초록 ▼

o 국민건강과 환경 및 생태계보호를 위해서 기존 화학농약을 대체할 수 있는 무공해 Br 생물농약균주의 산업화를 위한 연구개발을 위해 힘쓴 본 과제는 Bt 살충제의 생산성을 높이고, 이를 위한 균주육종 및 생산공정을 개발하였다.
o 생물농약 전문기업인 미국의 마이코젠사에 Bt 균주 및 유전자 관련 기술을 추출하였다.
o 모기유충에 살충활성이 있는 Bti 균주의 용혈활성을 가진 유전자를 제거하면서 외래 DNA 조각을 삽입하지 않은 환경 친화적 균주를 제작하였다.
o 모니터링용 Bti 균주를 이용한 현장적용 실험에서 돼지

o 국민건강과 환경 및 생태계보호를 위해서 기존 화학농약을 대체할 수 있는 무공해 Br 생물농약균주의 산업화를 위한 연구개발을 위해 힘쓴 본 과제는 Bt 살충제의 생산성을 높이고, 이를 위한 균주육종 및 생산공정을 개발하였다.
o 생물농약 전문기업인 미국의 마이코젠사에 Bt 균주 및 유전자 관련 기술을 추출하였다.
o 모기유충에 살충활성이 있는 Bti 균주의 용혈활성을 가진 유전자를 제거하면서 외래 DNA 조각을 삽입하지 않은 환경 친화적 균주를 제작하였다.
o 모니터링용 Bti 균주를 이용한 현장적용 실험에서 돼지의 사료에 섞여 먹였을 때 90% 이상이 돼지의 장을 통과하는 동안 생존하여 분변을 통해 배출되었고 배출된 Bt는 모기 유충에 대해 살출력을 나타냄을 확인함으로써 Bti 균주의 사료첨가 가능성을 확인하였다.
o 포자형성 과정에 의존적으로 생산되는 Bt 독소단백질의 생산성을 조절하기 위한 포자형서 조절인자를 처음으로 발견하여 그 특성을 분석하였다.
o Bt 전체 게놈의 95% 정도를 해독 완료하였으며 Bt 균이 생산하는 다양한 독소단백질, 효소, 2차 대사산물 등을 샌산하는 유전자를 확인하였다.
o 새로운 신호물질 분해 유전자를 발견하였는데 재조합 대장균을 이용한 실험에서 무름병을 억제할 수 있어 살충제로서의 기능뿐만 아니라 살균제로서의 개발가능성을 확인하였다.
o 위생해충 제어용 Bti 미생물제를 여러 종류의 유화제 형태로 만들어 현장적용 시험을 실시하였고 국내 현실에 적합함 위생해퉁 제어용 Bti 제형의 선전 및 시제품을 제작하였다.

Abstract ▼

Bacillus thuringiensis is a Gram-positive spore-forming bacterium characterized by producing a parasporal crystal, which consists of proteins, dugin the stationary phase of its growth cycle. The insecticidal activity of B. thuringiensis was attributed largely or completely (depending on the insect)

Bacillus thuringiensis is a Gram-positive spore-forming bacterium characterized by producing a parasporal crystal, which consists of proteins, dugin the stationary phase of its growth cycle. The insecticidal activity of B. thuringiensis was attributed largely or completely (depending on the insect) to the parasporal crystals. This observation led to the development of bioinscticieds based on B. thuringiesis for the control of certain insect species among the orders Lepidoptera, Diptera, and Coleoptera. The mode of action of the B. thuringiensis Cry proteins involves several steps such as solubilization of the crystal in the insect midgut, proteolytic processing of the protoxin, binding of the Cry toxin to midgut receptors, and insertion oif the toxin into the apical membrane to create ion channels or pores. THese are more recent reports of B. thuringiensis isolates active against other insect orders (Hymenoptera, Homoptera, Orthoptera, and Mallophaga) and against nematodes, mites, and protozoa. B. thuringiesis is already a useful alternative or supplement to synthetic chemical pesticide application in commercial agriculture, forest management, and mosquito control. It is also a key source of genes for transgenie expression to provide pest resistance in plants. To date, many insectidal srystal protein (cry) genes have been isolated from B. theringiensis. In 1998, Crickmore et al. proposed a nomenclature system according to their degree of evolutionary divergence as estimated by phylogenetic tree algorithms. They have been classified dryl to cry32 and cyt by degree of amino acid sequence homology. B. thuringiensis is now the most widely used biologically produced pest control agent. In 1995, worldwide sales of B. theringiensis were projected at $90 million, representing about 2% of the total global insenticide market. it was reported that the annual worldwide distribution of B. theringiensis amounts to 2.3 x 106 kg. As of early 1998, there were nearly 200 registered B. theringiensis products in the United States. While the use of biological pesticides in agriculture remains significantly behind that of synthetic chemical pesricides, several environment and safety considerations favor the future development of B. thuringiensis. Cry proteins that have been studied thus far are not pathogenic to mammals, birds, amplhubians, or reptiles, bu are very specific to the group of insects and invertebrates pests against which they have activity. Cry-based pesricieds generally have low costs for development and reistration. B. thuringiensis shbsp. israelensis, for example, had a development cost estimate at 1/40 that of a comparable novel synthetic chemical pesticide. Finally, the mode of action for the Cry proteins differs completely from the modes of antion of known synthetic chemical pesticides, making Cry proteins ket components of integrated perst management strategies aimed at preserving natural enemies of pest and managing insect resistance. Mosquito-borne disease affect millions of people worldwide each year. some species of mosquitoes can transmit diseased such as encephalitis, dengue fever, ans malaria to humans, and a variety of diseases to wildlife and domestic animals. Many countries have used chemical pesticides to combat mosquitoes and the public health hazzrds they present. however, the chemical pestidices is not effective to mosquito control and not favorable to ecosystem. Since Bt insraelensis is highly effective against mosquito larvae and favorable to ecosystem. biological control using Br pesticide is recommented. Bt. israelensis produces a parasporal crystal composing Cry4Aa, Cry4Ba, Crt11Aa and Cry1Aa. Whereas Cry4Aa, Cry4Ba and Cry11Aa have high host spcificity, the Cyt1Aa has no. Cyt1Aa is a potential hazardous toxin because of it hemoltic activity. We constructed a Bt israelensis strain having cyt1Aa deletion. This strain may use a biological pesticide with environmental safety. Most of the crystal protein genes in B. thuringiensis are transcribed at the sporulation stage because their promoters have consensus sequences recognized by RNA polmerases containing sporulation-specific sigma factors, $\delta^35$ and $\delta^28$, that share homology with $\delta$E and $\delta$K from B. subtilis, respectively. Since the expression of crystal protein genes is dependent on sporulation process, the sporulation frequency crystal protein. That is, the enhancement of sporulation effeiciency caused the increased production for crystal protein in B. thringienses. Among the Gram-positive bacteria, B. subtilis of which genome is completely sequences is well known its sporulation process. B. subtilis sense and respond to their environment by two-component signal transduction system composing sensor histidine kinase and response regulator. Sensor kinase autophosphorylate at a conserved histidine residue in response to the signal. The phosphorylated histine kinase catalyzed the transfer of the phosphate to an aspartyl of the response regualtor. The phosphorylated response regulator is an transcription factor govering the initiation of sporulation or competence. Response regulator aspartyl-phosphare (Rap) phosphatase modulate the sporulation and/or competence by detaching the phosphate from the phosphorylated response regulator. The activity of Rap phsphatase is inhibited by a peptide comprising the last five amino acids at the C-terminal end of the Phr protein encoded by small gene translationaly coupled with rap gene. However the regulation of sporulation such as Rap/Phr system has never found in any other spore-forming Gram-positive bacteria. In this study, a pair of genes, orf50fin and orf4find, involving sporulation regualtion in B. theringiensis was identified and characterized. A comparision of the deduced amino acid sequence of the orf50fin with those of proteins in the GenBank database using the BLAST network service also shows significant similarity to the Rap phosphatases of B. subtilis. The phr-like sequence (orf4fin) coding for a 43-amino-acid protein was also found in the C-terminus of orf50finwith translationally coupled. The roles of Orf50fin and Orf4find on sporulation of B. thuringiensis cells contaignin plasmind pBP58 or pBP59 or pBP60 were investigated. Plasmids pBP58, pB59 and pB60,which exist multicopy in Bacillus cells, contain both orf50fin and orf4fin and orf50fin only and ofr4fin only, respectively. The sporulation efficiency of B. thuringiensis subsp. irsaelensis cells containing pBP58 was similar with wild type cells. The cells harboring plasmid pB59 showed sporulation deficiency, whereas the cells containing plasmid pB60 showed significantly increased sporulation efficiency. The similar results were obtained in B. thuringiensis subsp. kutstaki. These results revealed that the Orf50fin and Orf4fin exerted negative and positive effects on sporulation of B. theringiensis, repectively.
The Orf4fin closely resembles Phr protein, a secretory protein, by presence of a positively charged amino terminus, a hydrophobid amino-terminal domain and a hydrophilic carboxy-terminal domain with a potential signal peptidase cleavage site separating the two domains to study whether the secretory proteins or peptides from B. theringiensis cells regulates intracellur Orf50fin activity, we tested the complementation of sporulation deficiency in B. thuringgiensis cells containing pBP59 by growth in sterilized culture supernatants of wild type cells or cells containing plasmid pBP60. The culture supernatants of wild type cells or cells containing plasmid pBP60 restored the capability of sporulation of B. thuringiensis cells containing plasmid pBP59. This result revealed that the secretory components suppressed intracellular Orf50fin activity, and the components might derive from Orf4fin. To confirm that the secretory derivatives of Ofr4fin regulate the Orf50fin activity, the chemically sysnthesized petide compostin C-terminus 19 amino acids of Orf4fin was added in culture superatants of B. theringiensis cells containing plamid pBP59. The result showed that the chemically sysnthesized Orf4fin derivative suppressed the sporulation deficiency caused by intracelluar Orf50fin activity. Thus it was speculated that the secretory derivative of Orf4fin might re-enter into the cells and inhibit the Orf50fin activity like mode of action of Phr. orf50fin-like genes are widley distributed in B. thuringiensis subsp. kurstaki HK1 and subsp. israelensis stains are most widely used as biological control agents. The importance of these two strains in industrial field led us to clone genes for regulation of sporulation. The sequence analysis of orf50isr was revealed that it encodes a 424-amino-acids protein having a predicted molecular weight of 50.34 kDa. A comparison of the deduced amino acid sequence of the Orf50isr with those of Orf50fin revealed that the Orf50isr protein was 79.5% indentical to that of Orf50fin. A small open reading frame (odf4isr) encoding a 43-amino-acids protein was found down stream of orf50isr. The deduced amino acid sequence of Ord4isr was 39.5% identical to that of Orf4fin. Like Orf4fin, Orf4isr is also considered as secretory protein. The orf50kur encodes a 424-amino-acids protein having a predicted molecular weight of 51.05 kDa. A comparison of the deduces amino acid sequence of the Orf50kur with those of Orf50fin and Orf50isr revelated that the Orf50kur protein was 67.1% and 68.6% identical to those of Orf50fin and Orf50isr, respectively. Interestingly, the amino acid sequence of Orf50kut was 96.2% identical to that of PreL of Lactobacillus sp. A small open reading frame (orf4kur) located downstream of orf50kur is also considered as a secretory protein, by presence of a positively charged amino terminun, a hydrophobic amino-terminal domian and a hydrlphilic carboxy-terminal domain with a potential signal peptidase cleavage site separating the two domoins. However, Orf4kur has differenct character from Orf4fin or Orf4isr. Oft4kur encodes an 84-amino-acids protein that is larger than Orf4fin or Orf4isr. Comparison of Orf4kur with other proteins showed that the dedued amino acid sequence of Orf4kur is 91.8% identical proteins to that of a protein (Orf9) encoded by a gene located downstream of preL of Lactobacillus sp. The orf50kur-orf4kur structure is closed resembled to preL-orf9. These results indicate that, at least, two types of regulators for sporulation exist in B. theringiensis strains.

목차 Contents

• 제1장 연구개발과제의 개요...18
• 제2장 국내외 기술개발 현황...20
• 제3장 연구개발수행 내용 및 결과...22
• 제1절 용혈활성이 제거된 Bti 균주의 개발...22
• 제2절 포자 형성 유전자의 클로닝 및 포자형성 제어...29
• 제3절 음식물 쓰레기를 이용한 Bti 균주 생산공정 개발...45
• 제4절 Bti 제제화...77
• 제5절 Bt 게놈 분석 및 신호전달 물질 분해 효소 유전자...84
• 제4장 목표달성도 및 관련분야에의 기여도...96
• 제5장 연구개발결과의 활용계획...99
• 제6장 연구개발과정에서 수집한 해외과학기술정보...101
• 제7장 참고문헌...106