The honey bee soluble acetylcholinesterase 1 (AmAChE1) is overexpressed under the overwintering and brood rearing-suppressed conditions. To investigate the role of AmAChE1 in regulating acetylcholine (ACh) titer, ACh concentrations in both the head (central nervous system) and abdomen (peripheral nervous system) were analyzed. ACh titer was significantly lower in both tissues of worker bees under the overwintering and brood rearing-suppressed conditions compared to control bees. Interestingly, the expression levels of choline acetyltransferase (AmChAT) and molecular marker genes of immune systems were significantly reduced in honey bee head under the same conditions. Taken together, ACh titer appears to be reduced via a cooperative interaction of the AmAChE1 overexpression and AmChAT underexpression and to be linked to reduced inmmune responses under the overwintering and brood rearing-suppressed conditions. The roles of AmAChE1 (with little catalytic activity) and AmChAT in the ACh homeostasis and signaling was discussed in the contexts of immune response and longevity regulation in honey bees.

Insect cuticle is a first physical barrier to protect their body from multifarious environments. Cuticle tanning (sclerotization and pigmentation) is a complex process involves hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), decarboxylation of DOPA to dopamine, N-acylation of dopamine to N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD), oxidation of NADA and NBAD to their corresponding quinones, and reactions between the quinones or quinone derivatives with cuticular proteins (CPs) resulting in protein cross-linking. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to NADA whose covalent-linkage of CPs is correlated with colorless cuticle (β-sclerotization). In this study, we analyzed functions of TcNAT1 on cuticle tanning of adult Tribolium castaneum by RNAi. Injection of dsRNA for TcNAT1 (dsTcNAT1) had no affect on animal development and growth. However, some of the resulting adults (~70%) showed split elytra that could not cover their abdomen, resulting in improper folding of their hindwings. Interestingly, body color of the mature adults was darker than that of control dsTcVer-treated adults because probably due to the buildup of abnormally high levels of dopamine, which is used for dopamine eumelanin pigment synthesis (black pigment). On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles located at the intervein regions, suggesting that NADA mediated β-sclerotization is occurred in these regions. Similarly, darker pigment was evident at veins of the hindwings of TcNAT1-deficient adults. These results suggest that TcNAT1 plays important roles in cuticle tanning of T. castaneum adult. To characterize enzymatic properties of TcNAT1, furthermore, recombinant TcNAT1 protein expressed in E. coli was purified by utilizing Ni-NTA affinity column chromatography. This work was supported by NRF (NRF-2012R1A2A1A01006467).

Insect cuticle/exoskeleton is a first physical barrier to protect their body from multifarious environments such as desiccation, natural enemies and entomopathogenic microorganisms. Cuticle tanning (sclerotization and pigmentation) is a vital procedure for generating suitable cuticle depending on body region by sclerotization and pigmentation in insects. Insect cuticle tanning is a complex process involves hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), decarboxylation of DOPA to dopamine, N-acylation of dopamine to N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD), oxidation of NADA and NBAD to their corresponding quinones, and reactions between the quinones or quinone derivatives with cuticle protein (CP) side chains resulting in protein cross-linking. One type of pigmentation (quinone tanning) is associated with the covalent linkage of CPs to the ring component of NBAD. In contrast, linkage of CPs to the side chain of NADA (b-sclerotization) is correlated with colorless cuticle. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to N-acetyl dopamine (NADA) in cuticle tanning pathway. In this study, we studied function of TcNAT1 on adult cuticle tanning by double stranded-RNA (dsRNA) mediated gene silencing. Injection of dsTcNAT1 had no affect on animal development, growth and molting such as larva to larva, larva to pupa and pupa to adult. However, some of the resulting adults (~70%) showed split elytra that could not cover their abdomen, resulting in improper folding of their hindwings. Interestingly, body color of the mature adults (older than 3 days) was darker than that of control dsTcVer treated adults because probably due to the buildup of abnormally high levels of dopamine, which is used for dopamine eumelanin pigment synthesis (black pigment) and dopamine quinone-mediated protein crosslinking. On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles that are located in the intervein regions, suggesting that NADA mediated b-sclerotization is occurred at these regions. Similarly, darker pigment was evident at veins of the hindwings of the dsTcNAT1-mature adults. These results suggest that TcNAT1 have important roles in sclerotization and pigmentation of adult body and wings (elytron and hindwing). This work was supported by NRF (NRF-2012R1A2A1A01006467).

제초제 성분인 phosphinothricin을 분해하는 phosphinothricin acetyltransferase (PAT) 단백질을 과발현시킨 제초제저항성 감 자가 개발되었다. 본 연구에서는 안전성평가 기준에 따라, PAT 단백질과 제초제저항성 감자에 대한 급성경구독성 및 아만성 독성 평가를 실시하였다. PAT 단백질을 재조합 대장균에서 분리정제 한 후, ICR계 쥐에 2,000 mg/kg weight 용량의 PAT 단백질을 먹이고 14일 동안 치사율과 체중증가를 관찰한 결과, 죽은 동물이 없었고, 체중증가, 사료 섭취 및 특별한 이상증상을 발견하지 못했다. 아만성 독성의 경우 제초제저항성 감자를 표준사료에 0, 1, 5% 섞어 90일 동안 경구 투여하였다. 이 경우에서도 사망한 개체는 없었으며, 조사한 여러 항목에 대해 특별한 이상증후를 관찰하지 못했다. 또한, 제초제저항성 감자를 먹인 동물군과 대조구로 일반감자를 먹인 동물 사이에서 유의 할 만한 차이는 발견되지 않았다. 이런 결과를 토대로 볼 때, 제초제저항성 감자와 제초제 저항성 단백질인 PAT는 동물에게 독소로 작용하지 않음을 확인하였다.

Serotonin N-acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasmexpressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis.

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared to seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.

본 연구는 개량 포플러 현사시 3호를 재료로 제초제 저항성 유전자 PAT으로 형질전환시 킨 다음 제초제 Basta처리에 의한 효율적인 형질전환체 선발을 목적으로 시험되었다. Basta 처리에 따른 캘러스유도, 부정아유도 및 액아유도 시험을 통해 조직치사 농도는 1.0mg/L로 결정할 수 있었으며, 이 농도처리에 의해 형질전환체의 조기선발에 사용하였다. 이 농도처리로 비형질전환체는 모두 고사되었지만, 형질전환체는 정상적으로 반응을 보이며, 생육이 가능하였다 .한편, 형질전환체에 제초제를 직접 살포한 결과, 정상적 생육이 가능했고, PAT activity측정 결과에서도 양성 반응을 나타내어 현사시 3호 세포내에 PAT유전자가 정상 발현됨이 확인되었다. 이로써 현사시에 PAT유전자 도입 및 발현을 위하여 제초제 Basta 처 리와 PAT assay가 매우 효과적인 것으로 나타났다.

제초제 bialaphos에 저항성인 phosphinothricin ace-tytransferase gene이 도입된 형질전환 감자 식물체에서 유전자의 간편한 확인방법을 확립하였다. 먼저 형질전환체와 대조식물체의 잎 disc를 취해 bialaphos 5mg/l를 처리한 액체배지에 치상하였을 때 배양 25일이면 대조식물체의 잎 disc는 완전히 고사되어 형질전환체와 구별이 가능하였다. 감자의 뿌리 생육 비교실험에서는 bialaphos 0.5mg/l의 농도에 agar를 0.6% 첨가한 선발배지에서 식물체의 정단부위를 치상하여 비교하였을 때 7-10일이면 뿌리의 생육차이를 이용하여 간편하게 형질전환여부를 판정할 수 있었다. 또한 감자의 잎 disc를 제초제 bialaphos 0.5mg/l를 처리한 액체배지에 치상하여 15일이 경과한 후 chlorophyll A의 함량을 측정하였을때 형질전환체는 대조식물체보다 2.5배 정도 많은 chlorophyll A를 가지고 있어 형질전환체의 조기 판별시 chlorophyll A함량에 의해 검증이 가능하였다. 한편, 이렇게 형질전환 여부가 확인된 감자 식물체를 이용하여 PAT효소의 활성을 측정한 결과 대조 식물체에서는 PAT효소의 활성을 보이지 않았으나 형질전환 식물체에서는 모두 효소의 활성이 높았다.

This experiment was conducted to introduce phosphinothricin acetyl -transferase(PAT) gene, resistant to basta and non-selective herbidide, into tobacco(Nicotiana tabacum cv.BY4). For shoot formation,tobacco leaf disks were placed on the MS medium supplemented with 2.0mg/L BA and 0.1mg/L NAA. In this medium condition, tobacco leaf disces were cocultivated with A. tumefaciens MP90 containing NPT IIand PAT resistant to kanamycin and Basta, respectively. Shoots were obtained in the medium containing antibiotics, and those were transferred to rooting medium supplemented with 0.1mg/L NAA and antibiotics. The plants obtaining roots were transplanted into soil. Phenotype of transgenic tobacco plant was mostly as normal plant. However, about 5% was abnormal plant, which did not set seeds. PCR analysis and southern blot were performed to determine transformation. As the results, it was confirmed that PAT gene was stably integrated into tobacco genome.When herbicide, basta, was sprayed to the plants confirmed by PCR, the transgenic plants showed normal growth, whereas normal plants died. Therefore, the result of this experiment show that tobacco transformation for the resistance to basta, non-selective herbicide, was successful because PAT gene was stably integrated into tobacco.