6 negatively controls innate immune responses against L. monocytogenes and can be a novel regulator of bacterial infection.

조력 T 세포 활성화와 분화에서 Sirt6의 역할 연구

유승록 전북대학교 일반대학원 2021 국내석사

The Sirtuin family belongs to Class III histone deacetylase (HDAC)which removes acetyl groups from histones and non-histone proteins. The function of SIRT6, one of the members of Sirtuin family, has been shown to be associated with aging metabolism, DNA repair, and immune responses. The role of SIRT6 in immune system is mostly focused on inflammation mediated diseases and macrophages were main target immune cells in those studies. Although previous studies suggested a possible role of SIRT6 in T cells, only limited studies have been performed and it is still unclear whether SIRT6 play a role in CD4+ T cell activation and differentiation. CD4+ T cells are critical mediators of the adaptive immune response to various pathogens. Upon recognition of antigens presented by antigen presenting cells, these cells are activated and differentiated into subsets of T helper (Th) cells including Th1, Th2, Th17, and iTreg, which modulate immune responses by producing cytokines. Therefore, understanding the networks of cytokines and transcription factors during CD4+ T cell differentiation is critical for diagnosis and treatment of many immune-related human diseases. Inappropriate CD4+ T cell response to pathogens can be associated with chronic infection and excessive responses result in tissue damages. In addition, unregulated CD4+ T cell differentiation can cause autoimmune or allergic diseases. Therefore, understanding the molecular mechanisms by which CD4+ T cell activation and differentiation is regulated is critical for development of novel therapeutics for immune-mediated diseases. The goal of this study is to demonstrate a possible role of SIRT6 in CD4+ T cell development, activation and differentiation. Even with SIRT6 deficiency in CD4+ T cells, there was no difference in the frequency of T cell development and activation effector T cells in the body. Interestingly, however, Treg and follicular T cells showed an increased frequency compared to WT. These results indicate that SIRT6 may be involved in immunoregulation of CD4+ T cells. In vitro, there was also no difference in early activation of CD4+ T cells, but there were differences in proliferation and differentiation. In the case of proliferation, when SIRT6 was deficient, proliferation with a slight defect was observed. In the case of differentiation, even if SIRT6 was deficient, there was no significant difference in Th1. However, in the case of Th2 and Treg, high frequencies were shown, whereas in the case of Th9 and Th17, low frequencies were shown. That is, it indicates that SIRT6 has a role in the differentiation of CD4+ T cells. These results suggest that a novel effect of SIRT6 could be discovered from an immunological point of view.

전혈을 이용한 돼지생식기호흡기증후군 분리주의 포괄적 면역세포 프로파일연구

살람사미룰 전북대학교 일반대학원 2021 국내박사

Porcine reproductive and respiratory syndrome (PRRS), a major viral disease of pigs, causes huge economic losses to the swine industry worldwide. Moreover, PRRSV strains have evolved to achieve high virulence features. Different pathogenic PRRSV strains lead to different possible outcomes. The continuous evolution and genetic diversity have posed challenges in understanding the immunopathogenesis of the disease. To evaluate the immune responses in peripheral blood usually, PBMCs are obtained and evaluated. PBMC isolation is a time-consuming procedure, introduces technical variability and a relatively large volume of blood is required. In contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires a lesser sample volume, laboratory training, and equipment. Herein, two consecutive studies were performed; in the first study whole blood (WB) assay was standardized to evaluate immune responses and in the second study two genetically different PRRSV strains were assessed for their immunomodulation in host and practicability of WB to replace PBMC for evaluation of immune responses was assessed. The first study aimed to develop a porcine WBA for in vitro evaluation of immune responses. Heparinized WB was serially diluted (non-diluted, 1/2, 1/4, 1/8, and 1/16) in RPMI-1640 media, having pre-added phorbol myristate acetate, and ionomycin (PMA/ION) and samples were kept in humidified air at 37°C and 5% CO2. After 24 hours, cells were stained for IFN- γ secreting T-cells, followed by flow cytometry, and the supernatant was analyzed for TNF-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (poly I:C), Streptococcus suis field isolate (S. suis FI), and S. suis reference strain (S. suis RS), heat killed S. suis (S. suis HK) and PRRSV. Our results demonstrated that on stimulation of WB by PMA/ION, cell surface immunofluorescent staining showed consistent results at all dilutions while in non-diluted WB, intracellular cytokine staining turned out to be obstinate. With an increase in dilution, the frequency of IFN-γ secreting T-cells and concentration of TNF-α in the supernatant of WB increased and were optimal at 1/8. On stimulation of WB with LPS or poly I:C, TNF-α and IL-10 cytokine levels increased significantly. Further, FI and RS induced the production of IL-10 while TNF-α was specifically induced by RS. Additionally, PRRSV strains increased the frequency of CTLs (cytotoxic T-cells), and IFN-γ was induced in the supernatant of re-stimulated samples, by homologous strains. In conclusion, we propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses. PRRSV causes great economic loss to the swine industry. Mostly PBMCs are used to evaluate immune responses while WB has been rarely used to evaluate immune responses in porcine. The isolation procedure of PBMC removes polymorphonuclear cells and other natural constituents of the serum. On the other hand, WB maintains the natural constituents and is less laborious to assess immune responses compared to PBMC. So, our second study aimed to evaluate the immune responses against two genetically different PRRSV strains isolated in Korea and the practicability of WB to replace PBMC for evaluation of immune responses. In this study, we challenged four weeks old piglets intramuscularly (IM) with two strains of PRRSV: 10D415 and NA10, and analysed immune cell profiles using WB, and PBMC. The results displayed that 10D415 and NA10 significantly induced NKp46+CD8+, and CTL responses, in comparison to NC. Further NA10 induced a higher CTL response compared to 10D415. Moreover, on evaluating γδ T-cells, results displayed that γδ+CD8- T-cells were downregulated significantly by NA10. The evaluation of association between WB and PBMC showed a strong correlation for cellular innate and adaptive immune cells. Conclusively, our results indicate that 10D415 and NA10 being genetically different altered immune responses differently. Moreover, WB and PBMC displayed comparable responses for different immune cell subsets. Taken together our results justify the uses of WB as an alternative for PBMC to study cellular immune responses modulation by PRRSV in the peripheral blood.

야생콩과 베타카로틴강화 GM콩의 교잡종에 대한 환경 위해성 평가

장예진 전북대학교 일반대학원 2021 국내박사

As the cultivation area for genetically modified (GM) crops is continuously increasing, there has been a growingthe concerns about the environmental release of GM crops are also growing. This study was conducted to evaluate and monitor the safety of GM soybeans and hybrid soybeans, which could occur due to hybridization between wild soybeans and GM soybeans when GM soybeans are unintentionally released to the environment. β-carotene-enhanced GM soybean and hybrid soybeans that have been artificially produced by wild soybeans and GM soybeans are used for this study. The hybridization rate between GM soybean and wild-type soybean was 0.21%. The hybrid soybean has intermediate characteristics of growth including seed phenotype between their parents. The hybrid soybean showed the a lower over-wintering dormancy rates than the wild-type soybean, and the GM soybean was shown to have low or no dormancy rates. One of the major factors for the safety evaluation of GM crops is the nutritional equivalence. In this study, the all the nutrients that showed no differences in hybrid soybeans compared to GM soybean and wild soybean and the contents of all nutrients were within the natural variation. It was found that the variation of nutrient composition was affected by environmental factors such as growing conditions. Therefore, this concluded that the nutritional safety of hybrid soybeans and GM soybeans are not different from cultivated soybean and wild soybean. Additionally, 40 bioactive compounds were identified using GC-TOFMS. These results can be used for the development of discriminant markers for GM and hybrid crops. The bacterial community analysis of rhizosphere is one of the approaches for evaluating the environmental risk assessment of GM crops. There is no significantly effects on the bacterial community of GM soybeans and hybrid soybeans. To confirm the horizontal gene transfer in GM soybeans or hybrid soybeans to the rhizosphere soil microorganisms, no gene was detected in genomic DNA in the soil. Also, we experimented to investigate whether GM soybean and hybrid soybean could survive and persist seed vitality and for assessing weediness. GM soybean and hybrid soybean did not survive with weeds. So, the results confirm that GM soybeans and hybrid soybeans cannot persist under uncultivated environments and competition with weeds.

배추의 염 스트레스를 경감시키는 Herbaspirillum sp. strain GW103의 유전체적 분석

이건웅 전북대학교 일반대학원 2013 국내박사

Mutual interactions between plant and rhizosphere bacteria can facilitate plant growth and reduce both biotic and abiotic stresses on plants. I hereby report the characteristics of Herbaspirillum sp. strain GW103 isolated from rhizosphere of Phragmites australis grown in reclaimed land. The strain GW103 exhibited increase in weight and length of shoot and root of Chinese cabbage (Brassica rapa L. ssp. pekinensis) under salt stress. The strain was capable of producing plant beneficial factors, such as auxin, siderophore, and ACC deaminase. In addition, the strain GW103 increased K+/Na+ ratio and decreased the proline content in Chinese cabbage roots, which could alleviate salt stress. When treated to Chinese cabbage, isolate GW103 displayed an ability for root colonization that was monitored by GFP-tagging method using a mini-Tn7 transposon. The whole genome sequences were determined by next generation sequencing. The genome annotation revealed the presence of the ACC deaminase gene and genes related to the biosynthesis pathways of auxin, siderophores, and urease which were known as plant beneficial factors. The genome also contained genes that encode factors required for penetration into plant cells, attachment on the surface during plant root colonization. In conclusion, current study strongly suggests that strain GW103 is a plant growth promoting bacteria and increase stress tolerance on plant via plant-microbe interaction.

휘발성 디메틸 디설파이드가 식물 발달 및 식물 병원균에 미치는 영향

Tyagi, Swati 전북대학교 일반대학원 2018 국내박사

미생물 기원의 휘발성 유기 화합물(미생물 휘발성 화합물, MVCs)은 식물과 병원체 사이의 상호작용을 포함한 대부분 생물체의 많은 생리학적 과정에 영향을 미친다. MVCs는 대기, 물, 토양을 통하여 멀리 이동을 할 수 있으며, 식물 및 관련 미생물들의 생리학적 과정에 영향을 끼칠 수 있다. 실질적인 예시로 MVCs는 식물 병원체를 억제하고 식물의 생장을 증진시킴에 따라 간접적인 식물 촉진제로써 역할을 할 수 있음이 보고되었다. 그러나 MVCs의 작용 기작 및 기능을 이해하기 위해서는 MVCs의 특성이 어떠한 영향을 미치는지에 대한 연구가 필요하다. 지난 20년간 MVCs 혼합물과 수용체에 대한 효과에 대해 연구되어 왔지만 기작에 대해서는 아직 밝혀진 바가 없다. 기존에 알려진 MVCs는 단일물질 또는 혼합물로써 여러 기작들을 조절할 수 있다. 본 연구에서는 식물과 식물 병원체(곰팡이, 세균) 및 미생물 기원의 휘발성 화합물 유사체인 이황화메틸 (Dimethyl disulfide, DMDs)을 활용하여 이들 사이에서 발생하는 식물 방어 작용 및 병원균의 독성과 관련된 후속 신호 전달 네크워크와 상호작용에 대해 조사하였다. 식물에 대한 DMDS의 영향을 알아보기 위해, Arabidospsis 모종을 다양한 농도의 휘발성 DMDS에 노출시키고 일주일 동안 배양하였다. DMDS에 노출된 Arabidospsis 모종은 처리하지 않은 식물에 비해 식물 생장 및 측근 발달이 상당히 개선된 것으로 나타났다. 근본적인 기작을 파악하기 위해, DMDS에 의한 뿌리 시스템 구조의 변화를 자세히 조사해보았다. 휘발성 DMDS를 처리한 경우에 식물에서 초기 옥신 반응 유전자 (SAUR-AC1, GH3.1 IAA14)와 옥신 신호 전달기의 발현 (DR5::GUS), 옥신 신호 강도가 향상되었다. 결론적으로 옥신 신호 전달 이 소실된 Arabidopsis의 유전자(tir1-1, arf7, arf7, arf19) 변성은 측근 형성 및 뿌리털 발달에 변화를 유발하지 못하였으나, DMDS는 Arabidopsis에서 옥신 신호 전달 경로를 통해 뿌리 시스템 구조를 수정하여 식물의 성장 및 발달에 기여한다는 것을 알 수 있었다. 생물학적 방제 분야에서 MVCs의 영향이 광범위하게 보고된 이후, 진행된 연구들은 곰팡이의 성장과 작용기전에 대한 외인성 DMDS의 효과를 이해하는데 초점이 맞추어져 있었다. 이와 관련하여 병원성 통제 인자로써 휘발성 DMDS의 가능성을 확인하기 위해 우리는 다수의 곰팡이 균에 DMDS를 노출시켰고 거의 모든 병원체가 DMDS에 의해 저해 영향을 받는다는 것을 알 수 있었다. 또한 Sclerotinia minor를 이용한 균핵병 실험을 수행하였으며, S. minor에 DMDS를 노출시킨 뒤 에르고스테롤, 생체량, 균핵 형성 및 발아, 균사 성장, 최소 억제양 (MIQ)과 같은 다양한 조사를 실행하였다. DMDS의 MIQ가 50 μM/mL 일 때, S. minor는 80%까지 성장이 감소되는 것을 확인할 수 있었고 성장 저해, 균핵 형성 및 발아, 생체량도 현저히 감소하는 것으로 나타났다. 또한 DMDS에 노출된 균사는 처리하지 않은 대조군에 비해 심각한 영향을 받는 것으로 나타났다. TEM (transmission electron microscopy), 형광 현미경, 주사현미경, 세포구성물의 방출을 통해 알아본 생리학적, 형태학적 및 초미세구조의 연구 결과는 막 손상 기작이 원인이라는 것을 증명하였다. 또한 탈메틸효소 유전자의 발현과 총 에르고스테롤 함량이 감소하였으며, 이는 세포막의 손상 및 에르고스테롤 생합성의 저해 때문인 것으로 나타났다. MVCs가 토마토 식물의 방어 관련 유전자의 발현을 증가시키고 식물체의 병 저항성을 감소시킨다는 것은 분명하지만 DMDS와 같은 외인성 화합물에 노출된 병원균의 독성 및 병원성의 변화는 간과되어 왔다. 따라서 휘발성 DMDS가 식물 생장 촉진에 미치는 영향을 파악하기 위해 시험관 내 실험(in vitro)과 실증(in vivo) 실험을 진행하였다. DMDS에 노출된 두 조건 모두에서 토마토 식물의 생장이 크게 개선되었다. 또한 병원체 저항성과 식물 생장 및 방어 관련 유전자 발현의 유도에 DMDS가 어떠한 영향을 끼치는지 알아보기 위해 DMDS를 처리한 것과 처리하지 않은 대조실험을 진행하였다. 그 결과, DMDS에 노출된 경우에 곰팡이 병원체에 대한 길항작용이 발생되어 식물을 보호하는 것으로 나타났다. 그리고 식물 방어 관련 유전자 (APX2, PA2, PR1, PR5), 옥신 (ARF5), 익스팬진 발현이 증가되었으나 에틸렌 생산과 관련된 유전자의 발현은 감소되는 것으로 나타났다. 또한 과산화수소 및 칼로오스 축적은 DMDS를 처리한 실험구가 처리하지 않은 식물에 비해 훨씬 더 강하게 나타났다. 이러한 연구결과는 DMDS, 토마토 식물(숙주) 및 병원균간 상호작용을 통하여 식물의 생장 증진 및 병원균에 대한 저항성이 높아질 수 있다는 새로운 가능성을 제시하였다. 다른 생체 시스템과 마찬가지로, 세균은 생물학적으로 독성 혼합물의 존재를 포함하여 주변 환경 조건을 감지하고 이에 반응을 하며, 이러한 반응에서 특정 세포 내 및 세포 외 신호를 탐지 단백질을 통해 감지하고 적응 반응을 조절하게 된다. 식물 병원성(Pseudomonas syringae, Ralstonia solanoseraum) 또는 다른 세균(Escherichia coli)에 대한 MVCs의 영향을 알아보기 위해 연구를 진행하였다. DMDS와 같은 특정 화합물의 독성 영향 및 식물과의 상호작용은 아직 밝혀지지 않았으므로 본 연구에서는 독성이 없는 수준에서 DMDS의 유무에 따른 P. syringae pv. tomato DC3000 (Strain DC3000)의 전체적인 유전자 발현을 전사체 분석을 통해 조사하였다. 그 결과 다양한 유전자가 DMDS에 노출되면 아미노산의 물질대사 및 에너지 생성, 운동성과 관련된 유전자들이 다르게 발현된다는 것을 알 수 있었다. 이 유전자들 중 일부는 일반적인 스트레스 반응과 관련이 있으며 다른 일부는 VOC에 대한 반응에만 국한 된다는 것을 알 수 있었다. 또한 DMDS에 노출 시켰을 때, 세균의 병독성에 영향을 미치고 토마토 식물에서 질병 증상을 54% 감소시킨다는 것을 알 수 있었다. 이를 통하여 DMDS는 병원성 세균을 통제하는데 효과적이었으며 DMDS에 대한 새로운 생물학적 효과를 확인할 수 있었다. 종합적으로 DMDS는 병원체 감염에 대한 식물의 저항성을 향상시킬 뿐만 아니라 식물의 방어, 발달, 생장에 관련한 기작에 영향을 준다는 것을 알 수 있었다. 또한 DMDS와 식물 및 식물 병원체(세균/곰팡이) 간에 서로 상호작용한다는 것을 알 수 있었고 DMDS는 옥신 신호를 이용하여 Arabidospsis의 뿌리 시스템 구조를 조절하는 반면, 막 손상 기작을 통해 곰팡이 병원균의 성장을 억제한다는 것을 알 수 있었다. S. minor에 감염된 토마토 식물의 발병도는 DMDS를 처리하였을 때 성장 및 저항유전자의 발현이 증가되면서 70% 감소하는 것으로 나타났다. DMDS가 처리된 P. syringae 세포의 전사체 분석을 통해 병독성 및 발달, 운동성, 생장과 관련된 유전자들의 발현에 DMDS가 큰 영향을 주는 것이 확인되었다. 그 외에도 DMDS의 처리는 P. syrinage 세포의 연축 운동성을 포함한 다양한 운동성을 감소시킬 뿐만 아니라 병원균의 독성을 감소시켰다. 그러나 식물과 병원균 사이에 DMDS가 미치는 연관성을 보다 잘 이해하기 위해서는 DMDS와 같은 휘발성 화합물을 받는 수용체와 활동스펙트럼 분석에 대해 보다 더 깊이 있는 추가적인 연구가 필요하다고 사료된다.

반려동물과 주변 환경에서 분리된 Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pseudintermedius의 역학적 특성 연구

이해성 전북대학교 일반대학원 2021 국내석사

Recently, research on antibiotic resistance based on the concept of One Health, which provides optimal health to all through the connection between humans, animals, and ecosystems, is earnest at domestic government research institutes. Companion animals living in the surrounding environment with humans can be a very important model for understanding One Health, which studies the transmission mechanism of antibiotic-resistant bacteria between individuals. This study is to identify antibiotic-resistant bacteria distributed in humans, companion animals, and the surrounding environment, and to evaluate the epidemiologic correlation between individuals and the environment. Through this, the purpose is to understand antibiotic-resistant bacteria distributed in companion animals, their owners, and the surrounding environment, and to preemptively prevent the spread of resistant bacteria in the community. In this study, from 2017 to 2019, specimens were collected from humans, companion animals, residential environments, and veterinary hospital environments in three areas: the metropolitan area, the central area, and the southern area. Staphylococcus aureus 139 strains, Staphylococcus epidermidis 776 strains, and Staphylococcus pseudintermedius 375 strains were identified in all collected samples. Among these strains, 85 strains of methicillin-resistant S. aureus, 443 strains of methicillin-resistant S. epidermidis, and 158 strains of methicillin-resistant S. pseudintermedius were identified. The methicillin-resistant staphylococci strains were specified in molecular epidemiological methods, such as Staphylococcal Cassette Chromosome mec (SCCmec) typing, multilocus sequence typing (MLST), and virulence factors. Based on the results, pair strains (clones) between human and animal were selected. For MRSA, SCCmec type IV. ST72 pair strains were found in two households with people, pet, and environment and MRSP pair clones were identified in one household. Through molecular epidemiologic results, whole genome sequencing results of strains identified as the same pair in humans, animals, and environment, CDS, tRNA, rRNA, and antibiotic resistance genes were all identified with the same genetic structure. In conclusion, it was confirmed that the staphylococcus aureus distributed in the space where humans and companion animals live together can spread antibiotic-resistant bacteria between individuals according to physical contact with the environment.

Biocontrol of popcorn disease in mulberry (Morus australis L.) using Bacillus thuringiensis C25

술타나 라지아 전북대학교 일반대학원 2016 국내박사

Mulberry (Morus spp.) plants are valued worldwide for their economic importance as high nutritive value with wide range of use in the human diet, medicine and sericulture industry. The annual productivity of mulberry fruits is greatly reduced in Asia since it is prone to popcorn disease. In Korea, two fungal species (Ciboria. shiraiana and Scleromitrula shiraiana) have been reported as the major causal organisms. However, their morphological features along with control measures are not yet studied well. In this thesis, Ciboria carunculoides (Siegler & Jenkins) Whetzel is reported to be present in Korea, which was confirmed by the isolation of fungal tissue followed by 5.8S rRNA sequencing. The detailed features of anamorphic stage is described, which exhibited ampuliform to navicular shaped conidiophores developed on short mycelia measured as (5.1) 9.57−13.34 × 1.2−1.58 μm. Conidia were one celled, pear shaped and précised as 1.69−3.44 × (1.54) 1.7−2.68 μm. C. carunculoides also exhibited the various morphological alterations which might be associated with the maintenance of cell viability and asexual propagations inside host tissues. On the other hand, in vitro antifungal assays were conducted to ascertain efficient biological control agents (BCAs) against C. shiraiana. Initially, C. shiraiana was isolated from mulberry drupelets showing the popcorn disease symptoms and its (a)sexual morphologies were delineated. Apothecia of C. shiraiana were successfully induced in vitro from the overwintered sclerotia, which was used as a system to monitor the antifungal activities of selected bacterial strains. Two bacterial isolates, Enterobacter sp. C5 and Bacillus thuringiensis C25 strongly suppressed the elongation and fresh weight accumulation of apothecia, width of hymenia, and formation of ascus, ascospore in C. shiraiana. In addition, ultrastructural studies revealed the degradation of hymenium cells by both bacterial strains. Ultimately, the treatment of mulberry trees with B. thuringiensis C25 mitigated the incidence of popcorn mulberry disease under field conditions. In conclusion, the current studies provided the advanced insight into fungal behavior and development of effective controlling methods against mulberry popcorn disease.

Mycobacterium tuberculosis에서 유래한 ADK의 발현, 정제 그리고 결정화

김다솜 전북대학교 일반대학원 2016 국내석사

Actinorhodopsin, Candidatus Rhodoluna planktonica strain MWH Dar1에서 유래한 잠재적 카로테노이드 결합 로돕신의 특성화와 구조에 관한 연구

김다솜 전북대학교 일반대학원 2021 국내박사

ActR(Actinorhodopsn) protein is the rhodopsin, which is structurally composed of seven transmembrane helices with retinal as a photoreceptor. The function of ActR has been verified as external proton pumping by absorption of light energy. The characteristic of ActR is that the amino acid sequence of ActR is partially conserved with Xanthorhodpsin including carotenoid binding residues. Therefore, ActR has been expected as putative carotenoid binding rhodopsin so far. In this thesis, the gene of Candidatus Rhodoluna planktonica strain MWH-Dar1 is recombined to E. coli expression system, purified using the affinity chromatography and analyze three-dimensional structure. Moreover, the absorption spectra, proton-pumping, pKa, and photocycle of ActR were verified in the photochemical study. For structural analysis of ActR, the crystallization of ActR was performed using the LCP method and then the crystal structure of ActR was analyzed at 3.2 Å. For this result, the crystal structure of ActR shows seven transmembrane helices including joint loops at the extracellular and intracellular side. Thus the crystal structure of ActR shows as pentamer in asymmetric unit. Interestingly, glucose ring, which is considered as a part of octylglucoside, plays a role as mediator for pentamerization, as well as a salt bridge and hydrophobic residues between interfaces of each protomer. For canthaxanthin binding analysis, the double plasmid expression system was used during the ActR expression in vivo. Several ActRs, (ActR, ActR-K, and ActR mutants-K (G196 residue mutants)) were expressed and purified to analyze canthaxanthin binding with ActR. Absorbance spectroscopy and HPLC analysis showed the binding of carotenoids to ActR. As a result, an outward proton-pumping function is identified as typical bacteriorhodopsin, and the photocycle rate of ActR is 100 ms. Thus the pKa and maximum absorbance of ActR is 6.09 and 535 nm, respectively, while the maximum absorbance of ActR-K is 530 nm. In summary, the ActR plays a role as rhodopsin as bacteriorhdoopsin. In the crystal packing of ActR, the structure shows as pentamer form in asymmetric unit. In the second putative antenna binding, especially, G196 residue is strongly considered as a key residue of the second antenna binding in ActR. In addition, based on the photochemical and structural result, we can explain the functional and structural relationship of ActR and putative carotenoid binding rhodopsin.