• 한국응용곤충학회지
• /
• 제36권4호
• /
• pp.341-350
• /
• 1997

Autographa californica 핵다각체병 바이러스(AcNPV)의 다각체 단백질과 Bacillus thuringiensis(Bt) cryIA(c) 내독소 단백질의 융합단백질을 생산하는 새로운 재조합 바이러스를 제작하고, 곤충세포주(Spodoptera frugiperda 9)에서 발현된 융합단백질의 특성을 분석하였다. Bt kurstaki HD-73의 cryIA(c) 내독소 단백질 유전자의 N-발단 AcNPV의 완전한 다각체 단백질 유전자의 앞쪽에 융합함에 의하여 또는 다닥체 단백질 유전자내의 제한효소 HindII부위에 삽입함에 의하여 다각체 단백질 유전자의 프로모터 조절하에 도입하였다. 이렇게 작성된 재조합 바이러스를 각각 Btrusl 또는 BtrusII라고 명명하였다. BtrusI은 분명히 단일 전사체를 보임에도 92kDa의 융합 단백질과 다각체 단백질의 두 단백질을 생산하였다. 또한 Btrusl에 의해 만들어진 융합 단백질은 다각체를 형성하지 않았다. 한편, BtrusII에 의해 감염된 곤충세포주에서는 33kDa의 다각체 단백질은 보이지 않았고 단지 융합 단백질만 생산하였으나 다각체는 형성하지 않았다. 따라서 Btrusl에 의해 생산된 융합 단백질의 독성을 조사하기 위하여, Btrusl으로 감염된 곤충세포주를 2령 누에(Bombyx mori)에 접종한 결과 융합 단백질에 의한 독성이 관찰되었다. 결론적으로 다각체 단백질과 Bt cryIA(c) 내독소 단백질에 의한 융합 단백질이 독성을 가지고 있음을 확인하였다.

• 한국미생물·생명공학회지
• /
• 제24권2호
• /
• pp.173-177
• /
• 1996

Bacillus thuringiensis subsp. morrisoni PG-14 is a gram-positive soil bacterium producing mosquitocidal parasporal inclusions composed of several crystal proteins. Among these crystal protein genes, cryIVD gene is one of major component which has 72 kDa in size. However, these parasporal inclusions sink quickly from the surface of water where mosquito larval feeding occurred. To develope mosquitocidal cyanobacterium, therefore, we constructed the expression vector, pCYASK 5-1 harboring cryIVD gene. The expression vector, pCYASK5-1 was transformed into the cyanobacterium Syne- chocystis PCC6803 reported as a natural mosquito larval food source and the transformants were selected with kanamycin. Expression of IVD gene in transformant was characterized by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis. The mosquitocidal activity of a transformant was determined with Culex tritaeniorhynchus. The results showed that, the transformed cyanobacterium is toxic to mosquito larvae and will be expected as a potential agent that is used for mosquito control.

• 한국응용곤충학회지
• /
• 제48권4호
• /
• pp.509-517
• /
• 2009

국내에서 분리된 Bacillus thuringiensis subsp. aizawai CAB109균주가 난방제 해충으로 알려진 담배거세미나방과 파밤나방에 동시에 높은 독성을 보이는 것으로 나타났다. B.t. CAB109 균주의 활성을 평가하기 위해 혈청형이 aizawai이면서 미생물농약으로 시판중인 TB-WP제품 및 SC제품과의 살충활성을 비교한 결과, B.t. CAB109균주, TB-WP제품, SC제품은 담배거세미나방 2령충에 대한 반수치사농도($LC_{50}$)가 각각 $1.3{\times}10^5cfu/ml$, $2.3{\times}10^6cfu/ml$, $5.2{\times}10^5cfu/ml$으로 나타났고 파밤나방 2령충에 대한 반수치사농도는 $1.8{\times}10^4cfu/ml$, $1.3{\times}10^6cfu/ml$, $1.5{\times}10^6cfu/ml$으로 나타나 두 종 해충 모두에서 B.t. CAB109 균주가 독성이 더 높은 것을 볼 수 있었다. B.t. CAB109균주가 이미 알려져 있는 aizawai와 비교해 차이가 나는 새로운 유전자를 소유하는지 확인하기 위해 Plasmid DNA를 추출하여 전기영동 한 결과 B.t. subsp. aizawai HD-133과 다른 패턴을 보이는 것을 확인 할 수 있었고 Cry1-Cry5의 primer를 사용하여 PCR을 진행한 결과 B.t. subsp. aizawai CAB109균주는 Cry1Aa, 1Ab, 1C, 1D를 B.t. subsp. aizawai HD-133은 Cry1Aa, 1Ab를 가지고 있음을 확인 할 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제18권6호
• /
• pp.1033-1039
• /
• 2008

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of otfl was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.

• Journal of Pharmaceutical Investigation
• /
• 제28권2호
• /
• pp.121-126
• /
• 1998

The solubility and physicochemical stability of caroverine hydrochloride (CRV), an antispasmodic, in buffered aqueous solutions were studied using a reverse phase high performance liquid chromatography. The solubilty of the drug at pH 2.76-5.40 was similar at the range 31.9-36.2 mg/ml $(34^{circ}C)$, but, at the pH higher than 6.0, markedly decreased. The use of polyethylene glycol 400 as a cosolvent did not increase the solubility at any compositions examined. Moreover. increasing molar concentration of aqueous phosphate buffer from 0 to 0.5 M remarkably decreased the solubility. The degradation of CRY followed the apparent first-order kinetics. The degradation was accelerated with decreasing pH and increasing storage temperature. The half-lives for the degradation of CRY (1.0 mg/ml) at pH 1.28. 4.01 and 5.93 $(45^{\circ}C)$ were 2.8, 31.4 and 124 hr. respectively. The pHs of incubated solutions were to some extent lowered perhaps due to the formation of acidic degradation products. The addition of disodium edetate (0.01%) to the CRY solution (pH 4.95) retarded 2.5 times the degradation rate at $45^{\circ}C$, but the use of sodium bisulfite (0.1%) accelerated 2.9 times the rate. The activation energy for the CRY solution (20 mg/ml. pH 5.4) containing 0.01% EDTA was calculated to be 5.98 kcal/mole. When the solution was stored under nitrogen displacement in ampoule, there was no significant degradation even after 3 months at $40^{\circ}C$, indicating that protection from oxidation by air (oxygen) is essential for the complete stabilization of CRY solution.

• Molecules and Cells
• /
• 제22권3호
• /
• pp.285-290
• /
• 2006

The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제1권2호
• /
• pp.115-122
• /
• 2000

A Bacillus thuringiensis designated NT0423, belonging to B. thuringiensis subsp. aizawai (H 7), was isolated from samples of dust and soil of sericultural farms. B. thuringiensis NT0423 having dualspecificity against Lepidoptera and Diptera produced bipyramidal inclusions consisting of two major polypeptides of approximately 130- and 70-kDa. Proteolytic processing by trypsin and gut juice of Bombyx mori yielded predominant proteins with molecular masses of about 66-kDa. The whole crystal protein of B. thuringiensis NT0423 immunologically was related to that of B. thuringiensis subsp. aizawai. PCR analysis showed that B. thuringiensis NT0423 has at least five crystal protein genes including cryIA(a), cryIA(b), cryIC, cryID and cryIIA, and southern blot was determined the location of each gene on intact and enzyme-digested plasmid DNA fragments. Except for cryIA(a) gene on the high molecular weight plasmid of 165-kb, all of four genes were located on the plasmid of 66-kb. The production of $\beta$-exotoxin from B. thuringiensis NT0423 was identified by the HPLC analysis. In addition, the $\beta$-exotoxin showed its ability to prevent pupation of treated larvae of house flies (Musca domestica) from developing into normal adults.

• 한국응용곤충학회지
• /
• 제32권3호
• /
• pp.300-306
• /
• 1993

Bacillus thuringiensis subsp. kurstaki HD-1으로부터 생산된 살충성 내독소 단백질을 coding하는 CryIIA 유전자를 클로닝하고 염기서열을 조사하였다. HD-1 균주의 12개 plasmid 중 225kb plasmid를 분리하여 CryIIA 유전자를 포함하는 5kb HindIII 절편을 hybridization하여 찾아냈다. 이 절편을 plasmid pUC19에 ligation하여 E. coli에 형질 전환하였다. 이 독소 유전자를 포함하는 4kb BamHI-HindIII 절편은 vector pT7-5에 ligation하여 pSKIIA라하였다. pSKIIA는 3개의 open reading frames(orf1, orf2, orf3)로 구성되어 있으며 염기서열은 3,952base로 되어 있었다. 이러한 3개의 orf 각각의 발현 여부를 확인하기 위하여 생물검정을 하였다. 그러나 orf1 또는 orf2에 의한 형질 전환체는 독성이 없는 것으로 나타났다. orf3를 포함하는 형질 전환체는 3종의 나비목 곤충(배추점나방, 담배거세미나방, 담배나방) 및 1종의 파리목 곤충(집모기) 유충에 대하여 독성을 나타내었다.

• 한국해양생명과학회지
• /
• 제9권1호
• /
• pp.47-52
• /
• 2024

Light is a major external environmental factor that influences the circadian rhythm of photosynthetic organisms and various physiological phenomena, such as growth, maturation, and behavior. The number of light-reaching organisms changes depending on the season and atmospheric conditions, and the intensity and wavelength of light differ depending on the organisms inhabiting the environment. Altered light changes the circadian rhythm of fish, which is controlled by clock genes, such as period 2 (Per2), cryptochrome 1 (Cry1), and melatonin. In this study, we set the zeitgeber time (ZT; 14 light-10 dark, LD) based on the actual sunrise and sunset times and examined Per2 and Cry1 activities, levels of aralkylamine N-acetyltransferase (AANAT), and melatonin in Pholis nebulosa, a drifting seaweed species exposed to irregular light. Per2 and Cry1 levels increased during the daytime and decreased after sunset. The AANAT levels decreased during the daytime and increased during the night. Melatonin concentration was highest around midnight (ZT21, 23:30), but exhibited similar concentrations during the daytime. While the activity of Per2, Cry1, and AANAT levels exhibited a typical circadian rhythm observed in most vertebrates, melatonin concentrations did not show a significant difference between the daytime and nighttime. These findings provide insights into the circadian rhythm patterns of organisms exposed to irregular light environments, such as P. nebulosa, which differ from those of typical fish species.

• 한국미생물·생명공학회지
• /
• 제17권2호
• /
• pp.142-147
• /
• 1989

나비목 유충에 독성이 강한 균으로 알려진 Bacillus thuringiensis serovar. kurstaki HD73 을 ethidium bromide(0.02$\mu\textrm{g}$/$m\ell$)처리와 자연적 curing에 의한 내독소 유전자의 위치를 확인하여 변이주간 내독소 생성능을 간이 선별할 수 있는 배지를 선발한 다음 국내 토양에서 분리한 KBS722 균주에 적응하여 그 내독소 단백질 유전자의 위치를 확인한 결과를 요약하면 다음과 같다. HD73-NRRL과 Dul-mage박사로 분양 받은 균주는 약 7.4, 7.1, 8.1, 11. 3, 75kb 및 크기가 75kb와 비슷하고 copy수가 적은 또 하나의 plasmid로 전부 6개의 plasmid를 보유하고 있었으며 IPL 균주는 약 4.0과 70kb plasmid를 더 보유하는 것으로 나타났다. 상기 HD 73 균주들의 내독소 단백질 크기는 모두 133KD 정도였고 HD73의 내독소 유전자는 변이주간 내독소의 현미경 검경과, immunoblotting plasmid DNA 의 전기영동결과 75kb상에 있는 것으로 나타났다. 이들 변이주들을 potato dextrose agar, starch agar, spizizen casamino acid glucose 와 nutrient agar 평판배지에 접종하여 균형태를 관찰하였을 때 내독소 비형성균(Cry-)은 starch agar 배지에서만 반투명하고 균 군락의 색깔이 엷은 회색을 띄었다. 한편 국내 분리균 KBS722를 novobiocin(3$\mu\textrm{g}$/$m\ell$)으로 plasmid를 curing시켜 상기 4가지 배지에 도달했을때 nutrient agar배지에서만 Cry 변이주가 반투명하고 엷은 회색을 나타내었다. KBS722의 내독소유전자는 약 225kb의 plasmid상에 있는 것으로 나타났으며 in vitro에서 쉽게 Cry$^+$주와 Cry$^-$주의 판별이 가능하였다.