KR100375675B1 - Synthetic insecticidal toxin gene for transformation of Brassica plants - Google Patents
Synthetic insecticidal toxin gene for transformation of Brassica plants Download PDFInfo
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- KR100375675B1 KR100375675B1 KR10-2000-0046327A KR20000046327A KR100375675B1 KR 100375675 B1 KR100375675 B1 KR 100375675B1 KR 20000046327 A KR20000046327 A KR 20000046327A KR 100375675 B1 KR100375675 B1 KR 100375675B1
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- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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Abstract
본 발명은 배추 등 배추과 작물의 중요해충인 배추좀나방(diamondback moth,Plutella xylostella)의 피해로부터 작물을 보호하기 위하여 배추좀나방에 살충력을 보이는 바실러스 츄린기엔시스(Bacillus thuringiensis)의 내독소 단백질 유전자cryIAc를 배추에서 잘 발현될 수 있도록 수정, 합성하는 방법, 이 방법에 의해 수정, 합성된cryIAc유전자, 이 유전자를 포함하는 발현벡터 및 이 발현 벡터에 의해 형질전환된 배추과 작물 또는 이들 종자에 관한 것이다.The present invention is cryiac endotoxin protein gene of Bacillus thuringiensis showing insecticidality to Chinese cabbage moth in order to protect crops from damage of diamondback moth ( Plutella xylostella ), an important pest of Chinese cabbage and other crops The present invention relates to a method of fertilizing and synthesizing so as to be well expressed in Chinese cabbage, cryIAc gene modified and synthesized by this method, an expression vector comprising the gene, and a cabbage family crop or transformed by the expression vector.
Description
본 발명은 배추 등 배추과 작물의 중요해충인 배추좀나방(diamondback moth,Plutella xylostella)의 피해로부터 작물을 보호하기 위하여 배추좀나방에 살충력을 보이는 바실러스 츄린기엔시스(Bacillus thuringiensis)의 내독소 단백질cryIAc유전자를 배추에서 잘 발현되도록 수정, 합성하는 방법, 이 방법에 의해 수정, 합성된cryIAc유전자, 이 유전자를 포함하는 발현 벡터 및 이 발현 벡터에 의해 형질전환된 배추과 작물 또는 이들의 종자에 관한 것이다.The present invention is an endotoxin protein cryIAc gene of Bacillus thuringiensis showing insecticidality to Chinese cabbage moth to protect crops from damage of diamondback moth ( Plutella xylostella ), an important pest of Chinese cabbage and other crops. The present invention relates to a method for modifying and synthesizing well in Chinese cabbage, a cryIAc gene modified and synthesized by this method, an expression vector including the gene, and a cabbage crop transformed by the expression vector or seeds thereof.
작물의 생산성 향상에 크게 기여해온 화학합성 농약의 장기간 사용 및 남용으로 인하여 환경오염, 생태계 파괴 등의 문제가 야기될 뿐만 아니라 농약에 대한 저항성을 갖는 병원균이나 곤충이 출현하게 되기 때문에, 이러한 문제점을 극복하기 위하여 무공해 내지 저공해 농약의 개발이 절실히 요구되고 있는 것이 현실이다.This problem is overcome because long-term use and abuse of chemical synthetic pesticides, which have contributed greatly to the productivity of crops, not only causes problems such as environmental pollution and ecosystem destruction, but also causes pathogens and insects to resist pesticides. In order to do so, the development of pollution-free or low pollution pesticides is urgently required.
이에 미생물을 원체로 한 미생물 농약을 개발하여 사용하고 있는데, 이중 대표적인 것이 나비목(Lepidopteran)이나 모기의 유충 등 특정해충에 대하여 병원성이 높으며 공업수준의 배양이 용이한바실러스 츄린기엔시스(Bacillus thuringiensis; 이하 "Bt"로 약함)를 원체로 한 미생물 농약이 BT제라 하여 유용하게 사용되고 있다. Bt는 아포를 형성하는 그램양성균으로 아포형성과 함께 특정 곤충에 치사효과가 있는 내독소를 생성한다. 이 내독소 단백질은 곤충의 중장내에 있는 단백질 분해 효소에 의해 활성화되어서 특정한 수용체에 결합하여 살충작용을 일으키므로 수용체를 가지고 있지 않은 사람이나 동식물 및 어류 등에는 무해하여 자연생태계에도 안전한 미래형 농약으로 알려지고 있다.Therefore, microbial pesticides have been developed and used. Among them , Bacillus thuringiensis, which has high pathogenicity to certain pests such as Lepidopteran and mosquito larvae, and is easy to cultivate at an industrial level, is used. Microbial pesticides based on "Bt" are commonly used as BT agents. Bt is a Gram-positive bacterium that forms follicles and produces endotoxins that have a lethal effect on certain insects along with follicle formation. This endotoxin protein is activated by proteolytic enzymes in the insect's mid- and long-term, binds to specific receptors and causes insecticidal action. Therefore, it is harmless to people who do not have receptors, animals and plants, fish, etc. have.
현재까지 많은 종류의 Bt 균주가 흙, 죽은 곤충, 먼지, 식물 등에서 분리되었으며(Appl. Environ. Microbiol. 57, 311-315), 분리된 균주로부터 많은 수의 내독소 단백질 유전자가 클로닝되어 알려지고 있는데, 이들 내독소 단백질은 나비목, 파리목, 딱정벌레목 등 다양한 곤충에 선택적 독성을 가지는 것으로 알려져 있으며, 그들의 아미노산 서열의 상동성에 따라 CryⅠ에서 CryⅩⅧ의 Cry 단백질과 Cyt로 분류된다.To date, many strains of Bt have been isolated from soil, dead insects, dust, and plants (Appl. Environ. Microbiol. 57, 311-315), and numerous endotoxin protein genes have been cloned from the isolated strains. These endotoxin proteins are known to have selective toxicity to various insects such as lepidoptera, fly, and coleopteran, and they are classified into Cry I and Cry protein from Cry I to Cyt according to the homology of their amino acid sequences.
현재 경제적 측면에서 문제가 되는 주요 해충들은 대부분 나비목에 속해 있어 CryⅠ계열의 내독소 단백질 유전자에 대한 연구가 가장 많이 수행되었다. CryⅠ계열의 유전자들은 다시 아미노산 서열의 상동성에 따라 CryⅠA에서 CryⅠK까지 분류되고 있으며, 이들은 다시 CryⅠAa, CryⅠAb, CryⅠAc, CryⅠAd 및 CryⅠAe 등으로 세분화된다.Most of the major pests that are currently problematic in economic terms belong to Lepidoptera, and the most research on endotoxin protein gene of Cry I family has been conducted. Cry I series genes are further classified into Cry IA through Cry IK according to the homology of amino acid sequences, which are further subdivided into CryIAa, CryIAb, CryIAc, CryIAd and CryIAe.
따라서, 본 발명자 또한 우리 나라 주요 채소작물인 배추에 발생되고 있는 주요해충인 배추좀나방을 이들 내독소 단백질을 이용하여 방제하고자 연구를 하였다. 그 결과, Bt의 내독소 단백질 유전자 중 배추좀나방에 특이적으로 높은 살충력을 보이는 CryⅠAc 유전자를 이용하여 배추좀나방을 방제하고자 하였다. 그러나, CryⅠAc 유전자를 그대로 배추에 형질전환하는 경우에는 발현효율이 매우 낮아 배추좀나방 등을 효과적으로 방제할 수 없는 문제점이 있기 때문에, CryⅠAc 유전자의 배추에서의 발현효율을 향상시킬 수 있는 새로운 방법이 요구되었다.Therefore, the present inventors also studied to control the Chinese cabbage moth, which is a major pest generated in Chinese cabbage, which is a major vegetable crop in Korea, using these endotoxin proteins. As a result, we tried to control Chinese cabbage moth using the Cry IAc gene showing a particularly high insecticidal activity against Chinese cabbage moth among the endotoxin protein gene of Bt. However, when the CryⅠAc gene is directly transformed into the cabbage, the expression efficiency is very low, so there is a problem in that it is not possible to control the Chinese cabbage moth effectively. Therefore, a new method for improving the expression efficiency in the cabbage of the CryⅠAc gene is required. It became.
이에, 본 발명자들은 배추에서 CryⅠAc 유전자의 발현효율을 향상시킬 수 있는 방법에 대하여 연구를 한 결과, CryⅠAc 유전자가 배추에서의 발현효율이 낮은 근본적인 이유는 원핵생물 유전자와 식물 유전자의 구조적 차이에 있다라는 사실,즉, CryⅠAc 유전자는 정상적인 식물 유전자에는 없는 전사(transcription) 조절 부분, 폴리아데닐레이션 부분, 인트론 부분 등과 유사한 염기배열을 갖고 있어 완전한 mRNA가 만들어지기 어려우며, CryⅠAc 유전자에서 사용하는 코돈이 식물 유전자에서 사용하는 코돈과 큰 차이가 있어서 이 유전자가 전사되어 mRNA가 만들어졌다고 할지라도 번역되는 과정에서 식물체가 흔히 사용하지 않는 코돈이 문제를 발생시킬 수 있다는 사실로부터, CryⅠAc 유전자를 배추 유전자와 유사하게 수정, 합성하여, 이를 배추 등의 배추과 작물에 형질전환시키면 발현효율이 향상되어 배추좀나방에 대한 저항성이 우수한 배추과 작물을 제공할 수 있다는 것을 발견하고 본 발명을 완성하게 되었다.Therefore, the present inventors studied a method for improving the expression efficiency of the Cry IAc gene in Chinese cabbage, the fundamental reason that Cry IAc gene is low in the Chinese cabbage expression is the structural difference between prokaryotic and plant genes In fact, the CryIAc gene has a nucleotide sequence similar to that of transcription control, polyadenylation, intron, etc., which are not found in normal plant genes, making it difficult to produce complete mRNAs. The CryⅠAc gene is similarly modified to cabbage genes because of the fact that the codons that are used are so different from the codons used that they may be transcribed and that mRNAs are produced. Synthesized it, cabbage crops such as Chinese cabbage When transgenic expression efficiency is improved, thereby completing the present invention and found that it is possible to provide excellent resistance to crop baechugwa cabbage moth.
따라서, 본 발명의 목적은 배추좀나방에 대하여 우수한 살충성을 갖는 합성 CryⅠAc 유전자를 제공하는 것이다.Accordingly, it is an object of the present invention to provide a synthetic CryIAc gene having excellent insecticidal properties against cabbage myth moths.
본 발명의 다른 목적은 상기 유전자를 합성하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for synthesizing the gene.
본 발명의 또 다른 목적은 상기 유전자를 포함하는 발현벡터를 제공하는 것이다.Still another object of the present invention is to provide an expression vector containing the gene.
본 발명의 또 다른 목적은 상기 발현벡터를 배추과 작물 및 종자에 도입하여, 배추좀나방에 대한 저항성을 갖는 형질전환 작물 및 종자를 제공하는 것이다.Still another object of the present invention is to introduce the expression vector into cabbage family crops and seeds, to provide a transformed crops and seeds having resistance to Chinese cabbage moth.
도 1은 합성cryIAc유전자의 1344 염기부터 1854 염기까지에 번역 종료 코돈 TAA 및 제한효소 KpnI (GGTACC)을 첨부한 PCR을 위한 프라이머의 배열 및 제한효소 자리를 나타낸 것이다.Figure 1 shows the sequence and restriction enzyme site of the PCR for PCR with the translation end codon TAA and restriction enzyme KpnI (GGTACC) from base 1344 to 1854 base of the synthetic cryIAc gene.
도 2는 합성cryIAc유전자의 864 염기부터 1351 염기까지의 PCR을 위한 프라이머의 배열 및 제한효소 자리를 나타낸 것이다.Figure 2 shows the alignment of the primers and restriction enzyme sites for PCR from 864 base to 1351 base of the synthetic cryIAc gene.
도 3은 합성cryIAc시작 코돈 ATG 앞에 CC를 첨부하고 900 염기까지의 PCR을 위한 프라이머의 배열 및 제한효소 자리를 나타낸 것이다.Figure 3 shows the alignment of the primer and restriction enzyme site for PCR up to 900 bases with CC attached to the synthetic cryIAc start codon ATG.
도 4는 도 1 내지 도 3의 프라이머들을 이용한 PCR 결과 합성cryIAc전체 염기배열에 해당하는 1865bp를 포함하는 PCR 단편을 클로닝한 플라스미드 7개 및 그들의 제한효소 지도이다.4 is a plasmid of 7 cloned PCR fragments containing 1865 bp corresponding to the synthetic cryIAc whole nucleotide sequence as a result of PCR using the primers of FIGS. 1 to 3 and their restriction enzyme maps.
도 5는 도 4의 PCR 단편 7개를 순서에 따라 연결하여 합성cryIAc를 완성하는 과정 중의 플라스미드 및 그들의 제한효소 지도이다.FIG. 5 is a plasmid and restriction maps of the PCR fragments of FIG. 4 connected in sequence to complete synthetic cryIAc . FIG.
도 6은 도 5의 합성cryIAc유전자의 시험관내에서의 발현을 위해 제작한 전이벡터 pBac8-Bt9 제작과정 및 그 벡터의 구조를 나타낸 것이다.Figure 6 shows the construction of the transition vector pBac8-Bt9 prepared for in vitro expression of the synthetic cryIAc gene of Figure 5 and the structure of the vector.
도 7은 도 6의 전이벡터 pBac8-Bt9를 이용해 선발한 재조합 바이러스 vBac-Bt9에 의한 합성cryIAc유전자 발현 단백질의 SDS-PAGE 및 웨스턴 블럿(western blot) 분석 결과를 나타낸다.Figure 7 shows the results of SDS-PAGE and Western blot analysis of the synthetic cryIAc gene expression protein by recombinant virus vBac-Bt9 selected using the transition vector pBac8-Bt9 of FIG.
도 8a 및 8b는 재조합 바이러스 vBac-Bt9에 의해 곤충세포에서 발현된 합성cryIAc단백질의 배추좀나방에 대한 살충성 검정 결과이다.8A and 8B show the results of a pesticidal assay for cabbage moths of synthetic cryIAc protein expressed in insect cells by recombinant virus vBac-Bt9.
도 9는 도 5의 플라스미드 pBt9의 합성cryIAc유전자를 포함하는 플라스미드 및 그들의 제한효소지도이다.9 is a plasmid containing the synthetic cryIAc gene of plasmid pBt9 of FIG. 5 and their restriction enzyme maps.
도 10은 도 9의 플라스미드를 사용하여 합성cryIAc유전자의 식물 발현을 위해 제작한 식물 발현 벡터 pGRBt20의 구조를 나타낸다.10 shows the structure of the plant expression vector pGRBt20 prepared for plant expression of the synthetic cryIAc gene using the plasmid of FIG. 9.
도 11a 및 11b는 도 10의 합성cryIAc유전자 식물 발현 벡터 pGRBt20으로 형질전환된 배추의 배추좀나방에 대한 살충성 검정 결과를 나타낸다.11A and 11B show the results of the insecticidal assay for cabbage moths of cabbage transformed with the synthetic cryIAc gene plant expression vector pGRBt20 of FIG. 10.
본 발명은 우리 나라에서는 물론 중국, 일본 등 동북아시아에서 널리 재배되는 주요 채소이며, 이미 세계적 식품이 된 김치의 기본 재료인 배추를 주요해충인 배추좀나방(diamondback moth,Plutella xylostella)의 피해로부터 보호하기 위하여 배추좀나방에 살충력을 보이는바실러스 츄린기엔시스의 내독소 단백질 CryⅠAc 유전자 배추에서 잘 발현되도록 수정, 합성한 유전자를 제공하는 것이다.The present invention is a main vegetable widely cultivated not only in Korea but also in Northeast Asia, such as China and Japan, and protects cabbage, a basic ingredient of kimchi, which has already become a global food, from the damage of diamondback moth ( Plutella xylostella ), a major pest. In order to provide a gene that has been modified and synthesized so as to be well expressed in the endotoxin protein Cry IAc gene cabbage of Bacillus thuringiensis exhibiting insecticidal properties to cabbage moth.
Bt 본래의 내독소 단백질 유전자 CryⅠAc는 3,537bp로 아미노산 1,178개를 코드하고 있는데, C-말단 부위의 약 1,653bp는 내독소 단백질의 결정화(crystallization)에만 관여하고, 독성에는 무관하기 때문에, 살충성 관련부위인 N-말단의 1,854bp를 수정, 합성하였다. 한편, 유전자의 변형시, CryⅠAc 유전자의 살충성 관련 부위의 코돈을 배추과 작물과 유사하게 바꾸되 아미노산 배열은 변화시키지 않도록 코돈을 변화시킨다.Bt's native endotoxin protein gene CryIAc is 3,537 bp, which encodes 1,178 amino acids. About 1,653 bp of the C-terminal region is involved in crystallization of endotoxin protein and is not related to toxicity. N-terminal 1,854bp of the site was modified and synthesized. On the other hand, when the gene is modified, CryIAc Codons are altered in a similar way to cabbage crops, but not in the amino acid sequence, so that the codons in the pesticidal sites of the gene are altered.
한편, 합성 CryⅠAc 유전자를 수정, 합성하는 방법은 하기의 단계를 포함한다;Meanwhile, synthetic CryⅠAc Methods for modifying and synthesizing genes include the following steps;
① Bt 본래의 내독소 단백질 유전자 CryⅠAc의 살충성 관련 부위(이하 'cryIAc'라 한다) 및 배추 유전자의 코돈 사용 빈도를 나타내는 표를 작성하는 단계;(1) preparing a table showing the codon usage of the insecticidal-related site (hereinafter referred to as ' cryIAc ') of the Bt original endotoxin protein gene CryIAc and the cabbage gene;
② 상기 표를 이용하여,cryIAc유전자의 염기서열을 수정하는 전략을 세우는 단계; 및② formulating a strategy for modifying the nucleotide sequence of the cryIAc gene using the above table; And
③ 상기 전략을 토대로, 56개의 합성 프라이머를 이용 순환(Recursive) PCR로cryIAc유전자를 합성하는 단계.③ synthesizing the cryIAc gene by recursive PCR using 56 synthetic primers based on the above strategy.
수정 합성된cryIAc유전자를 배큘로바이러스(baculovirus) 발현 벡터 pBacPAK8에 삽입하고, 곤충세포에서 발현시킨 후, 발현 단백질의 살충성을 검정한 결과, 배추좀나방에 대한 살충성이 우수함을 확인하였다.The modified synthesized cryIAc gene was inserted into a baculovirus expression vector pBacPAK8, expressed in insect cells, and tested for the insecticidality of the expressed protein.
아울러, 수정 합성된cryIAc유전자를 발현벡터 pCAMBIA1390 내의 CaMV 35S 프로모터하에 조절이 되도록 도입한 식물 발현 벡터를 제작하고, 배추를 형질전환 한 후, 하이그로마이신(hygromycine)으로 선발된 형질전환체 배추로 배추좀나방에 대한 살충성을 검정한 결과, 우수한 살충력을 확인할 수 있었다.In addition, a plant expression vector in which the modified synthesized cryIAc gene was regulated under the CaMV 35S promoter in the expression vector pCAMBIA1390 was prepared, transformed cabbage, and then transformed into cabbage with a transformed cabbage selected with hygromycine. As a result of the insecticidal test for the moth, the excellent insecticidal activity was confirmed.
이하 실시예를 들어 본 발명을 상세히 설명하지만 본 발명이 이들예로 만 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited only to these Examples.
한편, 하기 실시예들에서 일반적으로 사용되는 실험방법을 정리하면 다음과 같다. 하기 방법에서 특별히 언급되지 않은 DNA 실험들은 Sambrook 등의 " Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989"을 참조하였다.On the other hand, summarized the experimental method generally used in the following examples. For DNA experiments not specifically mentioned in the following method, see Sambrook et al., "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989".
(1) 리게이션(Ligations): PCR 결과물 또는 제한효소 처리된 단편들을 0.8% 아가로스 겔에 TAE 버퍼에서 전기영동한 후, 목적하는 DNA 단편을 포함하는 아가로스 겔을 잘라내고, QIAEX II gel extraction kit(QIAGEN, Germany)를 이용하여 공급자가 제공하는 방법에 의하여 DNA 단편을 정제하였다.(1) Ligations: PCR results or restriction enzyme treated fragments were electrophoresed in 0.8% agarose gel in TAE buffer, and then agarose gels containing the desired DNA fragments were cut out and QIAEX II gel extraction. DNA fragments were purified using a kit (QIAGEN, Germany) by a method provided by a supplier.
정제된 DNA 단편은 다시 아가로스 겔에서 전기영동하여 농도를 확인한 후, 목적하는 단편과 벡터를 1:1로 혼합하고 T4 DNA 리가제를 사용하여 15℃에서 밤새 리게이션시킨 후,E. coli에 형질전환하고, 앰피실린 (100㎍/㎖) 등 적당한 항생제 배지를 이용하여 클론을 선발하였다.The purified DNA fragments were again subjected to electrophoresis on an agarose gel to confirm the concentration, and then, the desired fragments and the vector were mixed 1: 1, ligation was performed at 15 ° C. using T4 DNA ligase overnight, and then, E. coli Transformants were cloned using appropriate antibiotic media such as ampicillin (100 μg / ml).
(2)E. coli형질전환:E. coli는 DH5??(GIBCO BRL)를 사용하였다. Sambrook 등에 따라 CaCl2방법으로 만든 competent cell 200㎕에 밤새 리게이션한 DNA 시료를 혼합한 후, 얼음에서 30분, 42℃에서 90초, 얼음에서 다시 10분동안 처리하였다, 그 다음, 액체 LB배지 800㎕를 첨가하여 150rpm으로 흔들어주며 1시간 동안 배양하고, 앰피실린 (100 ug/ml) 등 적당한 항생제를 함유하는 배지(LB 배지)에 도말한 후, 37 ℃에서 밤새 배양하였다. 필요에 따라서는 0.008% X-gal과 80mMIPTG(isopropylthio-beta-galactoside)를 사용하여 벡터의 beta-galactosidase 유전자 발현여부를 보고 목적 DNA 단편의 삽입여부를 확인하였다.(2) E. coli transformation: E. coli used DH5 ?? (GIBCO BRL). The DNA samples obtained by ligating overnight were mixed in 200 μl of competent cells made by CaCl 2 method according to Sambrook et al., And then treated for 30 minutes on ice, 90 seconds at 42 ° C., and another 10 minutes on ice. Then, liquid LB medium 800 μl was added and shaken at 150 rpm for 1 hour, incubated in medium containing a suitable antibiotic such as ampicillin (100 ug / ml) (LB medium), and then incubated overnight at 37 ° C. If necessary, 0.008% X-gal and 80mMIPTG (isopropylthio-beta-galactoside) were used to confirm the beta-galactosidase gene expression of the vector and to confirm the insertion of the target DNA fragment.
(3) 플라스미드 분리 및 삽입 단편 확인: 선택된 형질전환E. coli를 앰피실린 등 적당한 항생제를 포함하는 액체 배지 5㎖에 접종하고, 37℃, 250rpm에서 밤새 배양한 후, Sambrook 등에 따라 알칼리 방법으로 플라스미드를 분리하였다. 분리된 플라스미드는 적당한 제한효소로 처리한 후 전기영동하여 확인하거나, QIAGEN miniprep kit(QIAGEN, Germany)를 이용하여 플라스미드를 분리한 후, DNA 염기서열을 확인하였다.(3) Plasmid Isolation and Insertion Fragment Identification: The selected transformed E. coli was inoculated into 5 ml of liquid medium containing an appropriate antibiotic such as ampicillin, incubated overnight at 37 ° C. and 250 rpm, followed by plasmid by alkaline method according to Sambrook et al. Was separated. The separated plasmid was confirmed by electrophoresis after treatment with a suitable restriction enzyme, or DNA sequencing was confirmed after separation of the plasmid using a QIAGEN miniprep kit (QIAGEN, Germany).
실시예 1.Example 1. cryIAccryIAc 유전자 코돈 사용빈도 분석Gene codon usage analysis
CryIAc 유전자의 염기서열은 서열 번호 1과 같다. 이를 토대로cryIAc의 코돈 사용빈도를 조사하여 그 결과를 표 1에 나타내었다. 하기 표 1로부터,cryIAc유전자의 G+C 함량이 약 37.32%라는 것을 알 수 있다.The base sequence of the CryIAc gene is shown in SEQ ID NO: 1. Based on this, the codon usage of cryIAc was investigated and the results are shown in Table 1. From Table 1, it can be seen that the G + C content of the cryIAc gene is about 37.32%.
실시예 2. 배추 유전자의 코돈 사용빈도 분석Example 2 Codon Usage of the Chinese Cabbage Gene
하기 12개의 배추 유전자들의 코돈 사용 빈도를 조사하여, 그 결과를 표 2에 나타내었다.The codon usage frequency of the following 12 Chinese cabbage genes was examined, and the results are shown in Table 2.
1.Brassica campestris(clone BE5) napis mRNA. Genbank Accession No. M64631(서열번호 3) Brassica campestris (clone BE5) napis mRNA. Genbank Accession No. M64631 (SEQ ID NO: 3)
2.Brassica rapaubiquitin/ribosomal protein mRNA. Genbank Accession No. L21898(서열번호 5) Brassica rapa ubiquitin / ribosomal protein mRNA. Genbank Accession No. L21898 (SEQ ID NO: 5)
3.Brassica raparibosomal protein L37A(RPL37A) mRNA. Genbank Accession No. L21897(서열번호 7)3. Brassica rapa ribosomal protein L37A (RPL37A) mRNA. Genbank Accession No. L21897 (SEQ ID NO: 7)
4.Brassica campestrisL. ssppekinensis(clone bif98) metallothionein- like protein mRNA. Genbank Accession No. L31940(서열번호 9) Brassica campestris L. ssp pekinensis (clone bif98) metallothionein-like protein mRNA. Genbank Accession No. L31940 (SEQ ID NO: 9)
5.Brassica campestrisssp.pekinensis(clone bif11) photosystem II 10kDa polypeptide mRNA. Genbank Accession No. L31936(서열번호 11)5. Brassica campestris ssp. pekinensis (clone bif11) photosystem II 10kDa polypeptide mRNA. Genbank Accession No. L31936 (SEQ ID NO: 11)
6.Brassica rapa(clone bif25) protease inhibitor II mRNA. Genbank Accession No. L31937(서열번호 13)6. Brassica rapa (clone bif25) protease inhibitor II mRNA. Genbank Accession No. L31937 (SEQ ID NO: 13)
7.Brassica rapa(bif38) mRNA. Genbank Accession No. L31938(서열번호 15)7. Brassica rapa (bif38) mRNA. Genbank Accession No. L31938 (SEQ ID NO: 15)
8.Brassica rapa(clone bif72) kin mRNA. Genbank Accession No. L31939(서열번호 17)8. Brassica rapa (clone bif72) kin mRNA. Genbank Accession No. L31939 (SEQ ID NO: 17)
9.Brassica campestrisanther-specific mRNA. Genbank Accession No.L48179(서열번호 19)9. Brassica campestris anther-specific mRNA. Genbank Accession No. L48179 (SEQ ID NO: 19)
10.Brassica campestrisanther-specific(BNA237) mRNA. Genbank Accession No. L48180(서열번호 21)10. Brassica campestris anther-specific (BNA237) mRNA. Genbank Accession No. L48180 (SEQ ID NO: 21)
11.Brassica campestrisL.pekinensisypt-related protein mRNA. Genbank Accession No. L48181(서열번호 23)11. Brassica campestris L. pekinensis ypt-related protein mRNA. Genbank Accession No. L48181 (SEQ ID NO: 23)
상기 표 1과 표 2의cryIAc유전자와 배추 유전자의 코돈 사용빈도를 비교하면, 각 아미노산 코돈의 세 번째 염기에서 배추의 경우 전반적으로 G 또는 C의 사용 빈도가 높았는데, 특히 페닐알라닌(wt-cryIAc: TTT 83 %, TTC 17 %, 배추: TTT 45 %, TTC 55 %), 티로신(wt-cryIAc: TAT 86 %, TAC 14 %, 배추: TAT 37 %, TAC 63 %), 이소류신(wt-cryIAc: ATT 49 %, ATC 13 %, ATA 38 %, 배추: ATT 36 %, ATC 48 %, ATA 19 %), 아스파라긴(wt-cryIAc: AAT 71 %, AAC 29 %, 배추: AAT 40 %, AAC60 %) 및 아스파르트산(wt-cryIAc: GAT 80 %, GAC 20 %, 배추: GAT 59 %, GAC 41 %)들에서 특히 차이가 있다는 것을 알 수 있다.Comparing the codon usage of the cryIAc gene and the Chinese cabbage gene of Table 1 and Table 2, the use of G or C was generally higher in the third base of each amino acid codon, especially phenylalanine (wt- cryIAc : TTT 83%, TTC 17%, Chinese cabbage: TTT 45%, TTC 55%), tyrosine (wt- cryIAc : TAT 86%, TAC 14%, cabbage: TAT 37%, TAC 63%), isoleucine (wt- cryIAc : ATT 49%, ATC 13%, ATA 38%, Chinese cabbage: ATT 36%, ATC 48%, ATA 19%), Asparagine (wt- cryIAc : AAT 71%, AAC 29%, Chinese cabbage: AAT 40%, AAC60%) And aspartic acid (wt- cryIAc : 80% GAT, 20% GAC, cabbage: 59% GAT, 41% GAC).
한편, 조사한 배추 유전자들의 G+C 함량은 평균 48.5%로cryIAc유전자의 37.32%보다는 10% 이상 높았다.On the other hand, the G + C content of the cabbage genes examined was 48.5% on average, more than 10% higher than 37.32% of cryIAc genes.
실시예 3.Example 3. cryIAccryIAc 의 염기 서열의 수정 전략Strategy of Modification of Base Sequences
상기 표 2의 배추 유전자들의 코돈 이용 빈도를 이용하여 표 1의cryIAc유전자의 코돈을 바꾸되 아미노산의 서열에는 변화가 없도록 염기서열을 수정한다. 이때 진핵세포에서 유전자의 발현조절에 관여하는 DNA 염기서열과 유사한 부분, 예를 들어 TATA box나 CAAT box, 폴리아데닐레이션 시그널(AATAAA를 위시한 연속된 6개 이상의 AT 염기서열), 및 인트론(intron)이 될 가능성이 있는 부분(AGGT, 인트론 3'가능 부분)의 코돈을 바꾸어, 이 서열들이 제거되도록 한다. 한편, mRNA의 안정성과 관련이 있을 것으로 보고된 ATTTA 염기서열도 전부 제거하도록 한다.By using the codon usage frequency of the cabbage genes of Table 2cryIAcGene codon Change the nucleotide sequence so that there is no change in the sequence of amino acids. At this time, a portion similar to the DNA sequence involved in gene expression control in eukaryotic cells, such as TATA box or CAAT box, polyadenylation signal (sequential 6 or more AT sequences including AATAAA), and intron Change the codons of the part that is likely to be (AGGT, intron 3 'capable part) to remove these sequences. Meanwhile, all ATTTA sequences reported to be related to mRNA stability should be removed.
또한, 배추 유전자의 G+C 함량보다cryIAc유전자의 G+C 함량이 작으므로,cryIAc유전자의 G+C 함량을 배추 유전자의 G+C 함량과 유사하게 조절한다.In addition, since the G + C content of the cryIAc gene is smaller than the G + C content of the cabbage gene, the G + C content of the cryIAc gene is controlled to be similar to the G + C content of the cabbage gene.
또한, 유전자 합성을 위해 PCR을 한 후, 각 DNA 단편을 연결하기 위하여 필요한 제한효소 자리를 만들고 불필요한 제한효소 자리는 제거하도록 하며, 처음 부분에 번역 시작 코돈을 포함하여 제한효소 NcoI (CCATGG) 자리를 만들고, 종료 코돈(TAA) 뒤에 제한효소 KpnI (GGTACC) 자리를 추가한다.In addition, after PCR for gene synthesis, make restriction enzyme sites necessary to connect each DNA fragment, remove unnecessary restriction enzyme sites, and place the restriction enzyme NcoI (CCATGG) sites including the translation start codon at the beginning. The restriction enzyme KpnI (GGTACC) site is added after the termination codon (TAA).
그 결과, 수정된cryIAc의 코돈 사용 빈도를 조사하면 하기 표 3과 같다.As a result, the codon usage frequency of the modified cryIAc is investigated as shown in Table 3 below.
즉, 전체 코돈 618개 중 56.5%에 해당하는 349개 코돈이 변환되도록 하였고, G+C함량 또한 배추 유전자의 그것과 유사한 48.22% 조절하였다.That is, 349 codons corresponding to 56.5% of the total 618 codons were converted, and the G + C content was also regulated by 48.22% similar to that of the Chinese cabbage gene.
한편,cryIAc와 수정된cryIAc유전자를 비교하면, 표 4와 같다.On the other hand, comparing cryIAc and the modified cryIAc gene, it is shown in Table 4.
실시예 4.Example 4. cryIAccryIAc 의 유전자를 수정, 합성하기 위한 프라이머의 합성;Synthesis of primers for modifying and synthesizing a gene of;
cryIAc유전자를 수정 합성하기 위하여 순환(Recursive) PCR 방법을 사용하는데 , 이를 위해 상기 실시예 3의 전략에 따라 프라이머를 합성하였다.Recursive PCR is used to modify and synthesize the cryIAc gene. For this, primers were synthesized according to the strategy of Example 3 above.
즉, 프라이머는 하기에서와 같이 모두 3세트로 56개를 합성하는데, 50-mer47개, 49-mer 3개, 48-mer 2 개, 47-mer 1개, 44-mer 1개, 39-mer 1개, 32-mer 1개를 합성하였다. 각 프라이머는 순서대로 인접한 프라이머와 5' 및 3' 각각 14~18 염기씩 중첩되도록 하되, 중첩부분의 어닐닝(annealing) 온도가 45℃이상이 되도록 하였다. 한편, 본 발명에서 프라이머는 (주)BMS(서울 서초구 서초동 1717-55)에 제작을 의뢰하여 합성하였다.That is, the primers synthesize 56 in all three sets as follows: 50-mer47, three 49-mers, two 48-mers, one 47-mer, one 44-mer, and 39-mer One, one 32-mer was synthesized. Each primer was sequentially overlapped with adjacent primers by 14-18 bases of 5 'and 3', respectively, but the annealing temperature of the overlapping portion was 45 ° C. or higher. On the other hand, in the present invention, the primer was synthesized by commissioning BMS (1717-55, Seocho-dong, Seocho-gu, Seoul).
프라이머 세트 1번;Primer set 1;
cryIAc유전자의 1,344염기부터 1,854염기까지의 PCR을 위한 프라이머로서, 1,854염기에 번역종료 코돈 TAA 및 제한효소 KpnI(GGTACC)을 포함하고 있다. 이들의 PCR을 위한 배열 및 제한효소 자리는 도 1과 같다.As a primer for PCR from 1,344 bases to 1,854 bases of the cryIAc gene, 1,854 bases contain translation termination codon TAA and restriction enzyme KpnI (GGTACC). The arrangement and restriction enzyme sites for their PCR are shown in FIG. 1.
프라이머 세트 2번;Primer set 2;
cryIAc유전자의 864염기부터 1,351염기까지의 PCR을 위한 프라이머이다. 이들의 PCR을 위한 배열 및 제한효소 자리는 도 2와 같다.Primer for PCR from 864 base to 1,351 base of the cryIAc gene. The arrangement and restriction enzyme sites for their PCR are shown in FIG. 2.
프라이머 세트 3번;Primer set 3;
cryIAc유전자의 1염기부터 900염기까지의 PCR을 위한 프라이머로서, 시작코돈 앞에 CC를 포함하고 있다. 이들의 PCR을 위한 배열 및 제한효소 자리는 도 3과 같다.Primer for PCR from base 1 to base 900 of the cryIAc gene, including the CC before the start codon. The arrangement and restriction enzyme sites for their PCR are shown in FIG. 3.
실시예 5. 순환 PCR, PCR한 DNA 단편의 염기서열 확인 및 연결Example 5. Cyclic PCR, DNA Sequence Identification and Linking of PCR DNA Fragments
순환 PCR은 Prodromou와 Pearl의 방법(Protein engineering, 1992, 827-829)을 응용하였다.Cyclic PCR applied the method of Prodromou and Pearl (Protein engineering, 1992, 827-829).
즉, 목적하는 DNA 단편의 양쪽 끝 프라이머는 20pmol, 중간의 프라이머는 0.2pmol 농도가 되도록 첨가하고, 95℃ 2분, 45~55℃ 2분, 72℃ 1분으로 30회 PCR반응을 수행하였다. 이 때, 폴리머라제는 교정(proofreading) 기능을 수행하며, 블런트-말단(blunt-end)을 만드는 PWO 폴리머라제(Boehringer Mannheim) 또는 Pyrobest(TAKARA)를 사용하였다. 예상되는 크기의 PCR 단편을 분리하여, pBluescript SK(+)(Stratagene)의 EcoRV 또는 SmaⅠ에 삽입하여,E. coli에 형질전환하고, 단편이 삽입된 백색 콜로니를 선발하였다.That is, the primers at both ends of the desired DNA fragment were added at a concentration of 20 pmol and the intermediate primer at a concentration of 0.2 pmol, and the PCR reaction was performed 30 times at 95 ° C for 2 minutes, 45 to 55 ° C for 2 minutes, and 72 ° C for 1 minute. At this time, the polymerase was used as PWO polymerase (Boehringer Mannheim) or Pyrobest (TAKARA) to perform a proofreading function and to make a blunt-end. PCR fragments of expected size were isolated, inserted into EcoRV or SmaI of pBluescript SK (+) (Stratagene), transformed into E. coli , and white colonies with fragments inserted were selected.
선발된 백색 콜로니 10개를 QIAGEN miniprep kit를 이용하여 플라스미드를 분리하고, DNA Sequencing kit(BigDyeTMTerminator Cycle Sequencing Ready Reaction, Perkin-Elmer)를 사용하여 양방향에서 염기서열을 분석하여 계획된 염기서열과 일치하는 클론을 선택하였다.Plasmids were isolated from 10 selected white colonies using a QIAGEN miniprep kit, and sequenced in both directions using a DNA sequencing kit (BigDye ™ Terminator Cycle Sequencing Ready Reaction, Perkin-Elmer) to match the planned sequence. Clones were selected.
수정된cryIAc전체에 해당하는 1,865bp를 포함하게 되었을 때, PCR 단편 클론은 7개였다.When the 1,865bp corresponding to the entire modified cryIAc was included, there were 7 PCR fragment clones.
각 PCR 단편의 제작을 상술하면 다음과 같다(도 4 참조).Detailed description of the preparation of each PCR fragment is as follows (see Fig. 4).
1) p1-3 : primer 3-1부터 primer 3-12까지 12개 primer의 PCR 단편 약 420 bp로서, 수정cryIAc유전자의 처음부터 CfoI(그림 2의 419 염기)까지를 포함한다. pBluescript SK(+)의 EcoRV 자리에 클로닝하였다. p2-4의 CfoI 자리와 연결된다.1) p1-3: PCR fragments of 12 primers from primer 3-1 to primer 3-12 are about 420 bp and include CfoI (base 419 in Figure 2) from the beginning of the modified cryIAc gene. Cloning into the EcoRV site of pBluescript SK (+). is linked to the CfoI site of p2-4.
2) p2-4 : primer 3-11부터 primer 3-22까지 12개 primer의 PCR 단편 약 450bp로서, 수정cryIAc유전자의 AccI(381 염기)부터 XbaI(668 염기)까지를 포함한다. pBluescript SK(+)의 EcoRV 자리에 클로닝하였다. p1-3의 CfoI 자리 및 p5-4의 DraII 자리와 연결된다.2) p2-4: PCR fragment of 12 primers from primer 3-11 to primer 3-22 is about 450bp and includes from AccI (381 base) to XbaI (668 base) of the modified cryIAc gene. Cloning into the EcoRV site of pBluescript SK (+). the CfoI site of p1-3 and the DraII site of p5-4.
3) p5-4 : primer 3-19부터 primer 3-24까지 6개 primer의 PCR 단편 약 190 bp로서, 수정cryIAc유전자의 DraII(659 염기)부터 DsaI(841 염기)까지를 포함한다. pBluescript SK(+)의 EcoRV 자리에 클로닝하였다. p2-4의 DraII 자리 및 p7-3의 DsaI 자리와 연결된다.3) p5-4: PCR fragment of 6 primers from primer 3-19 to primer 3-24 is about 190 bp and includes DraII (659 base) to DsaI (841 base) of the modified cryIAc gene. Cloning into the EcoRV site of pBluescript SK (+). the DraII site of p2-4 and the DsaI site of p7-3.
4) p7-3 : primer 3-25, 3-26, 2-1, 2-1까지 4개 primer의 PCR 단편 약 170 bp로서, 수정cryIAc유전자의 DsaI(841 염기)부터 MroI(968 염기)까지를 포함한다. pBluescript SK(+)의 EcoRV 자리에 클로닝하였다. p5-4의 DsaI 자리 및 pBS10의 MroI 자리와 연결된다.4) p7-3: PCR fragment of 4 primers up to primers 3-25, 3-26, 2-1, and 2-1, about 170 bp, from DsaI (841 base) to MroI (968 base) of the modified cryIAc gene. It includes. Cloning into the EcoRV site of pBluescript SK (+). the DsaI site of p5-4 and the MroI site of pBS10.
5) pBS10 : primer 2-1부터 primer 2-8까지 8개 primer의 PCR 단편 약 240 bp로서, 수정cryIAc유전자의 MroI(968 염기)부터 AccI(1074 염기)까지를 포함한다. pBluescript SK(+)의 SmaI 자리에 클로닝하였다. p7-3의 MroI 자리 및 pP6의 AccI 자리와 연결된다.5) pBS10: PCR fragment of 8 primers from primer 2-1 to primer 2-8, about 240 bp, including MroI (968 base) to AccI (1074 base) of the modified cryIAc gene. It was cloned into the SmaI site of pBluescript SK (+). the MroI site of p7-3 and the AccI site of pP6.
6) pP6 : primer 2-7부터 primer 2-14까지 8개 primer의 PCR 단편 약 380 bp로서, 수정cryIAc유전자의 AccI(1074 염기)부터 SacI(1351 염기)까지를 포함한다. pBluescript SK(+)의 SmaI 자리에 클로닝하였다. p1-3의 CfoI 자리 및 p5-4의 DraII 자리와 연결된다.6) pP6: PCR fragment of 8 primers from primers 2-7 to primers 2-14, about 380 bp, including AccI (1074 base) to SacI (1351 base) of the modified cryIAc gene. It was cloned into the SmaI site of pBluescript SK (+). the CfoI site of p1-3 and the DraII site of p5-4.
7) p17 : primer 1-1부터 primer 1-16까지 16개 primer의 PCR 단편 약 520bp로서, 수정cryIAc유전자의 SacI(1351 염기)부터 1854 염기와 번역 종료 코돈 다음에 첨부된 KpnI(1865 염기)까지를 포함한다. pBluescript SK(+)의 EcoRV 자리에 클로닝하였다. pP6의 SacI 자리와 연결된다.7) p17: PCR fragments of 16 primers from primer 1-1 to primer 1-16, approximately 520 bp, from SacI (1351 base) of the modified cryIAc gene to 1854 base and KpnI (1865 base) following the translation termination codon. It includes. Cloning into the EcoRV site of pBluescript SK (+). is associated with the SacI site of pP6.
상기 PCR 단편의 연결을 상술하면 다음과 같다(도 5 참조).The connection of the PCR fragment is described in detail as follows (see FIG. 5).
1) p1-3를 SmaI과 CfoI로, p2-4를 CfoI과 EcoRI로, pBluescript SK(+)를 HincⅡ와 EcoRI로 절단하고, 각각 분리한 후 연결하여(three-way ligation) p13-24를 제작하였다.1) p1-3 was cut into SmaI and CfoI, p2-4 was cut into CfoI and EcoRI, pBluescript SK (+) was cut into HincII and EcoRI, and p13-24 was prepared by separating and connecting them respectively (three-way ligation). It was.
2) pP6을 ClaI과 SacI로, p17을 SacI과 XbaI로, pBluescript SK(+)를 ClaI과 XbaI으로 절단하고, 각각 분리한 후 연결하여(three-way ligation) p6-17을 제작하였다.2) pP6 was cut into ClaI and SacI, p17 was cut into SacI and XbaI, and pBluescript SK (+) was cut into ClaI and XbaI, and then separated and linked (three-way ligation) to prepare p6-17.
3) pBS10을 BamHI과 MroI로, p7-3을 BamHI과 MroI으로 절단하고, 각각 분리한 후 연결하여 pBS10-73을 제작하였다.3) pBS10 was cut into BamHI and MroI, and p7-3 was cut into BamHI and MroI, and then separated and connected to prepare pBS10-73.
4) p5-4를 DsaI과 EcoRI로, pBS10-73을 EcoRI과 DsaI으로 절단하고, 각각 분리한 후 연결하여 p54를 제작하였다.4) p5-4 was digested with DsaI and EcoRI, pBS10-73 was digested with EcoRI and DsaI, and separated and connected, respectively, to prepare p54.
5) p54를 ApaI과 DraII로, p13-24를 ApaI과 DraII로 절단하고, 각각 분리한 후 연결하여 p54-13을 제작하였다.5) p54 was cut into ApaI and DraII, p13-24 was cut into ApaI and DraII, and each was separated and connected to prepare p54-13.
6) p54-13을 AccI과 BamHI로, p6-17을 AccI과 BamHI으로 절단하고, 각각 분리한 후 연결하여 pBt9를 제작하였다.6) p54-13 was digested with AccI and BamHI, and p6-17 was digested with AccI and BamHI.
pBt9의 염기서열을 확인한 결과, 서열번호 25로 표시된다.When the nucleotide sequence of pBt9 was confirmed, it is shown by SEQ ID NO: 25.
실시예 6. 합성Example 6. Synthesis cryIAccryIAc 유전자의 시험관내 발현 및 살충성 검정In vitro expression and pesticidal assay of genes
6-1. 곤충세포배양6-1. Insect Cell Culture
곤충세포주는 Sf9(Spodoptera frugiperda,CloneTech)을 이용하였고, 10% FBS(fetal bovine serum; Gibco-BRL, USA)가 함유된 곤충세포주 배양용 TC-100 (Gibco-BRL, USA)배양액으로 Summers와 Smith의 방법(1987)에 따라 27℃ 항온기에서 초기분주농도를 플라스크(25㎠)당 2×106세포로하여 4~5일마다 계대배양하였다.Insect cell line was Sf9 ( Spodoptera frugiperda, CloneTech), and TC-100 (Gibco-BRL, USA) culture medium for insect cell line culture containing 10% FBS (fetal bovine serum; Gibco-BRL, USA) According to the method (1987), the initial dispensing concentration in a 27 ℃ thermostat was passaged every 4-5 days with 2 × 10 6 cells per flask (25 cm 2).
6-2. 재조합 전이벡터 작성6-2. Create Recombinant Transfer Vector
상기 실시예 5에서 제작한 pBt9 플라스미드를 제한효소 XhoI과 SmaI으로 절단하여 합성cryIAc유전자를 분리하고, 동일한 제한효소로 처리된 전이벡터 pBacPAK8(CloneTech, USA)에 삽입하여, 재조합 전이벡터 pBac8-Bt9를 작성하였다(도 6 참조)The pBt9 plasmid prepared in Example 5 was cleaved with restriction enzymes XhoI and SmaI to separate the synthetic cryIAc gene, and inserted into the transfer vector pBacPAK8 (CloneTech, USA) treated with the same restriction enzyme, and the recombinant transfer vector pBac8-Bt9 was inserted. Created (see FIG. 6).
6-3. 재조합 바이러스 제작 및 선발6-3. Recombinant virus production and selection
6-1에서 배양한 Sf9 세포주에 재조합 전이벡터 pBac8-Bt9의 DNA 500ng과Bsu36I으로 절단된 BacPAK6(Clontech, USA) viral DNA 5㎕를 혼합하고, 리포펙틴(lipofectin; 11㎎/㎖) 15㎕를 첨가하여 27℃에서 5시간 동안 동시감염시켰다. 이 후, 새 배양액으로 교체하고, 접종 5일 후 바이러스 병징을 관찰하여 감염유무를 확인하였다. 바이러스 순화를 위한 한계희석(end-point dilution)은 바이러스 접종액을 10-2~10-8배까지 희석하고, 96-웰 플레이트에 웰당 세포 1×104개의 농도로 분주된 배양세포에 각 희석액을 접종한 다음, 27℃에서 배양하였다. 접종 후 4일부터 바이러스 감염유무를 도립현미경으로 관찰하여 바이러스를 선발하고,동일한 방법으로 3회 이상의 순화과정을 거쳐 순수한 재조합 바이러스 vBac8-Bt9을 선발하였다500 μl of the recombinant transfer vector pBac8-Bt9 and 5 μl of BacPAK6 (Clontech, USA) viral DNA digested with Bsu36 I were mixed in the Sf9 cell line cultured in 6-1, and 15 μl of lipofectin (11 mg / ml). Was co-infected at 27 ° C. for 5 hours. Thereafter, the resultant was replaced with a new culture solution, and 5 days after the inoculation, the virus symptoms were observed to confirm the infection. End-point dilution for virus purification dilutes the virus inoculum by 10 −2 to 10 −8 folds and each dilution in cultured cells dispensed at a concentration of 1 × 10 4 cells per well in 96-well plates. And then incubated at 27 ℃. From the 4th day after inoculation, the virus was examined by inverted microscopy to select the virus, and the same method was used to select the pure recombinant virus vBac8-Bt9 after three or more purification processes.
6-4. 외래유전자 발현 확인6-4. Confirmation of foreign gene expression
Sf9 세포주를 35mm 직경의 디쉬에 1×106세포로 분주하고, wt-AcNPV (CloneTech)와 재조합 바이러스 vBac8-Bt9 감염액을 세포당 5PFU농도로 각각 접종한 후, 감염 3일째에 각각 수거하여, 단백질 전기 영동과 생물검정으로 외래유전자 발현을 확인하였다.Sf9 cell lines were divided into 35 mm diameter dishes into 1 × 10 6 cells, and the wt-AcNPV (CloneTech) and the recombinant virus vBac8-Bt9 infection solution were respectively inoculated at 5 PFU concentration per cell, and collected on the third day of infection. Protein electrophoresis and bioassay confirmed foreign gene expression.
6-4-1. 단백질 전기영동6-4-1. Protein Electrophoresis
바이러스 감염세포의 단백질에 대한 전기영동은 바이러스가 감염된 세포를 거두어 PBS완충액(137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)으로 2회 세척한 다음, 2배 농도의 단백질 시료 용액(0.0625M Tris-HCl(pH 6.8), 2% SDS, 10% 글리세롤, 5% β-머캅토에탄올, 0.001% 브로모페놀블루)을 같은 양으로 혼합하고, 100℃에서 10분간 처리하여 전기영동시료를 준비하였다. 전기영동은 Laemmli 방법 (1970)에 따라 SDS-폴리아크릴아마이드겔에서 수행하고 Commassie brilliant blue로 염색하여 관찰하였다(도 7(A) 참조).Electrophoresis of the virus-infected cells protein was performed by harvesting the virus-infected cells and washing twice with PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 ) and then doubling the concentration. Protein sample solution (0.0625M Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.001% bromophenol blue) in the same amount and mixed for 10 minutes at 100 ℃ The electrophoretic sample was prepared by treatment. Electrophoresis was performed on SDS-polyacrylamide gel according to Laemmli method (1970) and stained with Commassie brilliant blue (see FIG. 7 (A)).
6-4-2. 웨스턴 블럿 분석6-4-2. Western blot analysis
단백질 시료를 전기영동한 후 PVDF막에 전이시켰다. PVDF 막을 5% 스킴밀크용액으로 블로킹시키고, cryIAc 항체(Adgia 제품 : PSB05200)를 100:1의 비율로 5% 스킴-밀크 용액에 희석하여 반응시켰다. TBST 용액으로 3회 및 TBS 용액으로 1회세척하고, BP-A와 BP-B 1:1 혼합액(Adgia 제품)으로 발색시킨 후, 다시 TBS 용액으로 세척하였다(도 7(B) 참조).Protein samples were electrophoresed and then transferred to PVDF membranes. The PVDF membrane was blocked with 5% skim milk solution and the cryIAc antibody (Adgia: PSB05200) was diluted and reacted with 5% skim-milk solution at a ratio of 100: 1. Washed three times with TBST solution and once with TBS solution, developed with BP-A and BP-B 1: 1 mixture (Adgia), and washed again with TBS solution (see FIG. 7 (B)).
6-4-3. 생물검정6-4-3. Bioassay
배추좀나방에 대한 바이러스의 생물검정은 Evans(1981)의 표면 오염법 (surface contamination assay)에 따라 실시하였다. 즉, 2㎠ 크기로 자른 배추 잎을 2개 준비하고 한쪽에는 vBac-Bt9 감염세포 현탁액을, 다른 한쪽에는 wt-AcNPV 배양액을 각각 도말하였다. 그 다음, 6시간 이상 굶긴 3령 또는 4령의 배추좀나방 유충 20마리씩을 사용하여 각각의 배추잎을 섭식하도록 하였다. 섭식 18시간 경과후, 배추잎을 관찰하고, 그 결과를 도 8a 및 도 8b에 나타내었다.Bioassay of the virus against Chinese cabbage moth was performed according to the surface contamination assay of Evans (1981). That is, two cabbage leaves cut to a size of 2 cm 2 were prepared, one was coated with a vBac-Bt9 infected cell suspension, and the other was coated with wt-AcNPV culture. Each cabbage leaf was then fed with 20 larvae of three or four cabbage moths starved for more than six hours. After 18 hours of feeding, the cabbage leaves were observed, and the results are shown in FIGS. 8A and 8B.
도 8a 및 8b로부터, vBac-Bt9 감염세포 현탁액이 도말된 배추잎(9b)에서는 배추잎의 손상없이 배추좀나방이 모두 죽었음을 확인할 수 있었다.8A and 8B, it was confirmed that all of the cabbage moths died in the cabbage leaf 9b to which the vBac-Bt9 infected cell suspension was smeared.
실시예 7. 합성Example 7. Synthesis cryIAccryIAc 유전자의 식물 발현Plant expression of genes
7-1. 식물 발현 벡터 제작7-1. Plant Expression Vector Production
pBt9의 합성cryIAc(1.86kb)를 EcoRI으로 잘라내어 pBluescript SK(+)의 EcoRI 자리에 삽입하였다. 그 결과 합성cryIAc의 삽입 방향에 따라 벡터 pBt91과 pBt92를 얻었다(도 9 참조).Synthesis of pBt9 cryIAc (1.86 kb) was cut with EcoRI and inserted into the EcoRI site of pBluescript SK (+). As a result, the vectors pBt91 and pBt92 were obtained according to the insertion direction of the synthetic cryIAc (see FIG. 9).
그 다음, 벡터 pBt91을 HindⅢ와 NcoⅠ으로 절단하고, pCAMBIA3301(Center for the Application of Molecular Biology to International Agriculture, 호주)을 HindⅢ와 NcoⅠ으로 절단한 후, 0.8kb CaMV35S 프로모터를 분리하여 연결하여 p35SBt를 제작하였다. p35SBt를 HindⅢ와 BamHⅠ으로 절단하여 2.65kb(CaMV35S 프로모터/합성cryIAc)를 분리한 후, pCAMBIA1390를 HindⅢ와 BamHⅠ으로 절단한 부위에 삽입하여 식물 발현 벡터 pGRBt20을 제작하였다(도 10 참조).Subsequently, the vector pBt91 was cleaved with HindIII and NcoI, the pCAMBIA3301 (Center for the Application of Molecular Biology to International Agriculture, Australia) was cleaved with HindIII and NcoI, and the 0.8kb CaMV35S promoter was separated and connected to prepare p35SBt. . p35SBt was cleaved with HindIII and BamHI to separate 2.65 kb (CaMV35S promoter / synthetic cryIAc ), and then pCAMBIA1390 was inserted into the site cleaved with HindIII and BamHI to prepare a plant expression vector pGRBt20 (see FIG. 10).
7-2. 형질전환7-2. Transformation
상기 발현벡터 pGRBt20을 아그로박테리움 튜메페시엔스(Agrobacterium tumefaciens) LBA4404(Stratagene)에 형질전환시켰다. 형질전환은 An 등의 freeze-thaw 방법(G. An, P.R. Ebert, A. Mitra, and S.M. Ha, "Binary vectors" in Plant molecular biology manual. A3: 1-19. Kluwer Academic Publishers)을 사용하였다. 즉, 아그로박테리움 튜메페시엔스(Agrobacterium tumefaciens) LBA4404을 카나마이신(Kanamycin)이 50㎍/㎖ 함유된 YEP배지(물 1 리터당, Bacto-peptone 10g, Bacto-yeast extract 10g, NaCl 5g)에서 배양한 다음, 플라스미드를 분리하고, 제한효소 패턴을 분석하여, 아그로박테리움의 형질전환을 재확인한 후, 배추 형질 전환에 사용하였다.The expression vector pGRBt20 was transformed into Agrobacterium tumefaciens LBA4404 (Stratagene). Transformation was performed using Anze et al. Freeze-thaw method (G. An, PR Ebert, A. Mitra, and SM Ha, "Binary vectors" in Plant molecular biology manual. A3: 1-19. Kluwer Academic Publishers). That is, Agrobacterium tumefaciens LBA4404 was incubated in a YEP medium containing 50 µg / mL of Kanamycin (per liter of water, 10 g of Bacto-peptone, 10 g of Bacto-yeast extract, and 10 g of NaCl 5 g). The plasmids were isolated and the restriction enzyme patterns analyzed to confirm the transformation of Agrobacterium and then used for cabbage transformation.
7-3. 배추 형질 전환7-3. Cabbage transformation
배추는 샛노랑배추와 매력배추(농우종묘)를 사용하였다. 배추 종자를 70% 에탄올에 1분, 50% 클로락스에 20분동안 침지한 후, 150rpm으로 20분 동안 소독하고, 멸균수로 3~4회 세척하였다. 멸균된 배추의 종자들을 발아배지(MS기본배지, 3% 슈크로스, 0.3% 젤라틴, pH 5.8)에 옮겨 발아시켰다. 파종 5일 후, 떡잎이 완전히 전개되고, 배축의 길이가 약 4~5cm정도가 될 때까지 배양하였다. 배양 5일된 배추의 유묘로부터 하배축을 채취하였으며, 절단길이는 약 1cm정도로 하였다. 절단된 배축은 MS 고체배지나 MS 액체배지에서 1~2일 동안 전배양하였다. 액체배지를 사용할 때는 150rpm으로 흔들어 주면서 배양하였다.Chinese cabbage was used with yellow cabbage and attractive cabbage (bean seedling). Chinese cabbage seeds were soaked in 70% ethanol for 1 minute and 50% chlorax for 20 minutes, then sterilized at 150 rpm for 20 minutes and washed 3-4 times with sterile water. Seeds of sterilized cabbage were transferred to germination medium (MS base medium, 3% sucrose, 0.3% gelatin, pH 5.8) for germination. After 5 days of sowing, the cotyledon fully developed and incubated until the length of the hypocotyls was about 4-5 cm. The lower hypocotyl was collected from the seedlings of the cultured Chinese cabbage 5 days old, and the cut length was about 1 cm. The cut embryos were pre-cultured for 1 to 2 days in MS solid medium or MS liquid medium. When using the liquid medium was incubated with shaking at 150rpm.
상기 7-2의 pGRBt20으로 형질전환된아그로박테리움을 카나마이신이 첨가된 5㎖의 YEP액체배지에서 200rpm으로 흔들어주면서 2일 동안 배양하여, 종배양물(seed culture)을 만들었다. 그리고, 배추 형질 전환 1일전에아그로박테리움종배양물로부터 200㎕의 용액을 취하여 20㎖의 YEP 액체 배지로 옮겨 하루 동안 흔들어주면서 배양하였다. Agrobacterium transformed with pGRBt20 of 7-2 was incubated for 2 days with shaking at 200 rpm in a 5 ml YEP liquid medium to which kanamycin was added, thereby creating a seed culture. Then, 1 day before the Chinese cabbage transformation, 200 μl of the solution was taken from the Agrobacterium spp. Culture and transferred to 20 ml of YEP liquid medium and incubated with shaking for one day.
전배양된 배축을 20㎖의 MS 액체 배지가 들어 있는 코니칼튜브(cornical tube)에 넣고, 2㎖의아그로박테리움용액을 첨가한 다음, 이를 200rpm으로 20분 동안 흔들어 주면서 배축에 전반적으로 균이 도포되게 하였다. 그리고, MS 액체 배지는 버리고 과도한 MS용액은 멸균된 여과지로 제거하였다.아그로박테리움을 감염시킨 배추의 배축을 멸균된 9cm 여과지를 깐 MS 공배양(cocultivation)배지(MS 기본 배지, 3㎎/ℓ BA, 1㎎/ℓ NAA, 4㎎/ℓ AgNO3, 8g/ℓ plantagar, pH 5.6)에 약 50~100개씩 놓고 2 일동안 배양하였다.The precultured embryos were placed in a conical tube containing 20 ml of MS liquid medium, 2 ml of Agrobacterium solution was added and shaken at 200 rpm for 20 minutes to allow bacteria to grow in the embryo. Application was made. The MS liquid medium was discarded and excess MS solution was removed with sterile filter paper. A 9cm filter paper to sterilize the hypocotyl of the infection of the Agrobacterium cabbage laid MS co-culture (cocultivation) medium (MS basal medium, 3㎎ / ℓ BA, 1㎎ / ℓ NAA, 4㎎ / ℓ AgNO3, 8g / ℓ plantagar , pH 5.6) were put in about 50-100 each and incubated for 2 days.
공배양이 끝난 배축을 유전자가 전환된 세포를 선발하기 위하여 선발 배지(MS 기본 배지, 3㎎/ℓ BA, 1㎎/ℓNAA, 4㎎/ℓAgNO3, 8 g/ℓ plantagar, pH 5.8, 10 ㎎/ℓ하이그로마이신(hygromycin), 300㎎/ℓ 세포탁심(cefotaxim))에서 25℃, 광조건하에서 배양하며, 배양 후 매 4주 간격으로 계대배양을 하여 캘루스를 형성시킨다. 캘루스로부터 잎이 형성되면, 한 캘루스에서 잘 자란 잎(재분화된 잎의 길이가 1cm이상)을 선발, 이를 MS 기본 배지로 구성된 뿌리 유기 배지(MS 기본배지, 8㎎/ℓ한천, 300㎎/ℓ세포탁심, 10㎎/ℓ하이그로마이신, pH 5.8)에서 뿌리를 유기시켰다. 뿌리가 유기된 녹색 식물체들은 버미큐라이트, 퍼라이트, 피트모스(2:1:1)를 혼합한 상토에 옮겨심고, 1주일간 순화시킨 후, 토양 포트에 옮겨 온실에서 재배하였다.Selection medium (MS basal medium, 3 mg / l BA, 1 mg / lNAA, 4 mg / l AgNO 3 , 8 g / l plantagar, pH 5.8, 10 mg) for selection of gene-converted cells for coculture / l hygromycin (hygromycin, 300mg / l cefotaxim)) incubated under light conditions at 25 ℃, and cultured at intervals every four weeks after culture to form callus. When the leaves are formed from callus, the well-grown leaves (regrown leaves of 1 cm or more) are selected from the callus, and the root organic medium (MS basal medium, 8 mg / L agar, 300 mg) composed of MS basal medium is selected. roots were incubated at / l celltaxime, 10 mg / l hygromycin, pH 5.8). Green plants with roots were planted in a mixture of vermiculite, perlite, and peat moss (2: 1: 1), purified for one week, and then transferred to soil pots and grown in greenhouses.
합성cryIAc유전자가 전환된 형질전환 배추들은 4℃ 저온실에서 30~40일정도 저온 처리한 다음 온실로 옮겨 화분화를 유기시키고, 자가 수분시켜서 후대종자를 획득하였다.Transgenic cabbages transformed with the synthetic cryIAc gene were cold treated for 30-40 days in a low temperature room at 4 ° C., and then transferred to a greenhouse to induce pollen and self-pollinated to obtain seed seed.
7-4. 유전자전환 배추의 생물검정7-4. Bioassay of Transgenic Chinese Cabbage
형질전환된 배추의 배추좀나방에 대한 살충성을 검정하였다. 즉, 합성cryIAc유전자 전환 배추와 정상 배추의 잎을 절단한 후, 배추좀나방 3령 유충 10마리씩 3반복으로 조사한 결과, 합성cryIAc유전자 전환 배추의 경우 섭식 18시간 내에 모두 죽었으며 배추잎도 거의 손상되지 않은 반면(도 11b), 정상 배추잎의 경우는 많이 손상되고 유충이 전혀 죽지 않았다(도 11a).Insecticidal activity against the cabbage moths of the transformed cabbage was assayed. That is, the synthesis cryIAc gene conversion and then cutting the leaves of Chinese cabbage and the normal Chinese cabbage, cabbage moth third instar when the larvae 10 rats examined in three repeats results, synthetic cryIAc gene conversion cabbage were all dead within feeding 18 hours hardly damage Chinese cabbage leaves On the other hand (Figure 11b), the normal cabbage leaves were much damaged and the larvae did not die at all (Figure 11a).
본 발명의 결과물인 합성cryIAc유전자는 배추의 유전자와 유사하게 수정된 유전자로, 형질전환 배추에서 높은 효율로 발현되며 배추좀나방 유충에 높은 살충성을 보였다. 따라서 이 합성cryIAc유전자를 이용한 배추를 비롯한 배추과 작물의 형질전환체 개발을 통하여 배추를 비롯한 배추과 작물의 중요 해충인 배추좀나방의 방제에 농약 사용 및 노동력 절감 효과를 얻을 수 있다.Synthetic cryIAc gene, which is a result of the present invention, is a gene modified similarly to that of cabbage, and is expressed at high efficiency in transgenic cabbage and showed high insecticidality against Chinese cabbage moth larvae. Therefore, through the development of transformants of Chinese cabbage and other crops using the synthetic cryIAc gene, it is possible to obtain pesticide use and labor saving effects in the control of Chinese cabbage moth, which is an important pest of Chinese cabbage and other crops.
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