KR102418848B1 - Guide RNA for editing gigantea gene and use thereof - Google Patents
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Abstract
본 발명의 지잔티아 유전자 교정용 가이드 RNA, 상기 가이드 RNA를 포함하는 지잔티아 유전자 교정용 벡터, 및 이를 이용한 염 또는 건조 스트레스 저항성이 증진된 식물체의 제조방법에 관한 것으로, 본 발명은 고염 또는 건조 환경에서 배추 속 작물의 피해를 막을 수 있으며, 배추 육종 시 염 또는 건조 스트레스 저항성 증진 또는 개선된 주요 교배 계통으로 유용하게 이용될 수 있다.The guide RNA for Zyzantia gene editing of the present invention, a vector for Zyzantia gene editing comprising the guide RNA, and a method for producing a plant having improved resistance to salt or dry stress using the same, the present invention relates to a high salt or dry environment It can prevent damage to crops of the genus Chinese cabbage, and can be usefully used as a major crossbreeding line to improve resistance to salt or dry stress during breeding of cabbage.
Description
본 발명은 지잔티아 유전자에 상보적인 가이드 RNA 및 이의 용도에 관한 것이다. The present invention relates to a guide RNA complementary to a Zyzantia gene and uses thereof.
고등식물은 이동이 불가하기 때문에 그들의 일생동안 다양한 환경 요인들인 가뭄, 고염(high salt), 중금속, 냉해, 열충격 및 오존과 같은 스트레스에 직면하게 된다. 이러한 비생물학적 스트레스는 작물의 생장과 발달의 제한 요인이 되며 이들 스트레스 중에서 수분 부족은 작물생산 감소의 주요 원인으로 여겨지는 가장 심각한 환경 요인이다. 세계적으로 물의 소비는 계속적으로 증가되어 왔으며, 깨끗한 물의 이용가능성은 인간에게 뿐만 아니라 고등식물에게 또한 중요한 문제가 될 수 있다. 식물은 단기적 또는 장기적인 물 부족에 대해 내성을 증가시키기 위해 신호 네트워크 경로를 조절하거나 스트레스-반응성 유전자들을 유도하는 등의 다양한 방어 전략을 작동시킨다. 이러한 수분 스트레스에 대한 세포적 또는 유전적 방어 메카니즘은 널리 알려져 있다. 그러나 아직까지 고등식물에 있어서 스트레스에 대한 내성이나 민감성에 관여하는 스트레스-관련 유전자들의 생물학적 기능들에 대한 지식은 여전히 부족한 상태다. 그러므로 작물의 생산성을 증가시키기 위해 스트레스 반응 유전자들에 대한 기능 연구가 중요하다.Because higher plants are immobile, they face stresses such as drought, high salt, heavy metals, cold damage, thermal shock and ozone during their lifetime. These abiotic stresses are limiting factors for the growth and development of crops, and among these stresses, lack of water is the most serious environmental factor considered to be the main cause of the decrease in crop production. Worldwide water consumption has been continuously increasing, and the availability of clean water can be an important issue not only for humans but also for higher plants. Plants engage a variety of defense strategies, such as modulating signaling network pathways or inducing stress-responsive genes, to increase tolerance to short-term or long-term water scarcity. Cellular or genetic defense mechanisms against such water stress are well known. However, knowledge about the biological functions of stress-related genes involved in tolerance or sensitivity to stress in higher plants is still lacking. Therefore, functional studies on stress response genes are important to increase crop productivity.
유전자 교정(gene editing)은 원하는 염기서열을 인식하여 절단하도록 만들어지는 인공제한효소로 동물 또는 식물 세포내에서 정확히 원하는 유전체 염기서열을 정확히 절단하여 기존의 기술들에 비해 높은 효율로 유전체 상의 유전정보를 교정하는 분자도구이다. 실제로 유전자 교정 방법은 2002년 초파리에서 유전자 knock-out을 위하였던 다양한 동물뿐만 아니라, 애기장대, 담배, 벼, 옥수수 등의 식물에서 매우 효율적인 유전정보 조작을 성공적으로 유도할 수 있음이 증명되었다. 이러한 유전자 교정이 성공적으로 이루어지기 위해서 목적 유전자를 특이적으로 목적하는 효율이 높으면서, 비특이적인 지역을 변화시키지 않는 유전자 교정 방법이 요구되고 있다. Gene editing is an artificial restriction enzyme that is made to recognize and cut a desired nucleotide sequence. It precisely cuts the desired genomic nucleotide sequence in animal or plant cells, thereby cutting the genetic information on the genome with high efficiency compared to existing technologies. It is a molecular tool for correction. In fact, it was proved that the gene editing method can successfully induce highly efficient genetic information manipulation in plants such as Arabidopsis thaliana, tobacco, rice, and corn as well as various animals for gene knock-out in Drosophila in 2002. In order to successfully perform such gene editing, there is a need for a gene editing method that specifically targets a target gene and does not change a non-specific region with high efficiency.
한편, 배추는 김치의 주재료로서 한국의 4대 채소 가운데 하나이며 배추 속(Brassica)에 포함되어 있다. 배추 속(Brassica)에는 유채, 겨자 등 산업적 경제성이 우수한 작물이 포함되어 있어 외국에서도 중요한 작물이다. 배추 속(Brassica) 작물은 건조 환경에 의한 피해에 민감하며, 이로 인한 생육장애로 수확량이 감소되어 농가와 소비자에게 막대한 피해를 줄 수 있으므로 배추의 염 또는 건조 스트레스 저항성이 증진된 배추를 개발하는 연구가 요구되고 있다.On the other hand, Chinese cabbage is one of the four major vegetables in Korea as the main ingredient of kimchi and is included in the genus Brassica . The genus Brassica contains crops with excellent industrial economic efficiency, such as rapeseed and mustard, and is therefore an important crop in foreign countries. A study to develop Chinese cabbage with improved resistance to salt or drying stress, as crops of the genus Brassica are sensitive to damage caused by a dry environment, and the yield is reduced due to growth failure, which can cause enormous damage to farmers and consumers. is being requested
본 발명의 해결하고자 하는 과제는 서열번호2 또는 서열번호3과 동일하거나 상보적인 염기서열로 이루어진 지잔티아 유전자 교정용 가이드 RNA를 제공하는 것이다. An object to be solved by the present invention is to provide a guide RNA for Zyzantia gene editing consisting of a nucleotide sequence identical to or complementary to SEQ ID NO:2 or SEQ ID NO:3.
본 발명의 다른 과제는 상기 가이드 RNA를 포함하는 지잔티아 유전자 교정용 벡터를 제공하는 것이다. Another object of the present invention is to provide a vector for editing Zyzantia gene comprising the guide RNA.
본 발명의 또 다른 과제는 상기 가이드 RNA 또는 상기 유전자 교정용 벡터를 포함하는 지잔티아 유전자 교정용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for editing Zyzantia gene comprising the guide RNA or the vector for editing the gene.
본 발명의 또 다른 과제는 상기 지잔티아 유전자 교정용 벡터를 형질도입하여 지잔티아 유전자가 교정된 형질전환 식물체를 제공하는 것이다.Another object of the present invention is to provide a transgenic plant in which the Zyzantia gene is corrected by transducing the vector for editing the Zyzantia gene.
본 발명의 또 다른 과제는 서열번호2 또는 서열번호3과 동일하거나 상보적인 염기서열로 이루어진 가이드 RNA를 포함하는 지잔티아 유전자 교정용 벡터를 준비하는 단계 및 상기 벡터를 식물체에 형질도입하여 지잔티아 유전자를 교정하는 단계를 포함하는 염 또는 건조 스트레스 저항성이 증진된 식물체의 제조방법을 제공하는 것이다.Another object of the present invention is to prepare a vector for correcting Zyzantia gene comprising a guide RNA consisting of a nucleotide sequence identical to or complementary to SEQ ID NO:2 or SEQ ID NO:3, and transducing the vector into a plant to produce a Zyzantia gene It is to provide a method for producing a plant having improved resistance to salt or drying stress, comprising the step of correcting it.
상기의 과제를 해결하기 위해 본 발명의 하나의 양태로, 서열번호2 또는 서열번호3과 동일하거나 상보적인 염기서열로 이루어진 지잔티아 유전자 교정용 가이드 RNA를 제공한다. In order to solve the above problems, in one aspect of the present invention, there is provided a guide RNA for correcting Zyzantia gene consisting of the same or complementary nucleotide sequence to SEQ ID NO:2 or SEQ ID NO:3.
본 발명의 "가이드 RNA(guide RNA, gRNA)"란 RNA(ribonucleic acid)의 한 종류로, 인간이나 동식물의 특정 유전자를 교정하는 유전자 교정(genome editing)에서 교정하려는 DNA를 인식하여 찾아내는 RNA를 의미한다.The term "guide RNA (gRNA)" of the present invention is a type of RNA (ribonucleic acid), and refers to RNA that recognizes and finds DNA to be edited in genome editing that corrects specific genes in humans or animals and plants do.
유전자 교정 기술로는 유전자 가위(engineered nuclease)가 있으며, 유전자 가위의 종류로 CRISPR-Cas9이 있다.Gene editing technology includes engineered nuclease, and CRISPR-Cas9 is a type of gene editing technology.
본 발명에서 "CRISPR-Cas9"이란 미생물의 면역체계로 알려진 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) 시스템을 이용해 원하는 유전자 염기서열을 절단하도록 고안된 것으로서, 고정적 구성요소로서 Cas9 단백질을 포함하고, 가변적 구성요소로서 타겟 유전자에 특이적인 가이드 RNA를 포함한다. 이때 타겟 유전자의 조건은 23 bp 길이이고 두 개의 구아닌 염기(GG)로 끝나기만 하면 된다. 가이드 RNA가 타겟 유전자를 인식하면 가이드 RNA에 Cas9 단백질이 결합하여 뉴클레아제로 작용하여 타겟 유전자의 하류 약 3 bp에 위치한 두 개의 구아닌 염기(GG)를 인식하여 절단함으로써 DNA 이중가닥 손상(DNA double strand break, DSB)을 유발한다. 상기 CRISPR-Cas9 시스템에는 tracr RNA를 더 포함할 수 있으며, 상기 tracr RNA는 gRNA와 복합체를 형성하여 Cas9이 인식할 수 있는 구조를 형성하는 역할을 할 수 있다In the present invention, "CRISPR-Cas9" is designed to cut a desired gene sequence using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system known as the immune system of a microorganism. as a guide RNA specific to the target gene. In this case, the condition of the target gene is only to be 23 bp long and end with two guanine bases (GG). When the guide RNA recognizes the target gene, the Cas9 protein binds to the guide RNA and acts as a nuclease to recognize and cut two guanine bases (GG) located about 3 bp downstream of the target gene, resulting in DNA double strand damage (DNA double strand). break, DSB). The CRISPR-Cas9 system may further include tracr RNA, and the tracr RNA may form a complex with gRNA to form a structure that Cas9 can recognize.
상기 가이드 RNA는 배추 유래 지잔티아(GIGANTEA) 유전자의 특정서열을 표적으로 하며, 상기 지잔티아(GIGANTEA) 유전자는 서열번호1의 염기서열로 이루어진 것일 수 있다. The guide RNA may target a specific sequence of a Chinese cabbage-derived GIGANTEA gene, and the GIGANTEA gene may consist of the nucleotide sequence of SEQ ID NO: 1.
상기 지잔티아(GIGANTEA) 유전자는 생체 리듬 유전자이며, 염 또는 건조 스트레스와 같은 환경 스트레스에 대한 저항성 기작에 관여한다. 따라서 식물체에서 지잔티아(GIGANTEA)의 유전자 교정하여 지잔티아(GIGANTEA)의 발현을 조절하면 염 스트레스 저항성과 건조 스트레스 저항성을 증진 또는 개선시킬 수 있다. The GIGANTEA gene is a biorhythm gene and is involved in a resistance mechanism to environmental stress such as salt or drying stress. Therefore, by regulating the expression of GIGANTEA by gene editing of GIGANTEA in plants, salt stress resistance and drying stress resistance can be enhanced or improved.
본 발명의 일실시예에서 서열번호1의 배추 유래 지잔티아(GIGANTEA) 유전자에서 g1 내지 g5의 가이드 RNA의 타겟 유전자 영역을 선별하였고, 각 gRNA를 Cas9 및 PEG와 접종하고, 유전자 교정(변이율)을 분석하였다. In an embodiment of the present invention, target gene regions of guide RNAs of g1 to g5 were selected from the Chinese cabbage-derived GIGANTEA gene of SEQ ID NO: 1, and each gRNA was inoculated with Cas9 and PEG, and gene correction (variation rate) was analyzed.
분석결과, 5개의 gRNA 중 g1의 변이율이 33.58% 내지 47.04%로 가장 높게 나타났고, g3의 변이율이 6.17% 내지 21.95%로 그 뒤를 이었다. 반면, g2, g4 및 g5의 gRNA는 PEG 30%(v/v)에서 1.08 내지 3.81%, PEG 40%(v/v)에서 5.04 내지 10.97%의 변이율(indel ratio)을 나타내, g1(서열번호2) 및 g3(서열번호3)의 gRNA와 비교하여 변이율이 상대적으로 낮게 나타났다. As a result of the analysis, among the five gRNAs, the g1 mutation rate was the highest at 33.58% to 47.04%, followed by the g3 mutation rate at 6.17% to 21.95%. On the other hand, gRNAs of g2, g4 and g5 exhibit an indel ratio of 1.08 to 3.81% in PEG 30% (v/v) and 5.04 to 10.97% in PEG 40% (v/v), g1 (sequence No. 2) and g3 (SEQ ID NO: 3) showed a relatively low mutation rate compared to the gRNA.
즉, g1(서열번호2) 및 g3(서열번호3)에 특이적인 gRNA를 이용할 경우, g2(서열번호4), g4(서열번호5) 및 g5(서열번호6)에 특이적인 gRNA를 이용할 때와 비교하여 배추(Brassica rapa) 유래 지잔티아(GIGANTEA) 유전자에 삽입 또는 결손을 효율적으로 유도하여, 유전자를 교정할 수 있음을 확인하였다. That is, when using gRNA specific for g1 (SEQ ID NO: 2) and g3 (SEQ ID NO: 3), gRNA specific for g2 (SEQ ID NO: 4), g4 (SEQ ID NO: 5) and g5 (SEQ ID NO: 6) When using It was confirmed that the gene can be corrected by efficiently inducing an insertion or deletion in the GIGANTEA gene derived from Chinese cabbage (Brassica rapa) compared to that.
따라서 본 발명의 가이드 RNA는 서열번호2 또는 서열번호3과 동일하거나 상보적인 염기서열로 이루어진다.Therefore, the guide RNA of the present invention consists of a nucleotide sequence identical to or complementary to SEQ ID NO:2 or SEQ ID NO:3.
본 발명의 다른 양태로 서열번호2 또는 서열번호3과 동일하거나 상보적인 염기서열로 이루어진 지잔티아 유전자 교정용 가이드 RNA를 포함하는 지잔티아 유전자 교정용 벡터를 제공한다.In another aspect of the present invention, there is provided a vector for editing the Zyzantia gene comprising a guide RNA for editing the Zyzantia gene consisting of the same or complementary nucleotide sequence to SEQ ID NO:2 or SEQ ID NO:3.
본 발명에서 가이드 RNA에 대한 설명은 전술한 바와 같다. The description of the guide RNA in the present invention is as described above.
본 발명의 유전자 교정용 벡터는 상기 식물체 내 지잔티아(GIGANTEA) 유전자를 교정하기 위해 특이적으로 인식하는 가이드 RNA가 도입된 식물형질 전환용 벡터를 의미한다.The vector for gene editing of the present invention refers to a vector for plant transformation into which a guide RNA specifically recognized to correct the GIGANTEA gene in the plant is introduced.
본 발명에 따른 벡터는 cas9 단백질을 암호화하는 염기서열을 더 포함할 수 있다. The vector according to the present invention may further include a nucleotide sequence encoding a cas9 protein.
상기 벡터는 목적하는 유전자의 발현 억제 또는 유전자의 발현이 증진될 수 있도록, 발현조절 서열과 기능적으로 연결될 수 있다. 예를 들어, 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널, 인핸서 같은 발현 조절 요소 외에도 막 표적화 또는 분비를 위한 신호 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 또한, 벡터는 선택성 마커를 포함할 수 있으며, 벡터는 자가 복제하거나 숙주 DNA에 통합될 수 있다. The vector may be functionally linked with an expression control sequence so that the expression of a desired gene can be suppressed or the expression of a gene can be enhanced. For example, the vector includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, operator, initiation codon, stop codon, polyadenylation signal, and enhancer, and can be prepared in various ways depending on the purpose. . In addition, the vector may include a selectable marker, and the vector may be self-replicating or integrated into host DNA.
본 발명의 벡터는 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.The vector of the present invention may be prepared using a genetic recombination technique well known in the art, and for site-specific DNA cleavage and ligation, an enzyme generally known in the art may be used.
본 발명의 일실시예에서, 배추 내의 지잔티아(GIGANTEA) 유전자를 교정하기 위해 cas9 유전자를 포함하는 pHAtC 벡터(도 3)에 g1 또는 g3에 특이적인 gRNA 유전자를 삽입하여 지잔티아(GIGANTEA) 유전자 교정용 구조체(construction)를 제조하였다. 제조된 유전자 교정용 구조체(g1 또는 g3 구조체)로 배추를 형질전환하고 형질전환 배추의 유전가를 분석한 결과, g1 구조체의 경우 삽입-결손(indel) 유도율이 99.48%로 나타났으며, g3 구조체는 71.12%로 나타났다. 즉, g1 또는 g3 구조체의 형질도입이 배추의 지잔티아(GIGANTEA) 유전자의 서열변이(유전자 교정)을 효율적으로 유도함을 확인하였다. In one embodiment of the present invention, GIGANTEA gene correction by inserting a gRNA gene specific for g1 or g3 into a pHAtC vector (FIG. 3) containing a cas9 gene to correct the GIGANTEA gene in Chinese cabbage A construction was prepared. As a result of transforming Chinese cabbage with the prepared gene editing construct (g1 or g3 construct) and analyzing the genetic value of the transgenic Chinese cabbage, the g1 construct showed an insertion-deletion (indel) induction rate of 99.48%, and the g3 construct was 71.12%. That is, it was confirmed that the transduction of the g1 or g3 construct efficiently induces sequence variation (gene correction) of the GIGANTEA gene of Chinese cabbage.
본 발명의 또 다른 양태로 상기 가이드 RNA 또는 상기 유전자 교정용 벡터를 포함하는 지잔티아 유전자 교정용 조성물을 제공한다. In another aspect of the present invention, there is provided a composition for editing Zyzantia gene comprising the guide RNA or the vector for editing the gene.
본 발명의 지잔티아 유전자 교정용 조성물은 서열번호2 또는 서열번호3과 동일하거나 상보적인 염기서열로 이루어진 지잔티아 유전자 교정용 가이드 RNA 또는 이를 포함하는 유전자 교정용 벡터를 포함한다.The composition for editing Zyzantia gene of the present invention includes a guide RNA for Zyzantia gene editing consisting of a nucleotide sequence identical to or complementary to SEQ ID NO: 2 or SEQ ID NO: 3 or a vector for gene editing including the same.
본 발명에서 가이드 RNA, 벡터에 관한 설명은 전술한 바와 같다. The description of the guide RNA and the vector in the present invention is the same as described above.
본 발명의 일실시예에서 g1(서열번호2) 및 g3(서열번호3)에 특이적인 gRNA를 Cas9 단백질 및 PEG와 함께 배추 원형질체에 접종하여 지잔티아 유전자의 삽입-결손(indel)을 유도하여 지잔티아 유전자를 교정함을 확인하였다. In an embodiment of the present invention, g1 (SEQ ID NO: 2) and g3 (SEQ ID NO: 3) specific gRNAs together with Cas9 protein and PEG were inoculated into a Chinese cabbage protoplast to induce an insertion-deletion (indel) of the Zizantia gene to induce Jizan. It was confirmed that the Tia gene was corrected.
또한, 본 발명의 다른 실시예에서 g1(서열번호2) 또는 g3(서열번호3)에 특이적인 gRNA 및 cas9 단백질 유전자를 포함하는 구조체를 배추에 형질도입하고 지잔티아 유전자 변이를 확인한 결과, 지잔티아 유전자의 삽입-결손(indel)을 유도되어 지잔티아 유전자가 교정됨을 확인하였다. In addition, in another embodiment of the present invention, a construct containing gRNA and cas9 protein gene specific to g1 (SEQ ID NO: 2) or g3 (SEQ ID NO: 3) was transduced into Chinese cabbage and the result of confirming the Zyzantia gene mutation, Zyzantia It was confirmed that the gene insertion-deletion (indel) was induced to correct the Zizantia gene.
따라서 상기 조성물은 Cas9 단백질 또는 Cas9 단백질 형질도입용 벡터를 더 포함할 수 있다. Accordingly, the composition may further include a Cas9 protein or a vector for Cas9 protein transduction.
본 발명의 또 다른 양태로 상기 지잔티아 유전자 교정용 벡터를 형질도입하여 지잔티아 유전자가 교정된 형질전환 식물체를 제공한다. In another aspect of the present invention, there is provided a transgenic plant in which the Zyzantia gene is corrected by transducing the vector for correcting the Zyzantia gene.
본 발명에서 “벡터”에 관한 설명은 전술한 바와 같다. The description of “vector” in the present invention is the same as described above.
본 발명에서 “형질전환”은, 유전물질인 DNA를 다른 계통의 살아 있는 세포에 주입했을 때, DNA가 그 세포에 들어가 유전형질(遺傳形質)을 변화시키는 현상으로, 형질변환, 형전환, 또는 형변환 이라고도 한다.In the present invention, “transformation” refers to a phenomenon in which DNA enters the cell and changes the genetic trait when DNA, which is a genetic material, is injected into a living cell of another lineage. Also called conversion.
본 발명에서 상기 벡터로 식물체를 "형질전환"하는 것은 당업자에게 공지된 형질전환기술에 의해 수행될 수 있다. 구체적으로는, 아그로박테리움을 이용한 형질전환방법, 미세사출법(microprojectile bombardment), 일렉트로포레이션(electroporation), PEG-매개 융합법(PEG-mediated fusion), 미세주입법(microinjection), 리포좀 매개법(liposome-mediated method), 인-플란타 형질전환법(In planta transformation), 진공 침윤법(Vacuum infiltration method), 화아침지법(floral meristem dipping method), 및 아그로박테리아 분사법(Agrobacteria spraying method)을 이용할 수 있다.In the present invention, "transformation" of a plant with the vector can be performed by transformation techniques known to those skilled in the art. Specifically, transformation method using Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome-mediated method ( liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, and Agrobacteria spraying method can
본 발명에서 "식물체"는, 성숙한 식물체뿐만 아니라 성숙한 식물로 발육할 있는 식물 세포, 식물 조직 및 식물의 종자 등을 모두 포함하는 의미로서 이해된다.In the present invention, the term "plant" is understood to include not only mature plants, but also plant cells, plant tissues, and seeds of plants that can develop into mature plants.
본 발명에서 상기 식물체는 벼, 밀, 보리, 옥수수, 콩, 감자, 밀, 팥, 귀리 또는 수수를 포함하는 식량 작물류; 애기장대, 배추, 무, 고추, 딸기, 토마토, 수박, 오이, 양배추, 참외, 호박, 파, 양파 또는 당근을 포함하는 채소 작물류; 인삼, 담배, 목화, 참깨, 사탕수수, 사탕무우, 들깨, 땅콩 또는 유채를 포함하는 특용작물류; 사과나무, 배나무, 대추나무, 복숭아, 양다래, 포도, 감귤, 감, 자두, 살구 또는 바나나를 포함하는 과수류; 장미, 글라디올러스, 거베라, 카네이션, 국화, 백합 또는 튤립을 포함하는 화훼류; 및 라이그라스, 레드클로버, 오차드그라스, 알파알파, 톨페스큐 또는 페레니얼라이그라스를 포함하는 사료작물류로 이루어진 군으로부터 선택된 어느 하나이며, 구체적으로는 인삼, 담배, 목화, 참깨, 사탕수수, 사탕무우, 들깨, 땅콩 또는 유채를 포함하는 특용작물류이며, 더욱 구체적으로는 배추일 수 있다.In the present invention, the plant includes rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat or sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion or carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut or rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots or bananas; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies or tulips; And ryegrass, red clover, orchard grass, alpha alpha, tall fescue or any one selected from the group consisting of forage crops including perennial ryegrass, specifically ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, It is a special crop including perilla, peanut or rapeseed, and more specifically, may be Chinese cabbage.
본 발명의 일실시예에서 g1 또는 g3의 gRNA를 이용하여 배추에서 지잔티아(GIGANTEA)의 삽입-결손(indel)을 유도하여, 지잔티아(GIGANTEA) 유전자를 교정하였으며, 유전자가 교정된 배추의 염 또는 건조 스트레스 저항성에 미치는 영향을 분석하였다. NaCl(250mM)으로 고염 스트레스를 2주간 유도하고, 대조군과 유전자 교정 식물체(GI-g1, GI-g3)의 생장을 비교한 결과, 대조구인 지부(Chiifu)는 시들어 죽었으나, 유전자 교정 식물체(GI-g1, GI-g3)는 생장이 유지됨을 확인하였다(도 4 및 도 5). In one embodiment of the present invention, g1 or g3 gRNA was used to induce insertion-deletion (indel) of GIGANTEA in Chinese cabbage, and the GIGANTEA gene was corrected, and the gene was corrected salt of Chinese cabbage. Alternatively, the effect on drying stress resistance was analyzed. As a result of inducing high salt stress with NaCl (250 mM) for 2 weeks, and comparing the growth of the control and gene-corrected plants (GI-g1, GI-g3), the control, Chiifu, withered and died, but the gene-corrected plants (GI) -g1, GI-g3) was confirmed that the growth is maintained ( FIGS. 4 and 5 ).
또한, 대조구(지부)와 유전자 교정 식물체(GI-g3)에 2주간 건조 스트레스 유도하고, 물을 재공급한 후 생장을 비교 분석한 결과, 유전자 교정 배추(GI-g3)는 생장이 개재하여 정상 상태로 회복되었으나, 대조구인 지부는 시들어 회복되지 않음을 확인하였다In addition, as a result of comparative analysis of growth after induction of drying stress for 2 weeks in the control (branch) and the gene-modified plant (GI-g3) and re-supplying water, the gene-corrected Chinese cabbage (GI-g3) was normalized due to the intervening growth. It was recovered to the state, but it was confirmed that the branch, which is a control, withered and did not recover.
다음으로 지잔티아(GIGANTEA) 유전자의 서열변이가 배추의 정상적인 생장발달에 영향을 주는지 확인하고자, 대조구(지부)와 형질전환 배추의 종자(T1)를 파종하여 온실에서 생육하였다. 9월 초에 파종하여 가온하지 않는 비가림 온실에서 11월 말까지 생육하고 결구 상태를 확인한 결과, 유전자 교정 배추가 정상적으로 생육함을 확인하였다. Next, in order to confirm whether the sequence variation of the GIGANTEA gene affects the normal growth and development of Chinese cabbage, control (branch) and transgenic cabbage seeds (T1) were sown and grown in a greenhouse. It was sown in early September and grown until the end of November in a non-warmed greenhouse without heating.
즉, 형질전환하여 지잔티아(GIGANTEA) 유전자를 교정 배추가 정상적으로 생장 발달하며, 염 저항성 및 건조 저항성이 증진 및 개선됨을 확인하였다.That is, it was confirmed that the transformation and correction of the GIGANTEA gene were normalized for growth and development of Chinese cabbage, and salt resistance and drying resistance were enhanced and improved.
따라서 본 발명의 “식물체”는 지잔티아 유전자에 유전자 교정(서열변이), 구체적으로 유전자의 삽입 또는 결손이 유도되어, 염 또는 건조 스트레스 저항성이 증진된 것을 특징으로 한다. 구체적으로 지잔티아 유전자 교정에 의해 염 및 건조 스트레스 저항성이 동시에 증진된 것 일 수 있다. Therefore, the "plant" of the present invention is characterized in that the gene correction (sequence mutation), specifically, the insertion or deletion of the gene is induced in the Zizantia gene, and salt or drying stress resistance is improved. Specifically, salt and drying stress resistance may be simultaneously enhanced by Zyzantia gene correction.
본 발명의 또 다른 양태로 서열번호2 또는 서열번호3과 동일하거나 상보적인 염기서열로 이루어진 가이드 RNA를 포함하는 지잔티아 유전자 교정용 벡터를 준비하는 단계 및 상기 벡터를 식물체에 형질도입하여 지잔티아 유전자를 교정하는 단계를 포함하는 염 또는 건조 스트레스 저항성이 증진된 식물체의 제조방법을 제공한다. In another aspect of the present invention, the step of preparing a vector for correcting Zyzantia gene comprising a guide RNA consisting of a nucleotide sequence identical to or complementary to SEQ ID NO: 2 or SEQ ID NO: 3 and transducing the vector into a plant to generate a Zyzantia gene It provides a method for producing a plant having improved resistance to salt or dry stress, comprising the step of correcting.
본 발명에서 벡터, 식물체, 형질도입에 대한 설명은 전술한 바와 같다. In the present invention, the description of the vector, plant, and transduction is the same as described above.
전술한 실시예에서 지잔티아 유전자가 교정된 배추가 대조군 배추과 비교하여 염 및 건조 스트레스 저항성이 증진되는 것을 확인하였다. It was confirmed that the Zizantia gene-corrected Chinese cabbage in the above-mentioned Example has improved resistance to salt and drying stress compared to the control cabbage.
따라서 본 발명의 제조방법에 의해 제조된 식물체는 염 또는 건조 스트레스 저항성이 증진되며, 구체적으로 염 및 건조 스트레스 저항성이 동시에 증진될 수 있다. Accordingly, the plant produced by the method of the present invention has improved resistance to salt or drying stress, and specifically, resistance to salt and drying stress can be simultaneously enhanced.
본 발명에서 지잔티아 유전자를 교정은 식물체에 형질도입된 가이드 RNA에 의해 지잔티아 유전자를 특정영역에 삽입 또는 결손을 유도하는 것을 의미한다. 구체적으로, 유전자 가위 시스템의 cas9 단백질을 이용하여 유전자를 교정하는 것일 수 있다. In the present invention, correction of the Zyzantia gene means inducing insertion or deletion of the Zyzantia gene into a specific region by the guide RNA transduced into the plant. Specifically, the gene may be corrected using the cas9 protein of the gene editing system.
따라서 본 발명의 방법은 식물체에 cas9 단백질을 추가적으로 처리되거나 또는 cas9 단백질을 발현하는 벡터를 형질도입하는 단계를 포함할 수 있다. Accordingly, the method of the present invention may include the step of transducing the plant with a cas9 protein additionally treated or a vector expressing the cas9 protein.
또한, 본 발명의 유전자 교정용 벡터는 cas9 단백질를 암호화하는 유전자를 더 포함하여 가이드 RNA와 cas9 단백질를 동시에 형질도입 하는 것일 수 있다. In addition, the gene editing vector of the present invention may further include a gene encoding a cas9 protein to simultaneously transduce the guide RNA and the cas9 protein.
본 발명은 배추 내 BrGI 유전자를 교정하여 염 또는 건조 저항성을 증진시키는 것을 확인하였으므로, 고염 또는 건조 환경에서 배추 속 작물의 피해를 막을 수 있으며, 배추 육종 시 염 또는 건조 스트레스 저항성 증진 또는 개선된 주요 교배 계통으로 유용하게 이용될 수 있다.Since it was confirmed that the present invention improves the salt or drying resistance by correcting the BrGI gene in Chinese cabbage, it is possible to prevent damage to the Chinese cabbage crops in a high salt or dry environment, and to improve or improve resistance to salt or drying stress during breeding of Chinese cabbage It can be usefully used as a system.
도 1은 배추 지잔티아(GIGANTEA, GI) 유전자에서 g1 내지 g5의 gRNA의 타겟 부위를 나타낸 모식도이다.
도 2는 g1 또는 g3 gRNA에 의한 지잔티아(GIGANTEA, GI) 유전자의 염기서열 변이를 분석한 결과이다.
도 3은 pHAtC 벡터이다 .
도 4는 지부(대조군, Chiifu)과 유전자 교정식물체(GI-g1) 파종하고 2주간 키운 후의 사진(위)과, 그 후 250mM의 NaCl로 염 스트레스를 처리하여 11일 동안 키워 배추의 염 저항성을 평가한 사진이다.
도 5는 대조군과 유전자 교정식물체(GI-g1, GI-g3)에 염 스트레스(250mM의 NaCl)를 2주간 처리한 후, 각 배추의 생장을 비교한 사진이다.
도 6은 대조군과 유전자 교정 배추(GI-g3)에서 건조 저항성을 비교 평가한 것으로, 위는 14일간 건조 처리한 배추의 사진이고, 아래는 다시 물(rehydration)을 주고 배추의 생장을 비교한 사진이다.
도 7은 대조군과 유전자 교정 배추(GI-g3)에서 건조 저항성을 비교 평가한 것으로, 14일간 건조 처리한 배추의 사진이다.
도 8은 대조군과 유전자 교정 배추(GI-g1, GI-g3)의 생장 발달을 비교한 사진이다. 1 is a schematic diagram showing the target sites of gRNAs of g1 to g5 in the Chinese cabbage Gizantia (GIGANTEA, GI) gene.
2 is a result of analyzing the nucleotide sequence variation of the GIGANTEA (GI) gene by g1 or g3 gRNA.
3 is a pHAtC vector.
4 is a photograph (above) after sowing a branch (control group, Chiifu) and a gene-corrected plant (GI-g1) and growing for 2 weeks, and then treated with salt stress with 250 mM NaCl and grown for 11 days to increase salt resistance of Chinese cabbage This is an evaluated picture.
5 is a photograph comparing the growth of each Chinese cabbage after treatment with salt stress (250 mM NaCl) for 2 weeks in a control group and genetically modified plants (GI-g1, GI-g3).
Figure 6 is a comparison evaluation of drying resistance in the control group and the gene-corrected Chinese cabbage (GI-g3), the top is a picture of the cabbage dried for 14 days, and the bottom is a picture comparing the growth of the cabbage after rehydration. to be.
7 is a comparison evaluation of drying resistance in the control group and the genetically modified Chinese cabbage (GI-g3), and is a photograph of the Chinese cabbage dried for 14 days.
8 is a photograph comparing the growth development of the control group and the gene-corrected Chinese cabbage (GI-g1, GI-g3).
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
<실시예1> 배추 내 BrGI(<Example 1> BrGI in Chinese cabbage ( Brassica rapaBrassica rapa GIGANTEA) 유전자를 교정하는 가이드 RNA 선정 및 제작 GIGANTEA) selection and production of guide RNA to correct gene
배추(Brassica rapa) 유래 지잔티아(GIGANTEA) 유전자(서열번호 1; GenBank HQ615940.1)의 염기서열을 분석하였으며, 배추 지잔티아(GIGANTEA) 유전자만 특이적으로 인식하고 나머지 유전자는 인식하지 않도록 가이드 RNA 영역을 제한하여, 도 1과 같이 g1 내지 g5의 가이드 RNA의 타겟 유전자 영역을 선별하였으며, g1 내지 g5 영역에 특이적인 gRNA(가이드 RNA)의 제조하였다(표 1). The base sequence of the Chinese cabbage ( Brassica rapa ) derived GIGANTEA gene (SEQ ID NO: 1; GenBank HQ615940.1) was analyzed, and guide RNA to specifically recognize only the Chinese cabbage GIGANTEA gene and not recognize the rest of the genes By limiting the region, target gene regions of the guide RNAs of g1 to g5 were selected as shown in FIG. 1 , and gRNAs (guide RNAs) specific to the regions g1 to g5 were prepared (Table 1).
서열번호1SEQ ID NO: 1
1: atgacatcgc ctacttcatc tgagaggtgg accgatggtc ttcagttctc ttccttgtta1: atgacatcgc ctacttcatc tgagaggtgg accgatggtc ttcagttctc ttccttgtta
61 tggtctcccc cacgtgaccc tcaacaacat aaggatcaag tcgttgctta tgtcgaatac61 tggtctcccc cacgtgaccc tcaacaacat aaggatcaag tcgttgctta tgtcgaatac
121 tttggtcagt tcacatcaga gcaattccct gatgatattg ctgagttggt ccgtaatcag121 tttggtcagt tcacatcaga gcaattccct gatgatattg ctgagttggt ccgtaatcag
181 tatccctcaa ctgagaagcg acttttggat gatgtgttgg ctatgtttgt actccatcat181 tatccctcaa ctgagaagcg acttttggat gatgtgttgg ctatgtttgt actccatcat
241 cctgagcatg gtcatgctgt catccttccg attatctcat gtctcatcga tggcactcta241 cctgagcatg gtcatgctgt catccttccg attatctcat gtctcatcga tggcactcta
301 gtgtacagca aggaagctca tcccttcgcc tctttcattt ctttagtttc cccaaatagt301 gtgtacagca aggaagctca tcccttcgcc tctttcattt ctttagtttc cccaaatagt
361 gagaatgact attcagagca atgggctttg gcgtgcggag aaatcctacg catcttgact361 gagaatgact attcagagca atgggctttg gcgtgcggag aaatcctacg catcttgact
421 cattacaacc gtcccattta caagacggag cagcaaaatg gagaaacgga gagcaaagct421 cattacaacc gtcccattta caagacggag cagcaaaatg gagaaacgga gagcaaagct
481 tctactagtg ggtctcctac gctttcagag gctaaggctg tatcaccagg acagcatgaa481 tctactagtg ggtctcctac gctttcagag gctaaggctg tatcaccagg acagcatgaa
541 aggaaaccgc taaggccttt gtctccatgg atcagtgata tactactcgc tgctcccctt541 aggaaaccgc taaggccttt gtctccatgg atcagtgata tactactcgc tgctcccctt
601 ggtattagaa gtgactactt tcgttggtgt agtggtgtta tgggtaaata tgctgctgga601 ggtattagaa gtgactactt tcgttggtgt agtggtgtta tgggtaaata tgctgctgga
661 gagctcaagc cacctaccat tggtgagtgt ccatcacctc tggctactac aatgggttct661 gagctcaagc cacctaccat tggtgagtgt ccatcacctc tggctactac aatgggttct
721 actcgaggat ctggtaaaca tcctcaatat atgccttcga caccaagatg ggcggttgct721 actcgaggat ctggtaaaca tcctcaatat atgccttcga caccaagatg ggcggttgct
781 aatggagctg gtgtcatact gagtgtttgt gatgatgaag tcgctcggta tgagactgct781 aatggagctg gtgtcatact gagtgtttgt gatgatgaag tcgctcggta tgagactgct
841 acgttaacag cggttgctgt ccctgcactt ctgcttcctc ccccaacgac atccttagat841 acgttaacag cggttgctgt ccctgcactt ctgcttcctc ccccaacgac atccttagat
901 gagcatttag ttgctggcct tccagctctt gagccttatg cacgtttgtt tcacagatat901 gagcatttag ttgctggcct tccagctctt gagccttatg cacgtttgtt tcacagatat
961 tatgcgattg caactccaag tgctactcag agacttcttc ttggactctt ggaagcacca961 tatgcgattg caactccaag tgctactcag agacttcttc ttggactctt ggaagcacca
1021 ccgtcgtggg ctccagatgc acttgatgct gccgtacagc ttgtggagct tctccgagct1021 ccgtcgtggg ctccagatgc acttgatgct gccgtacagc ttgtggagct tctccgagct
1081 gctgaagatt atgcatctgg tgtaaggcta ccaaggaact ggatgcattt gcacttcttg1081 gctgaagatt atgcatctgg tgtaaggcta ccaaggaact ggatgcattt gcacttcttg
1141 cgtgcaatag gaatcgccat gtctatgagg gcaggcgttg ctgctgacgc tgcagctgct1141 cgtgcaatag gaatcgccat gtctatgagg gcaggcgttg ctgctgacgc tgcagctgct
1201 ttacttttcc gcatactgtc gcagccggca ctgctttttc ctccgctaag ccaagctgag1201 ttacttttcc gcatactgtc gcagccggca ctgctttttc ctccgctaag ccaagctgag
1261 ggagtagaaa tcaaacacgc tcctattggt ggctacggtt caaattacag aaagcagata1261 ggagtagaaa tcaaacacgc tcctattggt ggctacggtt caaattacag aaagcagata
1321 gaagttcctg cagcagaagc aaccattgaa gccactgcac aaggaatagc ctcaatgctt1321 gaagttcctg cagcagaagc aaccattgaa gccactgcac aaggaatagc ctcaatgctt
1381 tgtgctcacg gacctgaagt ggagtggagg atctgcacta tatgggaagc tgcctatgga1381 tgtgctcacg gacctgaagt ggagtggagg atctgcacta tatgggaagc tgcctatgga
1441 ttgatccctt taaactcctc agccgttgat ctccctgaga tcattgtcgc caccccactg1441 ttgatccctt taaactcctc agccgttgat ctccctgaga tcattgtcgc caccccactg
1501 cagcctccca tcttgtcatg gaacctatac atcccactcc tcaaagtact cgagtatctt1501 cagcctccca tcttgtcatg gaacctatac atcccactcc tcaaagtact cgagtatctt
1561 ccacgtggga gtccttccga agcatgcttg atgaagatat tcgtcgccac ggtggaaaca1561 ccacgtggga gtccttccga agcatgcttg atgaagatat tcgtcgccac ggtggaaaca
1621 atcctcagca ggactttccc gccagagact tctatcagga aagctagagc gagtttagcc1621 atcctcagca ggactttccc gccagagact tctatcagga aagctagagc gagtttagcc
1681 acgagatcat cagcgaccaa aaacctagct atggctgagc ttcgtgctat ggtccatgct1681 acgagatcat cagcgaccaa aaacctagct atggctgagc ttcgtgctat ggtccatgct
1741 ctcttcttgg aatcatgcgc tggcgtggag atagcgtcgc gcctgctttt cgttgtgttg1741 ctcttcttgg aatcatgcgc tggcgtggag atagcgtcgc gcctgctttt cgttgtgttg
1801 actgtgtgtg ttagccatga agcgcagtct agtgggagca agagacggag aagcgaagaa1801 actgtgtgtg ttagccatga agcgcagtct agtgggagca agagacggag aagcgaagaa
1861 gatgctactg cagaggagaa tcaagacaat caaactagta accgtaaaag taggaacgtc1861 gatgctactg cagaggagaa tcaagacaat caaactagta accgtaaaag taggaacgtc
1921 aagggacaag gacctgtggc ggcgtttgat tcgtacgttc tcgctgctgt ctgtgctctc1921 aagggacaag gacctgtggc ggcgtttgat tcgtacgttc tcgctgctgt ctgtgctctc
1981 gcctgtgagg ttcagctgta tcctatgatc tccggaggag ggaacttctc caactctgca1981 gcctgtgagg ttcagctgta tcctatgatc tccggaggag ggaacttctc caactctgca
2041 gtggctgcaa ccattacaaa gtctgtgaag ataaacggtt catctaacga gtacggagct2041 gtggctgcaa ccattacaaa gtctgtgaag ataaacggtt catctaacga gtacggagct
2101 gggattgact ctgcaatcaa gcacacacgc cgcatcttag cgattctcga ggcgctcttt2101 gggattgact ctgcaatcaa gcacacacgc cgcatcttag cgattctcga ggcgctcttt
2161 tcgttgaagc catcttctgt ggggactccg tggagttaca gctctagcga gatagttgct2161 tcgttgaagc catcttctgt ggggactccg tggagttaca gctctagcga gatagttgct
2221 gcggccatgg tcgcagctca catctccgaa ctgttcagac gctcaaaggc cttgacgcat2221 gcggccatgg tcgcagctca catctccgaa ctgttcagac gctcaaaggc cttgacgcat
2281 gccttgtctg gtttgatgag atgcaaatgg gacaaggaga ttcataagag agcgtcgtct2281 gccttgtctg gtttgatgag atgcaaatgg gacaaggaga ttcataagag agcgtcgtct
2341 ttgtataacc tcatcgatgt tcatagcaaa gttgtagcat ccatcgtcga caaagctgaa2341 ttgtataacc tcatcgatgt tcatagcaaa gttgtagcat ccatcgtcga caaagctgaa
2401 cccttagaag cgtaccttaa gaacgccccg gtccagaagg attcgctggc ttgtgttaac2401 cccttagaag cgtaccttaa gaacgccccg gtccagaagg attcgctggc ttgtgttaac
2461 tggaaacaac agaacaacac atcatcagca gcagggtttg gtacagcggc ggtgacgtcc2461 tggaaacaac agaacaacac atcatcagca gcagggtttg gtacagcggc ggtgacgtcc
2521 acgtcacgta atgaaatggc tccgagagga ggtaaccata agtatgctag gcattcagat2521 acgtcacgta atgaaatggc tccgagagga ggtaaccata agtatgctag gcattcagat
2581 gaaggctcag ggagtagatc gtcatcagat aagggcatca aagatctgct gttggatgct2581 gaaggctcag ggagtagatc gtcatcagat aagggcatca aagatctgct gttggatgct
2641 tctgatctag cgaatttcct cacggctgat aggctagcag ggttttaccg tggtacgcaa2641 tctgatctag cgaatttcct cacggctgat aggctagcag ggttttaccg tggtacgcaa
2701 gttcttttga ggtcgatact tgctgagaaa ccggagcttt ctttctccgt tgtttcgctg2701 gttcttttga ggtcgatact tgctgagaaa ccggagcttt ctttctccgt tgtttcgctg
2761 ttgtggcaca aactgatcgc ttctcctgag atccagccca cagccgaaag cacctctgct2761 ttgtggcaca aactgatcgc ttctcctgag atccagccca cagccgaaag cacctctgct
2821 cagcaaggat ggagacaggt agttgatgca ctatgcaatg tggtatctgc aacgccagca2821 cagcaaggat ggagacaggt agttgatgca ctatgcaatg tggtatctgc aacgccagca
2881 aaagcagctg cagccgttgt tcttcaggct gagagagagt tgcagccttg gatagccaaa2881 aaagcagctg cagccgttgt tcttcaggct gagagagagt tgcagccttg gatagccaaa
2941 gatgatgaag aaggtcagaa aatgtggaaa ataaaccaaa ggatagtgaa agtgatggtg2941 gatgatgaag aaggtcagaa aatgtggaaa ataaaccaaa ggatagtgaa agtgatggtg
3001 gaactcatga ggaatcatga caggcctgag tcactggtga ttctggcaag tgcatctgat3001 gaactcatga ggaatcatga caggcctgag tcactggtga ttctggcaag tgcatctgat
3061 ctccttctga gagcaactga tggaatgctt gttgatggag aagcttgtac attacctcaa3061 ctccttctga gagcaactga tggaatgctt gttgatggag aagcttgtac attacctcaa
3121 cttgagctac ttgaagctac agcaagagca atacagccag tgttagcttg gggaccatct3121 cttgagctac ttgaagctac agcaagagca atacagccag tgttagcttg gggaccatct
3181 ggactagcag tagttgacgg cttatcaaat ctattgaagt gtcgtctgcc ggcaacaata3181 ggactagcag tagttgacgg cttatcaaat ctattgaagt gtcgtctgcc ggcaacaata
3241 cggtgcctct cacacccaag tgcacacgta cgtgccttga gcacatcagt actacgagac3241 cggtgcctct cacacccaag tgcacacgta cgtgccttga gcacatcagt actacgagac
3301 atcatgaacc aaagcggcat aaccaccacc aaggcaactc caaaaccgcc gccaacaata3301 atcatgaacc aaagcggcat aaccaccacc aaggcaactc caaaaccgcc gccaacaata
3361 acaaccgaga agaacggaac ggacagcccg tcgtacaggt tcttcaacgc ggcggcaata3361 acaaccgaga agaacggaac ggacagcccg tcgtacaggt tcttcaacgc ggcggcaata
3421 gactggaaag cagacataca aaagtgtttg aactgggaag cgcacagctt gctgtcgacg3421 gactggaaag cagacataca aaagtgtttg aactgggaag cgcacagctt gctgtcgacg
3481 acaatgccga cacagtttct tgacacggcg gctcgggaat taggatgtac catatcaatg3481 acaatgccga cacagtttct tgacacggcg gctcgggaat taggatgtac catatcaatg
3541 tcgtcccaat aa3541 tcgtcccaat aa
표 1은 도 1의 g1 내지 g5의 가이드 RNA의 타겟 유전자 영역의 염기서열을 나타낸 것이다.Table 1 shows the nucleotide sequences of the target gene regions of the guide RNAs g1 to g5 of FIG. 1 .
제조한 각 gRNA의 서열변이 유도율을 확인하고자, 배추 원형질체에 cas9 단백질(UniProtKB/Swiss-Prot: Q99ZW2.1)과 함께 접종한 후, 염기서열을 분석하고 gRNA에 따른 유전자 교정효과를 비교하였다.In order to confirm the sequence variation induction rate of each gRNA prepared, the Chinese cabbage protoplasts were inoculated with cas9 protein (UniProtKB/Swiss-Prot: Q99ZW2.1), then the nucleotide sequence was analyzed and the gene editing effect according to the gRNA was compared.
배추 원형질체에 폴리에틸렌글리콜(PEG) 30%(v/v) 또는 PEG 40%(v/v) 조건으로 cas9 단백질과 gRNA를 함께 접종하여, 배추 원형질체에서 지잔티아(GIGANTEA, GI) 유전자를 교정하였고, 딥 시퀸싱을 통해 염기서열의 변이율(indel ratio)을 분석하였다. 표 2는 g1 내지 g5에 특이적인 gRNA를 접종한 후, 지잔티아 유전자의 변이율을 분석한 결과이다.The Chinese cabbage protoplasts were inoculated with cas9 protein and gRNA under polyethylene glycol (PEG) 30% (v/v) or PEG 40% (v/v) conditions, and the GIGANTEA (GIGANTEA, GI) gene was corrected in the Chinese cabbage protoplasts, The indel ratio of the nucleotide sequence was analyzed through deep sequencing. Table 2 shows the results of analyzing the mutation rate of the Zizantia gene after inoculation of gRNA specific to g1 to g5.
표 2에서 비처리 대조군(control)의 변이율은 모두 0.12% 이하로 나타났다. 즉 유전자 변이가 자연적으로는 거의 발생하지 않음을 확인하였다. g1 내지 g5에 특이적인 gRNA를 접종한 경우 모두, PEG 40%(v/v)로 처리한 경우가 PEG 30%(v/v)와 비교하여 변이율(indel ratio)이 높게 나타났다. 또한, 5개의 gRNA 중 g1의 변이율이 33.58% 내지 47.04%로 가장 높게 나타났고, g3의 변이율이 6.17% 내지 21.95%로 그 뒤를 이었다. 반면, g2, g4 및 g5의 gRNA는 PEG 30%(v/v)에서 1.08 내지 3.81%, PEG 40%(v/v)에서 5.04 내지 10.97%의 변이율(indel ratio)을 나타내, g1 및 g3의 gRNA와 비교하여 변이율이 상대적으로 낮게 나타났다. In Table 2, the variability of the untreated control group was all 0.12% or less. That is, it was confirmed that genetic mutations rarely occur naturally. In all cases inoculated with gRNA specific to g1 to g5, the case of treatment with PEG 40% (v/v) showed a higher indel ratio than that of PEG 30% (v/v). Also, among the five gRNAs, g1 showed the highest mutation rate of 33.58% to 47.04%, followed by g3 mutation rate of 6.17% to 21.95%. On the other hand, gRNAs of g2, g4 and g5 exhibit indel ratios of 1.08 to 3.81% in PEG 30% (v/v) and 5.04 to 10.97% in PEG 40% (v/v), g1 and g3 The mutation rate was relatively low compared to that of gRNA.
다음으로, 5개의 gRNA 중 변이율(indel ratio)이 높은 g1 및 g3를 선발하여 배추(Brassica rapa) 유래 지잔티아(GIGANTEA)의 유전자 서열을 분석하여 변이위치 및 서열을 확인하였다(도 2).Next, g1 and g3 having a high indel ratio among the five gRNAs were selected and the gene sequence of GIGANTEA derived from Brassica rapa was analyzed to confirm the mutation position and sequence (FIG. 2).
분석결과, g1의 gRNA를 처리한 배추 원형질체에서 지잔티아(GIGANTEA) 유전자(서열번호 1)의 32번째와 33번째 염기서열 사이에 T 또는 A가 삽입되었음을 확인하였다. As a result of the analysis, it was confirmed that T or A was inserted between the 32nd and 33rd nucleotide sequences of the GIGANTEA gene (SEQ ID NO: 1) in the Chinese cabbage protoplasts treated with g1 gRNA.
또한 g3의 gRNA를 처리한 배추 원형질체에서 지잔티아(GIGANTEA) 유전자(서열번호1)의 531번째와 532번째 염기서열 사이에 T 또는 A가 삽입되었음을 확인하였다.In addition, it was confirmed that T or A was inserted between the 531 and 532 nucleotide sequences of the GIGANTEA gene (SEQ ID NO: 1) in the g3 gRNA-treated Chinese cabbage protoplasts.
g1 및 g3에 특이적인 gRNA를 이용할 경우, g2, g4 및 g5에 특이적인 gRNA를 이용할 때와 비교하여 배추(Brassica rapa) 유래 지잔티아(GIGANTEA) 유전자에 삽입 또는 결손을 효율적으로 유도하여, 유전자를 교정할 수 있음을 확인하였다. When gRNA specific for g1 and g3 is used, compared to when gRNA specific for g2, g4 and g5 is used, insertion or deletion is efficiently induced in the GIGANTEA gene derived from Brassica rapa. It was confirmed that it can be corrected.
<실시예2> 유전자 교정용 벡터 제작 및 배추의 형질전환<Example 2> Gene editing vector production and transformation of Chinese cabbage
실시예 1에서 변이율이 높았던, g1 또는 g3에 특이적인 gRNA 유전자를 pHAtC 벡터(도 3)에 삽입하여 지잔티아(GIGANTEA) 유전자 교정용 구조체(construction)를 제조하였다. A gRNA gene specific for g1 or g3, which had a high mutation rate in Example 1, was inserted into the pHAtC vector (FIG. 3) to prepare a GIGANTEA gene editing construct.
pHAtC 벡터는 도 3과 같다. 도 3을 참조하면 pHAtC는 하이그로마이신 저항성 유전자(HygR) 및 Cas9 유전자를 포함한다. 제조된 유전자 교정용 구조체(construction)를 배추(Brassica rapa, pekinensis, spp Chiifu)에 형질전환하여 형질전환 배추 식물체를 제조하였다. 하이그로마이신을 이용하여 유전자 구조체가 삽입된 형질전환 개체를 선별하였다. 또한, 형질전환하지 않은 배추를 대조군을 사용하였다.The pHAtC vector is shown in FIG. 3 . Referring to FIG. 3 , pHAtC includes a hygromycin resistance gene (HygR) and a Cas9 gene. The prepared gene editing construct was transformed into Chinese cabbage ( Brassica rapa , pekinensis, spp Chiifu) to prepare a transgenic Chinese cabbage plant. Transgenic individuals into which the gene construct was inserted were selected using hygromycin. In addition, untransformed Chinese cabbage was used as a control.
2016년 Plant cell Rep. 35: 1943-1954의 형질전환 방법을 참조하여, 배추(Brassica rapa, ssp. pekinensis Chiifu 의 하배축(下胚軸)에 아그로박테리움(agrobacterium)을 이용하여 gRNA와 cas9를 포함하는 벡터를 접종하고 이를 재분화시키는 조직배양을 이용하였다.2016 Plant cell Rep . 35: Referring to the transformation method of 1943-1954, inoculated with a vector containing gRNA and cas9 using Agrobacterium in the hypocotyl of Chinese cabbage ( Brassica rapa , ssp. pekinensis Chiifu ) Tissue culture for redifferentiation was used.
<실시예3> 유전자 교정 배추의 건조 및 염 저항성 분석<Example 3> Analysis of drying and salt resistance of genetically modified Chinese cabbage
형질전환 배추 식물체(T0)의 DNA를 추출하고 딥 시퀸싱을 통해 형질전환 여부를 확인하였다. 형질전환이 확인된 식물체를 자가교배한 종자(T1)를 하이그로마이신 배지에 파종하여 살아남은 개체를 선별하고, 이들 개체를 이용하여 건조 및 염저항성 검정을 수행하였다.DNA of the transformed Chinese cabbage plant (T0) was extracted and transformation was confirmed through deep sequencing. The seeds (T1) obtained by self-crossing the plants for which the transformation was confirmed were sown in a hygromycin medium to select surviving individuals, and drying and salt resistance assays were performed using these individuals.
표 3은 g1 또는 g3 구조체로 형질도입한 형질전환 식물체의 GI 유전자 서열변이 유도율이다. g1 구조체의 경우 삽입-결손(indel) 유도율이 99.48%로 나타났으며, g3 구조체는 71.12%로 나타났다. 즉, g1 또는 g3 구조체의 형질도입이 배추의 지잔티아(GIGANTEA) 유전자의 서열변이(유전자 교정)을 효율적으로 유도함을 확인하였다. Table 3 shows the induction rate of GI gene sequence variation in transgenic plants transduced with g1 or g3 constructs. In the case of the g1 construct, the insertion-deletion (indel) induction rate was 99.48%, and the g3 construct was 71.12%. That is, it was confirmed that the transduction of the g1 or g3 construct efficiently induces sequence variation (gene correction) of the GIGANTEA gene of Chinese cabbage.
지잔티아(GIGANTEA) 유전자는 생체 리듬 유전자이며, 염 스트레스 저항성 기작에 관여한다. 따라서 배추에서 지잔티아(GIGANTEA)의 돌연번이로 발현을 억제하면 염 스트레스 저항성과 건조 스트레스 저항성이 증진될 수 있다. The GIGANTEA gene is a circadian rhythm gene and is involved in the mechanism of salt stress resistance. Therefore, by suppressing the abrupt expression of GIGANTEA in Chinese cabbage, salt stress resistance and drying stress resistance can be enhanced.
따라서 g1 또는 g3의 gRNA를 이용한 지잔티아(GIGANTEA) 유전자 교정이 배추의 염 또는 건조 스트레스 저항성에 미치는 영향을 평가하였다. Therefore, the effect of GIGANTEA gene correction using g1 or g3 gRNA on resistance to salt or drying stress was evaluated.
1. 염 스트레스 저항성 검정1. Salt Stress Resistance Assay
지잔티아(GIGANTEA) 유전자의 서열변이가 배추의 염 스트레스 저항성에 미치는 영향을 확인하고자, 대조군 배추와 서열변이 배추의 염 스트레스 저항성을 비교 평가하였다. 수경재배용 스폰지에 양액을 흡수시키고 종자(대조군 또는 T1)를 파종하고 2일 간격으로 양액을 갈아주었다. 2주 후부터는 NaCL를 250mM의 농도로 양액에 혼합하여 2일 간격으로 갈아주면서 배양한 결과, 대조구인 지부(Chiifu)는 시들어 죽었으나, 유전자 교정 식물체(GI-g1, GI-g3)는 생장이 유지됨을 확인하였다(도 4 및 도 5).To determine the effect of sequence variation of GIGANTEA gene on salt stress resistance of Chinese cabbage, the salt stress resistance of control Chinese cabbage and sequence-mutated Chinese cabbage was compared and evaluated. The nutrient solution was absorbed into the hydroponics sponge, the seeds (control group or T1) were sown, and the nutrient solution was changed every 2 days. After 2 weeks, NaCL was mixed with the nutrient solution at a concentration of 250 mM and cultured while changing at intervals of 2 days. As a result, the control, Chiifu, withered and died, but the gene-edited plants (GI-g1, GI-g3) were maintained in growth. was confirmed (FIGS. 4 and 5).
2. 건조 저항성 검정2. Dry Resistance Assay
대조구(지부)와 유전자 교정 식물체(GI-g3)를 토양에 파종하여 2주간 키운 후 14일간 물을 주지 않고 건조 시켰다(도 6 위). 그 후 다시 물을 공급하여 14일간 배추의 생장을 관찰하였다. 유전자 교정 배추는 생장이 개재하여 정상 상태로 회복되었으나, 대조구인 지부는 시들어 회복되지 않음을 확인하였다(도 6 아래, 도 7)Control (branch) and gene-corrected plants (GI-g3) were sown in soil and grown for 2 weeks, and then dried without watering for 14 days (Fig. 6 above). After that, water was supplied again and the growth of Chinese cabbage was observed for 14 days. Gene-corrected Chinese cabbage was restored to a normal state due to growth intervening, but it was confirmed that the branch, which is a control, did not wither and recover (Fig. 6 below, Fig. 7).
<실시예4> 유전자 교정 배추의 생장 발달 및 결구 확인<Example 4> Confirmation of growth development and colonization of gene-corrected Chinese cabbage
다음으로 지잔티아(GIGANTEA) 유전자의 서열변이가 배추의 정상적인 생장발달에 영향을 주는지 확인하고자, 대조구(지부)와 형질전환 배추의 종자(T1)를 파종하여 온실에서 생육하였다. 9월 초에 파종하여 가온하지 않는 비가림 온실에서 11월 말까지 생육하고 결구 상태를 확인한 결과, 유전자 교정 배추가 정상적으로 생육함을 확인하였다. Next, in order to confirm whether the sequence variation of the GIGANTEA gene affects the normal growth and development of Chinese cabbage, a control (branch) and a transgenic Chinese cabbage seed (T1) were sown and grown in a greenhouse. It was sown in early September and grown until the end of November in a non-warming greenhouse without heating.
즉, 유전자 교정 배추가 정상적으로 생장 발달하며, 염 저항성 및 건조 저항성이 증진 및 개선됨을 확인하였다.That is, it was confirmed that the gene-corrected Chinese cabbage grows and develops normally, and salt resistance and drying resistance are enhanced and improved.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Guide RNA for editing gigantea gene and use thereof <130> DP20190385 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 3552 <212> DNA <213> Artificial Sequence <220> <223> Brassica rapa GIGANTEA <400> 1 atgacatcgc ctacttcatc tgagaggtgg accgatggtc ttcagttctc ttccttgtta 60 tggtctcccc cacgtgaccc tcaacaacat aaggatcaag tcgttgctta tgtcgaatac 120 tttggtcagt tcacatcaga gcaattccct gatgatattg ctgagttggt ccgtaatcag 180 tatccctcaa ctgagaagcg acttttggat gatgtgttgg ctatgtttgt actccatcat 240 cctgagcatg gtcatgctgt catccttccg attatctcat gtctcatcga tggcactcta 300 gtgtacagca aggaagctca tcccttcgcc tctttcattt ctttagtttc cccaaatagt 360 gagaatgact attcagagca atgggctttg gcgtgcggag aaatcctacg catcttgact 420 cattacaacc gtcccattta caagacggag cagcaaaatg gagaaacgga gagcaaagct 480 tctactagtg ggtctcctac gctttcagag gctaaggctg tatcaccagg acagcatgaa 540 aggaaaccgc taaggccttt gtctccatgg atcagtgata tactactcgc tgctcccctt 600 ggtattagaa gtgactactt tcgttggtgt agtggtgtta tgggtaaata tgctgctgga 660 gagctcaagc cacctaccat tggtgagtgt ccatcacctc tggctactac aatgggttct 720 actcgaggat ctggtaaaca tcctcaatat atgccttcga caccaagatg ggcggttgct 780 aatggagctg gtgtcatact gagtgtttgt gatgatgaag tcgctcggta tgagactgct 840 acgttaacag cggttgctgt ccctgcactt ctgcttcctc ccccaacgac atccttagat 900 gagcatttag ttgctggcct tccagctctt gagccttatg cacgtttgtt tcacagatat 960 tatgcgattg caactccaag tgctactcag agacttcttc ttggactctt ggaagcacca 1020 ccgtcgtggg ctccagatgc acttgatgct gccgtacagc ttgtggagct tctccgagct 1080 gctgaagatt atgcatctgg tgtaaggcta ccaaggaact ggatgcattt gcacttcttg 1140 cgtgcaatag gaatcgccat gtctatgagg gcaggcgttg ctgctgacgc tgcagctgct 1200 ttacttttcc gcatactgtc gcagccggca ctgctttttc ctccgctaag ccaagctgag 1260 ggagtagaaa tcaaacacgc tcctattggt ggctacggtt caaattacag aaagcagata 1320 gaagttcctg cagcagaagc aaccattgaa gccactgcac aaggaatagc ctcaatgctt 1380 tgtgctcacg gacctgaagt ggagtggagg atctgcacta tatgggaagc tgcctatgga 1440 ttgatccctt taaactcctc agccgttgat ctccctgaga tcattgtcgc caccccactg 1500 cagcctccca tcttgtcatg gaacctatac atcccactcc tcaaagtact cgagtatctt 1560 ccacgtggga gtccttccga agcatgcttg atgaagatat tcgtcgccac ggtggaaaca 1620 atcctcagca ggactttccc gccagagact tctatcagga aagctagagc gagtttagcc 1680 acgagatcat cagcgaccaa aaacctagct atggctgagc ttcgtgctat ggtccatgct 1740 ctcttcttgg aatcatgcgc tggcgtggag atagcgtcgc gcctgctttt cgttgtgttg 1800 actgtgtgtg ttagccatga agcgcagtct agtgggagca agagacggag aagcgaagaa 1860 gatgctactg cagaggagaa tcaagacaat caaactagta accgtaaaag taggaacgtc 1920 aagggacaag gacctgtggc ggcgtttgat tcgtacgttc tcgctgctgt ctgtgctctc 1980 gcctgtgagg ttcagctgta tcctatgatc tccggaggag ggaacttctc caactctgca 2040 gtggctgcaa ccattacaaa gtctgtgaag ataaacggtt catctaacga gtacggagct 2100 gggattgact ctgcaatcaa gcacacacgc cgcatcttag cgattctcga ggcgctcttt 2160 tcgttgaagc catcttctgt ggggactccg tggagttaca gctctagcga gatagttgct 2220 gcggccatgg tcgcagctca catctccgaa ctgttcagac gctcaaaggc cttgacgcat 2280 gccttgtctg gtttgatgag atgcaaatgg gacaaggaga ttcataagag agcgtcgtct 2340 ttgtataacc tcatcgatgt tcatagcaaa gttgtagcat ccatcgtcga caaagctgaa 2400 cccttagaag cgtaccttaa gaacgccccg gtccagaagg attcgctggc ttgtgttaac 2460 tggaaacaac agaacaacac atcatcagca gcagggtttg gtacagcggc ggtgacgtcc 2520 acgtcacgta atgaaatggc tccgagagga ggtaaccata agtatgctag gcattcagat 2580 gaaggctcag ggagtagatc gtcatcagat aagggcatca aagatctgct gttggatgct 2640 tctgatctag cgaatttcct cacggctgat aggctagcag ggttttaccg tggtacgcaa 2700 gttcttttga ggtcgatact tgctgagaaa ccggagcttt ctttctccgt tgtttcgctg 2760 ttgtggcaca aactgatcgc ttctcctgag atccagccca cagccgaaag cacctctgct 2820 cagcaaggat ggagacaggt agttgatgca ctatgcaatg tggtatctgc aacgccagca 2880 aaagcagctg cagccgttgt tcttcaggct gagagagagt tgcagccttg gatagccaaa 2940 gatgatgaag aaggtcagaa aatgtggaaa ataaaccaaa ggatagtgaa agtgatggtg 3000 gaactcatga ggaatcatga caggcctgag tcactggtga ttctggcaag tgcatctgat 3060 ctccttctga gagcaactga tggaatgctt gttgatggag aagcttgtac attacctcaa 3120 cttgagctac ttgaagctac agcaagagca atacagccag tgttagcttg gggaccatct 3180 ggactagcag tagttgacgg cttatcaaat ctattgaagt gtcgtctgcc ggcaacaata 3240 cggtgcctct cacacccaag tgcacacgta cgtgccttga gcacatcagt actacgagac 3300 atcatgaacc aaagcggcat aaccaccacc aaggcaactc caaaaccgcc gccaacaata 3360 acaaccgaga agaacggaac ggacagcccg tcgtacaggt tcttcaacgc ggcggcaata 3420 gactggaaag cagacataca aaagtgtttg aactgggaag cgcacagctt gctgtcgacg 3480 acaatgccga cacagtttct tgacacggcg gctcgggaat taggatgtac catatcaatg 3540 tcgtcccaat aa 3552 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g1 <400> 2 ucaucugaga gguggaccga ugg 23 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g3 <400> 3 auuaucucau gucucaucga ugg 23 <210> 4 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g2 <400> 4 caguugaggg auacugauua cgg 23 <210> 5 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g4 <400> 5 gaaugacuau ucagagcaau ggg 23 <210> 6 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g5 <400> 6 gguuuccuuu caugcugucc ugg 23 <110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Guide RNA for editing gigantea gene and use thereof <130> DP20190385 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 3552 <212> DNA <213> Artificial Sequence <220> <223> Brassica rapa GIGANTEA <400> 1 atgacatcgc ctacttcatc tgagaggtgg accgatggtc ttcagttctc ttccttgtta 60 tggtctcccc cacgtgaccc tcaacaacat aaggatcaag tcgttgctta tgtcgaatac 120 tttggtcagt tcacatcaga gcaattccct gatgatattg ctgagttggt ccgtaatcag 180 tatccctcaa ctgagaagcg acttttggat gatgtgttgg ctatgtttgt actccatcat 240 cctgagcatg gtcatgctgt catccttccg attatctcat gtctcatcga tggcactcta 300 gtgtacagca aggaagctca tcccttcgcc tctttcattt ctttagtttc cccaaatagt 360 gagaatgact attcagagca atgggctttg gcgtgcggag aaatcctacg catcttgact 420 cattacaacc gtcccattta caagacggag cagcaaaatg gagaaacgga gagcaaagct 480 tctactagtg ggtctcctac gctttcagag gctaaggctg tatcaccagg acagcatgaa 540 aggaaaccgc taaggccttt gtctccatgg atcagtgata tactactcgc tgctcccctt 600 ggtattagaa gtgactactt tcgttggtgt agtggtgtta tgggtaaata tgctgctgga 660 gagctcaagc cacctaccat tggtgagtgt ccatcacctc tggctactac aatgggttct 720 actcgaggat ctggtaaaca tcctcaatat atgccttcga caccaagatg ggcggttgct 780 aatggagctg gtgtcatact gagtgtttgt gatgatgaag tcgctcggta tgagactgct 840 acgttaacag cggttgctgt ccctgcactt ctgcttcctc ccccaacgac atccttagat 900 gagcatttag ttgctggcct tccagctctt gagccttatg cacgtttgtt tcacagatat 960 tatgcgattg caactccaag tgctactcag agacttcttc ttggactctt ggaagcacca 1020 ccgtcgtggg ctccagatgc acttgatgct gccgtacagc ttgtggagct tctccgagct 1080 gctgaagatt atgcatctgg tgtaaggcta ccaaggaact ggatgcattt gcacttcttg 1140 cgtgcaatag gaatcgccat gtctatgagg gcaggcgttg ctgctgacgc tgcagctgct 1200 ttacttttcc gcatactgtc gcagccggca ctgctttttc ctccgctaag ccaagctgag 1260 ggagtagaaa tcaaacacgc tcctattggt ggctacggtt caaattacag aaagcagata 1320 gaagttcctg cagcagaagc aaccattgaa gccactgcac aaggaatagc ctcaatgctt 1380 tgtgctcacg gacctgaagt ggagtggagg atctgcacta tatgggaagc tgcctatgga 1440 ttgatccctt taaactcctc agccgttgat ctccctgaga tcattgtcgc caccccactg 1500 cagcctccca tcttgtcatg gaacctatac atcccactcc tcaaagtact cgagtatctt 1560 ccacgtggga gtccttccga agcatgcttg atgaagatat tcgtcgccac ggtggaaaca 1620 atcctcagca ggactttccc gccagagact tctatcagga aagctagagc gagtttagcc 1680 acgagatcat cagcgaccaa aaacctagct atggctgagc ttcgtgctat ggtccatgct 1740 ctcttcttgg aatcatgcgc tggcgtggag atagcgtcgc gcctgctttt cgttgtgttg 1800 actgtgtgtg ttagccatga agcgcagtct agtgggagca agagacggag aagcgaagaa 1860 gatgctactg cagaggagaa tcaagacaat caaactagta accgtaaaag taggaacgtc 1920 aagggacaag gacctgtggc ggcgtttgat tcgtacgttc tcgctgctgt ctgtgctctc 1980 gcctgtgagg ttcagctgta tcctatgatc tccggaggag ggaacttctc caactctgca 2040 gtggctgcaa ccattacaaa gtctgtgaag ataaacggtt catctaacga gtacggagct 2100 gggattgact ctgcaatcaa gcacacacgc cgcatcttag cgattctcga ggcgctcttt 2160 tcgttgaagc catcttctgt ggggactccg tggagttaca gctctagcga gatagttgct 2220 gcggccatgg tcgcagctca catctccgaa ctgttcagac gctcaaaggc cttgacgcat 2280 gccttgtctg gtttgatgag atgcaaatgg gacaaggaga ttcataagag agcgtcgtct 2340 ttgtataacc tcatcgatgt tcatagcaaa gttgtagcat ccatcgtcga caaagctgaa 2400 cccttagaag cgtaccttaa gaacgccccg gtccagaagg attcgctggc ttgtgttaac 2460 tggaaacaac agaacaacac atcatcagca gcagggtttg gtacagcggc ggtgacgtcc 2520 acgtcacgta atgaaatggc tccgagagga ggtaaccata agtatgctag gcattcagat 2580 gaaggctcag ggagtagatc gtcatcagat aagggcatca aagatctgct gttggatgct 2640 tctgatctag cgaatttcct cacggctgat aggctagcag ggttttaccg tggtacgcaa 2700 gttcttttga ggtcgatact tgctgagaaa ccggagcttt ctttctccgt tgtttcgctg 2760 ttgtggcaca aactgatcgc ttctcctgag atccagccca cagccgaaag cacctctgct 2820 cagcaaggat ggagacaggt agttgatgca ctatgcaatg tggtatctgc aacgccagca 2880 aaagcagctg cagccgttgt tcttcaggct gagagagagt tgcagccttg gatagccaaa 2940 gatgatgaag aaggtcagaa aatgtggaaa ataaaccaaa ggatagtgaa agtgatggtg 3000 gaactcatga ggaatcatga caggcctgag tcactggtga ttctggcaag tgcatctgat 3060 ctccttctga gagcaactga tggaatgctt gttgatggag aagcttgtac attacctcaa 3120 cttgagctac ttgaagctac agcaagagca atacagccag tgttagcttg gggaccatct 3180 ggactagcag tagttgacgg cttatcaaat ctattgaagt gtcgtctgcc ggcaacaata 3240 cggtgcctct cacacccaag tgcacacgta cgtgccttga gcacatcagt actacgagac 3300 atcatgaacc aaagcggcat aaccaccacc aaggcaactc caaaaccgcc gccaacaata 3360 acaaccgaga agaacggaac ggacagcccg tcgtacaggt tcttcaacgc ggcggcaata 3420 gactggaaag cagacataca aaagtgtttg aactgggaag cgcacagctt gctgtcgacg 3480 acaatgccga cacagtttct tgacacggcg gctcgggaat taggatgtac catatcaatg 3540 tcgtcccaat aa 3552 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g1 <400> 2 ucaucugaga gguggaccga ugg 23 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g3 <400> 3 auuaucucau gucucaucga ugg 23 <210> 4 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g2 <400> 4 caguugaggg auacugauua cgg 23 <210> 5 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g4 <400> 5 gaaugacuau ucagagcaau ggg 23 <210> 6 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> BrGI_g5 <400> 6 gguuuccuuu caugcugucc ugg 23
Claims (8)
cas9 단백질을 암호화하는 염기서열을 더 포함하는 벡터.3. The method of claim 2,
A vector further comprising a nucleotide sequence encoding a cas9 protein.
상기 식물체는 염 또는 건조 스트레스 저항성이 증진된 것인 식물체. 6. The method of claim 5,
The plant is a plant with enhanced resistance to salt or dry stress.
상기 식물체는 배추인 것인 식물체.7. The method of claim 6,
The plant is a Chinese cabbage.
상기 벡터를 식물체에 형질도입하여 지잔티아 유전자를 교정하는 단계를 포함하는,
염 또는 건조 스트레스 저항성이 증진된 식물체의 제조방법.Preparing a vector for correcting Zyzantia gene comprising a guide RNA consisting of the nucleotide sequence of SEQ ID NO: 3; and
Transducing the vector into a plant comprising the step of correcting the Zyzantia gene,
A method for producing a plant having improved resistance to salt or dry stress.
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