KR100910924B1 - Arabidopsis AtCDCP2 gene that negatively regulates on biosynthesis of jasmonic acid and production method of male sterile plant using the same - Google Patents
Arabidopsis AtCDCP2 gene that negatively regulates on biosynthesis of jasmonic acid and production method of male sterile plant using the same Download PDFInfo
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Abstract
본 발명은 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자 및 이를 이용한 웅성불임 식물체 제조방법에 관한 것이다. 본 발명에 의하면 안정성 있는 불임계를 확보할 수 있으며, 웅성불임계를 유지하기 위한 다른 유지계통이 필요 없고 식물호르몬에 의해 임성 회복이 가능한 웅성불임 식물을 생산할 수 있다. The present invention relates to the Arabidopsis AtCDCP2 gene related to plant hormone jasmonic acid biosynthesis inhibition and a method of producing a male sterile plant using the same. According to the present invention, it is possible to secure stable infertility, and it is possible to produce male infertility plants capable of recovering fertility by plant hormones without requiring another maintenance system for maintaining male infertility.
웅성불임, 애기장대, 자스몬산, 열개불능 Male infertility, Baby pole, Mount Jasmon, Incapacitated
Description
본 발명은 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자 및 이를 이용한 웅성불임 식물체 제조방법에 관한 것이다. 더욱 상세하게는 안정성 있는 불임계를 확보할 수 있으며, 웅성불임계를 유지하기 위한 다른 유지계통이 필요 없고 식물호르몬에 의해 임성 회복이 가능한 신규한 웅성불임 유발 유전자인 AtCDCP2 유전자 및 이를 이용한 웅성불임 식물체 제조방법에 관한 것이다. The present invention relates to the Arabidopsis AtCDCP2 gene related to plant hormone jasmonic acid biosynthesis inhibition and a method of producing a male sterile plant using the same. More specifically, the AtCDCP2 gene, which is a novel male infertility gene and a male infertility plant using the same, can secure stable infertility, and does not need another maintenance system for maintaining male infertility, and can recover fertility by plant hormones. It relates to a manufacturing method.
신품종 개발 산업은 생산성 향상에 의한 부족한 식량자원의 확보차원에서, 또한 장미 및 난 등과 같은 고부가가치 산업에서의 선점확보 측면에서 세계적으로 신품종 개발 주체가 되기 위한 경쟁이 치열한 추세이다. 현재 신기능 신품종 종자를 개발하고자 하는 육종가들은 교잡실험과 유전자 조작을 통해 신품종 개발 및 육성에 노력하고 있다. In the new breed development industry, there is a fierce competition to become a global new breeding agent in terms of securing food resources by improving productivity and securing a preoccupation in high value-added industries such as roses and eggs. At present, breeders who want to develop new varieties of new varieties are trying to develop and nurture new varieties through hybridization experiments and genetic manipulation.
신품종 종자는 대부분 모계순계와 부계순계를 교잡하여 잡종 강세 현상이 두 드러지게 나타내는 일대 잡종 (F1 hybrid)을 이용하는데, 이는 양친보다 생육이 왕성하고, 수량이 많고, 균일하며, 개화와 성숙기가 촉진되면서 불량 환경에 대한 저항성이 강해지기 때문이다. Most of the new varieties use the F 1 hybrid, which hybridizes the maternal and paternal ancestry to reveal hybrid accents, which are more vigorous, higher in yield, more uniform, and more flowering and mature than their parents. This is because the resistance to the bad environment becomes stronger as it is accelerated.
하지만 일대잡종의 채종에서 꽃이 비교적 크고 하나의 과실에 종자가 비교적 많이 들어 있는 박과채소(Cucurbitae) 등은 인공수분이 쉽기 때문에 비교적 간단히 잡종 종자를 얻을 수 있지만, 양파, 당근 등은 꽃이 작아 인공교배가 어려울 뿐만 아니라, 1회의 교배로 얻어지는 종자의 수가 매우 적어 인공교배로 잡종을 생산하는 것은 현실성이 없다. 더욱이 대부분을 차지하고 있는 양전화(hermaphrodite, 한 개의 꽃에 암술과 수술이 다 있는) 식물의 경우는 인공교배에 앞서 꽃망울 상태에서 모계의 수술을 제거해 주는 시간과 인력이 많이 소요되는 제웅(除雄, emasculation) 작업이 필수적이다. However, Cucurbitae, which is relatively large in flowers and contains a lot of seeds in a single fruit, can easily obtain hybrid seeds because of easy artificial moisture, but onions and carrots have small flowers. Not only is artificial breeding difficult, but the number of seeds obtained by one breeding is very small, and it is not practical to produce hybrids by artificial breeding. In addition, hermaphrodite plants, which occupy most of them, have pistils and stamens in one flower, which require a lot of time and labor to remove the maternal stamens from the flower buds before artificial breeding. emasculation) is essential.
만약 웅성불임계가 있다면 이를 모계로 사용하고 정상의 가임계를 부계로 사용하여 제웅 작업을 생략하고 방임수분에 의해 일대잡종의 채종이 가능하기 때문에, 웅성불임 식물의 개발은 신품종 종자 개발에 유용하게 사용될 수 있는 기구이다. 이러한 웅성불임은 수술의 결함으로 수정능력이 있는 화분을 생산하지 못하는 현상으로, 웅화나 약(pollen)의 기형화 또는 열개불능(꽃밥이 터지지 않아 꽃가루가 나오지 못하는 현상)으로 인해 나타난다.If there is a male sterility system, it can be used as a mother and a normal fertility system as a patriarch, eliminating the work of ergonomics, and the hybridization of large-scale hybrids is possible. It is a mechanism that can. This male infertility is a phenomenon of inability to produce fertilizing pots due to the defect of surgery, manifested due to malformation or decapacity (pollen) and pollen does not come out.
웅성불임성은 유전양식에 따라 핵유전형(genic male sterility)과 세포질유전형(cytoplasmic male sterility)으로 나눌 수 있다. 핵유전형 웅성불임은 생존에 불리하게 작용하기 때문에 발견되는 것으로, 주로 열성유전을 하며 열성동형(homozygously recessive)인 경우에 불임성을 띠게 되기 때문에 불임계의 유지 및 증식에 많은 노력이 소요되고, 또 작물에 따라서는 꽃이 작아 임성(稔性, fertility)의 판별이 힘들고, 꽃이 필 때까지 시간이 많이 소요되는 작물에서는 적용이 힘든 단점이 있다. Male infertility can be divided into genetic male sterility and cytoplasmic male sterility according to genetic patterns. Nuclear hereditary male infertility is found because it adversely affects survival. Since it is mainly recessive, it becomes infertile when it is recessive. Therefore, it takes a lot of effort to maintain and propagate infertility. Depending on the flower is small, it is difficult to determine fertility, and it is difficult to apply it to crops that take a long time to bloom.
한편 세포질 웅성불임성은 어느 가임계를 교배해도 100% 불임주가 나오기 때문에, 불임주에 여러 가지 가임주를 교배하여 50% 혹은 100% 불임주를 낼 수 있는 개체 및 계통, 즉 유지계통(maintainer)을 찾아내서 심어주어야 하기 때문에 현실성이 없다. 하지만 일대잡종 육종을 위해 웅성불임계 획득을 위한 방사선 및 화학 약품을 통한 인위적 웅성불임 유기도 시도된 바가 있으나, 안정성이 떨어져 실용화가 쉽지 않은 문제점이 있다.On the other hand, since cellular male infertility results in 100% infertility in any fertility system, it is possible to cross a variety of fertility strains to infertility strains, which can produce 50% or 100% infertility. It is not realistic because it must be found and planted. However, artificial male infertility through radiation and chemicals for obtaining male infertility has also been attempted for large-scale hybrid breeding, but there is a problem that it is not easy to be commercialized due to poor stability.
일대 잡종종자를 생산하기 위한 웅성불임 식물의 개발 및 연구에 많은 투자를 하고 있지만 현재까지 웅성불임 식물이 가지고 있는 문제점으로 인해 이에 관한 연구 성과는 매우 미비한 실정이다. 만약 이러한 웅성불임계 식물이 가지고 있는 단점들을 극복, 즉 안정성 있는 웅성불임계의 확보 및 다른 유지계통이 필요 없이 불임계를 유지할 수만 있다면, 이를 이용한 손쉬운 신품종 개발 및 고부가가치의 산업생산에 유용하게 이용할 수 있을 것이다. Although much investment has been made in the development and research of male sterile plants to produce large hybrid hybrids, the research results on the sterile plants have been insufficient. If you can overcome the shortcomings of these male sterile plants, that is, you can maintain the sterility without the need for stable male sterility and other maintenance systems, you can use them for easy development of new varieties and high value-added industrial production. Could be.
따라서 안정성 있는 웅성불임계를 확보할 수 있고 다른 유지계통이 필요 없이 불임계를 유지할 있는 신규한 유전자에 대한 요구가 끊임없이 존재하여 왔다.Therefore, there has been a constant need for new genes that can ensure stable male infertility and maintain infertility without the need for other maintenance systems.
본 발명은 종래 기술의 문제점 및 상기 요구를 해결하기 위해 안출된 것으로, 본 발명의 목적은 안정성 있는 웅성불임계를 확보할 수 있고 다른 유지계통이 필요 없이 불임계를 유지할 수 있는 신규한 웅성불임 유발 유전자를 제공하는 것이다.The present invention has been made to solve the problems and needs of the prior art, an object of the present invention is to induce a new male infertility that can ensure a stable male infertility and can maintain infertility without the need for other maintenance systems To provide genes.
본 발명의 다른 목적은 상기 웅성불임 유발 유전자를 포함한 재조합 벡터 및 이에 의해 형질전환된 식물체를 제공하는 것이다.Another object of the present invention is to provide a recombinant vector comprising the male infertility inducing gene and plants transformed thereby.
본 발명의 또 다른 목적은 상기 유전자의 웅성불임 유발 기작을 규명하여 임성을 정상적으로 회복할 수 있는 방법을 제공하는 것이다.Still another object of the present invention is to provide a method of restoring fertility normally by identifying a mechanism of causing male infertility of the gene.
상기 목적을 달성하기 위해, 본 발명자들은 애기장대 AtCDCP2 유전자의 DNA를 클로닝하여서, 식물체 형질 전환용 발현 벡터를 제작하였으며, 동일 벡터를 이용하여 애기장대 형질전환체를 제작하여 그 웅성불임 및 그 웅성불임 식물체의 식물 호르몬 자스몬산 함량의 감소 및 상기 식물호르몬 처리에 의한 임성 회복을 확인하여 본 발명을 완성하게 되었다.In order to achieve the above object, the present inventors cloned the DNA of Arabidopsis AtCDCP2 gene, and produced an expression vector for plant transformation, and produced the Arabidopsis transformant using the same vector, the male infertility and its male infertility. The present invention has been completed by confirming the reduction of the plant hormone jasmonic acid content of the plant and recovery of fertility by the plant hormone treatment.
따라서 본 발명은 서열번호 1의 염기서열을 갖는 애기장대에서 분리된 웅성불임 유발 유전자 AtCDCP2를 제공한다. Therefore, the present invention provides a male infertility inducing gene AtCDCP2 isolated from Arabidopsis edodae having the nucleotide sequence of SEQ ID NO: 1.
상기 유전자는 식물 호르몬 자스몬산 생합성을 저해하여 웅성불임을 유발하 는 것을 특징으로 한다. The gene is characterized by causing male sterility by inhibiting plant hormone jasmonic acid biosynthesis.
본 발명은 상기 웅성불임 유발 유전자를 포함한 재조합 벡터 및 이에 의해 형질전환된 식물체를 제공한다. The present invention provides a recombinant vector and a plant transformed by the male infertility inducing gene.
상기 재조합 벡터는 형질전환용 바이너리 벡터로서, 다양한 종류의 식물 형질전환용 벡터가 이용될 수 있다. The recombinant vector is a binary vector for transformation, and various kinds of plant transformation vectors may be used.
본 발명은 상기 유전자가 과다발현된 식물체에 자스몬산을 처리하여 임성을 정상적으로 회복하는 방법을 제공한다.The present invention provides a method for restoring fertility normally by treating jasmonic acid on a plant overexpressed with the gene.
이하, 첨부된 도면을 참조하면서 본 발명의 구성을 상세하게 설명하면 다음과 같다.Hereinafter, the configuration of the present invention with reference to the accompanying drawings in detail as follows.
애기장대의 전체 염기서열은 이미 모두 밝혀져 있다. 이들 염기서열을 바탕으로 각 유전자들의 위치와 중요한 도메인(domain), 엑손(exon), 인트론(intron) 등에 관한 정보도 이미 추정하여 공개되어 있지만, 아직도 특정 도메인이 없고 기능을 추정할 수 없는 유전자가 대다수이다. The entire sequence of Arabidopsis is already known. Based on these sequences, information on the location of each gene, important domains, exons, introns, etc. has already been estimated and published, but there are still genes that do not have specific domains and whose functions cannot be estimated. The majority.
본 발명자들은 아직까지 그 기능이 보고되지 않은 애기장대의 미지 유전자 중 웅성불임에 관련될 것으로 추정되는 유전자 AtCDCP2(At4g36910)를 선발하여 식물 형질전환용 바이너리 벡터를 이용하여 클로닝하고 염기서열(서열번호 1)을 확인하였다(도 1 및 도 2 참조). The present inventors selected a gene AtCDCP2 (At4g36910), which is estimated to be related to male infertility among unknown genes of Arabidopsis, whose function has not yet been reported, and cloned using a binary vector for plant transformation (SEQ ID NO: 1). ) (See FIGS. 1 and 2).
상기 바이너리 벡터를 이용하여 본 발명에 의한 AtCDCP2 유전자를 애기장대 내에서 과다발현 시켰을 때 수술의 열개 (anther dehiscence) 불능으로 인한 웅성불임이 야기된다(도 3 및 도 4 참조). AtCDCP2 according to the present invention using the binary vector When the gene is overexpressed in the Arabidopsis, male infertility is caused due to the inability of anther dehiscence (see FIGS. 3 and 4).
본 발명에 의한 유전자를 포함하는 재조합 벡터를 제조하고 이 재조합 벡터를 식물세포 내로 도입시키기 위하여, 본 발명이 속하는 분야의 통상의 지식을 가진 자에게 알려진 다양한 기술을 사용할 수 있다(참고 문헌: Clough SJ and Bent AF, 1998. Floral dip:a simlified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-43). 예를 들어 아그로박테리움-매개 형질전환을 이용할 수 있다.In order to prepare a recombinant vector comprising the gene according to the present invention and to introduce the recombinant vector into plant cells, various techniques known to those of ordinary skill in the art to which the present invention pertains can be used (Reference: Clough SJ and Bent AF, 1998. Floral dip: a simlified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J 16: 735-43). For example, Agrobacterium-mediated transformation can be used.
또한, 본 발명은 서열번호 1의 염기서열을 갖는 DNA 단편을 증폭하기 위한 서열번호 2 및 서열번호 3으로 표시되는 PCR용 프라이머(표 1 참조)를 제공한다.The present invention also provides a primer for PCR (see Table 1) represented by SEQ ID NO: 2 and SEQ ID NO: 3 for amplifying a DNA fragment having a nucleotide sequence of SEQ ID NO: 1.
한편 이 식물체들은 정상적인 약 (pollen)을 가지고 있고, 또한 이러한 웅성불임의 원인은 식물 호르몬인 자스몬산 (jasmonic acid)의 생합성이 적게 일어나서 발생한다(도 5a 및 도 5b 참조).On the other hand, these plants have a normal drug (pollen), and also the cause of this male infertility is caused by the low biosynthesis of the plant hormone jasmonic acid (see Figs. 5a and 5b).
나아가, 다른 화약 약품이 아닌 상기 자스몬산 식물호르몬을 인위적 처리하여 임성이 정상적으로 회복된다(도 6 참조).Furthermore, fertility is normally restored by artificially treating the Jasmonic Plant Hormone instead of other chemicals (see FIG. 6).
따라서 본 발명에 의한 유전자로 형질전환시켜서 얻어진 웅성불임 식물체는 안정성이 있으며, 웅성불임계를 유지하기 위한 다른 유지계통이 필요 없다. 따라서 본 발명은 신품종 개발을 위한 유용한 방법으로 사용되어 질 수 있다. Therefore, male sterile plants obtained by transforming with the gene of the present invention is stable, there is no need for other maintenance system for maintaining male sterility. Therefore, the present invention can be used as a useful method for developing new varieties.
상술한 바와 본 발명에 의한 유전자 AtCDCP2을 이용하면 종래의 웅성불임 식물 제조방법에 비해 안정성이 있으며, 웅성불임계를 유지하기 위한 다른 유지계통 이 필요 없고 식물호르몬에 의해 임성 회복이 가능하여 웅성불임 식물을 제조할 수 있다. 따라서 본 발명은 손쉬운 신품종 개발 및 고부가가치의 산업생산에 유용하게 이용할 수 있을 것이다.By using the gene AtCDCP2 according to the present invention as described above, it is more stable than the conventional male sterile plant manufacturing method, and does not need other maintenance system for maintaining male sterility, and it is possible to recover fertility by plant hormones. Can be prepared. Therefore, the present invention can be usefully used for easy development of new varieties and high value added industrial production.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.
[실시예 1. 본 발명에 의한 신규 유전자인 AtCDCP2의 클로닝]Example 1. Cloning of AtCDCP2 , a novel gene according to the present invention
양 말단에 XbaI (5' 말단)와 BamHI site (3’말단)를 포함하는 프라이머 (표 1)를 이용하여 AtCDCP2 (At4g36910) 유전자의 ORF (open reading frame)를 애기장대 (Arabidopsis thaliana ecotype Columbia)의 총RNA(total RNA)로부터 제작된 cDNA 풀(pool)에서 PCR 기법으로 증폭하였다.An open reading frame (ORF) of the AtCDCP2 (At4g36910) gene was obtained by using primers containing Xba I (5 'end) and BamHI site (3' end) at both ends of Arabidopsis thaliana ecotype Columbia. Amplification was performed by PCR technique in a cDNA pool prepared from total RNA.
상기 PCR 산물의 말단을 각 제한 효소로 절단한 후, 레프트보더(Left boarder)와 라이트보더(right boarder) 사이에 CaMV (Cauline flower mosaic virus) 35S 프로모터를 가지고 있는 식물 형질전환용 바이너리벡터(binary vector)인 pCAMBIA2300에 클로닝하였다 (도 1). 이 클론을 해독(sequencing)한 결과 정확한 염기서열이 클로닝(cloning)되었음을 확인할 수 있었다 (도 2). After cutting the ends of the PCR product with each restriction enzyme, a binary vector for plant transformation having a CaMV (Cauline flower mosaic virus) 35S promoter between a left boarder and a right boarder ) Was cloned into pCAMBIA2300 (FIG. 1). As a result of sequencing the clone, it was confirmed that the exact nucleotide sequence was cloned (FIG. 2).
[실시예 2. 본 발명에 의한 유전자인 AtCDCP2의 과다발현 식물체의 웅성 불임 확인] Example 2 Male Infertility Confirmation of Overexpressing Plants of AtCDCP2 Gene
실시예 1에서 제작된 벡터를 아그로박테리움 (Agrobacterium GV3101)에 냉-해동 (Freeze-thaw) 방법 (An, G. 1987, Methods in Enzymology)으로 도입하였다. 형질전환된 아그로박테리아는 꽃침지방법(flower dipping, Clough 와 Bent, 1998, The Plant Journal)으로 애기장대를 형질전환 시켰다. 형질전환 시킨 애기장대를 세 세대를 걸쳐 선발(screening)하여 T3 line을 확보하였다. The vector produced in Example 1 was introduced into Agrobacterium GV3101 by the Freeze-thaw method (An, G. 1987, Methods in Enzymology). The transformed Agrobacteria were transformed into Arabidopsis by flower dipping (Clough and Bent, 1998, The Plant Journal). The transgenic Arabidopsis was screened for three generations to secure a T 3 line.
먼저 형질전환 식물체와 정상적인 야생형(wild-type) 식물체로부터 총RNA(total RNA)를 추출하여 cDNA로 전환(conversion)후 RT-PCR 분석으로 형질전환 식물체가 야생형(wild-type) 식물체보다 명확하게 AtCDCP2의 유전자가 과다 발현되고 있음을 확인하였다(도 3 참조). 이때 내적대조군(Internal control)으로는 eIF4a1를 사용하였으며, PCR 반응은 Gene Amp® PCR System 2700 (Perkin Elmer, USA)을 사용하여, 95℃에서 2분간 초기 변성 후, 터치-다운(touch-down) PCR 방법으로 5회[95℃ 40초, 60℃ → 55℃(매 cycle당 1℃씩 감소) 15초, 72℃ 1분 30초] 실시하고, 정상적인 PCR 방법으로 30회 [95℃ 40초, 55℃ 15초, 72℃ 1분 30초]와 72℃에서 30초를 수행하였다. First, total RNA is extracted from transgenic plants and normal wild-type plants, converted to cDNA, and then converted to cDNA by RT-PCR analysis. It was confirmed that the gene is overexpressed (see Fig. 3). At this time, eIF4a1 was used as internal control, and PCR reaction was performed using Gene Amp ® PCR System 2700 (Perkin Elmer, USA), after initial denaturation at 95 ° C. for 2 minutes, and then touch-down. 5 times of PCR [95 ℃ 40 seconds, 60 ℃ → 55 ℃ (decreased by 1 ℃ per cycle) 15 seconds, 72 ℃ 1 minute 30 seconds], 30 times by the normal PCR method [95 ℃ 40 seconds, 55 ° C. 15 sec, 72 ° C. 1 min 30 sec] and 72 ° C. for 30 sec.
본 발명에 의한 AtCDCP2 과다 발현 식물체의 표현형을 관찰해 본 결과, 정상적으로 임성을 가지고 있는 야생형(wild-type) 식물체와 비교하여 확실하게 불임이 야기되고 있음을 확인하였다 (도 4의 A). 그리고 꽃을 해부하여 현미경으로 관찰한 결과, 수술이 열개 (anther dehiscence)되어야 하는 시점에서도 AtCDCP2 과다 발현 식물체는 열개가 되지 않고 있음을 확인하였다 (도 4의 B). AtCDCP2 according to the present invention As a result of observing the phenotype of the overexpressing plant, it was confirmed that infertility was clearly induced as compared with the wild-type plant having normal fertility (FIG. 4A). The dissection of the flower under a microscope showed that AtCDCP2 even when the surgery had to be dehiscence. It was confirmed that the overexpressing plants did not become dehiscence (FIG. 4B).
[실시예 3. 본 발명에 의한 유전자 AtCDCP2 과다발현 식물체내에서 자스몬산 생합성 저해 확인]Example 3 Confirmation of Jasmonic Acid Biosynthesis Inhibition in Genes AtCDCP2 Overexpressing Plants According to the Present Invention
기존 몇몇 논문에서 애기장대 식물에서 자스몬산의 생합성이 저해되면, 수술의 열개가 늦어짐으로써 불임이 야기된다고 보고된 적이 있다. 도 5a는 현재까지 보고된 자스몬산 생합성 경로를 나타내고, DDE1 유전자는 자스몬산 생합성 경로의 마지막 단계에 관여하고 있으며, 이 유전자의 감소는 자스몬산 합성량의 감소를 의미한다. Several previous papers have reported that the inhibition of biosynthesis of Jasmonic acid in Arabidopsis plants causes infertility by delaying the dehiscence of surgery. Figure 5a shows the Jasmonic acid biosynthetic pathway reported so far, the DDE1 gene is involved in the last step of the Jasmonic acid biosynthetic pathway, the decrease means a decrease in the amount of Jasmonic acid synthesis.
이에 의거하여 본 발명에서도 AtCDCP2 과다발현 식물체의 꽃들을 개화하기 전과 후로 나누어 채취한 후, 각각에서 자스몬산 생합성에 관여하는 12-oxophytodienoic reductase(DDE1/OPR3) 유전자의 발현을 확인하였다. 그 결과 도 5b에 도시된 바와 같이, 본 발명에 의한 AtCDCP2 과다발현 식물체내에서 DDE1 유전자의 발현이 정상적인 야생형 식물체내에서 보다 감소하고 있음을 확인하였다. Based on this, in the present invention, the flowers of AtCDCP2 overexpressing plants were collected before and after flowering, and the expression of 12-oxophytodienoic reductase (DDE1 / OPR3) genes involved in the biosynthesis of jasmonic acid in each was confirmed. As a result, it was confirmed that the reduction in the present invention than AtCDCP2 overexpressed wild-type plant, plant vivo expression of the normal gene DDE1 in vivo by, as shown in Figure 5b.
[실시예 4. 본 발명에 의한 유전자 AtCDCP2 과다발현 식물체의 인위적 자스몬산 처리를 통한 임성 회복 확인]Example 4 Confirmation of Fertility Recovery through Artificial Jasmonic Acid Treatment of Gene AtCDCP2 Overexpressing Plants According to the Present Invention
본 발명에 의한 유전자 AtCDCP2 과다발현 식물체는 웅성불임을 보이면서 화기(花器) 내의 자스몬산 합성량이 적게 일어나기 때문에 외부에서 인위적으로 자스몬산을 처리하였을 때 임성이 회복되는지를 확인하는 실험을 수행하였다. The gene AtCDCP2 overexpressed plant according to the present invention was shown to be male sterility, so the amount of Jasmonic acid synthesis in the fire (Flower) occurs less than the experiment was performed to determine whether the fertility recovered when artificially treated with Jasmonic acid.
먼저 AtCDCP2 과다발현 식물의 꽃봉오리(flower bud cluster)에 500 ㎛ 자스몬산을 처리하고 대조군(control)으로써 아무것도 처리하지 않거나 물 (water)만 처리하여 임성 회복 여부를 확인하였다. First, the buds of AtCDCP2 overexpressed plants were treated with 500 μm Jasmonic acid and treated with nothing as a control or treated with only water to determine whether fertility was recovered.
그 결과 도 6에서 확인할 수 있듯이, control 처리구에서는 여전히 웅성불임을 보이고 있는 반면에, 자스몬산 처리 후 일주일이 지난 식물체는 부분적으로 임성이 회복되는 현상을 확인할 수 있었고 (흰색 화살표), 또한 처리 후 이틀째 꽃을 현미경으로 관찰해 보았을 때 열개 불능의 수술의 열개되어져 있음을 확인하였다. 이로써 AtCDCP2 과다발현 식물체의 웅성불임 표현형을 가임으로 회복시킬 수 있음을 확인할 수 있었다.As a result, as shown in Figure 6, the control treatment was still showing male sterility, while a week after the treatment of Jasmonic acid was found to partially recover the fertility phenomenon (white arrow), also two days after treatment When the flower was observed under a microscope, it was confirmed that the dehiscence was dehiscence. It was confirmed that male sterility phenotype of AtCDCP2 overexpressed plants can be recovered by fertility.
도 1은 본 발명에 의한 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자의 클로닝에 사용되는 pCAMBIA2300 벡터의 레프트보더와 라이트보더 카세트를 나타내는 도면.1 is a diagram showing a left border and a light border cassette of a pCAMBIA2300 vector used for cloning the Arabidopsis AtCDCP2 gene associated with plant hormone Jasmonic acid biosynthesis inhibition according to the present invention.
도 2는 본 발명에 의한 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자 ORF의 전체 cDNA 염기서열을 나타내는 도면.Figure 2 shows the entire cDNA sequence of the Arabidopsis AtCDCP2 gene ORF related to plant hormone jasmonic acid biosynthesis inhibition according to the present invention.
도 3은 본 발명에 의한 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자가 과다발현된 식물체에서 상기 AtCDCP2 유전자의 전사발현양상을 나타내는 도면.Figure 3 is a view showing the transcriptional expression patterns of the AtCDCP2 gene in plants overexpressed Arabidopsis AtCDCP2 gene related to plant hormone jasmonic acid biosynthesis inhibition according to the present invention.
도 4는 본 발명에 의한 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자가 과다발현된 식물체에서 웅성불임 표현형을 보여주는 식물체 사진.Figure 4 is a plant photograph showing a male infertility phenotype in plants overexpressed Arabidopsis AtCDCP2 gene related to plant hormone jasmonic acid biosynthesis inhibition according to the present invention.
도 5a는 DDE1 유전자 감소가 본 발명과 관련된 자스몬산 합성량 감소를 의미함을 보인 자스몬산 생합성 경로의 도면.FIG. 5A is a diagram of a Jasmonic acid biosynthetic pathway showing that a decrease in DDE1 gene means a reduction in the amount of Jasmonic acid synthesis associated with the present invention. FIG.
도 5b는 본 발명에 의한 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자가 과다발현된 식물체 꽃들을 개화 전과 후로 나누어 자스몬산 생합성에 관여하는 DDE1 유전자 발현 양상을 보인 도면.Figure 5b is a diagram showing the expression of DDE1 gene involved in Jasmonic acid biosynthesis by dividing the plant flowers overexpressed Arabidopsis AtCDCP2 gene overexpression of plant hormone Jasmonic acid biosynthesis according to the present invention before and after flowering.
도 6은 본 발명에 의한 식물 호르몬 자스몬산 생합성 저해 관련 애기장대 AtCDCP2 유전자가 과다발현된 식물체에 자스몬산 처리를 통해 웅성불임 표현형이 회복되는 것을 보여주는 사진.Figure 6 is a photograph showing that the male sterility phenotype is recovered through the treatment of the plant hormone Jasmonic acid biosynthesis inhibition related Arabidopsis AtCDCP2 gene overexpression in the plant over the Jasmonic acid.
<110> Korea University Industrial and Academic Collaboration Foundation <120> Arabidopsis AtCDCP2 gene that negatively regulates on biosynthesis of jasmonic acid and production method of male sterile plant using the samee <130> P11-070828-01 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 711 <212> DNA <213> Arabidopsis thaliana <400> 1 atggacgccg tcctttactc tgttccactc tccttcactc ccctacgcgc atcatcctct 60 ccttcctcgc cgtatcttct tctgccgagg tttctctccg ttcagccatg tcacaaattc 120 accttctctc gaagcttccc ttccaaatcc cggattccct cagcttcttc cgccgccggt 180 tccacgttga tgacgaattc ctcttcgcca agaagtggag tgtacactgt tggtgagttc 240 atgacaaaga aagaggactt gcacgtggtg aaacctacga ctactgtgga tgaagctctg 300 gaactccttg tggagaatag aatcactgga tttcctgtaa ttgacgaaga ctggaaattg 360 gttgggcttg tttcagatta tgacttgttg gctttggact ccatatctgg tagtggaaga 420 acagaaaatt ccatgttccc tgaggttgac agcacctgga aaactttcaa tgctgtgcaa 480 aagctactca gcaaaaccaa tgggaagctt gttggagatt taatgacacc agctccacta 540 gttgttgagg aaaaaaccaa cctggaagat gctgctaaaa tattgcttga gacaaaatat 600 cgccggctcc ctgtggtaga ttctgatggc aaattggttg gtatcatcac aagaggaaac 660 gtggttagag ccgcgcttca aataaagcgc tctggtgata ggaatgcttg a 711 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 2 ggctctagac aatggacgcc gtcctttac 29 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 3 taaggattca agcattccta tcaccagagc gc 32 <110> Korea University Industrial and Academic Collaboration Foundation <120> Arabidopsis AtCDCP2 gene that negatively regulates on biosynthesis of jasmonic acid and production method of male sterile plant using the samee <130> P11-070828-01 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 711 <212> DNA <213> Arabidopsis thaliana <400> 1 atggacgccg tcctttactc tgttccactc tccttcactc ccctacgcgc atcatcctct 60 ccttcctcgc cgtatcttct tctgccgagg tttctctccg ttcagccatg tcacaaattc 120 accttctctc gaagcttccc ttccaaatcc cggattccct cagcttcttc cgccgccggt 180 tccacgttga tgacgaattc ctcttcgcca agaagtggag tgtacactgt tggtgagttc 240 atgacaaaga aagaggactt gcacgtggtg aaacctacga ctactgtgga tgaagctctg 300 gaactccttg tggagaatag aatcactgga tttcctgtaa ttgacgaaga ctggaaattg 360 gttgggcttg tttcagatta tgacttgttg gctttggact ccatatctgg tagtggaaga 420 acagaaaatt ccatgttccc tgaggttgac agcacctgga aaactttcaa tgctgtgcaa 480 aagctactca gcaaaaccaa tgggaagctt gttggagatt taatgacacc agctccacta 540 gttgttgagg aaaaaaccaa cctggaagat gctgctaaaa tattgcttga gacaaaatat 600 cgccggctcc ctgtggtaga ttctgatggc aaattggttg gtatcatcac aagaggaaac 660 gtggttagag ccgcgcttca aataaagcgc tctggtgata ggaatgcttg a 711 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 2 ggctctagac aatggacgcc gtcctttac 29 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 3 taaggattca agcattccta tcaccagagc gc 32
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