KR20080103716A - Anther specific promoter derived from acapulo lily, recombinant vector, transgenic plant and preparation method - Google Patents

Anther specific promoter derived from acapulo lily, recombinant vector, transgenic plant and preparation method Download PDF

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KR20080103716A
KR20080103716A KR1020070050797A KR20070050797A KR20080103716A KR 20080103716 A KR20080103716 A KR 20080103716A KR 1020070050797 A KR1020070050797 A KR 1020070050797A KR 20070050797 A KR20070050797 A KR 20070050797A KR 20080103716 A KR20080103716 A KR 20080103716A
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specific promoter
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서은정
한봉희
예병우
우종규
유희주
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대한민국(관리부서:농촌진흥청)
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Abstract

A specific promoter derived from Lilium hybrid cv. Acapulco, a recombinant vector and a transgenic plant is provided to improve quality of transformant by expressing many proteins of a target gene, to increase an efficiency, to obtain an exact expression and to be used for cross breeding after introducing a luxury gene. A plant specific promoter includes a nucleotide having a homoeology over 70 % with a base sequence of a sequence number 1 or a complement thereof. A base sequence of a sequence number 2 is included in the promoter. A vector includes the plant specific promoter. The plant is asexuality-propagated by a method of culturing plant tissue transformed to the vector, cuttage or meristele.

Description

나리 유래의 약에 특이적인 프로모터, 재조합 벡터, 형질전환체 및 그의 제조 방법{ANTHER SPECIFIC PROMOTER DERIVED FROM ACAPULO LILY, RECOMBINANT VECTOR, TRANSGENIC PLANT AND PREPARATION METHOD}TECHNICAL SPECIFIC PROMOTER DERIVED FROM ACAPULO LILY, RECOMBINANT VECTOR, TRANSGENIC PLANT AND PREPARATION METHOD

도 1은 페튜니아를 형질전환하기 위한 재조합 벡터의 지도이다.1 is a map of a recombinant vector for transforming petunias.

도 2는 페튜니아를 형질전환한 후 서든 분석한 결과이다. 도 2에서, C(E)는 EcoRI으로 절단한 대조군 DNA이고, # 16, 20, 21, 53 및 62는 페튜니아 형질전환체의 DNA를 EcoRI으로 절단한 것이다.2 is a result of sudden analysis after the transformation of petunia. In FIG. 2, C (E) is control DNA digested with EcoRI and # 16, 20, 21, 53 and 62 are digested with EcoRI DNA of petunia transformants.

도 3은 페튜니아를 형질전환한 후 GUS 발현을 분석한 결과이다.Figure 3 shows the results of analyzing GUS expression after the transformation of petunia.

도 4는 페튜니아를 형질전환한 후 GUS 발현을 현미경으로 확인한 결과이다.Figure 4 shows the results of microscopic confirmation of GUS expression after the transformation of petunia.

기술분야Field of technology

본 발명은 식물 약 특이 프로모터에 관한 것으로, 보다 구체적으로는, 단자엽 및 쌍자엽 식물의 약에서 유전자의 발현을 유도할 수 있는 프로모터 및 상기 프 로모터를 이용한 대상 유전자 및/또는 단백질을 식물 약에 다량 발현시킬 수 있는 방법에 관한 것이다.The present invention relates to a plant drug specific promoter, and more particularly, a plant capable of inducing the expression of a gene in a monocotyledonous and dicotyledonous plant and a large amount of the target gene and / or protein using the promoter in the plant medicine. It relates to a method that can be expressed.

종래기술Prior art

식물의 형질전환시 사용되는 프로모터들은, 대체로 식물의 전체 조직 또는 세포에서 주로 발현되는 프로모터들이다. 이러한 프로모터들은 조직-선택성 또는 기관-선택성이 결여되어 있어, 발현시킨 유전자가 전체 식물에서 발현되는 문제가 있다. 또한 프로모터의 특성상 도입한 유전자의 발현을 목적하는 기관에 국한되어 충분히 발현시키지 못하므로 형질전환체의 경제성이 떨어지게 된다. 그러나, 현재 기관 특이적인 특성을 갖는 식물에서 분리된 프로모터에 대한 연구는 미비한 실정이다.Promoters used in the transformation of plants are usually promoters that are mainly expressed in the entire tissues or cells of the plant. These promoters lack tissue-selectivity or organ-selectivity, which causes a problem that the expressed gene is expressed in whole plants. In addition, due to the nature of the promoter, the expression of the introduced gene is limited to the target organ and cannot be sufficiently expressed, thereby reducing the economical efficiency of the transformant. However, currently, studies on promoters isolated from plants having organ-specific characteristics are insufficient.

현재, 형질전환에서 가장 흔히 사용되는 프로모터로는 35S CaMV가 있으며, 그외에도 Ubi(Ubiquitin: 옥수수, 1993), Act1(actin; 벼, 1994), OsCc1(cytichrome C, 2002) 등이 있다. 그러나, 이들 프로모터들은 유전자가 식물체의 전 기관에서 발현되도록 작동한다. Currently, the most commonly used promoters in transformation include 35S CaMV, and Ubi (Ubiquitin: maize, 1993), Act1 (actin; rice, 1994), OsCc1 (cytichrome C, 2002). However, these promoters work to ensure that genes are expressed in all organs of the plant.

특정한 기관(예, 약, 꽃잎, 뿌리, 약 등)이나 특정 시기(예, 병원체의 공격)에서만 발현되어야 하는 유전자(예, 화색, 웅성 불임, 화형, 특정 대사관련 물질, 방어물질 등)를 형질전환시킬 경우, 특정 기관이나 시기에만 작동하는 프로모터가 반드시 필요한 실정이다. 이러한 프로모터로는 벼(약, 종자), 애기장대(종자, 꽃잎, 뿌리, 약 등), 담배(약), 감자(상처) 등에서 보고된 바 있으며, 최근에도 계속 적으로 발표되고 있다. 그러나, 이들 프로모터 역시 이종 작물간의 적용은 어려운 문제가 있다.Genes that must be expressed only at specific organs (e.g., medicines, petals, roots, drugs, etc.) or at specific times (e.g., pathogen attack) When converting, it is necessary to have a promoter that operates only at a specific institution or time. Such promoters have been reported in rice (medicine, seeds), Arabidopsis (seeds, petals, roots, medicine, etc.), tobacco (medicine), potatoes (wounds), and the like. However, these promoters are also difficult to apply between different crops.

상기 종래기술의 문제점을 해결하기 위하여 안출된 것으로서, 본 발명의 목적은 식물 약에서만 특이적으로 유전자의 발현을 유도하는 프로모터를 제공하는 것이다.In order to solve the problems of the prior art, an object of the present invention is to provide a promoter for inducing the expression of genes specifically in plant medicine.

본 발명의 다른 목적은 식물 약에서 특이적으로 발현되는 유전자의 프로모터를 사용하여 목적 유전자의 단백질을 다량 발현시켜 형질전환체의 품질을 향상시키거나 대상 단백질을 약에 저장하거나 또는 생산하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method of improving the quality of a transformant or storing or producing a target protein in a drug by expressing a large amount of a protein of a gene of interest using a promoter of a gene specifically expressed in a plant drug. It is.

본 발명의 다른 목적은 식물 약 특이 발현성을 갖는 벡터를 제공하는 것이다.Another object of the present invention is to provide a vector having plant drug specific expression.

본 발명의 또 다른 목적은 식물의 약에서 특이적으로 대상 유전자를 발현시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for specifically expressing a gene of interest in a plant medicine.

본 발명의 또 다른 목적은 식물의 약 프로모터를 이용하여 웅성불임 및/또는 교배육종법을 제공하는 것이다.It is still another object of the present invention to provide a male infertility and / or mating breeding method using the medicinal promoter of the plant.

본 발명의 또 다른 목적은 대상 단백질이 식물 약에서 특이적으로 발현되는 형질전환체를 제공하는 것이다.Another object of the present invention is to provide a transformant in which the protein of interest is specifically expressed in plant medicines.

상기 목적을 달성하기 위하여, 본 발명은 서열번호 1의 염기서열과 적어도 70% 이상의 상동성을 갖는 뉴클레오티드 또는 이의 상보체를 포함하는 식물 약 조직 특이 프로모터를 제공한다. In order to achieve the above object, the present invention provides a plant drug tissue specific promoter comprising a nucleotide having at least 70% or more homology with the nucleotide sequence of SEQ ID NO: 1.

또한 본 발명은 상기 식물 약 특이 프로모터의 3' 방향에 대상 단백질에 대한 유전자가 발현되도록 연결된 벡터를 제조하고, 상기 벡터를 식물에 형질전환하여 식물 약에서 상기 대상 유전자의 발현을 유도하는 것을 포함하는 식물 약에서의 대상 단백질 생산방법을 제공한다.In another aspect, the present invention comprises preparing a vector linked to express the gene for the target protein in the 3 'direction of the plant drug specific promoter, and transforming the vector into a plant to induce the expression of the target gene in the plant medicine Provided are methods for producing target proteins in plant medicines.

또한 본 발명은 상기 식물 약 특이 프로모터를 포함하는 벡터를 제공한다.The present invention also provides a vector comprising the plant drug specific promoter.

또한 본 발명은 상기 벡터로 형질전환된 식물 조직 배양법에 의한 무성번식되는 식물을 제공한다. The present invention also provides a plant that is asexually propagated by the plant tissue culture method transformed with the vector.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명자들은, 식물의 조직 또는 기관 특이적인 유전자 발현을 조절할 수 있는 프로모터들을 발굴함으로써, 식물의 특정 조직 또는 기관에 국한시켜 목적 유전자를 특이적으로 발현 또는 저해할 수 있는 시스템을 확립하고자 하였다. 이에, 본 발명자들은 약 특이적인 프로모터를 연구하던 중, 단자엽 식물인 나리(Lilium hybrid cv. Acapulco)의 게놈 라이브러리를 통해 약 특이적인 프로모터를 클로닝하였고, 이를 쌍자엽 식물인 페츄니아에서 실험한 결과 안정적으로 약 특이적인 프로모터로서 기능하는 것을 발견하여, 이를 토대로 본 발명을 완성하였다.The present inventors have attempted to establish a system capable of specifically expressing or inhibiting a gene of interest by confining promoters capable of regulating plant tissue or organ specific gene expression. Therefore, the present inventors cloned the drug-specific promoter through the genome library of Lilium hybrid cv. Acapulco, a monocotyledonous plant, while studying the drug-specific promoter, and stably tested the drug-specific promoter in petunia. It has been found to function as a specific promoter and the present invention has been completed based on this.

본 발명에서 언급하는 "식물 약 특이 프로모터"는 상기 프로모터의 3' 위치 에 연결되어 발현이 조절되는 유전자를 식물 약에서 특이적으로 발현시키는 프로모터를 의미하며, 식물 약 이외의 식물 조직에서는 상기 유전자의 발현이 거의 이루어지지 않는 것을 의미한다. "Plant drug specific promoter" referred to in the present invention means a promoter that is linked to the 3 'position of the promoter to specifically express a gene whose expression is regulated in plant medicine, and in plant tissues other than plant medicine, It means little expression.

본 발명의 약 특이 프로모터는 단자엽 식물인 나리에서 분리되었으나, 쌍자엽 식물인 페츄니아에서도 성공적으로 기능하는 것으로 확인하였으므로, 상기 약 특이 프로모터는 단자엽 및 쌍자엽 식물 모두에 사용가능하다.Although the drug-specific promoter of the present invention has been isolated from Lilium, which is a monocotyledonous plant, it has been found to function successfully in the petunia, which is a dicotyledonous plant.

상기 식물은 통상의 단자엽 식물, 쌍자엽 식물, 초본 식물 또는 목본 식물일 있으며, 바람직하기로는 농작물, 화훼작물, 채소작물 또는 과수작물이며, 더욱 바람직하기로는 무성생식법이 공지된 식물이다. 식물의 일예로는, 벼, 밀, 보리, 귀리, 기장, 조, 수수, 옥수수, 율무, 아스파라거스, 양파, 대나무, 호밀, 사탕수수, 파인애플, 바나나, 토마토, 담배, 목화, 포도, 고추, 상추, 콩과 식물, 양배추, 브로콜리, 해바라기, 당근, 가지, 시금치, 오이, 호박, 감자, 고구마, 사과, 복숭아, 배, 포플러, 소나무 및 아라비돕시스 등이 있으나, 이에 한정되는 것은 아니다. 식물의 무성생식법으로는 삽목, 분주, 포기번식, 조직배양, 화분배양 등이 있다. 조직 배양법은 식물의 잎의 일부를 취하여 배지에서 배양한 후 식물 뿌리 형성 배지에서 배양하여 뿌리를 형성시킨 후 이를 토양에 식수하는 방법이다. The plant may be a common monocotyledonous plant, dicotyledonous plant, herbaceous plant or woody plant, preferably a crop, a flower crop, a vegetable crop or an orchard crop, and more preferably a plant in which asexual reproduction is known. Examples of plants include rice, wheat, barley, oats, millet, crude, sorghum, corn, barley, asparagus, onions, bamboo, rye, sugar cane, pineapple, bananas, tomatoes, tobacco, cotton, grapes, peppers, lettuce , Legumes, cabbage, broccoli, sunflowers, carrots, eggplants, spinach, cucumbers, pumpkins, potatoes, sweet potatoes, apples, peaches, pears, poplars, pines and arabidopsis, but are not limited thereto. Asexual reproduction of plants includes cutting, dispensing, abandonment, tissue culture, and pollen culture. Tissue culture method is a method of taking a part of the leaves of the plant and cultivating in the medium, followed by culturing in the plant root formation medium to form the root and drinking it in the soil.

본 발명의 식물 약 특이 프로모터는 서열번호 1에 기재한 염기서열과 적어도 70 % 이상의 상동성을 갖으며, 약 특이적인 프로모터 기능을 가지는 뉴클레오티드이다. 바람직하기로는, 상기 프로모터는 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 또는 99 % 이상의 상동성을 갖으며, 약 특이적인 프로모터 기능을 가지는 뉴클레오티드이다. 상기 식물 약 조직 특이 프로모터의 서열의 예로는 서열번호 1 또는 서열번호 2의 핵산 서열을 포함하나, 이로 한정되는 것은 아니다.The plant drug specific promoter of the present invention is a nucleotide having at least 70% or more homology with the nucleotide sequence set forth in SEQ ID NO: 1 and having a drug specific promoter function. Preferably, the promoter is a nucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology and about specific promoter function. Examples of the sequence of the plant drug tissue specific promoter include, but are not limited to, the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

서열번호 1:SEQ ID NO 1:

aa ctcggtggttaa ctcggtggtt

tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt ggaaataaactacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt ggaaataaac

ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat ccgaggttatttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat ccgaggttat

cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc cctataaaaccgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc cctataaaac

ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg ctctgcattcctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg ctctgcattc

tagtccgact ccgatagtat cttcggtttagtccgact ccgatagtat cttcggtt

서열번호 2:SEQ ID NO 2:

1 gtcgaccaag ggcaagccga ggcctccaca tttcttatca ttagtgtgtg gtctgagtac 1 gtcgaccaag ggcaagccga ggcctccaca tttcttatca ttagtgtgtg gtctgagtac

6l aattaggcca tcatgctaat ctaccaagtt cacccagata tgtccctgaa taggtcagca6l aattaggcca tcatgctaat ctaccaagtt cacccagata tgtccctgaa taggtcagca

121 gacaaagccc tgaccttcaa gttggacatc ataagccctg gctaactcct aggtgtgtcc121 gacaaagccc tgaccttcaa gttggacatc ataagccctg gctaactcct aggtgtgtcc

181 ctgaataggt tggcagtcag agatccgacc ttcaggccaa acttcctaag gccctactaa181 ctgaataggt tggcagtcag agatccgacc ttcaggccaa acttcctaag gccctactaa

241 ctcccaggca tttaagtccc taaattggtt atcgggcaaa gcctcgacct tcaggccaga241 ctcccaggca tttaagtccc taaattggtt atcgggcaaa gcctcgacct tcaggccaga

301 cttcctaaga tctggctaac tcccaggcat tcaagttcct aaatgagtgg gcggacagag301 cttcctaaga tctggctaac tcccaggcat tcaagttcct aaatgagtgg gcggacagag

361 ctctgacctt caggcaagac ttcttaagtc ctgactaact cccaggcatg tccctgaata361 ctctgacctt caggcaagac ttcttaagtc ctgactaact cccaggcatg tccctgaata

421 ggtcggtggg caaaactcct aagccctatc taactcccaa gtgtgtcccc gaacgagtca421 ggtcggtggg caaaactcct aagccctatc taactcccaa gtgtgtcccc gaacgagtca

481 gcgggtagag ctctgatctt cagaccagac ttcctaagca ttggctaact cctaagggca481 gcgggtagag ctctgatctt cagaccagac ttcctaagca ttggctaact cctaagggca

541 tccttgaatg ggtcaacggg cagagctctg accttcaggc tggactttct aagccctagc541 tccttgaatg ggtcaacggg cagagctctg accttcaggc tggactttct aagccctagc

601 taactctcaa gtattcatgt ccctgaacgg gcggcgggc aaagccctga ccttcaggct601 taactctcaa gtattcatgt ccctgaacgg gcggcgggc aaagccctga ccttcaggct

661 attcatgtcc ctgaacgggt cggcggcaa agccctgacc ttcaggctag atatcctaag661 attcatgtcc ctgaacgggt cggcggcaa agccctgacc ttcaggctag atatcctaag

721 ccctggctaa cccccaagtg tgtccctgaa tgggttgatg agcagatctc caaccttcat721 ccctggctaa cccccaagtg tgtccctgaa tgggttgatg agcagatctc caaccttcat

781 gccagacttc ctaagccttg gctaactccc aggCattcaa gtcccttaat gggtcagtgg781 gccagacttc ctaagccttg gctaactccc aggCattcaa gtcccttaat gggtcagtgg

841 gcagagatcc gaccttcaga ccggactttt taaaccttgg caaactccta ggggtgttcc841 gcagagatcc gaccttcaga ccggactttt taaaccttgg caaactccta ggggtgttcc

901 tgaacttgtt ggcgggcaga gctccgacct tcaggccaga ctttccaagc gctagctaac901 tgaacttgtt ggcgggcaga gctccgacct tcaggccaga ctttccaagc gctagctaac

961 tcccagggcc attcaaagcc cttgaattag tctatgggca tagccccgga cctttagttc961 tcccagggcc attcaaagcc cttgaattag tctatgggca tagccccgga cctttagttc

1021 aggcattcca agccctgact atactcccag gcacaataaa cccctgagtc gatctgcgag1021 aggcattcca agccctgact atactcccag gcacaataaa cccctgagtc gatctgcgag

1081 catagcaccg atcttcaggc tgggcatccc aaaccctggc tatactccca ggcatataag1081 catagcaccg atcttcaggc tgggcatccc aaaccctggc tatactccca ggcatataag

1141 tcccagatgc caaaactttt cttcttgcat cgatcccaaa attctcaggc caagcgagca1141 tcccagatgc caaaactttt cttcttgcat cgatcccaaa attctcaggc caagcgagca

1201 ttctccctca atccaccttg ccttctcctt aagaagcctg ggcaagaagg agggacaata1201 ttctccctca atccaccttg ccttctcctt aagaagcctg ggcaagaagg agggacaata

1261 gaccaaELgat caaaggatca actccgtgct gacttatccc cgacttctta gcacatgtgc1261 gaccaaELgat caaaggatca actccgtgct gacttatccc cgacttctta gcacatgtgc

1321 agggtcaaca aggcgtctcc gacaaactca tacacgggca tacaggctgg cccatgccct1321 agggtcaaca aggcgtctcc gacaaactca tacacgggca tacaggctgg cccatgccct

1381 ggtccaaacg acatcgaagg acagtctcct ccggctccgg cgggaagacc cgtgtgccca1381 ggtccaaacg acatcgaagg acagtctcct ccggctccgg cgggaagacc cgtgtgccca

1441 atcggtgggt atccctagga tatccatgcc cgacacccac aggatgaccc gtgtgccagg1441 atcggtgggt atccctagga tatccatgcc cgacacccac aggatgaccc gtgtgccagg

1501 acaaactaca caataaccag taccttaact gacttctcga cataacatca catatcgcat 1501 acaaactaca caataaccag taccttaact gacttctcga cataacatca catatcgcat

1561 ttaatgtgct gacctaggag ttgacgtgtt gtcccttctg ttggatcgct aggctacgta1561 ttaatgtgct gacctaggag ttgacgtgtt gtcccttctg ttggatcgct aggctacgta

1621 tgctcaacaa atagtcacac cgacctaagt gatttgacga ctcataggca tatgcgtgga1621 tgctcaacaa atagtcacac cgacctaagt gatttgacga ctcataggca tatgcgtgga

1681 tccttggaac tcgactccac agtcacccct agtgcatata aaaagcctcc tccactcccc1681 tccttggaac tcgactccac agtcacccct agtgcatata aaaagcctcc tccactcccc

1741 tttgaggggg taagctttac agactctttg cacgtatata atatgtccgg atcggagcac1741 tttgaggggg taagctttac agactctttg cacgtatata atatgtccgg atcggagcac

1801 aggtactctg ctcacctaag agcggtcagc tgcccagtat cgtagaaact cgacactcac1801 aggtactctg ctcacctaag agcggtcagc tgcccagtat cgtagaaact cgacactcac

1861 tctcacatcg gaggagtgtc gcggggatac gtccatgcga ccatgtttag gtgtttcttg1861 tctcacatcg gaggagtgtc gcggggatac gtccatgcga ccatgtttag gtgtttcttg

1921 tgctcgcagg tctcgacgac gacctcatcc gtaccagcct gagctctccc agttctccaa1921 tgctcgcagg tctcgacgac gacctcatcc gtaccagcct gagctctccc agttctccaa

1981 ccactacgat ggaacgtttg tctccagttc gaccctgcga gcgccctgct cacccaggtc1981 ccactacgat ggaacgtttg tctccagttc gaccctgcga gcgccctgct cacccaggtc

2041 ggagctccac tagctcgtag cccggatctg actgtctcta cgcacacgcc cggctagagc2041 ggagctccac tagctcgtag cccggatctg actgtctcta cgcacacgcc cggctagagc

2101 tctaccagct cgcagcctgg atccgaccat ctccgcgtcc atgacaggcc agagctccac2101 tctaccagct cgcagcctgg atccgaccat ctccgcgtcc atgacaggcc agagctccac

2161 tagctcgccg gacaaatcct cgacgcccat gcccgtggtt ggccatttcc cgcatcctcg2161 tagctcgccg gacaaatcct cgacgcccat gcccgtggtt ggccatttcc cgcatcctcg

2221 cctccgaaca aaactcaaca agccaggaat ctgaagcgta gtgtcacgtc agaccccgct2221 cctccgaaca aaactcaaca agccaggaat ctgaagcgta gtgtcacgtc agaccccgct

2281 gaagaaggac gtcatcacaa ccaatttatt ttgacgtcat caataagtaa tcttaaatct2281 gaagaaggac gtcatcacaa ccaatttatt ttgacgtcat caataagtaa tcttaaatct

2341 gcattctcca aaacatatat ttttgtcatt gaacttatta agtattagac atttctttta2341 gcattctcca aaacatatat ttttgtcatt gaacttatta agtattagac atttctttta

2401 cttggtcatt gatgtcgccg aaatcattcc actgaactct aacaccaaat tgttcaacta2401 cttggtcatt gatgtcgccg aaatcattcc actgaactct aacaccaaat tgttcaacta

2461 tatctaattc taaaaataag attatatgca acacttcggt accaatttgt tcggctatgt2461 tatctaattc taaaaataag attatatgca acacttcggt accaatttgt tcggctatgt

2521 caaactctac aaataaaatt aataaaatat atgtgatcga ccaattattc aaagagatca2521 caaactctac aaataaaatt aataaaatat atgtgatcga ccaattattc aaagagatca

2581 taatcggtcc caatgttaga aggtacacaa aatcgttgtc ccaaaaacaa ctcggtggtt2581 taatcggtcc caatgttaga aggtacacaa aatcgttgtc ccaaaaacaa ctcggtggtt

264l tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt ggaaataaac264l tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt ggaaataaac

2701 ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat ccgaggttat2701 ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat ccgaggttat

2761 cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc cctataaaac2761 cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc cctataaaac

2821 ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg ctctgcattc2821 ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg ctctgcattc

2881 tagtccgact ccgatagtat cttcggttct aaca2881 tagtccgact ccgatagtat cttcggttct aaca

일 예로, 서열번호 1의 핵산 서열을 포함하는 약 특이 프로모터는 약 조직에서 우세적으로 유전자의 전사를 수행한다. 다른 예로, 서열번호 2의 핵산 서열을 포함하는 약 특이 프로모터는 약 조직에서 우세적으로 발현되지만, 일부의 경우에는 자방 조직서도 유전자를 발현시킨다.In one embodiment, the drug specific promoter comprising the nucleic acid sequence of SEQ ID NO: 1 performs transcription of the gene predominantly in the drug tissue. In another embodiment, the drug specific promoter comprising the nucleic acid sequence of SEQ ID NO: 2 predominantly expresses in the drug tissue, but in some cases also expresses the gene in the room tissue.

본 발명에 따른 약 특이 프로모터는 형질전환으로 약에 화분을 제거하거나 색소를 교환하는 등의 특정 형질을 조절할 수 있다. 이에 따라, 채소나 화훼 등의 교배 육종에 이용될 수 있는 모본 식물을 육성하거나, 개약하는 화분으로 인해 상품성이 떨어지는 화훼작물의 단점을 개선할 수도 있다. 이를 위해, 본 발명에 따른 약 특이 프로모터에 대상 유전자 및/또는 단백질을 발현가능하도록 연결시켜, 형질전환함으로써, 원하는 형질을 갖는 식물을 제조할 수 있다. The drug specific promoter according to the present invention may control specific traits such as removing pollen or exchanging pigments in the drug by transformation. Accordingly, it is possible to cultivate the parent plants that can be used for breeding and breeding of vegetables or flowers, or to improve the disadvantages of flower crops that are less commercially available due to potted plants. To this end, a plant having a desired trait can be prepared by linking the gene and / or protein of interest to the drug specific promoter according to the present invention so that it can be expressed and transformed.

이를 위하여, 본 발명에서는 상기 식물 약 특이 프로모터를 포함하는 벡터를 제공한다. 상기 벡터는 프로모터 3' 방향에 대상 단백질을 암호화하는 핵산 서열을 발현 가능하게 연결된 형태로 포함할 수 있으며, 또한 복사기점(origen of replication), 프로모터 조절 부위 예컨대 인핸서, 전사 종결부위, 선별 마커 또는 전사 조절 인자를 더욱 포함할 수 있다. 바람직하기로는 상기 벡터는 5' 에서 3' 방향으로, 식물 약 특이 프로모터, 대상 단백질 코딩 부위 및 전사종결부위를 포함하며, 통상의 벡터에 프로모터를 상기 식물 약 특이 프로모터로 치환시킨 것일 수 있다. 상기 통상의 벡터로는 pUC 계열의 벡터, 예컨대, pBR322, pBI121, pCAMBIA, Gateway vector, Ti-plasmid 및 이에 파생된 벡타일 수 있으나 이에 한정되는 것은 아니다.To this end, the present invention provides a vector comprising the plant drug specific promoter. The vector may include a nucleic acid sequence encoding a protein of interest in the 3 'direction of the promoter in a form that is expressively linked, and also includes an origin of replication, a promoter regulatory region such as an enhancer, a transcription termination region, a selection marker, or a transcription. It may further comprise regulatory factors. Preferably, the vector includes a plant drug specific promoter, a target protein coding site, and a transcription termination site in a 5 'to 3' direction, and the promoter may be substituted with the plant drug specific promoter in a conventional vector. The conventional vector may be a pUC family vector such as pBR322, pBI121, pCAMBIA, Gateway vector, Ti-plasmid, and a vector derived therefrom, but is not limited thereto.

상기 "핵산 서열을 발현 가능하게 연결된"은, 핵산 서열의 전사 및 번역이 프로모터에 의하여 조절될 수 있는 것을 의미한다.Said "expressively linked nucleic acid sequence" means that the transcription and translation of the nucleic acid sequence can be regulated by a promoter.

상기 "프로모터 3' 방향"은 프로모터의 3' 말단에 대상 단백질을 암호화하는 핵산 서열이 직접 연결되거나 또는 인접하여 연결됨을 의미한다.The "promoter 3 'direction" means that the nucleic acid sequence encoding the protein of interest is directly linked or adjacent to the 3' end of the promoter.

상기 선별 마커는, 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질전환된 세포를 비형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 글리포세이트(glyphosate) 또는 포스피노트리신(phosphinothricin)과 같은 제초제 저항성 유전자, 카나마이신(Kanamycin), G418, 블레오망신(Bleomycin), 하이그로마이신(hygromycein), 클로람페니콜(chloramphenicol)과 같은 항생제 내성 유전자가 있으나, 이로 한정되는 것은 아니다.The selection marker is a nucleic acid sequence having properties that can be selected by conventional chemical methods, and all genes that can distinguish the transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, bleomycin, hygromycein, and chloramphenicol. There are, but are not limited to, resistance genes.

상기 전사종결부위는 통상의 전사종결부위를 사용할 수 있으며, 그 예로는 노팔린(nopaline) 합성 종결부위, 파세올린(phaseoline) 종결자, 아그로박테리움 투메파시엔스(agrobacterium tumefaciens)의 옥토핀(Octopine) 유전자의 종결자 등이 있으나, 이로 한정되는 것은 아니다.The transcription termination site may be a conventional transcription termination site, and examples thereof include a nopaline synthesis termination site, a phaseoline terminator, and octopine of agrobacterium tumefaciens. ) Terminators of genes, but are not limited thereto.

상기 전사 조절 인자는, 통상의 당업계에 공지된 것일 수 있으며, 표적 식물에 따라 적절히 조합하여 사용할 수 있다.The transcriptional regulator may be known in the art, and may be used in appropriate combination depending on the target plant.

상기 대상 단백질은 모든 종류의 식물 또는 동물 유래의 단백질일 수 있으며, 바람직하기로는 사용하는 식물 유래의 단백질일 수 있다. 그 예로, 색소 관련 단백질과 같은 화분의 표현형과 관련있는 단백질이거나, 교배 육종에 이용되는 단 백질, 색소 관련 단백질, 예컨대 안토시아닌 색소류, 화분 생성에 관련된 단백질, 예컨대 지질전달 관련 단백질 등, 화분의 생식과 관련된 단백질, 또는 개약 관련 단백질들일 수 있지만, 상기 대상 단백질은 kanamycin, β-glucrinidase(Gus), nopaline synthase, 및 Cauliflower Mosaic Virus promoter 로 이루어진 군으로부터 선택되고, 이로 한정되는 것은 아니다.The target protein may be a protein derived from all kinds of plants or animals, and preferably, a protein derived from a plant to be used. For example, it is a protein associated with a phenotype of pollen, such as a pigment-related protein, or a protein used for cross breeding, a pigment-related protein such as anthocyanin pigments, a protein related to pollen production, such as a lipid transfer-related protein, and the like. Or subject related proteins, the subject protein is selected from, but is not limited to, kanamycin, β-glucrinidase (Gus), nopaline synthase, and Cauliflower Mosaic Virus promoter.

상기 벡터는 본 발명이 속하는 기술분야의 당업자라면 공지의 방법에 따라 용이하게 제조가능하다. The vector can be easily prepared according to known methods by those skilled in the art.

본 발명의 식물 약 특이 프로모터를 포함하는 벡터는, 통상의 방법에 따라 식물에 도입하여 형질전환된 식물을 제조할 수 있다. 형질전환 방법으로는 칼슘/폴리에틸렌 글리콜법을 이용한 원형질체의 형질전환법, 전기 천공법, 미량주사법, (코팅된) 입자 충격 투입법, 유전자 총, 및 물리적 도입 등이 있으나, 이에 한정되지 않으며, 식물의 종류에 따라 적절히 조절할 수 있다. 이와 같은 직접적인 유전자 형질전환 방법이외에도, 바이러스 벡터(예, 콜리플라워 모자이크 바이러스(CaMV)로부터 수득됨) 및 박테리아 벡터(예, 아그로박테리움 속으로부터 수득됨) 등의 벡터를 포함하는 형질전환 시스템을 널리 이용할 수 있다. 제조한 형질전환 식물을 선별 및/또는 스크리닝한 후, 형질전환된 원형질체, 세포 또는 식물 부분을 공지의 방법에 따라 완전한 식물로 재생시킬 수 있다(Horsch, R. b. et al., 1985). 형질전환법 및/또는 재생기술의 선택은 당업자에게 자명한 사항이다.A vector comprising the plant drug specific promoter of the present invention can be introduced into a plant according to a conventional method to produce a transformed plant. Transformation methods include, but are not limited to, transformation of protoplasts using calcium / polyethylene glycol method, electroporation, microinjection, (coated) particle bombardment, gene gun, and physical introduction, but are not limited thereto. It can be adjusted appropriately according to the type of. In addition to such direct gene transformation methods, transformation systems including vectors such as viral vectors (eg from Cauliflower Mosaic Virus (CaMV)) and bacterial vectors (eg from the genus Agrobacterium) are widely used. It is available. After selection and / or screening of the resulting transgenic plants, the transformed protoplasts, cells or plant parts can be regenerated into complete plants according to known methods (Horsch, R. b. Et al., 1985). The choice of transformation and / or regeneration techniques is apparent to those skilled in the art.

식물에서 약은 관상과 생식 이 두가지를 모두 가지고 있는 중요한 기관으로, 관상 측면에서 약의 꽃밥이 터짐으로 해서 관상적인 가치가 낮아져 상품성이 저해 되는 경우가 많다. 이때, 본 발명의 약 특이 프로모터의 조절하에 개약관련 유전자가 발현되도록 형질전환주를 제조함으로써, 정상적인 모양을 갖추면서도 꽃밥이 터지지 않는 상품성이 우수한 화훼작물을 육성할 수 있다. 또한, 채소 작물의 교배시, 교배 대상 작물중 모계 쪽의 작물의 약은 일반적으로 제웅작업을 거쳐 교배가 이루어지는데, 이때, 본 발명의 약 특이 프로모터의 조절하에 약 생식 관련 유전자가 발현되도록 함으로써, 별다른 제웅과정없이도 간단하게 교배를 수행할 수 있는 이점이 있다.In plants, medicine is an important organ that has both coronary and reproductive, and the anther of the drug in the coronal side, the ornamental value is often lowered and the commercial value is often impaired. At this time, by producing a transgenic strain so that the expression-related gene is expressed under the control of the drug specific promoter of the present invention, it is possible to cultivate a flower crop having a normal shape and excellent commercial property without anther. In addition, when the crops of the vegetable crops, the crops of the maternal side of the crops to be mated are generally cross-breeding through the cross-breeding operation, at this time, by allowing the drug-related genes to be expressed under the control of the drug-specific promoter of the present invention There is an advantage that can perform a simple cross without the need of de-emulsion.

하기 실시예를 들어 본 발명을 상세히 설명한다. 하기 실시예들은 본 발명을 예시하기 위한 것일 뿐, 본 발명의 보호 범위를 한정하는 것은 아니다.The present invention is described in detail with reference to the following examples. The following examples are merely to illustrate the invention, but not to limit the protection scope of the invention.

실시예 1 - 게놈 라이브러리의 제조 및 게놈 유전자 분리Example 1 Preparation of Genomic Library and Genomic Gene Isolation

오리엔탈 나리 아카풀코(Lilium hybrid cv. Acapulco)의 잎으로부터 머레이 및 톰슨(Rapid isolation of high- molecular-weight plant DNA. 1980)의 방법에 따라 게놈 DNA를 분리하고, 이를 이용하여 Lambda Fix II/XhoI 키트(Startagene) 및 ZAP-cDNA Gigapack cloning kit(Stratagene)으로 제조사의 지침에 따라 실시하여 게놈 라이브러리를 제작하였다.Isolate genomic DNA from the leaves of Oriental Lilium hybrid cv.Acapulco according to the method of Rapid isolation of high-molecular-weight plant DNA (1980), and using the Lambda Fix II / XhoI kit ( Startagene) and ZAP-cDNA Gigapack cloning kit (Stratagene) were run according to the manufacturer's instructions to prepare a genomic library.

꽃잎과 약에서 발현이 되는 ALCHS2 cDNA를 RediprimeTM II DNA 레이블링 시스템(Amersham)을 이용하여, 제조사의 지침에 따라 [α-32P]dCTP로 표지하여 프로 브를 제조하였다. 이 프로브로 상기 제조한 게놈 라이브러리를 스크리닝하여 gALCHS7(Genome Acapulco Lily Chalcone Synthase 7: 약 15. 0 kbp)를 분리하였고, 이를 EcoR I로 절단하여, ALCHS2를 포함하는 6.2 kbp의 클론을 확보하였다.Probes were prepared by labeling ALCHS2 cDNA expressed in petals and drugs with [α- 32 P] dCTP according to the manufacturer's instructions using the Rediprime II DNA labeling system (Amersham). The genome library prepared above was screened with this probe to isolate gALCHS7 (Genome Acapulco Lily Chalcone Synthase 7: about 15.0 kbp), which was digested with EcoR I to obtain a 6.2 kbp clone containing ALCHS2.

실시예 2: CHS 게놈 유전자의 염기서열 분석 및 전사 조절 부위 조사 Example 2 Sequencing and Transcription Regulation Sites of CHS Genomic Genes

약 6.0 kbp의 gALCHS7 클론의 염기서열을 분석하기 위하여, 수종의 제한효소로 절단하여 제한효소 지도를 작성하였다. 이 지도에 따라서, salI과 EcoRI으로 절단하여, pBluescript II SK(+) 벡터로 서브클로닝한 다음, ABI Prime 31O Genetic Anaiyzer(PE Apphed Biosystems)로 분석하여 전장 염기서열을 수득하였다. 총 6203 bp의 염기서열은 genescan (http:// genes. mit. edu)을 이용하여 엑손과 인트론 부위를 확인하였고, PLACE 데이터베이스(PLAnt Cis-acting regulatory DNA Elememt: http//www.dna.affrc.go.jp)에 전송하여 전사 조절 부위를 분석하였다. 그 결과는 하기 표 1에 나타낸다(하기 위치는, ATG를 기준으로 기재함)In order to analyze the nucleotide sequence of a gALCHS7 clone of about 6.0 kbp, restriction enzyme maps were generated by cleavage with several restriction enzymes. According to this map, digested with salI and EcoRI, subcloned with pBluescript II SK (+) vector, and analyzed by ABI Prime 31O Genetic Anaiyzer (PE Apphed Biosystems) to obtain full length sequences. A total of 6203 bp sequences were identified by exon and intron sites using genescan (http: // genes. Mit. Edu), and PLACE database (PLAnt Cis-acting regulatory DNA Elememt: http // www.dna.affrc. go.jp) to analyze the transcriptional regulatory sites. The results are shown in Table 1 below (the following positions are described based on ATG).

인자 또는 부위 명칭Factor or site name 위치location 서열order 특징Characteristic GT1CONSENSUSGT1CONSENSUS -362-362 GRWAAWGRWAAW 펙테이트 리아제(pectate lyase)에 대한 상동성을 포이는 토바코 후기 화분 유전자 g10의 프로모터에서 발견되는 GTGA 모티프GTGA motif found in the promoter of Tobacco late pollen gene g10 that harbors homology to pectate lyase GTGANTG10GTGANTG10 -1264, -224-1264, -224 GTGAGTGA 광-조절 유전자 다수에서의 보존적인 GT-1 결합부Conservative GT-1 Binding in Multiple Photo-regulatory Genes INRNTPSADBINRNTPSADB -1716, -1057-1716, -1057 YTCANTYYYTCANTYY TATA 박스가 없는 토바코 psaDb 유전자 프로모터에서 발견된 "Inr(initiator)" 요소"Inr (initiator)" element found in tobacco psaDb gene promoter without TATA box INTRONLOWERINTRONLOWER -1605-1605 TGCAGGTGCAGG 3' 인트론-엑손 스플라이스 정션: 신물 인트론 하위 서열3 'intron-exon splice junction: new intron subsequence MYB2ATMYB2AT -1397, -132-1397, -132 TAACTGTAACTG ATMYB2에 대한 결합부, 아라비돕시스 MYB 상동체(TAACTG)Binding to ATMYB2, Arabidopsis MYB Homolog (TAACTG) MYBCOREMYBCORE -1315-1315 CNGTTRCNGTTR 플라보노이드 생합성 조절에 관여하는 페튜니아 MYB 결합 단백질Petunia MYB Binding Protein Involved in the Regulation of Flavonoid Biosynthesis MYBPZMMYBPZM -936-936 CCWACCCCWACC 보존적 옥수수 P(myb 상동체) 결합부의 코어, 옥수수 P 유전자는 케르넬 페릭(kernel peric)의 붉은 색소 형성The core of the conserved maize P (myb homolog) junction, the maize P gene, forms the red pigment of Kernel peric POLLEN1LELAT52POLLEN1LELAT52 -1069-1069 AGAAAAGAAA 토마토 lat52 유전자의 화분 특이적인 활성화에 븐응하는 조절 요소Regulatory Factors Response to Pollen-specific Activation of the Tomato lat52 Gene QELEMENTZMZM13QELEMENTZMZM13 -2811-2811 AGGTCAAGGTCA 발현 증강에 관여하는 옥수수(Z.m.) ZM13 유전자 프로모터에서 발견된 모티프Motif found in maize (Z.m.) ZM13 gene promoter involved in enhanced expression ROOTMOTIFTAPOX1ROOTMOTIFTAPOX1 -556-556 ATATTATATT rolD 및 뿌리 특이적인 밀 페록시다제 프로모터에서 발견된 모티프Motif found in rolD and root specific wheat peroxidase promoter CAAT BOXCAAT BOX -168-168 CAATCAAT 진핵 유전자의 5'-플랭킹 부위에서 발현되는 일반적인 서열General sequence expressed at the 5'- flanking region of eukaryotic genes

실시예 3: 벡터 제작Example 3: Vector Fabrication

6.2 kbp의 게놈 클론(gALCHS7)에서 5'-플랭킹 부위의 270 bp(서열번호 1)를 PCR을 이용하여 증폭한 다음 벡터 제작에 사용하였다. 상기 게놈 뵀Х隙◎주형으로 gSalI(정방향 프라이머: 5'-AACTCGGTGGTCGACACTCAGGGG-3') 및 gXbaI(역방향 프라이머: 5'-ATTTCTAGAACCGAAGATACTA-3') 프라이머로 증폭시킨 다음, SalI 및 XbaI으로 절단 및 분리하고, 동일한 제한효소로 절단한 벡터 pBI101에 연결하여, gALCHS7-7/GUS 벡터를 제작하였다(도 1) . In a 6.2 kbp genomic clone (gALCHS7), 270 bp (SEQ ID NO: 1) of the 5'- flanking region was amplified by PCR and used for vector construction. The genome gХ 隙 ◎ template was amplified with gSalI (forward primer: 5'-AACTCGGTGGTCGACACTCAGGGG-3 ') and gXbaI (reverse primer: 5'-ATTTCTAGAACCGAAGATACTA-3') primers, cleaved and isolated with SalI and XbaI, The gALCHS7-7 / GUS vector was constructed by connecting to the vector pBI101 digested with the same restriction enzyme (FIG. 1).

실시예 4: 형질전환 및 전환체 분석Example 4 Transformation and Convertor Analysis

Horsh 등(A Simple and general method for transferring genes into plants, 1985)의 방법을 변형한 리프 디스크(leaf disk) 방법으로, 아그로박테리움 균주 C58C1을 이용하여, 페튜니아(Dreams Red, PanAmerican Seed)로의 형질전환을 실시하였다. A leaf disk method modified from the method of Horsh et al. (A Simple and general method for transferring genes into plants, 1985), and transformation into Petunia (Dreams Red, PanAmerican Seed) using Agrobacterium strain C58C1 Was carried out.

구체적으로는, 기내에서 파종한 페튜니아 식물체를 총장 7 cm 정도로 키운 다음 1번에서 3번 엽만을 채취하여, 가로 및 세로 길이를 약 5 mm로 절단하고, 호르몬이 포함된 재분화 배지에 치상하여 1일간 배양하였다. 28 ℃에서 2일간 배양한 아그로박테리움 균주는 5,000 rpm으로 10분간 원심분리하여 그 침전물을 액체 배지에 희석하였다. 균 희석액과 절단한 잎을 혼합하여 10분간 접종한 후 재분화 배지에 치상하여 암상태에서 2일간 공동 배양을 실시하였다. 이틀 후, 공동 배양한 잎을 세포탁심 200 mg/L를 포함하는 액체 배지에 넣어 세척한 다음, 형질전환체 선별배지에 치상하였다. 형질전환체 선별 방법은 카나마이신(5O mg/L)이 포함된 배지에서 4주간 키운 슈트(shoot)를 다시 카나마이신(100 mg/L) 포함 배지로 옮겨, 3주간 배양하여 선별하였다. 선별한 형질전환체는 카나마이신이 포함되지 않은 발근 배지로 옮겨 약 4- 6주간 발근을 유도한 뒤 온실로 옮겨 순화시켰다. 선별한 형질전환체의 잎으로부터 DNeasy Plant Mini Kit(Qiagen, Germany)를 이용하여 게놈 DNA를 분리한 다음, NPTII 프라이머(프라이머 1: 5'- GAG GCT ATT CGG CTA TGA CTG - 3', 프라이머 2: 5' - ATC GGG AGC GGC GAT ACC GTA - 3')로 증폭하여 형질 전환 여부를 확인하였다. NPTII 프라이머로 형질전환 여부가 확인된 5 개체는 서던 방법(1975)으로, 서든 혼성화 반응을 수행하였다. 분리한 게놈 DNA 10 ㎍을 EcoR I으로 절단한 후, 0.7 % 아가로즈 젤에서 1 V/cm으로 18시간 동안 0.5 X TBE 완충용액으로 전기영동을 실시하였다. 베타-글루쿠로니다제(GUS)를 RediprimeTM II DNA Labeling system(Amersham)을 이용하여, 제조사의 지침에 따라 [α-32P] dCTP로 표지한 프로브를 서든 혼성화 반응에 사용하였다. 그 결과(도 2), 3개체에서 베타-글루쿠로니다제(GUS)의 삽입을 확인하였으며, 이후 이 개체는 증식 후 온실에서 순화하였다. Specifically, petunia plants sown in the cabin are grown to a total length of about 7 cm, and only 1 to 3 leaves are harvested, cut to about 5 mm in length and length, cut into hormone-containing regeneration media for 1 day. Incubated. Agrobacterium strains cultured at 28 ° C. for 2 days were centrifuged at 5,000 rpm for 10 minutes to dilute the precipitate in a liquid medium. The bacterial dilutions and the cut leaves were mixed and inoculated for 10 minutes, and then cultivated in the regeneration medium and co-cultured in the dark for 2 days. Two days later, the co-cultured leaves were washed in a liquid medium containing 200 mg / L of Cytoxim and washed in transformant selection medium. In the transformant selection method, a shoot grown for 4 weeks in a medium containing kanamycin (50 mg / L) was transferred to a medium containing kanamycin (100 mg / L) and cultured for 3 weeks. Selected transformants were transferred to the rooting medium containing no kanamycin and induced rooting for about 4-6 weeks before being transferred to the greenhouse for purification. Genomic DNA was isolated from the leaves of the selected transformants using DNeasy Plant Mini Kit (Qiagen, Germany), followed by NPTII primers (primer 1: 5'- GAG GCT ATT CGG CTA TGA CTG-3 ', primer 2: 5'-ATC GGG AGC GGC GAT ACC GTA-3 ') to confirm the transformation. Five individuals confirmed to be transformed with NPTII primers were subjected to Southern hybridization by Southern method (1975). 10 μg of the isolated genomic DNA was digested with EcoR I and subjected to electrophoresis with 0.5 × TBE buffer for 18 hours at 1 V / cm in a 0.7% agarose gel. Beta-glucuronidase (GUS) using a Rediprime II DNA Labeling system (Amersham), a probe labeled with [α- 32 P] dCTP was used for a sudden hybridization reaction according to the manufacturer's instructions. As a result (FIG. 2), the insertion of beta-glucuronidase (GUS) was confirmed in three individuals, which were then purified in the greenhouse after proliferation.

실시예Example 5:  5: GUSGUS 발현 분석 Expression analysis

GUS 활성은 페튜니아 형질전환체의 각 기관을 이용하여 조사하였다. 페튜니아 꽃에서 개약 전 단계의 잎, 꽃잎,약, 암술,자방을 면도칼로 자른 다음, 12시간 동안 37 ℃에서 Gallagher(GUS Protocols: Using the GUS gene as a reporter of gene expression, 1992)의 방법을 약간 변형하여 만든 GUS 용액에 침지하였다. 이때, 형질전환되지 않은 조직에서의 내생 GUS의 유사 발현을 억제하기 위하여, 20% 메탄올을 첨가하여 사용하였다(An improved assay for β-glucoronudase transfomed cells: methanol almost completely suppresses a putative endogenous β-glucoronudase activity, 1990).GUS activity was examined using each organ of petunia transformants. Cut the leaves, petals, pills, pistils, and stalks of the petunia flower at the pre-opening stage with a razor, and then use Gallagher (GUS Protocols: Using the GUS gene as a reporter of gene expression, 1992) at 37 ° C for 12 hours It was immersed in the modified GUS solution. At this time, 20% methanol was added to suppress the similar expression of endogenous GUS in untransformed tissue (An improved assay for β-glucoronudase transfomed cells: methanol almost completely suppresses a putative endogenous β-glucoronudase activity, 1990).

GUS 발현 분석 결과(도 3), 시험에 사용한 전체 형질전환체 중 2개의 약에서 강한 발현을 확인할 수 있었다. 그 외에 다른 잎이나 꽃잎 수술대 등의 기관에서는 형질전환하지 않은 식물체와 동일하게, 어떠한 가시적인 GUS 발현도 확인할 수 없었다. 서던이 확인된 3개의 개체 중 나머지 1개의 개체에서는 개약전 약 외에 어 떠한 기관에서도 GUS 발현을 확인할 수 없었다.As a result of GUS expression analysis (FIG. 3), strong expression was confirmed in two drugs among all the transformants used for the test. Other organs, such as leaf and petal operating tables, were unable to identify any visible GUS expression, as did plants without transformation. The remaining one of the three confirmed Southern populations was unable to confirm GUS expression in any organ other than the pre-drug drug.

실시예Example 6:  6: DarkDark -- fieldfield 기법을 이용한  Using techniques GUSGUS 발현 분석 Expression analysis

Dark-field로 관찰하기 위하여, 다음과 같은 두 가지 방법을 조합하여 시료를 고정하였다(Initiation patterns of flower and floral Organ development in Arabidopsis thaliana, Development, 1996: Method in short protocols in molecular biology, Unit 14.2-3). GUS 활성을 보인 2개체의 약을 GUS로 염색한 후, 상온에서 하기 용액으로 12시간 침지하여 고정시켰다(50% 에탄올, 5% 아세트산 및 3.7% 포름알데하이드). 일련의 에탄올 희석물(70%, 85%, 95%)을 이용하여 탈수한 다음, 크실렌(xylene)과 에탄올 희석물(1:3, 1:1, 3:1)을 이용하여 각각 1시간씩 침지시킨 다음, 100% 크실렌으로 각 1시간씩 2회 더 갈아주었다. 약 5 - 10 ml의 크실렌을 넣은 후 동량의 파라핀(paraplast, Fisher)을 첨가하여, 60 ℃ 오븐에서 12시간 두었다. 녹은 파라핀은 버리고 새로운 파리핀을 붓는 과정을 2- 3회 반복한 다음, 틀에 부어 고정한 후 절단에 알맞은 모양으로 다듬어 두었다. 파라핀 절단기기(Leica)를 이용하여 10 - 15 ㎛로 절단하고, 폴리-프렙 슬라이드(sigma)에 고정하여 37 ℃에서 12시간 정도 건조시킨다. 현미경 관찰을 위해, 시료가 고정된 슬라이드를 100% 크실렌에 12시간 녹인 다음 크실렌과 에탄올 희석물(1: 3, 1: 1, 3: 1)을 이용하여 탈수한 후, Permount(Fisher)를 도포하여 커버 슬립을 덮어 고정하였다. 이를 건조한 다음, dark-field가 장착된 현미경에서 100배 비율로 관찰하였다. 관찰 결과, 화분이 충분히 발달한 약에서는, 화분 뿐만 아니라 약의 지지조직(endothethium, epidermis)에서 강한 GUS 발현이 관찰되었으며(도 4), 다른 기관 즉, 자방이나 암술에서는 GUS 발현과 유사하게 어떠한 눈에 띄는 발현이 관찰되지 않았다. 따라서, gALCHS7-7 프로모터는 약과 약의 지지조직에서만 작동하는 프로모터임이 확인되었다.For dark field observation, samples were fixed by combining two methods (Initiation patterns of flower and floral Organ development in Arabidopsis thaliana, Development, 1996: Method in short protocols in molecular biology, Unit 14.2-3 ). Two drugs showing GUS activity were stained with GUS, and then fixed by immersion with the following solution at room temperature for 12 hours (50% ethanol, 5% acetic acid and 3.7% formaldehyde). Dehydrate with a series of ethanol dilutions (70%, 85%, 95%) and then 1 hour each with xylene and ethanol dilutions (1: 3, 1: 1, 3: 1) After soaking, the mixture was further changed twice with 1% of 100% xylene. About 5-10 ml of xylene was added and the same amount of paraffin (paraplast, Fisher) was added and placed in an oven at 60 ° C. for 12 hours. The molten paraffin was discarded and the new paraffin was poured 2-3 times, then poured into a mold, fixed and trimmed to a shape suitable for cutting. Using a paraffin cutter (Leica) was cut to 10-15 ㎛, fixed on a poly-prep slide (sigma) and dried for about 12 hours at 37 ℃. For microscopic observation, the slide on which the sample was fixed was dissolved in 100% xylene for 12 hours, then dehydrated using xylene and ethanol dilution (1: 3, 1: 1, 3: 1), and then applied Permount (Fisher). The cover slip was covered and fixed. After drying, it was observed at a ratio of 100 times on a microscope equipped with a dark field. Observations showed that in drugs with sufficiently developed pollen, strong GUS expression was observed not only in pollen but also in the supportive tissues of the drug (endothethium, epidermis) (FIG. 4). No significant expression was observed. Thus, it was confirmed that the gALCHS7-7 promoter is a promoter that operates only in the drug and its supportive tissue.

상기한 바와 같은, 본 발명의 약 특이적인 프로모터는 형질전환으로 특정 형질을 도입하고자 하는 경우에, 효율 증대와 함께 정확한 발현체를 얻을 수 있어, 약에 특정 유전자를 도입한 후 교배 육종에 이용할 수 있다. 아울러, 관상 식물에 적용하여 정상적인 모양을 갖추면서도 꽃밥이 터지지 않는 상품성이 우수한 화훼작물을 육성할 수 있으며, 채소 작물에 이용하여 약 특이 프로모터의 조절하에 약 생식 관련 유전자가 발현되도록 함으로써, 별다른 제웅과정 없이도 간단하게 교배를 수행할 수 있는 작물을 육성할 수 있다.As described above, the drug-specific promoter of the present invention can obtain an accurate expression with increased efficiency when introducing a specific trait by transformation, and can be used for breeding breeding after introducing a specific gene into the drug. have. In addition, it can be applied to ornamental plants to cultivate flower crops that have a normal shape, but not anther, and have excellent commercial properties.In addition, vegetable reproductive genes can be expressed under the control of drug-specific promoters for vegetable crops. It is possible to cultivate crops that can be simply crossbreed without.

<110> Rural Development Administration <120> ANTHER SPECIFIC PROMOTER DERIVED FROM ACAPULO LILY, RECOMBINANT VECTOR, TRANSGENIC PLANT AND PREPARATION METHOD <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 279 <212> DNA <213> Lilium hybrid cv. Acapulco <400> 1 aactcggtgg tttacactca ggggacttgc tacgaaccaa attttaaaac tgccagtatg 60 gtggaaataa acttctctcc gcctcttctg tccttgggga ataagattca aaccccttca 120 atcgaggtta tcgatgccgc cacgttgctt agttaactgc ttgttcccaa ctatccatct 180 ccctataaaa cctttcgccc catacaacct tcacttcatc tctctccatc cactacacct 240 gctctgcatt ctagtccgac tccgatagta tcttcggtt 279 <210> 2 <211> 1604 <212> DNA <213> Lilium hybrid cv. Acapulco <400> 2 gcacatgtgc agggtcaaca aggcgtctcc gacaaactca tacacgggca tacaggctgg 60 cccatgccct ggtccaaacg acatcgaagg acagtctcct ccggctccgg cgggaagacc 120 cgtgtgccca atcggtgggt atccctagga tatccatgcc cgacacccac aggatgaccc 180 gtgtgccagg acaaactaca caataaccag taccttaact gacttctcga cataacatca 240 catatcgcat ttaatgtgct gacctaggag ttgacgtgtt gtcccttctg ttggatcgct 300 aggctacgta tgctcaacaa atagtcacac cgacctaagt gatttgacga ctcataggca 360 tatgcgtgga tccttggaac tcgactccac agtcacccct agtgcatata aaaagcctcc 420 tccactcccc tttgaggggg taagctttac agactctttg cacgtatata atatgtccgg 480 atcggagcac aggtactctg ctcacctaag agcggtcagc tgcccagtat cgtagaaact 540 cgacactcac tctcacatcg gaggagtgtc gcggggatac gtccatgcga ccatgtttag 600 gtgtttcttg tgctcgcagg tctcgacgac gacctcatcc gtaccagcct gagctctccc 660 agttctccaa ccactacgat ggaacgtttg tctccagttc gaccctgcga gcgccctgct 720 cacccaggtc ggagctccac tagctcgtag cccggatctg actgtctcta cgcacacgcc 780 cggctagagc tctaccagct cgcagcctgg atccgaccat ctccgcgtcc atgacaggcc 840 agagctccac tagctcgccg gacaaatcct cgacgcccat gcccgtggtt ggccatttcc 900 cgcatcctcg cctccgaaca aaactcaaca agccaggaat ctgaagcgta gtgtcacgtc 960 agaccccgct gaagaaggac gtcatcacaa ccaatttatt ttgacgtcat caataagtaa 1020 tcttaaatct gcattctcca aaacatatat ttttgtcatt gaacttatta agtattagac 1080 atttctttta cttggtcatt gatgtcgccg aaatcattcc actgaactct aacaccaaat 1140 tgttcaacta tatctaattc taaaaataag attatatgca acacttcggt accaatttgt 1200 tcggctatgt caaactctac aaataaaatt aataaaatat atgtgatcga ccaattattc 1260 aaagagatca taatcggtcc caatgttaga aggtacacaa aatcgttgtc ccaaaaacaa 1320 ctcggtggtt tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt 1380 ggaaataaac ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat 1440 ccgaggttat cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc 1500 cctataaaac ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg 1560 ctctgcattc tagtccgact ccgatagtat cttcggttct aaca 1604 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer, gSall <400> 3 aactcggtgg tcgacactca gggg 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer, gXbaI <400> 4 atttctagaa ccgaagatac ta 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaggctattc ggctatgact g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atcgggagcg gcgataccgt a 21 <110> Rural Development Administration <120> ANTHER SPECIFIC PROMOTER DERIVED FROM ACAPULO LILY, RECOMBINANT          VECTOR, TRANSGENIC PLANT AND PREPARATION METHOD <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 279 <212> DNA 213 Lilium hybrid cv. Acapulco <400> 1 aactcggtgg tttacactca ggggacttgc tacgaaccaa attttaaaac tgccagtatg 60 gtggaaataa acttctctcc gcctcttctg tccttgggga ataagattca aaccccttca 120 atcgaggtta tcgatgccgc cacgttgctt agttaactgc ttgttcccaa ctatccatct 180 ccctataaaa cctttcgccc catacaacct tcacttcatc tctctccatc cactacacct 240 gctctgcatt ctagtccgac tccgatagta tcttcggtt 279 <210> 2 <211> 1604 <212> DNA 213 Lilium hybrid cv. Acapulco <400> 2 gcacatgtgc agggtcaaca aggcgtctcc gacaaactca tacacgggca tacaggctgg 60 cccatgccct ggtccaaacg acatcgaagg acagtctcct ccggctccgg cgggaagacc 120 cgtgtgccca atcggtgggt atccctagga tatccatgcc cgacacccac aggatgaccc 180 gtgtgccagg acaaactaca caataaccag taccttaact gacttctcga cataacatca 240 catatcgcat ttaatgtgct gacctaggag ttgacgtgtt gtcccttctg ttggatcgct 300 aggctacgta tgctcaacaa atagtcacac cgacctaagt gatttgacga ctcataggca 360 tatgcgtgga tccttggaac tcgactccac agtcacccct agtgcatata aaaagcctcc 420 tccactcccc tttgaggggg taagctttac agactctttg cacgtatata atatgtccgg 480 atcggagcac aggtactctg ctcacctaag agcggtcagc tgcccagtat cgtagaaact 540 cgacactcac tctcacatcg gaggagtgtc gcggggatac gtccatgcga ccatgtttag 600 gtgtttcttg tgctcgcagg tctcgacgac gacctcatcc gtaccagcct gagctctccc 660 agttctccaa ccactacgat ggaacgtttg tctccagttc gaccctgcga gcgccctgct 720 cacccaggtc ggagctccac tagctcgtag cccggatctg actgtctcta cgcacacgcc 780 cggctagagc tctaccagct cgcagcctgg atccgaccat ctccgcgtcc atgacaggcc 840 agagctccac tagctcgccg gacaaatcct cgacgcccat gcccgtggtt ggccatttcc 900 cgcatcctcg cctccgaaca aaactcaaca agccaggaat ctgaagcgta gtgtcacgtc 960 agaccccgct gaagaaggac gtcatcacaa ccaatttatt ttgacgtcat caataagtaa 1020 tcttaaatct gcattctcca aaacatatat ttttgtcatt gaacttatta agtattagac 1080 atttctttta cttggtcatt gatgtcgccg aaatcattcc actgaactct aacaccaaat 1140 tgttcaacta tatctaattc taaaaataag attatatgca acacttcggt accaatttgt 1200 tcggctatgt caaactctac aaataaaatt aataaaatat atgtgatcga ccaattattc 1260 aaagagatca taatcggtcc caatgttaga aggtacacaa aatcgttgtc ccaaaaacaa 1320 ctcggtggtt tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt 1380 ggaaataaac ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat 1440 ccgaggttat cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc 1500 cctataaaac ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg 1560 ctctgcattc tagtccgact ccgatagtat cttcggttct aaca 1604 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer, gSall <400> 3 aactcggtgg tcgacactca gggg 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer, gXbaI <400> 4 atttctagaa ccgaagatac ta 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaggctattc ggctatgact g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atcgggagcg gcgataccgt a 21  

Claims (8)

서열번호 1의 염기서열과 70 % 이상의 상동성을 갖는 뉴클레오타이드 또는 그의 상보체를 포함하는 식물 약 특이 프로모터.A plant drug specific promoter comprising a nucleotide or a complement thereof having a homology of 70% or more with the nucleotide sequence of SEQ ID NO: 1. 제 1항에 있어서, 상기 프로모터는 서열번호 2의 염기서열을 포함하는 것을 특징으로 하는 프로모터.The promoter according to claim 1, wherein the promoter comprises a nucleotide sequence of SEQ ID NO: 2. 제 1항 또는 제 2항에 따른 식물 약 특이 프로모터의 3' 방향에 대상 단백질에 대한 유전자가 발현되도록 연결된 벡터를 제조하고, 상기 벡터를 식물에 형질전환하여 식물 약에서 상기 대상 유전자 및/또는 단백질의 발현을 유도하는 것을 포함하는 대상 단백질이 약에서 특이적으로 발현되는 식물의 제조 방법.A vector is prepared in which the gene for the target protein is expressed in the 3 'direction of the plant drug specific promoter according to claim 1 or 2, and the vector is transformed into a plant to convert the plant gene and / or protein in the plant medicine. A method for producing a plant, wherein the protein of interest comprising the induction of the expression of the specific expression in the drug. 제 3항에 있어서, 상기 대상 단백질은 kanamycin, β-glucrinidase(Gus), nopaline synthase, 및 Cauliflower Mosaic Virus promoter 로 이루어진 군으로부터 선택되는 것을 특징으로 하는 방법.The method of claim 3, wherein the target protein is selected from the group consisting of kanamycin, β-glucrinidase (Gus), nopaline synthase, and Cauliflower Mosaic Virus promoter. 제 1항에 따른 식물 약 특이 프로모터를 포함하는 벡터.A vector comprising a plant drug specific promoter according to claim 1. 제 5항에 따른 벡터로 형질전환된 식물 조직 배양법, 삽목, 또는 분주에 의한 무성번식되는 식물.Plants that are asexually propagated by plant tissue culture, cutting, or aliquot transformed with the vector according to claim 5. 제 5항의 식물로부터 수득되는 식물의 단편, 식물 세포 및 식물의 후손으로 이루어진 군으로부터 선택되는 것을 포함하는 식물.A plant comprising a fragment of the plant obtained from the plant of claim 5, a plant cell and a descendant of the plant. 제 6항에 있어서, 상기 식물은 농작물, 화훼작물, 채소작물 및 과수작물로 이루어진 군으로부터 선택되는 것을 특징으로 하는 식물.7. The plant according to claim 6, wherein the plant is selected from the group consisting of crops, flower crops, vegetable crops and fruit trees.
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US5750868A (en) 1994-12-08 1998-05-12 Pioneer Hi-Bred International, Inc. Reversible nuclear genetic system for male sterility in transgenic plants
AR021056A1 (en) 1998-11-03 2002-06-12 Syngenta Participations Ag DNA THAT INCLUDES A SPECIFIC GENE OF RICE AND TRANSGENIC PLANT TRANSFORMED WITH THE SAME
JP4210746B2 (en) 2003-05-12 2009-01-21 独立行政法人農業生物資源研究所 Acupuncture-specific genes and promoters of the genes, and their use

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CN106011141A (en) * 2016-07-05 2016-10-12 昆明理工大学 Lilium regale inducible promoter and application thereof
CN106011141B (en) * 2016-07-05 2018-10-23 昆明理工大学 Ming River lily inducible promoter and its application
CN112708625A (en) * 2021-03-16 2021-04-27 昆明理工大学 Lilium regale inducible promoter PG1 and application thereof
CN112708625B (en) * 2021-03-16 2023-06-16 昆明理工大学 Lilium regale inducible promoter PG1 and application thereof

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