Honey bee, Apis mellifera L., have been widely used as a model organism for biological science because of its highly developed sociality, specialized labor division and passive population management. In order to examine the expression patterns of genes putatively involved in social development in honey bee, quantitative real-time PCR (qRT-PCR) that has been widely used to investigate the expression level of target gene can be used in honey bee study. However, the selection and validation of optimal reference genes is a crucial step prior to running qRT-PCR. In the present study, therefore, the seasonal expression stability of five candidate reference genes in the abdomen of forager and nurse was investigated using three programs (geNorm, NormFinder and BestKeeper), and selected reference genes were validated by the normalization of expression level of vg encoding vitellogenin. Although three programs revealed slightly different gene stability values, overall the combination of two genes (rpS18 and gapdh encoding ribosomal protein S18 and glyceraldehyde-3-phosphate dehydrogenase, respectively) was resulted in the most suitable use for normalization of the target gene in forager. However, a single gene, either rpL32 or rpS18 in the nurse or either rpL32, rpS18, or gapdh in the comparison between foragers and nurses, were suggested to be applied for normalization of seasonal and labor-specific gene expression by qRT-PCR.

The fruit fly, Drosophila melanogaster, is a good model organism in various areas of biological science. Since D. melanogaster has been thought to be adapted to the chemical stress environment caused by the overripen, decay and fermented fruits, identification of the genes involved in chemical tolerance and investigation of their expression patterns are essential for better understanding of the physiological evolution in D. melanogaster. For investigation of the gene expression level, quantitative real-time PCR (qRT-PCR) can be applied to quantify gene expression level and selection of reliable reference gene(s) for normalization is an accurate step. In the present study, therefore, we validated the expression stabilities of ten candidate reference genes using three softwares (geNorm, NormFinder and BestKeeper) in D. melanogaster exposed to different concentrations of acetic acid, ethanol and 2-phenylethanol. Although three programs resulted in slightly different gene stability ranks, but overall tbp encoding TATA box binding protein was most stable gene in acetic acid and ethanol exposed fly, while nd encoding NADH dehydrogenase was the most suitable reference gene in the case of 2-phenylethanol treatment. In the comparison of three chemical treatment condition, nd was also suggested to be most optimal reference gene. In addition, optimal number of reference gene for accurate normalization was calculated by geNorm pairwise analysis, and selection of multiple reference genes was suggested to be better for target gene normalization method than use of a single reference gene.

It has been well known that IKK-β, -ε and –γ play a pivotal role in IMD pathway. In this study, TmIKK-ε was identified and their functions in countering pathogenic infections were investigated. We identified TmIKK-ε gene which including 2,196 bp nucleotides (encoding 731 amino acid residues). Domain analysis of TmIKK-ε indicates that there is one Serine/Threonine protein kinases catalytic domain. TmIKK-ε gene was highly expressed in 2 day-old pupal stage and the expression was gradually decreased until 1 day-old adults. Then the expression was slightly increased until 4 day-old adult stage. Tissue specific expression of TmIKK-ε mRNA was high in the gut, integuments and hemocytes in last instar larvae, and fat body, Malpighian tubules and testis in 5-daysold adult. In hemocytes, TmIKK-ε was drastically induced by E. coli injection after 3 h and by S. aureus at 3 and 12 h-post injection. In gut, expression level of TmIKK-ε was high at 6 h-post injection of microbial injection. Expression of TmIKK-ε in fat body was drastically induced by E. coli at 3 and 24 h-post injection while it was not significantly induced by S. aureus and C. albicans. To understand the immunological role of TmIKK-ε, gene specific RNAi and mortality assay were performed. TmIKK-ε RNAi caused increased larval mortality against E. coli, not S. aureus and C. albicans. Finally, to investigate the induction patterns of Tenebrio fourteen AMP genes in response TmIKK-ε RNAi, three microorganisms were treated into TmIKK-ε-silenced T. molitor larvae. Nine out of fourteen AMP genes were not induced by microbial challenge in TmIKK-β dsRNA-injected group. Taken together, our results indicate that TmIKK-ε may regulates nine antimicrobial peptide genes in response to microbial challenge in T. molitor fat body.

Autophagy is an important self-eating process to eliminate damaged or unused organelles. We identified nine autophagy-related genes (Atg) including AaAtg-1, -3, -4b, -4d, -5, -6, -8, -12 and -13 from the Asian tiger mosquito, Aedes albopictus. Developmental expression patterns indicate that mRNA levels of AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 were highly expressed in egg, whereas expression of AaAtg8 was high in 1stand3rdinstarlarvalstages. TissuespecificexpressionofthesegenesindicatesthatAaAtg1 was highly expressed in thorax and midgut in blood-fed adult female mosquitoes (BF), and head and thorax in sugar fed adult female mosquitoes (SF). Transcript level of AaAtg3 was high in thorax in BF, but head, thorax and Malpighian tubules in SF. AaAtg4b, -4d mRNA levels were significantly high in Malpighian tubules in BF, and head in SF, respectively. AaAtg-5 and -6 transcripts were highly expressed in head in BF, and expression of AaAtg-8 was high in Malpighian tubules in BF. Levels of AaAtg-12 and -13 mRNAs were significantly high in head and midgut in BF. Induction patterns of AaAtg genes against pathogens showed that AaAtg-1, -3, -4b, -8, -12 and -13 were strongly induced at 6 h-post injection of S. aureus, and mRNA levels of AaAtg-1, -3 and -13 were significantly induced by E. coli challenge after 3 h-post injection in SF abdominal carcass. In SF midgut, AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 transcripts were drastically induced at 9 h-injection of E. coli and S. aureus, while expression of AaAtg-8 was highly induced by S. aureus and C. albicans at 9 h-post injection. Each AaAtg gene was slightly induced by E. coli, S. aureus or C. albicans at different time points in abdominal carcass in BF. Interestingly, AaAtg-8 was not induced by microbial challenge. While eight other Atg genes except AaAtg-8 were highly influenced by S. aureus at 6 and 9 h-post injection, E. coli at 3 h-post-treatment, and 3, 6, and 9 h-post inoculation. In the future, we will characterize the functional roles of autophagy during mosquito-microbes interaction.

붉은불개미는 남미가 원산인 외래해충으로써 기후변화로 인한 기온 상승과 국가간 교역량의 증가를 통해 전세계적으로 서식범위를 확장해 나가고 있다. 국내에서는 붉은불개미가 2017년 9월 부산 감만항에서 처음 발견된 이후 올해까지 4차례 추가로 발견되어 국내 침입 및 정착 가능성이 점차 증가하고 있다. 곤충에서 인슐린 신호체계는 일반적으로 산란, 생장 및 발육, 대사계, 스트레스 저항성에 관여하는 것으로 널리 알려져 있다. 본 연구에서는 붉은불개미의 인슐린에 대한 생물학적인 기능을 이해하기 위해 인슐린 수용체(insulin receptor)의 발현을 억제한 후 나타나는 생리적 변화를 관찰하였다. 그 결과 체내빙결점(supercooling point)에 영향을 미쳐 붉은불개미의 저온생리에 영향을 준다는 것을 확인할 수 있었다.

붉은불개미는 세계 100대 침입외래 해충 중의 하나로 우리나라에서는 관리 해충으로 1996년도에 지정되었다. 2017년 9월28일 부산항 감만 부두에서 국내 최초로 발견된 후, 2018년에 부산항, 평택항 과 인천항에서 발견이 되었고, 9월18일에 대구의 아파트 공사장에서 내륙에서는 처음으로 발견이 되었다. 대구에서 발견된 붉은불개미도 아파트 건설을 위하여 중국에서 수입한 석재에 혼합되어 수입된 토양과 식물체와 같이 혼입된 것으로 추측이 된다. 그러므로, 현재까지 국내에서 붉은불개미가 번식을 하여 토착화되었다는 증거는 없다. 현재까지 붉은불개미에 대한 종 판명은 전문가에 의한 형태적인 분류가 대부분이다. 그래서, 붉은불개미에 대한 확증을 위해서는 채집된 개체를 검역기관으로 이송하여 분자진단을 실시해야 하므로, 최소 이틀의 시간이 필요하다. 현재 항원항체 반응을 이용한 붉은불개미 진단 키트가 해외에서 개발되어 판매되고 있지만, 낮은 민감도로 최소 3~5개의 충체가 필요한 실정이다. 현재 판매되고 있는 진단키트가 가지는 문제점은 이를 대신할 새로운 키트의 개발이 필요함을 보여주고 있다. 신속한 붉은불개미의 검역현장에서 진단을 가능하게하기 위한 노력 중의 하나로 우리는 붉은불개미의 복부에서 발현되는 유전체에 대한 연구를 수행하였다. 본 발표에서는 그에 대한 현재까지의 연구 진행과 결과를 설명하도록 하겠다. (본 연구는 농림축산 검역본부의 연구비 지원으로 이루어졌음).

Laodelphax striatellus is an important pest of rice due to not only sucking rice seedlings, but also transmitting serious plant viruses. Among various kinds of insecticide groups (carbamates, organophosphorus, neonicotinoids, etc.), carbofuran, a systemic carbamate insecticide, has been most extensively used to control rice pests including L. striatellus, resulting in widespread carbamate resistance in Korea and other Northeast Asia countries. To identify the genes associated with carbofuran resistance, we obtained a 14-fold higher resistant strain (SEL9) from the mixed-field population (SEL0) by consecutive selection. A transcriptome-based analysis was conducted and differentially expressed genes (DEG) were compared between the SEL9 and SEL0 strains. A total of 96,185,150 reads were analyzed, of which 62,860,430 reads were mapped. From these reads, 15,356 transcripts were annotated. A total of 327 up-regulated and 275 down-regulated genes were identified in the resistant SEL9 strain compared to SEL0 strain by DEG analysis. Gene ontology (GO) analysis was performed using DEGs showing statistical significance (P < 0.05). The GO analysis identified 1,320 genes in biological process group, 1,100 genes in cellular component group and 428 genes in molecular_function group.

과일이나 농작물의 부패 및 발효 환경에서는 Methanol, Ethanol, Acetic acid을 비롯한 다양한 화학물질들이 생산된다. Drosophila melanogaster는 이러한 발효·부패 환경에 서식하면서 일정 농도 이상의 다양한 화학물질에 지속적으로 노출되어 생존하도록 적응되어온 것으로 생각된다. 다양한 화학물질이 포함한 환경에 안정적으로 서식하기 위해서는 D. melanogaster는 화학물질에 능동적으로 반응하여 해독 유전자나 대사 관련 유전자의 발현량을 변화 시킴으로써 발효·부패 환경에서 생성되는 화학물질에 대한 높은 내성을 가지고 있을 것으로 판단된다. 현재까지 유전자의 발현량 측정을 위해 real-time PCR를 이용하여 reference gene의 발현량을 기준으로 정량화하는 방법이 가장 널리 사용되고 있다. 그러나 조직별, 환경별, 발달단계를 비롯한 다양한 조건에서 안정적으로 발현되는 reference 유전자 선정이 필수적으로 선행되어야 하므로 본 연구에서는 발효·부패 환경에서 생산되는 두 화학물질인 Methanol과 Ethyl Acetate에 노출된 D. melanogaster에서 안정적으로 발현되는 reference gene을 찾는 연구를 실시하였다. 본 연구에서는 다양한 농도의 Methanol과 Ethyl Acetate을 D. melanogaster에 노출시킨 후 RNA 추출과 cDNA 합성을 실시였고, 5가지 후보 reference gene (hsp22, nd, rpL18, tbp and ef-1b)의 안정적 발현 여부를 qRT-PCR을 통해 조사하였으며, 유전자 발현의 안정성을 측정하는 3가지 프로그램(geNorm, NormFinder, BestKeeper)을 이용해 비교·분석하였다. 본 학회에서는 연구의 과정과 그 결과를 발표하고자 한다.

4,4’-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control in Africa. Previous DDT use has resulted in predisposition of resistance, and with continued use resistance will increase further in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, has been widely used for elucidating insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold); however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of Gal4/UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has identified the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (mdr50, mdr65, and mrp1) in increased sensitivity to DDT. These findings have been further validated in 91-R flies using a nanoparticle-enhanced RNAi strategy, directly implication these genes in DDT resistance in 91-R flies.

Cold, salt and heat are the most critical factors that restrict full genetic potential, growth and development of crops globally. However, clarification of genes expression and regulation is a fundamental approach to understanding the adaptive response of plants under unfavorable environments. In this study, we applied an annealing control primer (ACP) based on the GeneFishing approach to identify differentially expressed genes (DEGs) in Italian ryegrass (cv. Kowinearly) leaves under cold, salt and heat stresses. Two-week-old seedlings were exposed to cold (4°C), salt (NaCl 200 mM) and heat (42°C) treatments for six hours. A total 8 differentially expressed genes were isolated from ryegrass leaves. These genes were sequenced then identified and validated using the National Center for Biotechnology Information (NCBI) database. We identified several promising genes encoding light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, Lolium multiflorum plastid, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase. These genes were potentially involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance in plants. However, this study provides new insight regarding molecular information about several genes in response to multiple abiotic stresses. Additionally, these genes may be useful for enhancement of abiotic stress tolerance in fodder crops as well a crop improvement under unfavorable environmental conditions.

Cold, salt and heat are most critical factors that restrict full genetic potential, growth and development of crops worldwide.. In this study, we applied an annealing control primer (ACP) based GeneFishing approach to identify differentially expressed genes (DEGs) in annual ryegrass (cv. Kowinearly) leaves under cold, salt and heat stresses. Two-week-old seedlings were exposed to cold (4°C), salt (NaCl 200 mM) and heat (42 °C) treatments for 6 h. A total 8 differentially expressed genes were isolated form ryegrass leaves. These genes were sequenced then identified and validated form National Center for Biotechnology Information (NCBI) database. We identified several promising genes encoding light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase. These genes were potentially involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance in plants. These genes might be useful for the enhancement of abiotic stress tolerance in fodder crops along with crop improvement under unfavorable environmental conditions.

The purpose of this study is to expression pattern of melanogenesis associate genes on cultured melanocyte layer cells in Korean Brindle Cattle(Dark, Brindle and Yellow) were analyzed to evaluate the effects of sex hormones on the control of melanogenesis pathways. Korean Brindle Cattle(Dark, Brindle and Yellow) melanocyte in the skin cells was collected. after the addition of estrogen and testosterone, the culture was analyzed for expression of cell activity and melanin genes for 72 hours. For the analysis of estrogen in different coat color other than the melanogenesis-related genes it is increasingly yellow showed low expression. in particular, the cells of the brindle coat color is low active and expression of genes. However, the testosterone was low, the expression of cell activity inhibiting MMP-2. the expression of melanin genes actually showed a tendency to increase gradually, which is testosterone compared with the estrogen to be considered that affect the skin cell layer brindle coat color. In this study, stimulation with estrogen triggered the inhibition of MC1R of the melanocyte in brindle coat color, but testosterone is induced MC1R in melanocyte. Therefore, considered the eumelanin or phaeomelanin activation are controlled caused by differential expression of sex hormones on melanocyte in Korean Brindle Cattle.

기후 변화로 인한 수온의 상승은 어류 서식지에 영향을 미친다. 수온의 변화는 어류 생리 거의 모든 부분에 영향을 미치는 것으로 알려져 있다. 기후 변화에 따른 수온의 상승은 산소 용해도의 감소 및 산소 운반 헤모글로빈의 결합 능 력의 감소로 인해 저산소증을 초래할 수 있다. 본 연구는 대서양 연어 (Salmo salar) 치어 성장의 최적수온 (15°C)보다 고 수온 (20°C)에 사육 시, 대서양 연어 치어의 건강상태를 평가 하기 위해 수행되었다. 평가 방법은 NGS RNAseq 분석방법을 이용하여 생체지표유전자를 개발하고, RT-qPCR 분석을 이용하여 생체지표유전자의 발현양상을 조사하는 것이다. 개발한 생체지표유전자로는 interferon alpha-inducible protein 27-like protein 2A transcript variant X3, protein L-Myc- 1b-like, placenta growth factor-like transcript variant X1, fibroblast growth factor receptor-like 1 transcript variant X1, transferrin, intelectin, thioredoxin-like, c-type lectin lectoxin-Thr1- like, ladderlectin-like 및 calponin-1 등이다. 선택된 생체지표 유전자는 NGS RNAseq 분석을 통해 수온변화에 민감하게 발현한 유전자들이며, RT-qPCR 분석을 통한 이들 유전자의 발현 양상은 NGS RNAseq 분석을 통한 발현 양상과 매우 유사하게 나타났다.

돼지의 생시체중은 생존율과 폐사율에 밀접한 관련이 있어 양돈산업에서 자돈 관리와 직결된 중요한 경제형질이다. 본 연구는 Genome-Wide Association Study(GWAS) 분석을 통해 순종 랜드레이스의 생 시체중과 관련된 위치상 후보유전자 탐색을 실시하였다. 생시체중의 유의적 관련 분석 결과, genomewide suggestive level에서 유의성 있는 single nucleotide polymorphism(SNP) marker는 3번 염색체 (ASGA0098921, P=2.41×10-5)와 4번 염색체(H3GA0013451, P=2.47×10-5)에서 각각 1개씩 동정되었다. 이들 SNP marker가 위치한 3개의 유전자(LOC110260055, LOC100156472, LOC100157689)들은 순종 랜드레이스 생시체중의 위치상 후보 유전자이며, 이들 유전자 정보를 이용해서 순종 랜드레이스 생시 체중을 선발할 수 있는 기초 연구 자료가 될 것으로 사료된다.

As a transcription factor, the CCAAT/enhancer binding protein (C/EBP) induces the expression of specific genes by interacting with their promoters. C/EBP can trigger to express other genes, because it can open chromatin structure after binding to DNA, then lead to another transcription factors. In this study, we investigated the features and post-exercise expression patterns of two genes in the C/EBP family, C/EBPβ and C/EBPδ. The basic region leucine zipper motif (bZIP) is a crucial region of C/EBP, and analysis of synonymous and non-synonymous substitution ratios revealed that bZIP regions were well conserved in mammalian species. Quantitative real-time RT-PCR data showed that C/EBPβ and C/EBPδ expression increased significantly in the muscle after exercise, but did not do so in the blood. We therefore propose that exercise-induced increased expression of C/EBPβ and C/EBPδ may stimulate gene expression in physiological pathways in muscle cells.

Small hive beetle (Aethina tumida) (SHB) is an invasive species to most northern hemisphere countries, including Korea. In an attempt to obtain basic information for efficient management of SHB, genes encoding conventional insecticide targets [voltage-sensitive sodium channel α-subunit (VSSC) and acetylcholinesterase (AChE)] were annotated and characterized following the analysis of whole transcriptomes of adults and larvae. A single VSSC gene was identified but no apparent mutations associated with pyrethroid resistance were detected. Genes encoding two AChEs (AtAChE1 and AtAChE2) were identified from the SHB transcriptome. AtAChE1 was determined to be the main catalytic enzyme, thereby being a toxicologically more relevant target. No apparent mutations associated with resistance to organophosphorus and carbamate insecticides was identified in the AtAChE1 gene, whereas the S238G mutation, originally identified from the Colorado potato beetle, was detected in the AtAChE2 gene.

Honey bee has been widely used as a model insect for biological sciences because of its sociality and specialized labor division. For the investigation of the seasonal and labor-dependent expression patterns of genes putatively involved in its sociality, quantitative real-time PCR (qRT-PCR) can be applied to quantify gene expression level and selection of reliable reference gene(s) for normalization is an accurate step. In this study, using three softwares (geNorm, NormFinder and BestKeeper), we evaluated seasonal expression stabilities of four reference genes that have been widely used for qRT-PCR in forager and nurse heads. Among four candidates, two genes, rpS18 and gapdh, were suggested to be the optimal reference genes for qRT-PCR.

The ability of antimicrobial resistant Salmonella strains to cause invasive disease can be attributed to various virulence genes. In this study, the virulence genes located in SPI-1, SPI-2, SPI-5, SPI-11 were found in all antimicrobial-resistant Salmonella isolates. This suggests that these genes play important roles in Salmonella invasion, growth, or survival in the host. The association between the presence of virulence genes and antimicrobial resistance was assessed using the Spearman’s correlation coefficient, and there is a positive association between the gatC, tcfA, hylE, spiA, pagC, msgA, invA, sipB, prgH, spaN, orgA, tolC, iroN, sitC, lpfC, and sopB genes, and resistance to CF, NA and S. This suggests that the association between antimicrobial agents and virulence genes has been shown to vary with the types of antibiotics that are commonly used in different countries. These different associations can be explained by the mechanisms underlying pathogenicity and the acquisition of resistance genes by Salmonella.

Characteristics of induced pluripotent stem (iPS) cells are consistent with those of embryonic stem (ES) cells. However, exogenous genes integrated by using retrovirus delivery systems cannot be completely removed from the cells. In a recent report, activation-induced cytosine deaminase (AID) and thymine DNA glycosylase (TDG) can induce pluripotency state in mouse differentiated cells through the process of DNA demethylation. Thus, we hypothesized that the two reprogramming factors may convert efficiently bovine differentiated cells into pluripotency state. So, genes of AID and TDG were integrated into pCMV6-AC-IRES-GFP-Puro expression vector, which was transfected into bovine differentiated cells. As results, the colonies derived from AID+TDG-induced bovine cells were formed on day 7 after culture. The number of AP positively colonies in AID+TDG-induced bovine cells was significantly higher than in AID-induced bovine cells (p<0.05). Additionally, expression of pluripotent genes (OCT-3/4, NANOG, SOX2) was slightly increased in AID+TDG-induced bovine cells, as compared to AID-induced bovine cells. Protein expressions of OCT-3/4, NANOG and SOX2 in AID+TDG-induced bovine cells were slightly increased rather than AID-induced bovine cells. Finally, DNA demethylation in the promoter regions of pluripotent markers in AID+TDG-induced bovine cells was increased than that of AID-induced bovine cells. In conclusion, pluripotent stem cells could be efficiently produced from bovine differentiated cells by using non-integrating delivery system with the reprogramming factors (AID and TDG).