KR102674998B1 - CaFAF1 genes and Method for improving the resistance to the drought and salt stress using CaFAF1 in plants - Google Patents
CaFAF1 genes and Method for improving the resistance to the drought and salt stress using CaFAF1 in plants Download PDFInfo
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Abstract
본 발명은 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 단백질, 이를 암호화하는 유전자, 및 이를 이용한 식물체의 건조 또는 염 스트레스 저항성 증진방법 등에 관한 것으로서, 상기 유전자의 발현이 침묵된 식물체는 건조 스트레스에 대한 내성은 증가한 반면 염 스트레스에 대한 내성은 감소하고, 상기 유전자가 과발현된 식물체는 건조 스트레스에 대한 내성이 감소하는 것을 확인하여 완성된 것이다. 즉, 본 발명에 따른 CaFAF1은 건조 스트레스 저항성의 음성 조절자이자 염 스트레스 저항성의 양성 조절자로 기능할 수 있으므로, 상기 단백질의 발현 조절을 통해 인류가 이용할 수 있는 작물 등을 적절히 개량할 수 있으며, 특히 식물의 생산성을 향상시키는데 유용하게 활용할 수 있을 것으로 기대된다.The present invention relates to the CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) protein, the gene encoding it, and a method for enhancing dry or salt stress resistance in plants using the same. Plants in which the expression of the gene is silenced have increased tolerance to dry stress. On the other hand, tolerance to salt stress is reduced, and plants overexpressing the above gene are confirmed to have reduced tolerance to drying stress. In other words, CaFAF1 according to the present invention can function as a negative regulator of dry stress resistance and a positive regulator of salt stress resistance, so that crops that can be used by humans can be appropriately improved by controlling the expression of the protein, especially It is expected that it will be useful in improving plant productivity.
Description
본 발명은 고추 녹광 품종 유래 신규 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 유전자, 상기 유전자에 의해 암호화되는 단백질, 및 이를 이용한 식물체의 건조 또는 염 스트레스 저항성 증진방법 등에 관한 것이다.The present invention relates to a novel CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) gene derived from the pepper green light variety, a protein encoded by the gene, and a method for improving dry or salt stress resistance of plants using the same.
지구온난화는 극심한 기후 변화를 야기하여 식물들의 정상적인 생장 및 발달을 저해하는 환경적 스트레스를 유발하며, 이에 대하여 식물체는 추위, 고염, 및 가뭄과 같은 불리한 환경에서 생존하기 위한 정교한 방어기작을 진화시켜왔다. 예컨대, 가뭄(drought)은 수분 부족 환경에 의해 일어나며 식물의 정상적인 성장에 심각한 영향을 미쳐 결국 곡물의 수확량 감소를 초래하는데, 식물은 이러한 건조 스트레스에 적응하기 위해 기공폐쇄, 앱시스산 (abscisic acid; 이하 ABA)의 합성 및 축적과 같은 다양한 방어 기작을 활성화한다.Global warming causes extreme climate change and causes environmental stress that inhibits the normal growth and development of plants. In response to this, plants have evolved sophisticated defense mechanisms to survive in adverse environments such as cold, high salt, and drought. For example, drought is caused by a moisture-deficient environment and has a serious impact on the normal growth of plants, ultimately leading to a decrease in grain yield. In order to adapt to this drying stress, plants close stomata and use abscisic acid (hereinafter referred to as “abscisic acid”). Activates various defense mechanisms such as synthesis and accumulation of ABA).
ABA는 비생물적 스트레스 특히, 건조 스트레스에 대하여 식물을 보호하기 위한 조절 인자이며, 식물 생장 및 발달에 매우 중요한 역할을 한다고 알려졌다. 보고된 바에 의하면, ABA를 미리 처리하고 스트레스를 준 식물은 그렇지 않은 식물에 비해 스트레스에 잘 저항하는 반면 ABA를 생성하지 못하거나, ABA에 반응하지 못하는 돌연변이 식물들은 스트레스에 약한 것으로 알려졌다. 따라서 ABA의 반응에 관여하는 단백질들을 이용하면, 환경 스트레스에 대한 저항성이 향상된 식물을 개발할 수 있을 것으로 기대되고 있다.ABA is a regulatory factor to protect plants against abiotic stress, especially drying stress, and is known to play a very important role in plant growth and development. Reportedly, plants that were previously treated with ABA and stressed resist stress better than plants that were not treated with ABA, while mutant plants that do not produce ABA or cannot respond to ABA are known to be weak to stress. Therefore, it is expected that plants with improved resistance to environmental stress can be developed by using proteins involved in the ABA response.
ABA 신호는 삼투 적응 및 뿌리 수리 전도도(hydraulic conductivity)의 조절에 관여하는 전사인자와 E3 리가아제(E3 ligase)와 같은 다양한 스트레스 관련 유전자들의 발현을 조절한다고 알려졌으나, 완전한 ABA 신호전달경로는 아직 규명되지 않았다. 식물 세포에서 ABA 신호전달 개시를 위해서는 ABA 수용체가 존재해야 하고 신호를 하위 단백질로 전달해야 한다. 현재까지 ABA 수용체로써 PYR/PYL/RCARs가 ABA를 인식하는 역할을 한다고 알려졌다. 수용체가 ABA를 인식하면 표적 단백질의 인산화를 통해 특정 유전자들의 발현 및 이온 채널 활성화를 촉진시키며, 이는 식물체가 불리한 환경에 적응할 수 있도록 한다.ABA signaling is known to regulate the expression of various stress-related genes, such as E3 ligase and transcription factors involved in osmotic adaptation and regulation of root hydraulic conductivity, but the complete ABA signaling pathway has not yet been identified. It didn't work. To initiate ABA signaling in plant cells, an ABA receptor must be present and transmit the signal to downstream proteins. To date, it has been known that PYR/PYL/RCARs play a role in recognizing ABA as ABA receptors. When the receptor recognizes ABA, it promotes the expression of specific genes and the activation of ion channels through phosphorylation of target proteins, allowing plants to adapt to unfavorable environments.
오늘날 사막화가 진행됨에 따라 물 부족이 농업과 환경에 큰 문제점을 초래하고 있으며, 이에 물을 적게 사용하여도 건조한 환경에서 견디고 살 수 있는 식물의 개발이 필요한 실정이다. 이러한 기술이 개발되어 작물에 적용되면 농업 생산량이 매우 증가할 것으로 기대되며, 특히 건조한 지역의 경우, 건조 저항성이 향상된 식물, 즉 증산 작용을 낮출 수 있는 식물들은 생존에 유리하므로, 농업 생산성 향상에 기여할 수 있을 뿐 아니라, 환경이 매우 건조한 지역에서 환경정화에도 유용할 수 있다.Today, as desertification progresses, water shortage is causing major problems in agriculture and the environment, and there is a need to develop plants that can survive and live in dry environments even when using less water. When these technologies are developed and applied to crops, agricultural production is expected to greatly increase. Especially in dry areas, plants with improved desiccation resistance, that is, plants that can reduce transpiration, are advantageous for survival and will contribute to improving agricultural productivity. In addition, it can be useful for environmental purification in areas with very dry environments.
최근에는 식물체의 건조 및 고염 스트레스에 대한 저항성을 증진시키기 위한 방법이 주요 연구 대상이 되고 있으며, 이와 관련하여 건조 및/또는 염 스트레스 내성 및 생장 촉진 관련 유전자와 이에 따른 형질전환 식물체에 대한 연구가 이루어지고 있으나 아직 미비한 실정이다.Recently, methods to improve the resistance of plants to dryness and high salt stress have become a major research subject, and in this regard, research has been conducted on genes related to dryness and/or salt stress tolerance and growth promotion and the resulting transgenic plants. We are losing, but it is still insufficient.
본 발명자들은 식물의 분열조직 성장 등을 조절하는 FAF 유전자가 식물체의 비생물학적 스트레스 반응에 관여하는지 알아보기 위해 연구하던 중, 고추의 FAF 유전자인 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 유전자를 동정하였으며, 상기 유전자 발현이 식물의 건조 스트레스 및 염 스트레스에 대한 저항성과 관련이 있음을 확인하여 본 발명을 완성하였다. While conducting research to determine whether the FAF gene, which regulates meristem growth in plants, is involved in the abiotic stress response of plants, the present inventors identified the FAF gene of pepper, CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1), and The present invention was completed by confirming that gene expression is related to the resistance of plants to dry stress and salt stress.
이에, 본 발명의 목적은 서열번호 1의 아미노산 서열로 이루어진 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 단백질을 제공하는 것이다.Accordingly, the purpose of the present invention is to provide CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) protein consisting of the amino acid sequence of SEQ ID NO: 1.
본 발명의 다른 목적은 상기 CaFAF1 단백질을 암호화하는 CaFAF1 유전자를 제공하는 것이다.Another object of the present invention is to provide a CaFAF1 gene encoding the CaFAF1 protein.
본 발명의 또 다른 목적은 상기 CaFAF1 유전자를 포함하는 재조합 벡터를 제공하는 것이다.Another object of the present invention is to provide a recombinant vector containing the CaFAF1 gene.
본 발명의 또 다른 목적은 CaFAF1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는, 식물체의 건조 스트레스 저항성 증진용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for enhancing drought stress resistance in plants, which includes an expression or activity inhibitor of CaFAF1 protein as an active ingredient.
본 발명의 또 다른 목적은 상기 CaFAF1 단백질의 발현 또는 활성을 억제하는 단계를 포함하는, 식물체의 건조 스트레스 저항성 증진 방법을 제공하는 것이다.Another object of the present invention is to provide a method for enhancing drought stress resistance in plants, comprising the step of inhibiting the expression or activity of the CaFAF1 protein.
본 발명의 또 다른 목적은 상기 CaFAF1 단백질, 이를 암호화하는 CaFAF1 유전자, 및 상기 유전자를 포함하는 재조합 벡터로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 식물체의 염 스트레스 저항성 증진용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for enhancing salt stress resistance in plants, comprising as an active ingredient at least one selected from the group consisting of the CaFAF1 protein, the CaFAF1 gene encoding the same, and a recombinant vector containing the gene. .
본 발명의 또 다른 목적은 식물체에서 CaFAF1을 과발현시키는 단계를 포함하는 식물체의 염 스트레스 저항성 증진 방법을 제공하는 것이다.Another object of the present invention is to provide a method for enhancing salt stress resistance in plants, including the step of overexpressing CaFAF1 in plants.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art to which the present invention belongs from the description below. There will be.
본 발명은 서열번호 1의 아미노산 서열로 이루어진 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 단백질을 제공한다.The present invention provides CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) protein consisting of the amino acid sequence of SEQ ID NO: 1.
또한, 본 발명은 상기 CaFAF1 단백질을 암호화하는 CaFAF1 유전자를 제공한다.Additionally, the present invention provides a CaFAF1 gene encoding the CaFAF1 protein.
본 발명의 일 구현예에서, 상기 CaFAF1 유전자는 서열번호 2의 염기서열로 이루어진 것일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the CaFAF1 gene may consist of the base sequence of SEQ ID NO: 2, but is not limited thereto.
본 발명의 다른 구현예에서, 상기 CaFAF1 유전자는 고추로부터 분리된 것일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the CaFAF1 gene may be isolated from pepper, but is not limited thereto.
또한, 본 발명은 상기 CaFAF1 유전자를 포함하는 재조합 벡터를 제공한다.Additionally, the present invention provides a recombinant vector containing the CaFAF1 gene.
본 발명의 일 구현예에서, 상기 재조합 벡터는 건조 스트레스 저항성 증진용일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the recombinant vector may be used to improve drying stress resistance, but is not limited thereto.
또한, 본 발명은 CaFAF1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는, 식물체의 건조 스트레스 저항성 증진용 조성물을 제공한다.In addition, the present invention provides a composition for enhancing drought stress resistance in plants, comprising an expression or activity inhibitor of CaFAF1 protein as an active ingredient.
본 발명의 일 구현예에서, 상기 CaFAF1 단백질은 서열번호 1의 아미노산 서열로 이루어진 것일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the CaFAF1 protein may consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
또한, 본 발명은 서열번호 1의 아미노산 서열로 이루어진 CaFAF1 단백질의 발현 또는 활성을 억제하는 단계를 포함하는, 식물체의 건조 스트레스 저항성 증진 방법을 제공한다.In addition, the present invention provides a method for enhancing drought stress resistance in plants, comprising the step of inhibiting the expression or activity of the CaFAF1 protein consisting of the amino acid sequence of SEQ ID NO: 1.
또한, 본 발명은 상기 방법에 의해 건조 스트레스 저항성이 증진된 형질전환 식물체를 제공한다.Additionally, the present invention provides transgenic plants with improved drought stress resistance by the above method.
본 발명의 일 구현예에서, 상기 형질전환 식물체는 고추일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the transgenic plant may be pepper, but is not limited thereto.
본 발명의 일 구현예에서, 상기 재조합 벡터는 식물체의 염 스트레스 저항성 증진용일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the recombinant vector may be used to improve salt stress resistance in plants, but is not limited thereto.
또한, 본 발명은 본 발명에 따른 재조합 벡터로 형질전환된 식물을 제공한다.Additionally, the present invention provides plants transformed with the recombinant vector according to the present invention.
또한, 본 발명은 상기 CaFAF1 단백질, 이를 암호화하는 CaFAF1 유전자, 및 상기 유전자를 포함하는 재조합 벡터로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 식물체의 염 스트레스 저항성 증진용 조성물을 제공한다.In addition, the present invention provides a composition for enhancing salt stress resistance in plants, comprising as an active ingredient one or more selected from the group consisting of the CaFAF1 protein, the CaFAF1 gene encoding the same, and a recombinant vector containing the gene.
또한, 본 발명은 하기 단계를 포함하는, 식물체의 염 스트레스 저항성 증진 방법을 제공한다:Additionally, the present invention provides a method for enhancing salt stress resistance in plants, comprising the following steps:
(S1) CaFAF1 단백질을 암호화하는 유전자를 식물체에 형질전환 하는 단계; 및(S1) Transforming a gene encoding the CaFAF1 protein into a plant; and
(S2) 상기 형질전환된 식물체에서 CaFAF1을 과발현시키는 단계.(S2) Step of overexpressing CaFAF1 in the transformed plant.
또한, 본 발명은 상기 방법에 의해 염 스트레스 저항성이 증진된 형질전환 식물체를 제공한다.Additionally, the present invention provides transgenic plants with improved salt stress resistance by the above method.
본 발명은 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 단백질, 이를 암호화하는 유전자, 및 이를 이용한 식물체의 건조 또는 염 스트레스 저항성 증진방법 등에 관한 것으로서, 상기 유전자의 발현이 침묵된 식물체는 건조 스트레스에 대한 내성은 증가한 반면 염 스트레스에 대한 내성은 감소하고, 상기 유전자가 과발현된 식물체는 건조 스트레스에 대한 내성이 감소하는 것을 확인하여 완성된 것이다. 즉, 본 발명에 따른 CaFAF1은 건조 스트레스 저항성의 음성 조절자이자 염 스트레스 저항성의 양성 조절자로 기능할 수 있으므로, 상기 단백질의 발현 조절을 통해 인류가 이용할 수 있는 작물 등을 적절히 개량할 수 있으며, 특히 식물의 생산성을 향상시키는데 유용하게 활용할 수 있을 것으로 기대된다.The present invention relates to the CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) protein, the gene encoding it, and a method for enhancing dry or salt stress resistance in plants using the same. Plants in which the expression of the gene is silenced have increased tolerance to dry stress. On the other hand, tolerance to salt stress is reduced, and plants overexpressing the above gene are confirmed to have reduced tolerance to drying stress. In other words, CaFAF1 according to the present invention can function as a negative regulator of dry stress resistance and a positive regulator of salt stress resistance, so that crops that can be used by humans can be appropriately improved by controlling the expression of the protein, especially It is expected that it will be useful in improving plant productivity.
도 1은 웹툴인 SMART (http://smart.embl-heidelberg.de)를 이용하여 4가지 고추 FAF 유전자에서 확인한 단백질 도메인 분석 결과를 나타낸다. 각 유전자에서 보존된 FANTASTIC FOUR 도메인을 검출했다.
도 2a ClustalW2로부터 다른 식물종의 동종 단백질 서열을 확보하여 다중 서열 분석을 수행한 결과이다. 붉은색 표시는 FANTASTIC FOUR 도메인을 나타낸다.
도 2b는 CaFAF1 및 CA01g13720, CA01g13730, 및 CA11g16000 유전자의 동일성 및 유사성을 정리한 표이다.
도 3은 애기장대의 상동 단백질을 이용한 고추 FAF 유전자의 계통발생 분석 결과이다. 막대는 Posisson 보정 방법으로 계산된 진화적 거리 (evolutionary distance)를 나타낸다.
도 4는 고추의 조직별 FAF 유전자의 유도 수준 (induction level)을 확인한 결과이다. 발현 값은 Pepper Actin1 (CaACT1) 유전자에 대해 정규화하였으며, 각 조직에서 CaFAF1의 발현 수준을 1.0으로 설정하였다.
도 5a는 고추 식물에 가뭄 스트레스를 가한 후 네 가지 FAF 유전자 (CA01g13720, CA01g13730, CA06g22610, 및 CA11g16000)의 발현 수준을 확인한 결과이다.
도 5b는 고추 식물에 고염 스트레스를 가한 후 상기 네 가지 FAF 유전자의 발현 수준을 확인한 결과이다.
도 6은 고추 묘목 및 성숙한 고추 식물의 다양한 조직에서 CaFAF1 유전자 발현을 확인한 결과이다. 어린 잎에서의 각 유전자의 발현 수준을 1.0으로 설정한 후 상대적 발현량을 계산했다 (YL, 어린 잎; RT, 뿌리; ST, 줄기; CT, 자엽; FEL, 완전히 확장된 잎 (fully expanded leaf); PT, 잎자루; TR, 직근 (tap root); LR, 곁뿌리 (lateral root); FL, 꽃; GRF, 녹색 열매 (green fruit); SD, 종자.
도 7은 비생물학적 스트레스 (600 mM의 만니톨; 10℃의 저온; 100 μM의 ABA, 또는 100 μM의 H2O2) 처리 후 시간에 따른 CaFAF1 전사체 발현 변화를 확인한 결과를 나타낸다. Actin1 (CaACT1)에 대해 발현 값을 정규화하여 나타냈으며, 스트레스 처리 후 0시간 시점에서의 CaFAF1 발현 수준을 1.0으로 설정했다.
도 8은 CaFAF1의 세포 내 국소화 실험 결과를 나타낸다. Agroinfiltration 방법을 사용해 Pro35S:CaFAF1-GFP 작제물을 니코티아나 벤타미아나 식물의 잎 표피세포에서 일시적으로 발현시켰다. 0.8 M의 만니톨과 10분간 배양하여 표피세포의 원형질분리를 유도했다 (N, 핵; C, 세포질; CW, 세포벽; PM, 원형질막). 흰색 막대 = 20μm.
도 9는 대조군 (TRV2:00) 및 CaFAF1-침묵 고추 (TRV:CaFAF1)의 상대적 CaFAF1 발현량을 qRT-PCR로 확인한 결과를 나타낸다. CaACT1 유전자에 대해 발현 값을 정규화하여 상대적 발현량을 나타냈다.
도 10은 4주령의 대조군 및 CaFAF1-침묵 고추 식물에 14일간 단수하여 가뭄 스트레스를 가하고, 3일간 재급수한 뒤 식물 표현형을 관찰한 결과이다. 3일간의 재급수 후 생존율을 계산했다.
도 11은 대조군 및 CaFAF1-침묵 고추 식물에 가뭄 스트레스를 가한 후 식물 잎에서 스트레스 반응 관련 유전자 (CaNCED3, CaOSR1, CaRAB18, 및 CaDREB1)의 발현 변화를 qRT-PCR로 확인한 결과이다. 대조군 식물의 가뭄 스트레스 처리 0시간 시점에서의 각 유전자의 발현량을 1.0으로 설정하였으며, CaACT1 유전자에 대해 정규화하여 상대적 발현량을 나타냈다.
도 12는 대조군 및 CaFAF1-침묵 고추 식물의 수분 손실 정도를 비교하기 위해 각 식물에서 잎을 분리하여 시간에 따른 잎의 생중량 손실 정도를 비교한 결과를 나타낸다.
도 13은 4주령의 대조군 및 CaFAF1-침묵 고추 식물로부터 잎 껍질을 확보한 후 0, 10, 또는 20 μM의 ABA를 포함하는 기공 개방 용액 (stomatal opening solution, SOS) 완충액에서 2시간 동안 배양한 뒤 기공개도를 비교하기 위해 기공을 촬영한 것이다.
도 14는 ABA 처리에 따른 대조군 및 CaFAF1-침묵 고추 식물의 기공개도를 비교하기 위해, 현미경으로 임의의 100개 기공을 촬영한 뒤 기공 구멍의 너비 대비 길이 비율을 측정하여 나타낸 것이다.
도 15는 50 μM의 ABA를 처리하거나 처리하지 않은 대조군 및 CaFAF1-침묵 고추 식물의 잎 표면 온도를 확인하기 위해 열 화상 이미지를 촬영한 결과를 나타낸다.
도 16은 50 μM의 ABA를 처리하거나 처리하지 않은 대조군 및 CaFAF1-침묵 고추 식물의 잎 표면 온도를 정량적으로 확인한 결과로, 각 계통의 15개의 식물의 잎 온도로부터 평균 온도를 계산했다.
도 17은 대조군 (WT) 및 CaFAF1-과발현 (Pro35S:CaFAF1) 애기장대 식물에서 잎의 CaFAF1 발현 수준을 확인한 결과이다. CaACT1 유전자에 대해 발현 값을 정규화하여 상대적 발현량을 나타냈다.
도 18은 3주령의 대조군 및 CaFAF1-과발현 애기장대 식물에 14일간 단수하여 가뭄 스트레스를 가하고, 3일간 재급수한 뒤 식물 표현형을 관찰한 결과이다. 3일간의 재급수 후 생존율을 계산했다.
도 19는 대조군 및 CaFAF1-과발현 애기장대 식물에 가뭄 스트레스를 가한 후 가뭄-반응성 마커 유전자 RAB18, RD29B, NCED3, 및 DREB2A의 발현을 qRT-PCR로 확인한 결과를 나타낸다. 가뭄 스트레스 처리 0시간 시점의 야생형 식물의 각 유전자들의 발현을 1.0으로 설정하였으며, CaACT1 유전자에 대해 발현 값을 정규화하여 상대적 발현량을 나타냈다.
도 20은 대조군 및 CaFAF1-과발현 애기장대 식물의 수분 손실 정도를 비교하기 위해 각 식물에서 잎을 분리하여 시간에 따른 잎의 생중량 손실 정도를 비교한 결과를 나타낸다.
도 21은 3주령의 대조군 및 CaFAF1-과발현 애기장대 식물로부터 잎 껍질을 수확하여 0, 10, 또는 20 μM의 ABA를 포함하는 기공 개방 용액 (SOS) 완충액에서 2시간 동안 배양한 뒤 기공개도를 비교하기 위해 기공을 촬영한 결과를 나타낸다.
도 22는 ABA 처리에 따른 대조군 및 CaFAF1-과발현 애기장대 식물의 기공개도를 비교하기 위해, 현미경으로 임의의 100개 기공을 촬영한 뒤 기공 구멍의 너비 대비 길이 비율을 측정하여 나타낸 것이다.
도 23은 대조군 및 CaFAF1-과발현 애기장대 식물의 뿌리를 제거하여 탈수 스트레스 처리를 가한 후 잎 표면 온도를 확인하기 위해 열 화상 이미지를 촬영한 결과이다.
도 24는 CaFAF1 침묵에 의한 식물체의 염분 스트레스 내성 변화를 확인하기 위해, agroinfiltration 2주 후 정상 환경에서 배양된 대조군 및 CaFAF1-침묵 고추 식물을 물, 200 mM NaCl 용액, 또는 250 mM NaCl 용액이 담긴 플라스틱 용기에서 배양한 뒤 표현형을 관찰한 결과이다.
도 25는 대조군 및 CaFAF1-침묵 고추 식물에 250 mM의 NaCl 용액을 처리한 후 스트레스 관련 유전자 CaDREB1, CaOSR1, CaP5CS, 및 CaRAB18의 발현을 qRT-PCR로 확인한 결과이다. 염분 스트레스 처리 0시간 시점의 대조군 식물의 각 유전자들의 발현을 1.0으로 설정하였으며, CaACT1 유전자에 대해 발현 값을 정규화하여 상대적 발현량을 나타냈다.
도 26은 3주령의 대조군 및 CaFAF1-침묵 고추 식물에 200 mM의 NaCl 용액을 처리한 뒤 전해질 누출 정도를 측정한 결과이다.
도 27은 3주령의 대조군 및 CaFAF1-침묵 고추 식물에 200 mM의 NaCl 용액을 처리한 뒤 지질 과산화 산물인 말론알데히드 수준을 측정한 결과이다.
도 28은 3주령의 대조군 및 CaFAF1-침묵 고추 식물에 200 mM의 NaCl 용액을 처리한 뒤 프롤린 축적 정도를 측정한 결과이다.
도 29는 본 발명의 실시예에서 사용된 클로닝, PCR, 및 VIG 프라이머 서열을 정리한 표이다.
도 30은 CaFAF1 유전자의 뉴클레오티드 서열 및 CaFAF1 단백질의 아미노산 서열을 나타낸다.Figure 1 shows the results of protein domain analysis identified in four pepper FAF genes using the web tool SMART ( http://smart.embl-heidelberg.de ). A conserved FANTASTIC FOUR domain was detected in each gene.
Figure 2a shows the results of performing multiple sequence analysis by securing homologous protein sequences from other plant species from ClustalW2. The red mark indicates the FANTASTIC FOUR domain.
Figure 2b is a table summarizing the identity and similarity of CaFAF1 and CA01g13720, CA01g13730, and CA11g16000 genes.
Figure 3 shows the results of phylogenetic analysis of the pepper FAF gene using homologous proteins from Arabidopsis thaliana. Bars represent evolutionary distances calculated with the Posisson correction method.
Figure 4 shows the results of confirming the induction level of the FAF gene in each tissue of pepper. Expression values were normalized to the Pepper Actin1 (CaACT1) gene, and the expression level of CaFAF1 in each tissue was set to 1.0.
Figure 5a shows the results of confirming the expression levels of four FAF genes ( CA01g13720, CA01g13730, CA06g22610, and CA11g16000 ) after applying drought stress to pepper plants.
Figure 5b shows the results of confirming the expression levels of the four FAF genes after applying high salt stress to pepper plants.
Figure 6 shows the results of confirming CaFAF1 gene expression in various tissues of pepper seedlings and mature pepper plants. The expression level of each gene in young leaves was set to 1.0, and then the relative expression level was calculated (YL, young leaf; RT, root; ST, stem; CT, cotyledon; FEL, fully expanded leaf) ; PT, petiole; TR, tap root; FL, flower; SD, seed.
Figure 7 shows the results of confirming changes in CaFAF1 transcript expression over time after treatment with abiotic stress (600 mM mannitol; low temperature of 10°C; 100 μM ABA, or 100 μM H 2 O 2 ). Expression values were normalized to Actin1 (CaACT1), and the CaFAF1 expression level at 0 hours after stress treatment was set to 1.0.
Figure 8 shows the results of an intracellular localization experiment of CaFAF1. The Pro35S:CaFAF1-GFP construct was transiently expressed in leaf epidermal cells of Nicotiana benthamiana plants using an agroinfiltration method. Plasma separation of epidermal cells was induced by incubation with 0.8 M mannitol for 10 minutes (N, nucleus; C, cytoplasm; CW, cell wall; PM, plasma membrane). White bar = 20 μm.
Figure 9 shows the results of confirming the relative CaFAF1 expression level of control (TRV2:00) and CaFAF1- silenced pepper (TRV: CaFAF1 ) by qRT-PCR. Expression values were normalized to the CaACT1 gene to show relative expression levels.
Figure 10 shows the results of observing plant phenotypes after applying drought stress to 4-week-old control and CaFAF1- silenced pepper plants by cutting water for 14 days and re-watering them for 3 days. Survival rates were calculated after 3 days of refeeding.
Figure 11 shows the results of qRT-PCR confirming the expression changes of stress response-related genes ( CaNCED3 , CaOSR1 , CaRAB18 , and CaDREB1 ) in plant leaves after applying drought stress to control and CaFAF1- silenced pepper plants. The expression level of each gene at 0 hours of drought stress treatment of control plants was set to 1.0, and the relative expression level was expressed by normalizing to the CaACT1 gene.
Figure 12 shows the results of separating leaves from each plant to compare the degree of water loss in control and CaFAF1- silenced pepper plants and comparing the degree of fresh weight loss of leaves over time.
Figure 13 shows leaf skins obtained from 4-week-old control and CaFAF1- silenced pepper plants and incubated for 2 hours in stomatal opening solution (SOS) buffer containing 0, 10, or 20 μM ABA. The stomata were photographed to compare the degree of stomata opening.
Figure 14 shows the ratio of length to width of pores after photographing 100 random pores under a microscope to compare the stomata opening of control and CaFAF1- silenced pepper plants according to ABA treatment.
Figure 15 shows the results of taking thermographic images to confirm the leaf surface temperature of control and CaFAF1 -silenced pepper plants treated with or without 50 μM ABA.
Figure 16 shows the results of quantitative confirmation of the leaf surface temperature of control and CaFAF1 -silenced pepper plants treated with or without 50 μM ABA. The average temperature was calculated from the leaf temperatures of 15 plants of each line.
Figure 17 shows the results of confirming the CaFAF1 expression level in leaves in control (WT) and CaFAF1- overexpressing ( Pro35S:CaFAF1 ) Arabidopsis plants. Expression values were normalized to the CaACT1 gene to show relative expression levels.
Figure 18 shows the results of observing plant phenotypes after applying drought stress to 3-week-old control and CaFAF1- overexpressing Arabidopsis plants by cutting water for 14 days and re-watering them for 3 days. Survival rates were calculated after 3 days of refeeding.
Figure 19 shows the results of qRT-PCR confirming the expression of drought-responsive marker genes RAB18 , RD29B , NCED3 , and DREB2A after applying drought stress to control and CaFAF1- overexpressing Arabidopsis plants. The expression of each gene in wild-type plants at 0 hours of drought stress treatment was set to 1.0, and the expression value was normalized to the CaACT1 gene to show the relative expression level.
Figure 20 shows the results of comparing the degree of fresh weight loss of leaves over time by separating leaves from each plant to compare the degree of water loss between control and CaFAF1- overexpressing Arabidopsis plants.
Figure 21 compares the degree of stomatal opening after harvesting leaf bark from 3-week-old control and CaFAF1- overexpressing Arabidopsis plants and culturing them for 2 hours in stomatal opening solution (SOS) buffer containing 0, 10, or 20 μM ABA. The results of photographing the pores for this purpose are shown.
Figure 22 shows the ratio of length to width of pore openings after photographing 100 random pores under a microscope to compare the degree of stomatal opening of control and CaFAF1- overexpressing Arabidopsis plants according to ABA treatment.
Figure 23 shows the results of thermal imaging images taken to check the leaf surface temperature after removing the roots of control and CaFAF1- overexpressing Arabidopsis plants and applying dehydration stress treatment.
Figure 24 shows control and CaFAF1 -silenced pepper plants cultured in a normal environment 2 weeks after agroinfiltration to confirm changes in salt stress tolerance of plants due to CaFAF1 silencing, in water, 200 mM NaCl solution, or plastic containing 250 mM NaCl solution. This is the result of observing the phenotype after culturing in a container.
Figure 25 shows the results of qRT-PCR confirming the expression of stress-related genes CaDREB1 , CaOSR1 , CaP5CS , and CaRAB18 after treating control and CaFAF1- silenced pepper plants with 250 mM NaCl solution. The expression of each gene in the control plant at 0 hours of salt stress treatment was set to 1.0, and the expression value was normalized to the CaACT1 gene to show the relative expression level.
Figure 26 shows the results of measuring the degree of electrolyte leakage after treating 3-week-old control and CaFAF1- silenced pepper plants with a 200 mM NaCl solution.
Figure 27 shows the results of measuring the level of malonaldehyde, a lipid peroxidation product, after treating 3-week-old control and CaFAF1- silenced pepper plants with a 200 mM NaCl solution.
Figure 28 shows the results of measuring the degree of proline accumulation after treating 3-week-old control and CaFAF1- silenced pepper plants with a 200 mM NaCl solution.
Figure 29 is a table summarizing cloning, PCR, and VIG primer sequences used in the examples of the present invention.
Figure 30 shows the nucleotide sequence of the CaFAF1 gene and the amino acid sequence of the CaFAF1 protein.
본 발명은 건조 스트레스에 대한 음성적 조절인자 (negative regulator)이자 염 스트레스에 대한 양성적 조절인자 (positive regulator)인 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1)에 관한 것으로서, 상기 유전자의 발현이 침묵된 식물체는 가뭄 스트레스에 대한 내성은 증가하는 반면 염 스트레스에 대한 내성은 감소하고, 상기 유전자가 과발현된 식물체는 가뭄 스트레스 내성이 감소하는 것을 확인하여 완성된 것이다.The present invention relates to CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1), which is a negative regulator for drying stress and a positive regulator for salt stress, and plants in which the expression of the gene is silenced are susceptible to drought. It was confirmed that tolerance to stress increases while tolerance to salt stress decreases, and plants overexpressing the above gene have decreased drought stress tolerance.
이에, 본 발명은 건조 스트레스에 대한 음성적 조절인자이자 염 스트레스에 대한 양성적 조절인자인, 서열번호 1의 아미노산 서열로 이루어진 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 단백질, 및 이를 암호화하는 CaFAF1 유전자를 제공한다.Accordingly, the present invention provides a CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) protein consisting of the amino acid sequence of SEQ ID NO: 1, which is a negative regulator for drying stress and a positive regulator for salt stress, and the CaFAF1 gene encoding the same. .
본 발명에 있어서, 상기 CaFAF1 유전자는 서열번호 2의 염기서열로 이루어져 있으며, 상기 CaFAF1 유전자에 의해 암호화되는 펩타이드는 바람직하게는 서열번호 1의 아미노산 서열로 이루어질 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the CaFAF1 gene consists of the base sequence of SEQ ID NO: 2, and the peptide encoded by the CaFAF1 gene may preferably consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
본 발명에 있어서, 상기 CaFAF1 유전자는 고추로부터 분리된 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the CaFAF1 gene may be isolated from pepper, but is not limited thereto.
본 발명에 있어서 비생물학적 스트레스 또는 환경적 스트레스 등에 대한 저항성 (내성)의 “음성 조절인자 (negative regulator)”란 발현 또는 활성이 증가할수록 식물체의 스트레스 저항성을 감소시키는 유전자 및/또는 단백질을 의미하며, “양성 조절인자 (positive regulator)”란 발현 또는 활성이 증가할수록 식물체의 스트레스 저항성을 향상시키는 유전자 및/또는 단백질을 의미한다. 즉, 본 발명의 유전자인 CaFAF1은 가뭄 스트레스 내성에 대한 음성 조절인자이자 염 스트레스 내성에 대한 양성 조절인자로 기능하는 단백질을 코딩하므로, CaFAF1 유전자 발현이 억제되면 건조 스트레스에 대한 내성은 증가하고 염 스트레스에 대한 내성은 감소하며, 반대로 CaFAF1 유전자가 과발현되면 건조 스트레스에 대한 내성은 감소하고 염 스트레스에 대한 내성은 증가할 수 있다.In the present invention, a “negative regulator” of resistance (tolerance) to abiotic stress or environmental stress, etc. refers to a gene and/or protein that reduces the stress resistance of a plant as its expression or activity increases, “Positive regulator” refers to genes and/or proteins that improve the stress resistance of plants as their expression or activity increases. In other words, CaFAF1 , the gene of the present invention, encodes a protein that functions as a negative regulator for drought stress tolerance and a positive regulator for salt stress tolerance, so when CaFAF1 gene expression is suppressed, tolerance to dry stress increases and salt stress resistance to is decreased, and conversely, if the CaFAF1 gene is overexpressed, tolerance to drying stress may decrease and tolerance to salt stress may increase.
본 발명자들은 식물체의 비생물학적 스트레스 반응에 관여하는 유전자에 대해 연구하던 중, 고추로부터 CaFAF1 (Capsicum annuum FANTASTIC FOUR 1) 유전자를 동정하였고, 건조 및 염 스트레스에 대한 식물체의 내성과 상기 유전자의 연관성을 규명하고자 하였다.While researching genes involved in the abiotic stress response of plants, the present inventors identified the CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) gene from pepper and identified the relationship between the gene and the plant's tolerance to dryness and salt stress. I wanted to do it.
구체적으로, 본 발명의 일 실시예에서는 네 가지 고추 유래 FAF (FANTASTIC FOUR) 유전자를 동정한 후 가뭄 및 고염 스트레스 처리에 따른 상기 네 가지 유전자들의 발현 변화를 비교한 결과, CA06g22610 (Capsicum annuum FANTASTIC FOUR 1; CaFAF1) 유전자가 가뭄 및 고염 환경에서 발현 패턴이 유의미하게 달라지는 것을 확인하여, CaFAF1을 가뭄 및 고염 스트레스 반응 조절인자의 유력한 후보로 선별하였다 (실시예 1).Specifically, in one embodiment of the present invention, four pepper-derived FAF (FANTASTIC FOUR) genes were identified and the expression changes of the four genes were compared according to drought and high salt stress treatments. As a result, CA06g22610 (Capsicum annuum FANTASTIC FOUR 1) ; CaFAF1 ) gene was confirmed to have significantly different expression patterns in drought and high salt environments, and CaFAF1 was selected as a strong candidate for a regulator of drought and high salt stress responses (Example 1).
본 발명의 다른 실시예에서는 CaFAF1의 분자적 특성을 분석한 결과 상기 단백질은 고추의 직근 (tap root)에서 특히 높은 수준으로 발현되고, 세포질에 위치한다는 것을 확인하였다 (실시예 2).In another example of the present invention, the molecular characteristics of CaFAF1 were analyzed and it was confirmed that the protein was expressed at a particularly high level in the tap root of pepper and was located in the cytoplasm (Example 2).
본 발명의 또 다른 실시예에서는 고추에서 CaFAF1 유전자 발현을 침묵시킨 후 가뭄 스트레스에 대한 반응을 관찰한 결과, CaFAF1-침묵 고추 식물은 가뭄 환경에서 생존율이 증가하고, 스트레스-관련 마커 유전자들의 발현이 증가하며, 기공을 통한 수분 손실이 감소하는 것을 확인한 바, CaFAF1 유전자가 고추의 가뭄 스트레스에 대한 내성의 음성 조절인자로 기능함을 확인하였다 (실시예 3).In another embodiment of the present invention, the response to drought stress was observed after silencing CaFAF1 gene expression in peppers, and the results showed that CaFAF1- silenced pepper plants had an increased survival rate in a drought environment and increased expression of stress-related marker genes. As it was confirmed that water loss through stomata was reduced, it was confirmed that the CaFAF1 gene functions as a negative regulator of tolerance to drought stress in pepper (Example 3).
본 발명의 또 다른 실시예에서는 애기장대에서 CaFAF1 유전자를 과발현 시킨 후 가뭄 스트레스에 대한 반응을 관찰한 결과 CaFAF1-과발현 애기장대는 가뭄 환경에서 생존율이 감소하고, 스트레스-관련 마커 유전자들의 발현이 감소하며, 기공을 통한 수분 손실이 증가하는 것을 확인한 바, CaFAF1 유전자가 고추의 가뭄 스트레스에 대한 내성의 음성 조절인자로 기능함을 검증하였다 (실시예 4).In another embodiment of the present invention, the response to drought stress was observed after overexpressing the CaFAF1 gene in Arabidopsis thaliana. As a result, the survival rate of CaFAF1- overexpressing Arabidopsis thaliana was reduced in a drought environment, and the expression of stress-related marker genes was reduced. , As it was confirmed that water loss through stomata increases, it was verified that the CaFAF1 gene functions as a negative regulator of tolerance to drought stress in pepper (Example 4).
본 발명의 또 다른 실시예에서는 고추에서 CaFAF1 유전자 발현을 침묵시킨 후 고염 스트레스에 대한 반응을 관찰한 결과, CaFAF1-침묵 고추 식물은 고염 환경에서 생존율이 감소하고, 스트레스-관련 마커 유전자들의 발현이 감소하며, 염분 스트레스 마커인 전해질 누출, 지질 과산화, 및 프롤린 생합성 등이 증가한 것을 확인한 바, CaFAF1 유전자가 고추의 염 스트레스에 대한 내성의 양성 조절인자로 기능함을 확인하였다 (실시예 5).In another embodiment of the present invention, the response to high-salt stress was observed after silencing CaFAF1 gene expression in peppers, and the results showed that CaFAF1- silenced pepper plants had a reduced survival rate in a high-salt environment and decreased expression of stress-related marker genes. In addition, it was confirmed that salt stress markers such as electrolyte leakage, lipid peroxidation, and proline biosynthesis increased, and it was confirmed that the CaFAF1 gene functions as a positive regulator of tolerance to salt stress in pepper (Example 5).
본 발명은 CaFAF1 유전자를 포함하는 재조합 벡터를 제공한다.The present invention provides a recombinant vector containing the CaFAF1 gene.
바이러스에 의해 유도되는 유전자 침묵 기술인 VIGS (virus-induced gene silencing)는 바이러스 유도 RNA 침묵 (virus-induced RNA silencing)이라고도 불린다. 다양한 식물과 진균, 곤충류, 선충류, 어류, 쥐 등에서 잘 알려진 전사 후 유전자 침묵현상의 일종으로 바이러스가 식물체에 침입하여 복제하는 과정에서 감염 식물체에 바이러스 유전체와 상동성이 있는 내성유전자가 발현될 때 내성유전자와 바이러스 유전체의 발현과 복제가 함께 억제되는 현상이라고 할 수 있다. 즉, 바이러스 게놈의 cDNA에 기주에서 유래된 특정 유전자의 일부 혹은 전체를 삽입하고 감염성 RNA로 제작하여 식물체에 감염시키면, 상응하는 기주의 RNA가 목표가 되어, 감염된 기주 식물체에서는 목적 유전자의 발현이 억제되거나 소실된다. 그 결과 목적 유전자의 기능을 간접적으로 추정할 수 있다. 바이러스 유도 유전자 침묵 (VIGS) 기작은 바이러스에 대항하여 RNA가 매개하는 식물 방어 기작의 일종이라고 알려져 있으며, 1) 전사 후 유전자 침묵 2) RNA 전환 3) 뉴클레오티드 서열 특이성이라는 특징을 가진다.Virus-induced gene silencing (VIGS), a gene silencing technology induced by a virus, is also called virus-induced RNA silencing. It is a type of post-transcriptional gene silencing phenomenon that is well known in various plants, fungi, insects, nematodes, fish, mice, etc. Resistance occurs when a resistance gene homologous to the viral genome is expressed in the infected plant during the process of virus invasion and replication in the plant. It can be said to be a phenomenon in which the expression and replication of genes and viral genomes are suppressed together. In other words, if part or all of a specific gene derived from the host is inserted into the cDNA of the viral genome, made into infectious RNA, and then infected into a plant, the corresponding host RNA becomes the target, and the expression of the target gene is suppressed or suppressed in the infected host plant. It disappears. As a result, the function of the target gene can be indirectly estimated. Virus-induced gene silencing (VIGS) is known to be a type of RNA-mediated plant defense mechanism against viruses, and has the following characteristics: 1) post-transcriptional gene silencing, 2) RNA conversion, and 3) nucleotide sequence specificity.
본 발명에서는, CaFAF1의 ORF 영역을 삽입한 VIGS용 형질전환 벡터 TRV (Tobacco Rattle Virus)를 아그로박테리움 (Agrobacterium)에 삽입하고, 상기 아그로박테리움을 식물체에 침투시킴으로써 CaFAF1 유전자의 발현을 성공적으로 억제한 바 있다.In the present invention, the transformation vector TRV (Tobacco Rattle Virus) for VIGS, which inserts the ORF region of CaFAF1, is inserted into Agrobacterium , and the expression of the CaFAF1 gene is successfully suppressed by infiltrating the Agrobacterium into the plant. It has been done.
본 발명에서 용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로서 인위적인 수단에 의해 세포 내 재도입된 것이다.As used herein, the term "recombinant" refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the natural form of the cell, either in sense or antisense form. Additionally, recombinant cells can express genes found in cells in their natural state, but the genes have been modified and reintroduced into the cells by artificial means.
상기 용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 본 발명에서 재조합 벡터의 바람직한 예는 아그로박테리움 투메파시엔스 (Agrobacterium tumefaciens)와 같은 적당한 숙주에 존재할 때 그 자체의 일부를 식물 세포로 전이시킬 수 있는 플라스미드 벡터, pTRV1 또는 pTRV2일 수 있으나 이에 한정되는 것은 아니며, 본 발명에 따른 DNA를 식물 숙주에 도입시키는데 이용될 수 있는 다른 적합한 벡터는 이중 가닥 식물 바이러스 (예를 들면, CaMV) 및 단일 가닥 바이러스, 게미니 바이러스 등으로부터 유래 될 수 있는 것과 같은 바이러스 벡터, 예를 들면 비완전성 식물 바이러스 벡터로부터 선택될 수 있다. 상기 벡터는 식물 숙주를 적당하게 형질전환하는 것이 어려울 때 유리하게 사용할 수 있다. The term “vector” is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can reproduce independently in host cells. A preferred example of a recombinant vector in the present invention may be a plasmid vector, pTRV1 or pTRV2, which can transfer a part of itself to a plant cell when present in a suitable host such as Agrobacterium tumefaciens, but is limited thereto. However, other suitable vectors that can be used to introduce the DNA according to the present invention into a plant host include viruses such as those that may be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc. Vectors may be selected from, for example, non-intact plant virus vectors. The vector can be advantageously used when it is difficult to properly transform a plant host.
용어 "재조합 벡터" 또는 "재조합 발현 벡터"는 세균 플라스미드, 파아지, 효모 플라스미드, 식물 세포 바이러스, 포유동물 세포 바이러스, 또는 다른 벡터를 의미한다. 대체로, 임의의 플라스미드 및 벡터는 숙주 내에서 복제 및 안정화할 수 있다면 사용될 수 있다. 상기 발현 벡터의 중요한 특성은 복제 원점, 프로모터, 마커 유전자 및 번역 조절 요소 (translation control element)를 가지는 것이다.The term “recombinant vector” or “recombinant expression vector” means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. Broadly speaking, any plasmid and vector can be used as long as it can replicate and stabilize within the host. Important characteristics of the expression vector are that it has an origin of replication, a promoter, a marker gene, and a translation control element.
상기 유전자 서열 및 적당한 전사/번역 조절 신호를 포함하는 발현 벡터는 당업자에 주지된 방법에 의해 구축될 수 있다. 상기 방법은 시험관 내 재조합 DNA 기술, DNA 합성 기술 및 생체 내 재조합 기술 등을 포함한다. 상기 DNA 서열은 mRNA 합성을 이끌기 위해 발현 벡터 내의 적당한 프로모터에 효과적으로 연결될 수 있다. 또한 발현 벡터는 번역 개시 부위로서 리보좀 결합 부위 및 전사 터미네이터를 포함할 수 있다.An expression vector containing the above gene sequence and appropriate transcription/translation control signals can be constructed by methods well known to those skilled in the art. The methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to an appropriate promoter within an expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.
본 발명의 재조합 벡터의 또 다른 바람직한 예는 아그로박테리움 투머파시엔스와 같은 적당한 숙주에 존재할 때 그 자체의 일부, 소위 T-영역을 식물 세포로 전이시킬 수 있는 Ti-플라스미드 벡터이다. 다른 유형의 Ti-플라스미드 벡터 (EP 0 116 718 B1호 참조)는 현재 식물 세포, 또는 잡종 DNA를 식물의 게놈 내에 적당하게 삽입시키는 새로운 식물이 생산될 수 있는 원형질체로 잡종 DNA 서열을 전이시키는데 이용되고 있다. Ti-플라스미드 벡터의 특히 바람직한 형태는 EP 0 120 516 B1호 및 미국 특허 제4,940,838호에 청구된 바와 같은 소위 바이너리(binary) 벡터이다. 본 발명에 따른 DNA를 식물 숙주에 도입시키는데 이용될 수 있는 다른 적합한 벡터는 이중 가닥 식물 바이러스(예를 들면, CaMV) 및 단일 가닥 바이러스, 게미니 바이러스 등으로부터 유래될 수 있는 것과 같은 바이러스 벡터, 예를 들면 비완전성 식물 바이러스 벡터로부터 선택될 수 있다. 그러한 벡터의 사용은 특히 식물 숙주를 적당하게 형질전환하는 것이 어려울 때 유리할 수 있다.Another preferred example of the recombinant vector of the present invention is the Ti-plasmid vector, which is capable of transferring part of itself, the so-called T-region, into plant cells when present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences into plant cells or protoplasts from which new plants can be produced that properly integrate the hybrid DNA into the plant's genome. there is. A particularly preferred form of Ti-plasmid vectors are the so-called binary vectors as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host include viral vectors, such as those that may be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc. For example, it may be selected from non-intact plant virus vectors. The use of such vectors can be particularly advantageous when it is difficult to properly transform plant hosts.
발현 벡터는 하나 이상의 선택성 마커를 포함할 수 있다. 상기 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, 블레오마이신 (Bleomycin), 하이그로마이신 (hygromycin), 클로람페니콜 (chloramphenicol)과 같은 항생제 내성 유전자가 있으나, 이에 한정되는 것은 아니다.Expression vectors may include one or more selectable markers. The marker is a nucleic acid sequence that has characteristics that can be generally selected by chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, and chloramphenicol. There is a resistance gene, but it is not limited to this.
본 발명의 재조합 벡터에서, 프로모터는 CaMV 35S, 액틴, 유비퀴틴, pEMU, MAS 또는 히스톤 프로모터일 수 있으나, 이에 제한되지 않는다. "프로모터"란 용어는 구조 유전자로부터의 DNA 업스트림의 영역을 의미하며 전사를 개시하기 위하여 RNA 폴리머라아제가 결합하는 DNA 분자를 말한다. "식물 프로모터"는 식물 세포에서 전사를 개시할 수 있는 프로모터이다. "지속적 (constitutive) 프로모터"는 대부분의 환경 조건 및 발달 상태 또는 세포 분화 하에서 활성이 있는 프로모터이다. In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter. The term "promoter" refers to the region of DNA upstream from a structural gene and refers to the DNA molecule to which RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation.
본 발명의 재조합 벡터에서, 통상의 터미네이터를 사용할 수 있으며, 그 예로는 노팔린 신타아제 (NOS), 벼 α-아밀라아제 RAmy1 A 터미네이터, 파세올린 (phaseoline) 터미네이터, 아그로박테리움 투메파시엔스 (Agrobacterium tumefaciens)의 옥토파인 (Octopine) 유전자의 터미네이터 등이 있으나, 이에 한정되는 것은 아니다. 터미네이터의 필요성에 관하여, 그러한 영역이 식물 세포에서의 전사의 확실성 및 효율을 증가시키는 것으로 일반적으로 알려져 있다. In the recombinant vector of the present invention, common terminators can be used, examples of which include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ) , but is not limited to the terminator of the Octopine gene. Regarding the necessity of terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells.
식물의 형질전환은 DNA를 식물에 전이시키는 임의의 방법을 의미한다. 그러한 형질전환 방법은 반드시 재생 및 (또는) 조직 배양기간을 가질 필요는 없다. 식물 종의 형질전환은 이제는 쌍자엽 식물뿐만 아니라 단자엽 식물양자를 포함한 식물 종에 대해 일반적이다. 원칙적으로, 임의의 형질전환 방법은 본 발명에 따른 잡종 DNA를 적당한 선조 세포로 도입시키는데 이용될 수 있다. 방법은 원형질체에 대한 칼슘/폴리에틸렌 글리콜 방법 (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), 원형질체의 전기천공법 (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), 식물 요소로의 현미주사법 (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), 각종 식물 요소의 (DNA 또는 RNA-코팅된) 입자충격법 (Klein T.M. et al., 1987, Nature 327, 70), 식물의 침윤 또는 성숙 화분 또는 소포자의 형질전환에 의한 아그로박테리움 투메파시엔스 매개된 유전자 전이에서 (비완전성) 바이러스에 의한 감염 (EP 0 301 316호) 등으로부터 적당하게 선택될 수 있다. 본 발명에 따른 바람직한 방법은 아그로박테리움 매개된 DNA 전달을 포함한다.Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily require a regeneration and/or tissue culture period. Transformation of plant species is now common for plant species including monocots as well as dicots. In principle, any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. The method is the calcium/polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373). electroporation (Shillito RD et al., 1985 Bio/Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) ), particle bombardment of various plant elements (DNA or RNA-coated) (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumefacsi by infiltration of plants or transformation of mature pollen or spores. It can be appropriately selected from ens- mediated gene transfer, infection by a (non-complete) virus (EP 0 301 316), etc. A preferred method according to the invention involves Agrobacterium mediated DNA transfer.
본 발명에 있어서, 상기 재조합 벡터는 바람직하게는 재조합 식물 발현 벡터일 수 있다.In the present invention, the recombinant vector may preferably be a recombinant plant expression vector.
또한, 본 발명은 본 발명에 따른 방법에 의해 건조 저항성이 증진된 형질전환 식물체를 제공한다.Additionally, the present invention provides transgenic plants with improved desiccation resistance by the method according to the present invention.
본 발명의 또 다른 구현예에서, 본 발명에 따른 재조합 벡터는 식물체의 염 스트레스 저항성 증진용일 수 있다.In another embodiment of the present invention, the recombinant vector according to the present invention may be used to improve salt stress resistance in plants.
또한, 본 발명은 상기 재조합 벡터로 형질전환된 식물을 제공한다.Additionally, the present invention provides plants transformed with the above recombinant vector.
또한, 본 발명은 하기 단계를 포함하는, 식물체의 염 스트레스 저항성 증진 방법을 제공한다:Additionally, the present invention provides a method for enhancing salt stress resistance in plants, comprising the following steps:
(S1) CaFAF1 단백질을 암호화하는 유전자를 식물체에 형질전환 하는 단계; 및(S1) Transforming a gene encoding the CaFAF1 protein into a plant; and
(S2) 상기 형질전환된 식물체에서 CaFAF1을 과발현시키는 단계.(S2) Step of overexpressing CaFAF1 in the transformed plant.
또한, 본 발명은 상기 식물체의 염 스트레스 저항성 증진 방법에 의해 염 스트레스 저항성이 증진된 형질전환 식물체를 제공한다.In addition, the present invention provides a transgenic plant with improved salt stress resistance by the method for improving the salt stress resistance of the plant.
본 발명에서 상기 식물(체)는 벼, 밀, 보리, 옥수수, 콩, 감자, 팥, 귀리, 수수를 포함하는 식량 작물류; 애기장대, 배추, 무, 고추, 딸기, 토마토, 수박, 오이, 양배추, 참외, 호박, 파, 양파, 당근을 포함하는 채소 작물류; 인삼, 담배, 목화, 참깨, 사탕수수, 사탕무우, 들깨, 땅콩, 유채를 포함하는 특용 작물류; 사과나무, 배나무, 대추나무, 복숭아, 양다래, 포도, 감귤, 감, 자두, 살구, 바나나를 포함하는 과수류; 장미, 글라디올러스, 거베라, 카네이션, 국화, 백합, 튤립을 포함하는 화훼류; 및 라이그라스, 레드클로버, 오차드그라스, 알파파, 톨페스큐, 페레니얼라이그라스를 포함하는 사료 물류 등일 수 있으며, 가장 바람직하게는 애기장대 또는 고추이나, 이에 한정되는 것은 아니다.In the present invention, the plants (sieves) include food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; Vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pear trees, jujube trees, peaches, persimmons, grapes, tangerines, persimmons, plums, apricots, and bananas; Flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, and tulips; and feed logistics including ryegrass, red clover, orchard grass, alfalfa, tall fescue, and perennial ryegrass, most preferably Arabidopsis thaliana or pepper, but is not limited thereto.
본 발명의 다른 구현예에서, 본 발명은 CaFAF1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는, 식물체의 건조 스트레스 저항성 증진용 조성물을 제공한다.In another embodiment of the present invention, the present invention provides a composition for enhancing drought stress resistance in plants, comprising an expression or activity inhibitor of CaFAF1 protein as an active ingredient.
상기 억제제는 CaFAF1 유전자의 서열, 상기 서열에 상보적인 서열 또는 상기 유전자 서열의 단편에 대한 mRNA에 상보적으로 결합하여 발현을 억제하는 siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), 리보자임 (ribozyme), DNAzyme, PNA (peptide nucleic acids), 안티센스 뉴클레오티드 중 어느 하나인 것이 바람직하고, CaFAF1 단백질에 상보적으로 결합하여 활성을 억제하는 화합물, 펩티드 (Peptide), 펩티드 미메틱스 (Peptide Minetics), 항체, 또는 앱타머 (aptamer) 중 어느 하나인 것이 바람직하나, 이에 한정되는 것은 아니다.The inhibitor is siRNA (small interference RNA), shRNA (short hairpin RNA), and miRNA (microRNA) that suppresses expression by binding complementary to the sequence of the CaFAF1 gene, a sequence complementary to the sequence, or mRNA for a fragment of the gene sequence. ), ribozyme, DNAzyme, PNA (peptide nucleic acids), or antisense nucleotide, preferably a compound that binds complementary to CaFAF1 protein and inhibits its activity, peptide, peptide mimetics (Peptide Minetics), an antibody, or an aptamer is preferred, but is not limited thereto.
본 발명에 따른 조성물에서, 상기 CaFAF1 단백질은 서열번호 1의 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.In the composition according to the present invention, the CaFAF1 protein may consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
본 발명의 또 다른 구현예에서, 서열번호 1의 아미노산 서열로 이루어진 CaFAF1 단백질의 발현 또는 활성을 억제하는 단계를 포함하는, 식물체의 건조 스트레스 저항성 증진 방법을 제공한다. 상기 CaFAF1 단백질의 발현 또는 활성을 억제하는 단계는 전술한 siRNA, shRNA, miRNA, 리보자임, DNAzyme, PNA, 안티센스 뉴클레오티드, 화합물, 펩티드, 펩티드 미메틱스, 항체, 또는 앱타머 등을 이용하여 제한 없이 이루어질 수 있다.In another embodiment of the present invention, a method for enhancing drought stress resistance in plants is provided, comprising the step of inhibiting the expression or activity of the CaFAF1 protein consisting of the amino acid sequence of SEQ ID NO: 1. The step of inhibiting the expression or activity of the CaFAF1 protein can be carried out without limitation using the above-mentioned siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA, antisense nucleotide, compound, peptide, peptide mimetics, antibody, or aptamer. It can be done.
또한, 본 발명은 본 발명에 따른 CaFAF1 단백질, 이를 암호화하는 CaFAF1 유전자, 및 상기 유전자를 포함하는 재조합 벡터로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 식물체의 염 스트레스 저항성 증진용 조성물을 제공한다.In addition, the present invention provides a composition for enhancing salt stress resistance in plants, comprising as an active ingredient at least one selected from the group consisting of the CaFAF1 protein according to the present invention, the CaFAF1 gene encoding the same, and a recombinant vector containing the gene. .
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.
[실험예][Experimental example]
실험예 1. 식물 재료 및 성장 조건Experimental Example 1. Plant materials and growth conditions
본 발명에서는 고추 (Capsicum annuum L., cv. Nockwang), 애기장대 (Arabidopsis thaliana), 및 담배 (Nicotiana benthamiana)가 사용됐다. 애기장대의 종자는 4℃에서 2일 동안 춘화처리 (vernalization) 후, 수크로스 (sucrose) (Biopure, Cambridge, MA, USA) 1% 및 마이크로 한천 (micro agar) (Duchefa biochemie) 8 %가 포함된 MS 배지 (Murashige and Skoog medium, Duchefa biochemie, Haarlem, The Netherlands)에서 발아시켰다. 고추 종자는 29 ℃의 어두운 곳에서 5일간 발아시켰다. 담배 종자, 애기장대 및 고추 묘목은 증기 소독된 배합토 [피트모스(peat moss):펄라이트(perlite):질석(vermiculite)=5:3:2, v/v/v], 사토(sand), 및 양토 (loam soil) 1:1:1(v/v/v)에서, 24 ℃, 상대 습도 60 % 조건의 백색 형광 (130 μmol photons m-2S-1)하에서 성장시켰다.In the present invention, pepper ( Capsicum annuum L., cv. Nockwang), Arabidopsis thaliana , and tobacco ( Nicotiana benthamiana ) were used. Arabidopsis seeds were vernalized for 2 days at 4°C, then cultured in a mixture containing 1% sucrose (Biopure, Cambridge, MA, USA) and 8% micro agar (Duchefa biochemie). Germination was performed on MS medium (Murashige and Skoog medium, Duchefa biochemie, Haarlem, The Netherlands). Pepper seeds were germinated in a dark place at 29°C for 5 days. Tobacco seeds, Arabidopsis thaliana, and pepper seedlings were grown in steam-sterilized soil mix [peat moss:perlite:vermiculite=5:3:2, v/v/v], sand, and loam. (loam soil) 1:1:1 (v/v/v), grown under white fluorescence (130 μmol photons m-2S-1) at 24°C and 60% relative humidity.
실험예 2. 바이러스-유도성 유전자 침묵 (Virus-induced gene silencing, VIGS)Experimental Example 2. Virus-induced gene silencing (VIGS)
CaFAF1-침묵 고추 식물은 담배얼룩바이러스(Tobacco rattle virus; TRV) 기반 바이러스 유도 유전자 침묵 시스템을 사용하여 생성했다. CaFAF1-cDNA의 299(2-300) bp의 단편을 pTRV2 벡터에 삽입하고 동결-해동 방법 (freeze-thaw method)을 사용하여 아그로박테리움 투메파시엔스 (Agrobacterium tumefaciens) 균주 GV3101에 도입하였다. 음성대조군인 TRV2:00 또는 TRV2:CaFAF1을 운반하는 형질전환된 아그로박테리아를 TRV1과 함께 고추의 완전히 자란 자엽 (fully expanded cotyledons)에 침투시켰다 (각 균주별 OD600=0.2). 식물체들은 생장 및 바이러스가 증식할 수 있도록 16시간 낮/8시간 밤으로 광주기를 설정한 24℃의 생육실에 두었다. CaFAF1 -silenced pepper plants were generated using a Tobacco rattle virus (TRV)-based virus-induced gene silencing system. A 299 (2-300) bp fragment of CaFAF1 -cDNA was inserted into the pTRV2 vector and introduced into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method. As a negative control, TRV2:00 or TRV2:transformed Agrobacteria carrying CaFAF1 were infiltrated into fully expanded cotyledons of pepper together with TRV1 (OD 600 = 0.2 for each strain). Plants were placed in a growth room at 24°C with a photoperiod of 16 hours day/8 hours night to allow growth and virus proliferation.
클로닝, PCR, 및 VIGS에 사용된 프라이머는 하기 표 1에 나타냈다.Primers used for cloning, PCR, and VIGS are shown in Table 1 below.
실험예 3. 세포 내 국소화 (Subcellular localization)Experimental Example 3. Subcellular localization
세포 내에서 CaFAF1 단백질의 위치를 확인하기 위해, 콜리 플라워 모자이크 바이러스 (CaMV) 35S 프로모터 및 녹색 형광 단백질 (GFP) 태그를 포함하는 이원 벡터 p326-GFP에 종결코돈이 없는 CaFAF1 유전자의 암호화 영역을 삽입하여 35S: CaFAF1-GFP 작제물 (construct)을 제조하였다. 이어서 GFP-표지된 CaFAF1를 도입한 Agrobacterium tumefaciens 균주 GV3101을 균주 p19 (1 : 1 비율, OD600 = 0.5)와 혼합하여 유전자 침묵을 방지한 후, 완전히 팽창된 5 주령의 담배잎에 함께 침투시켜, 상기 작제물의 일시적 발현을 유도하였다. 2일 내지 3일 후 공초점 현미경 (510 UV/Vis Meta; Zeiss, Oberkochen, Germany)으로 GFP 신호를 관찰하였다. To determine the localization of the CaFAF1 protein within the cell, the coding region of the CaFAF1 gene without a stop codon was inserted into the binary vector p326-GFP containing the cauliflower mosaic virus (CaMV) 35S promoter and green fluorescent protein (GFP) tag. 35S: CaFAF1-GFP construct was prepared. Subsequently, Agrobacterium tumefaciens strain GV3101, which introduced GFP-tagged CaFAF1 , was mixed with strain p19 (1:1 ratio, OD 600 = 0.5) to prevent gene silencing, and then co-infiltrated into fully expanded 5-week-old tobacco leaves. Transient expression of the construct was induced. After 2 to 3 days, the GFP signal was observed using a confocal microscope (510 UV/Vis Meta; Zeiss, Oberkochen, Germany).
실험예 4. RNA 추출 및 RNA 추출 및 실시간 역전사 중합 효소 연쇄 반응 (q-RT PCR)Experimental Example 4. RNA extraction and real-time reverse transcription polymerase chain reaction (q-RT PCR)
CaFAF1 전사체 발현 수준을 확인하기 위해, Rneasy Mini 키트 (Qiagen, USA)를 사용하여 고추 및 애기장대 잎으로부터 추출한 총 RNA를 사용했다. 모든 시료는 TRI-Reagent®-RNA/DNA/Protein 시약 (Molecular Research Center, USA)을 처리하여 DNA와 단백질을 제거한 후, 2-propanol을 처리하여 RNA를 침전시켰다. 주형을 위한 1 μg 총 RNA는 Synergy HTX Multi-Mode Microplate Reader를 사용하여 정량화 되었다. cDNA는 iScript™ cDNA synthesis kit(Bio-Rad, Hercules, CA, USA) 및 Transcriptor first strand cDNA synthesis kit(Roche)을 사용하여 합성하였다. qRT-PCR 분석을 위해, 상기 방법으로 합성한 cDNA를 iQ™SYBR Green Supermix(Bio-Rad) 및 적절한 프라이머와 함께 FX96 Touch™ Real-Time PCR detection system (Bio-Rad)을 이용하여 증폭시켰다. 내부 대조군으로는 CaACT1 (hot pepper actin1 gene)을 사용하였다. 각 반응은 세 번의 독립적인 실험으로 수행되었다.To determine CaFAF1 transcript expression levels, total RNA extracted from pepper and Arabidopsis leaves using the Rneasy Mini kit (Qiagen, USA) was used. All samples were treated with TRI-Reagent ® -RNA/DNA/Protein reagent (Molecular Research Center, USA) to remove DNA and proteins, and then treated with 2-propanol to precipitate RNA. 1 μg total RNA for template was quantified using Synergy HTX Multi-Mode Microplate Reader. cDNA was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) and Transcriptor first strand cDNA synthesis kit (Roche). For qRT-PCR analysis, the cDNA synthesized by the above method was amplified using the FX96 Touch™ Real-Time PCR detection system (Bio-Rad) with iQ™SYBR Green Supermix (Bio-Rad) and appropriate primers. CaACT1 (hot pepper actin1 gene) was used as an internal control. Each reaction was performed in three independent experiments.
실시예 5. 스트레스 처리Example 5. Stress Treatment
5-1. 만니톨, H5-1. Mannitol, H 22 OO 22 , 및 저온 스트레스 처리, and cold stress treatment
고추 식물에서 비생물학적 스트레스에 따른 CaFAF1 발현 수준을 관찰하기 위해, 탈수를 위해 1차 잎을 떼어내고, 6엽 단계 (six-leaf stage)의 고추 식물에 만니톨 (mannitol), H2O2 또는 저온 (10 ℃) 처리를 하였다. 잎은 각각 0, 1, 3, 6, 9, 12, 또는 24시간 스트레스 처리 후에 수확하였다. To observe the expression level of CaFAF1 according to abiotic stress in pepper plants, primary leaves were removed for dehydration, and six-leaf stage pepper plants were treated with mannitol, H 2 O 2 or low temperature. (10°C) treatment was performed. Leaves were harvested after 0, 1, 3, 6, 9, 12, or 24 hours of stress treatment, respectively.
5-2. ABA, 탈수 스트레스, 가뭄 스트레스 및 NaCl 처리5-2. ABA, dehydration stress, drought stress and NaCl treatment
ABA 처리 전후에 따른 고추 식물에서의 CaFAF1 발현 수준을 관찰하기 위해 6엽 단계의 고추 식물에 특정 농도의 ABA 또는 대조군 용액을 분사한 후 CaFAF1의 발현 변화를 확인하였다.To observe the expression level of CaFAF1 in pepper plants before and after ABA treatment, a specific concentration of ABA or control solution was sprayed on pepper plants at the 6-leaf stage, and then the expression change of CaFAF1 was confirmed.
탈수 스트레스 반응과 CaFAF1의 연관성을 확인하기 위해 3주령의 야생형 식물 및 CaFAF1-과발현 애기장대 식물에서 뿌리를 제거하여 탈수 스트레스를 가한 후 CaFAF1 발현을 확인하였다.To confirm the relationship between dehydration stress response and CaFAF1, roots were removed from 3-week-old wild-type plants and CaFAF1- overexpressing Arabidopsis plants, and CaFAF1 expression was confirmed after applying dehydration stress.
NaCl 및 가뭄 스트레스 처리를 위해, 고추 식물을 특정 농도의 NaCl 용액으로 관개한 후, 상처 나지 않도록 조심스럽게 토양에서 분리하고, 3mm 여과 종이 위에서 두어 탈수시켰다. 각각의 처리 후 적절한 시간에 잎을 회수하고, RNA 분리 및 RT-PCR 분석을 실시하였다.For NaCl and drought stress treatment, pepper plants were irrigated with a NaCl solution of a certain concentration, then separated from the soil carefully to avoid injury, and placed on 3 mm filter paper to dehydrate. Leaves were collected at an appropriate time after each treatment, and RNA isolation and RT-PCR analysis were performed.
가뭄 스트레스는 2주 내지 4주령의 야생형 및 CaFAF1 침묵 고추를 무작위로 심은 후 14일간 급수를 중단하여 가뭄 스트레스를 주고, 2 내지 3일간 재급수하여 식물이 회복하도록 하였다. 수분손실을 측정하기 위해, 1차 잎과 2차 잎을 분리하여 플라스틱 계량 접시에 넣었다. 잎은 상대습도가 40 %인 조건에서 보관되었다. 그 후, 1 시간을 기준으로 0 내지 8 시간의 생중량을 측정하였다.Drought stress was administered by randomly planting 2- to 4-week-old wild-type and CaFAF1- silenced peppers and stopping watering for 14 days, then re-watering for 2 to 3 days to allow the plants to recover. To measure water loss, primary and secondary leaves were separated and placed in a plastic measuring dish. Leaves were stored at 40% relative humidity. Afterwards, the fresh weight was measured from 0 to 8 hours based on 1 hour.
실험예 6. 열화상 분석Experimental Example 6. Thermal image analysis
잎의 온도를 측정하기 위해, 특정 농도의 ABA를 고추와 애기장대에 뿌렸다. 열 영상은 적외선 카메라 (FLIR systems; T420)을 사용하여 얻었으며, 식물의 잎 온도는 FLIR Tools+ ver 5.13 software를 이용해 측정하였다.To measure leaf temperature, specific concentrations of ABA were sprayed on pepper and Arabidopsis thaliana. Thermal images were obtained using an infrared camera (FLIR systems; T420), and plant leaf temperatures were measured using FLIR Tools+ ver 5.13 software.
실험예 7. 기공개도의 생물학적 정량Experimental Example 7. Biological determination of pore opening degree
기공개도(stomata apertures)의 측정을 위해 고추와 애기장대 잎의 배축 표피를 분리하고, 24 ℃에서 2.5시간 동안 기공 개구 용액 (stomatal opening solution, SOS; 50 mM KCl, 10 mM MES-KOH, 10 mM CaCl2, pH 6.15) 위에 띄웠다. 기공 개구된 표피는 24℃에서 2.5시간 동안 ABA (10 및 20 μM)가 없거나 또는 포함된 SOS로 옮겨졌다. 기공 구경을 측정하기 위해 Nikon Eclipse 80i 현미경으로 샘플당 100개의 기공을 무작위로 관찰하고 이미지 J 1.46r 소프트웨어 (http://imagej.nih.gov/ij)를 사용하여 개별 기공의 폭과 길이를 기록했다. 각 실험은 독립적으로 3 번 수행했다.To measure stomata apertures, the hypocotyl epidermis of pepper and Arabidopsis leaves was separated and incubated in stomatal opening solution (SOS; 50 mM KCl, 10 mM MES-KOH, 10 μm) for 2.5 hours at 24°C. mM CaCl 2 , pH 6.15). The pore-opened epidermis was transferred to SOS without or with ABA (10 and 20 μM) for 2.5 hours at 24°C. To measure pore aperture, 100 pores per sample were randomly observed under a Nikon Eclipse 80i microscope and the width and length of individual pores were recorded using Image J 1.46r software (http://imagej.nih.gov/ij). did. Each experiment was performed independently three times.
실시예 8. Example 8. CaFAF1 CaFAF1 유전자가 과발현된 형질전환 애기장대 제조Production of transgenic Arabidopsis thaliana with overexpressed genes
CaFAF1 유전자가 과발현된 형질전환 애기장대 식물을 생산하기 위해, CaFAF1의 cDNA 전장 서열을 pENTR/D-TOPO 벡터 (Invitrogen, Carlsbad, CA, USA)에 클로닝 한 후 pK2GW7에 재클로닝 하였다. 꽃가루 딥 방법 (floral dip method)을 통하여 아그로박테리움 매개 형질전환을 사용하였다. 형질전환된 애기장대를 선택하기 위해, 종자를 25 ug ml-1 포스피노트리신 (phosphinothricin)을 함유하는 MS 고체 배지에서 성장시켰다.To produce transgenic Arabidopsis plants overexpressing the CaFAF1 gene, the full-length cDNA sequence of CaFAF1 was cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA) and then recloned into pK2GW7. Agrobacterium-mediated transformation was used through the pollen dip method. To select transformed Arabidopsis, seeds were grown on MS solid medium containing 25 ug ml -1 phosphinothricin.
실시예 9. 통계적 분석Example 9. Statistical analysis
통계적 분석은 유전형 사이의 유의한 차이를 결정하기 위해서 student's t-test를 이용하여 수행되었다. 데이터는 3회의 독립적인 실험의 평균 ± 표준 오차를 나타내며, P value가 0.05 이하일 때 유의한 수준의 차이로 판단하였다(*P<0.05, ***P<0.001).Statistical analysis was performed using student's t-test to determine significant differences between genotypes. Data represent the mean ± standard error of three independent experiments, and a P value of 0.05 or less was considered a significant difference (* P <0.05, *** P <0.001).
[실시예][Example]
실시예 1. 가뭄 및/또는 고염-반응 관련 유전자 Example 1. Drought and/or high salt-response related genes FANTASTIC FOUR (FAF)FANTASTIC FOUR (FAF) 유전자의 분리 isolation of genes
식물은 고착성 (sessile) 유기체로서, 분열조직 (meristem) 화라성과 관련된 발달 및 성장 반응을 통해 가뭄, 염분, 저온 등을 포함한 환경적 스트레스에 대처한다. 식물-특이적 FANTASTIC FOUR (FAF) 유전자 중 일부는 CLVATA3-WUSCHEL 피드백 루프 (feedback loop)를 조절하여 분열조직 기능의 유지 및 싹 (shoot)의 분열조직 크기를 조절하는 것으로 보고되었다. 이와 같은 배경기술을 바탕으로, FAF 유전자가 비생물학적 스트레스 반응과 관련이 있는지 확인하였다. Plants are sessile organisms and cope with environmental stresses, including drought, salinity, and low temperature, through developmental and growth responses involving meristem florescence. Some of the plant-specific FANTASTIC FOUR (FAF) genes have been reported to regulate the CLVATA3-WUSCHEL feedback loop to maintain meristem function and control shoot meristem size. Based on this background technology, we confirmed whether the FAF gene is related to abiotic stress response.
식물의 FAF 유전자에 의한 비생물학적 스트레스 반응 조절 여부를 확인하기 위해, 본 발명자들은 고추 유전체 CA01g13720, CA01g13730, CA06g22610, 및 CA11g16000로부터 네 가지 FAF 유전자를 분리했다. 웹툴인 SMART (http://smart.embl-heidelberg.de)을 이용해 도메인 분석을 수행한 결과, 상기 유전자들의 예측 단백질은 435 내지 520개 아미노산으로 이루어지며, 중앙 (51 내지 52번 아미노산)에 매우 보존된 FAF 도메인을 포함한다는 것을 확인했다 (도 1). 또한, 애기장대 (Arabidopsis)의 FAF 단백질 서열을 이용한 계통발생 분석 (phylogenetic analysis)을 수행했다. 구체적으로 MEGA-X 소프트웨어를 이용한 이웃-결합 (neighbor-joining) 방법을 통해 애기장대 식물의 상동 단백질로 고추 FAF 단백질의 계통발생 분석을 수행했다. 그 결과, 모든 고추 종의 FAF 단백질이 ABA Co-Receptor1 (AtEAR1)의 Arabidopsis Enhancer와 그룹화되는 것을 확인했다 (도 3). To determine whether abiotic stress responses are regulated by FAF genes in plants, the present inventors isolated four FAF genes from the pepper genomes CA01g13720, CA01g13730, CA06g22610, and CA11g16000 . As a result of domain analysis using the web tool SMART ( http://smart.embl-heidelberg.de ), the predicted proteins of the above genes consist of 435 to 520 amino acids, with a very large amino acid located in the center (amino acids 51 to 52). It was confirmed that it contains a conserved FAF domain (Figure 1). Additionally, phylogenetic analysis was performed using the FAF protein sequence of Arabidopsis. Specifically, phylogenetic analysis of the pepper FAF protein as a homologous protein in Arabidopsis plants was performed using the neighbor-joining method using MEGA-X software. As a result, it was confirmed that FAF proteins from all pepper species were grouped with the Arabidopsis Enhancer of ABA Co-Receptor1 (AtEAR1) (Figure 3).
이어서, 최상부 (apex), 잎, 꽃, 줄기, 및 뿌리를 포함한 식물의 다양한 조직별 CaFAF 유전자 발현 수준을 확인하기 위해, 정량적 역전사 중합효소연쇄반응 (quantitative reverse transcription-PCR, qRT-PCR)을 수행했다. 그 결과, 도 4에 나타낸 바와 같이, CA06g22610 유전자는 실험에 사용한 고추 조직 모두에서 가장 높은 발현을 보인 바, CA06g22610 유전자가 매우 풍부한 FAF 유전자임을 알 수 있었다. Subsequently, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the CaFAF gene expression level in various tissues of the plant, including the apex, leaves, flowers, stems, and roots. did. As a result, as shown in Figure 4, the CA06g22610 gene showed the highest expression in all pepper tissues used in the experiment, indicating that the CA06g22610 gene was a very abundant FAF gene.
다음으로, 가뭄 및 고염 환경에서 고추 잎에서의 CaFAF 유전자의 발현 변화를 관찰했다. 가뭄 또는 고염 (250 mM의 NaCl) 스트레스를 가한 후 FAF 유전자들의 발현 패턴을 비교한 결과 CA06g22610는 가뭄 스트레스 3시간 후 유의한 발현 유도 (induction)를 보였으며, 나머지 3개 유전자는 가뭄 스트레스 1시간 후 발현이 유의하게 증가하였다 (도 5a). 반면, 고염 스트레스의 경우 다른 발현 양상이 나타난 바, 나머지 유전자들의 발현은 고염 스트레스가 처리됨에 따라 발현이 점차 감소한 반면, CA06g22610는 고염 스트레스 처리 후 2시간 동안 발현이 유의하게 증가한 것으로 나타났다 (도 5b). 상기 데이터를 기반으로, 가뭄 및 고염 스트레스 반응 조절인자의 후보로 CA06g22610 (CaFAF1)를 선택하였다.Next, we observed expression changes of the CaFAF gene in pepper leaves under drought and high salt environments. As a result of comparing the expression patterns of FAF genes after applying drought or high salt (250mM NaCl) stress, CA06g22610 showed significant expression induction after 3 hours of drought stress, and the remaining three genes showed significant expression induction after 1 hour of drought stress. Expression increased significantly (Figure 5a). On the other hand, in the case of high-salt stress, a different expression pattern was observed. While the expression of the remaining genes gradually decreased as high-salt stress was treated, the expression of CA06g22610 showed a significant increase for 2 hours after high-salt stress treatment (Figure 5b) . Based on the above data, CA06g22610 (CaFAF1) was selected as a candidate for a regulator of drought and high salt stress responses.
실시예 2. Example 2. CaFAF1 CaFAF1 유전자의 분자적 특성 분석Molecular characterization of genes
CaFAF1 유전자의 조직 특이적 발현을 분석하기 위해, 고추 묘목 (seedlings) 및 성숙한 식물의 다양한 조직으로부터 cDNA를 확보하여 qRT-PCR 분석을 수행했다. 그 결과, 종자를 제외한 모든 조직에서 CaFAF1 유전자의 발현이 관찰됐으며, 특히 직근 (tap root) 및 줄기에서 발현이 높은 것을 확인할 수 있었다 (도 6). To analyze tissue-specific expression of the CaFAF1 gene, cDNA was obtained from various tissues of pepper seedlings and mature plants and qRT-PCR analysis was performed. As a result, expression of the CaFAF1 gene was observed in all tissues except seeds, and expression was particularly high in tap roots and stems (Figure 6).
이어서, 만니톨 (mannitol), 저온, 앱시스산 (abscisic acid; ABA), 및 H2O2를 포함한 다양한 비생물학적 스트레스를 고추 식물에 가한 후 CaFAF1 유전자의 발현 변화를 확인했다. 그 결과, 도 7에 나타낸 바와 같이, 만니톨 처리시 1시간 내에 CaFAF1 유전자 발현이 급격히 감소한 반면, 저온 및 H2O2의 처리는 CaFAF1 전사체 발현을 더욱 유도하는 것으로 나타났다. ABA를 처리한 경우, 3시간 처리했을 때 외에는 유의미한 발현 변화가 관찰되지 않았다.Subsequently, changes in the expression of the CaFAF1 gene were confirmed after applying various abiotic stresses, including mannitol, low temperature, abscisic acid (ABA), and H 2 O 2 , to pepper plants. As a result, as shown in Figure 7, CaFAF1 gene expression was rapidly decreased within 1 hour upon mannitol treatment, while treatment with low temperature and H 2 O 2 appeared to further induce CaFAF1 transcript expression. When treated with ABA, no significant expression changes were observed except when treated for 3 hours.
CaFAF1 단백질의 기능을 알아보기 위해서는 단백질의 세포 내 위치를 파악해야 하므로, 세포 내 국소화 (subcellular localization) 실험을 수행했다. 웹-기반 툴인 WOLF-PSORT를 이용해 분석한 결과, CaFAF1 단백질은 핵 및 세포질에 위치하는 것으로 예측됐다. 특히, GFP-태그된 CaFAF1을 니코티아나 벤타미아나 (Nicotiana benthamiana) 식물의 잎 표피세포에서 일시적으로 발현시켰을 때, GFP 형광 신호는 핵에서의 DAP1 신호와는 중첩되지 않았으며, 세포질에서만 관측됐다 (도 8). 특히, 상기 단백질의 국소화 (localization)는 0.8 M의 만니톨 처리에 의한 표피세포의 원형질분리 유도를 통해 더욱 뒷받침되었다. 원형질 분리가 일어난 세포의 세포벽으로부터 희미해진 GFP 신호를 통해, CaFAF1가 세포질 단백질임을 확인할 수 있었다.In order to investigate the function of the CaFAF1 protein, the intracellular location of the protein must be identified, so a subcellular localization experiment was performed. As a result of analysis using the web-based tool WOLF-PSORT, CaFAF1 protein was predicted to be located in the nucleus and cytoplasm. In particular, when GFP-tagged CaFAF1 was transiently expressed in leaf epidermal cells of Nicotiana benthamiana plants, the GFP fluorescence signal did not overlap with the DAP1 signal in the nucleus and was observed only in the cytoplasm ( Figure 8). In particular, the localization of the protein was further supported by the induction of plasmolysis of epidermal cells by treatment with 0.8 M mannitol. Through the faded GFP signal from the cell wall of the cell where protoplasm separation occurred, it was confirmed that CaFAF1 was a cytoplasmic protein.
실시예 3. Example 3. CaFAF1-CaFAF1- 침묵 고추 식물의 가뭄 스트레스 내성 증진 확인Confirmation of enhanced drought stress tolerance in silent pepper plants
상기 실시예들을 통해 가뭄 및 고염 스트레스가 CaFAF1 유전자의 발현 증가를 유도하는 것을 확인하였으므로, VIGS 실험을 통해 CaFAF1 단백질이 가뭄 및 고염 스트레스에 대해 식물에서 어떻게 반응하는지 관찰하였다. 먼저, CaFAF1 유전자의 발현이 침묵된 고추 식물 (CaFAF1-침묵 고추; TRV2:CaFAF1)을 제조하고, qRT-PCR을 통해 CaFAF1 유전자의 발현 수준을 확인했다. 그 결과, 도 9에 나타낸 바와 같이, 대조군 (TRV2:00)에 비해 CaFAF1-침묵 고추에서 CaFAF1 유전자 발현이 약 50%로 확연히 감소한 것을 확인했다. Since it was confirmed through the above examples that drought and high salt stress induce increased expression of the CaFAF1 gene, we observed how the CaFAF1 protein responds to drought and high salt stress in plants through VIGS experiments. First, pepper plants in which the expression of the CaFAF1 gene was silenced ( CaFAF1- silenced pepper; TRV2: CaFAF1 ) were prepared, and the expression level of the CaFAF1 gene was confirmed through qRT-PCR. As a result, as shown in Figure 9, it was confirmed that CaFAF1 gene expression was significantly reduced by about 50% in CaFAF1- silenced peppers compared to the control group (TRV2:00).
이어서, CaFAF1 유전자 침묵이 고추 식물의 가뭄 반응에 어떤 영향을 미치는지 확인하기 위해 4주령의 대조군 고추 및 CaFAF1-침묵 고추에 14일간 급수를 중단하여 가뭄 스트레스를 가하고, 3일간 재급수하여 각 식물의 표현형 변화를 관찰했다. 도 10에 나타낸 바와 같이, 정상 환경에서는 두 식물간에 표현형 차이가 없었으나, 14일 동안의 가뭄 스트레스를 가했을 때 TRV2:00에 비해 TRV2:CaFAF1의 시들음 정도가 덜한 것을 확인하였다. 이와 같은 표현형 차이는 가뭄 스트레스 후 3일 동안 재급수한 뒤에 더욱 확연해졌다. 재급수 후의 생존율을 계산한 결과, 대조군 식물의 생존율은 26%에 불과한 반면 CaFAF1-침묵 고추의 생존율은 73%에 달했다.Subsequently, to determine how CaFAF1 gene silencing affects the drought response of pepper plants, 4-week-old control peppers and CaFAF1- silenced peppers were subjected to drought stress by stopping watering for 14 days, and then re-watering for 3 days to determine the phenotype of each plant. Changes were observed. As shown in Figure 10, there was no phenotypic difference between the two plants in a normal environment, but when drought stress was applied for 14 days, it was confirmed that the degree of wilting of TRV2: CaFAF1 was less than that of TRV2:00. These phenotypic differences became more pronounced after rewatering for 3 days after drought stress. Calculation of the survival rate after re-watering showed that the survival rate of control plants was only 26%, while the survival rate of CaFAF1- silenced peppers reached 73%.
CaNCED3, CaOSR1, CaRAB18, 및 CaDREB1와 같은 스트레스-관련 마커 유전자들의 발현이 식물체의 가뭄 스트레스 저항과 관련이 있음이 여러 연구를 통해 보고된 바, 고추에서의 CaFAF1 발현 침묵이 상기 유전자들의 발현 변화를 유도하는지 확인했다. qRT-PCR을 통해 대조군 및 CaFAF1-침묵 고추의 CaNCED3, CaOSR1, CaRAB18, 및 CaDREB1 유전자 발현 수준을 확인한 결과, CaNCED3을 제외한 모든 유전자가 대조군에 비해 CaFAF1-침묵 고추에서 발현이 증가한 것으로 나타났다 (도 11). 상기 결과들은 모두 식물에서 CaFAF1 유전자의 발현 침묵이 가뭄 스트레스에 대한 내성을 증진시킨다는 것을 보여준다.Several studies have reported that the expression of stress-related marker genes such as CaNCED3 , CaOSR1 , CaRAB18 , and CaDREB1 are related to drought stress resistance in plants, and silencing CaFAF1 expression in pepper induces changes in the expression of these genes. I checked to see if it worked. The expression levels of CaNCED3 , CaOSR1 , CaRAB18 , and CaDREB1 genes in control and CaFAF1- silenced peppers were confirmed through qRT-PCR, and the results showed that all genes except CaNCED3 showed increased expression in CaFAF1- silenced peppers compared to the control group (Figure 11) . All of the above results show that silencing the expression of the CaFAF1 gene in plants improves tolerance to drought stress.
따라서, CaFAF1-침묵 고추의 가뭄 저항성 증가가 수분 보유 능력의 변화에 의한 것인지 확인하기 위해, 대조군 및 CaFAF1-침묵 고추에서 분리된 잎의 생중량 손실 (fresh weight loss)을 측정했다. 그 결과, 같은 시점에서 CaFAF1-침묵 고추 잎의 생중량은 대조군에 비해 더 높은 것으로 나타난 바, 수분 손실이 적게 일어난 것을 확인할 수 있었다 (도 12). Therefore, to determine whether the increased drought resistance of CaFAF1- silenced peppers was due to changes in water retention capacity, fresh weight loss of leaves isolated from control and CaFAF1- silenced peppers was measured. As a result, at the same time point, the fresh weight of CaFAF1- silenced pepper leaves was higher than that of the control group, confirming that less water loss occurred (FIG. 12).
식물은 증산작용에 의한 수분 손실을 방지하기 위해 기공을 닫는데, 식물 호르몬인 ABA가 기공 개폐를 조절한다. 따라서, 대조군 및 CaFAF1-침묵 고추에 ABA를 처리한 뒤 기공개도를 관찰하였다. 그 결과, ABA를 처리하지 않았을 때에는 대조군 및 CaFAF1-침묵 고추간에 기공 크기에 유의미한 차이가 없었으며, ABA를 처리했을 때 두 식물 모두에서 기공 폐쇄가 정상적으로 일어났다 (도 13 및 도 14). 그러나, ABA를 처리한 뒤 기공 크기를 비교했을 때 대조군에 비해 CaFAF1-침묵 고추 잎의 기공 크기가 현저히 작은 것을 관찰할 수 있었다. Plants close their stomata to prevent water loss due to transpiration, and the plant hormone ABA regulates the opening and closing of stomata. Therefore, the degree of stomatal opening was observed after treating control and CaFAF1- silenced peppers with ABA. As a result, when ABA was not treated, there was no significant difference in pore size between control and CaFAF1- silenced peppers, and when ABA was treated, stomatal closure occurred normally in both plants (Figures 13 and 14). However, when comparing the pore size after ABA treatment, it was observed that the pore size of CaFAF1- silenced pepper leaves was significantly smaller than that of the control.
잎의 기공이 닫히면, 수분 증발에 의한 냉각 효과가 감소하므로 잎 표면 온도가 증가한다. 이에, 대조군 및 CaFAF1-침묵 고추에 ABA를 처리한 뒤 잎의 표면 온도를 관찰했다. 도 15 및 도 16에 나타낸 바와 같이, ABA를 처리하였을 때 두 식물의 잎 표면 온도 모두 급격히 증가하긴 하였으나, CaFAF1-침묵 고추의 잎의 온도가 대조군에 비해 더욱 높은 것을 확인할 수 있었다. When the stomata of the leaf close, the cooling effect of water evaporation is reduced and the leaf surface temperature increases. Therefore, control and CaFAF1- silenced peppers were treated with ABA and the surface temperature of the leaves was observed. As shown in Figures 15 and 16, when ABA was treated, both the leaf surface temperatures of both plants increased rapidly, but the temperature of the leaves of the CaFAF1- silenced pepper was confirmed to be higher than that of the control.
상기 결과들은 CaFAF1 유전자의 침묵이 기공을 통한 증발 수분 손실을 억제하고, 가뭄 스트레스 반응 유전자의 발현을 촉진함으로써 식물의 가뭄 스트레스 저항성을 향상시킨다는 것을 보여준다.The above results show that silencing the CaFAF1 gene improves drought stress resistance in plants by suppressing evaporative water loss through stomata and promoting the expression of drought stress response genes.
실시예 4. Example 4. CaFAF1-CaFAF1- 과발현 애기장대 식물의 가뭄 스트레스 내성 감소 확인Confirmation of decreased drought stress tolerance in overexpressing Arabidopsis plants
다음으로, 가뭄 스트레스와 CaFAF1의 관련성을 추가로 검증하기 위해, 강력한 지속적 콜리플라워 모자이크 바이러스 35S 프로모터 (constitutive cauliflower mosaic virus 35S promoter)를 이용하여 CaFAF1 유전자를 과발현하는 애기장대 식물을 제조하여 (Pro35S:CaFAF1) 실험을 진행했다. CaFAF1-과발현 애기장대 식물의 표현형 분석을 위해, 2가지의 독립적인 T3 동형 접합성 형질전환 자손 (homozygous transgenic progenies)을 선택했다. 도 17은 상기 선택된 두 가지 형질전환체 식물의 CaFAF1 발현 수준을 보여준다.Next, to further verify the relationship between drought stress and CaFAF1, Arabidopsis plants overexpressing the CaFAF1 gene using the strong constitutive cauliflower mosaic virus 35S promoter were prepared ( Pro35S:CaFAF1 ) The experiment was conducted. For phenotypic analysis of CaFAF1- overexpressing Arabidopsis plants, two independent T 3 homozygous transgenic progenies were selected. Figure 17 shows CaFAF1 expression levels of the two selected transformant plants.
정상적인 성장 조건에서는 야생형 식물과 CaFAF1-과발현 애기장대 식물 계통 사이에 유의미한 표현형 차이가 없었다. 따라서, CaFAF1 단백질의 과발현이 가뭄 내성에 어떻게 영향을 미치는지 확인하기 위해, 정상적인 조건에서 3주 동안 배양된 야생형 식물 및 CaFAF1-과발현 애기장대 식물들을 무작위로 배치한 뒤, 14일 동안 급수를 중단하여 가뭄 스트레스를 가하고, 3일 동안 재급수하여 식물의 표현형을 비교관찰 하였다. 그 결과, 도 18에 나타낸 바와 같이, 가뭄 스트레스 환경에서 CaFAF1-과발현 애기장대 식물은 대조군 식물에 비해 더욱 시든 것을 확인하였으며, 재급수했을 때 대조군 식물에 비해 CaFAF1-과발현 애기장대 식물의 성장 재개가 더딘 것을 확인할 수 있었다. 또한, 재급수 후 생존율을 계산했을 때, 대조군 (약 70.03%)에 비해 CaFAF1-과발현 애기장대 식물의 생존율 (약 38.88% 및 약 21.72%)이 현저히 낮은 것을 확인할 수 있었다. Under normal growth conditions, there were no significant phenotypic differences between wild-type and CaFAF1- overexpressing Arabidopsis plant lines. Therefore, to determine how overexpression of CaFAF1 protein affects drought tolerance, wild-type plants and CaFAF1- overexpressing Arabidopsis plants cultured under normal conditions for 3 weeks were randomly placed, and then watering was stopped for 14 days to tolerate drought. Stress was applied and re-watered for 3 days to compare and observe plant phenotypes. As a result, as shown in Figure 18, it was confirmed that in a drought stress environment, CaFAF1- overexpressing Arabidopsis plants withered more than control plants, and when re-watered, growth resumption of CaFAF1- overexpressing Arabidopsis plants was slower than that of control plants. could be confirmed. In addition, when calculating the survival rate after re-watering, it was confirmed that the survival rate of CaFAF1- overexpressing Arabidopsis plants (about 38.88% and about 21.72%) was significantly lower than that of the control group (about 70.03%).
또한, CaFAF1-과발현에 따른 가뭄-반응성 마커 유전자들의 발현 변화를 확인하기 위해, 야생형 식물 및 CaFAF1-과발현 애기장대 식물에 가뭄 스트레스를 가한 후 RAB18, RD29B, NCED3, 및 DREB2A 유전자들의 발현을 비교한 결과, 탈수된 야생형 식물에 비해 탈수된 CaFAF1-과발현 애기장대 식물에서 상기 유전자들의 발현이 감소한 것으로 나타났다 (도 19). In addition, in order to confirm changes in the expression of drought-responsive marker genes due to CaFAF1- overexpression, the expression of RAB18 , RD29B , NCED3 , and DREB2A genes was compared after applying drought stress to wild-type plants and CaFAF1 -overexpressing Arabidopsis plants. , the expression of the above genes was found to be reduced in dehydrated CaFAF1- overexpressing Arabidopsis plants compared to dehydrated wild-type plants (Figure 19).
이어서, 야생형 식물 및 CaFAF1-과발현 애기장대 식물로부터 잎을 분리한 후 잎의 생중량을 측정했다. 도 20에 나타낸 바와 같이, 같은 시점에서 대조군 식물에 비해 CaFAF1-과발현 애기장대 식물에서 분리된 잎의 생중량이 더 낮은 것으로 나타났다. 즉, 이는 CaFAF1-과발현이 더 높은 증발 수분 손실을 유도한다는 것을 시사한다. Subsequently, leaves were separated from wild-type plants and CaFAF1- overexpressing Arabidopsis plants, and the fresh weight of the leaves was measured. As shown in Figure 20, the fresh weight of leaves isolated from CaFAF1- overexpressing Arabidopsis plants was found to be lower compared to control plants at the same time point. In other words, this suggests that CaFAF1- overexpression leads to higher evaporative water loss.
또한, 야생형 식물 및 CaFAF1-과발현 애기장대 식물 잎의 기공개도를 비교하였다. ABA에 의한 기공폐쇄를 관찰하기 위해, 3주령의 야생형 식물 및 CaFAF1-과발현 애기장대 식물로부터 잎 껍질 (leaf peels)을 수확하고, 10 μM의 ABA로 보충된 SOS 버퍼에서 배양한 뒤 기공개도를 비교했다. ABA를 처리하지 않은 식물의 경우 기공 크기에 유의미한 차이가 없었으나, ABA를 처리한 경우, 야생형 식물에 비해 CaFAF1-과발현 애기장대 식물의 잎의 기공 크기가 더욱 큰 것을 확인할 수 있었다 (도 21 및 도 22). Additionally, the stomatal opening degree of leaves of wild-type plants and CaFAF1- overexpressing Arabidopsis plants was compared. To observe ABA-induced stomatal closure, leaf peels were harvested from 3-week-old wild-type plants and CaFAF1- overexpressing Arabidopsis plants, cultured in SOS buffer supplemented with 10 μM ABA, and then stomatal opening degrees were compared. did. In the case of plants not treated with ABA, there was no significant difference in pore size, but in the case of ABA treatment, it was confirmed that the pore size of leaves of CaFAF1- overexpressing Arabidopsis plants was larger than that of wild-type plants (Figures 21 and 21 22).
다음으로, 탈수 조건에서 CaFAF1 과발현에 따른 잎의 온도 변화를 분석하기 위해, 3주령의 야생형 식물 및 CaFAF1-과발현 애기장대 식물에서 뿌리를 제거하여 탈수 스트레스를 가하였다. 도 23에 나타낸 바와 같이, 탈수 스트레스를 가하기 전에는 야생형 식물 및 CaFAF1-과발현 애기장대 식물의 잎의 온도에 유의미한 차이가 없었다. 그러나 가뭄 스트레스 처리 15분 후, CaFAF1-과발현 애기장대 식물은 야생형 식물에 비해 잎 온도가 낮은 것으로 나타났다. Next, to analyze leaf temperature changes due to CaFAF1 overexpression under dehydration conditions, roots were removed from 3-week-old wild-type plants and CaFAF1- overexpressing Arabidopsis plants and subjected to dehydration stress. As shown in Figure 23, there was no significant difference in the temperature of leaves of wild-type plants and CaFAF1- overexpressing Arabidopsis plants before applying dehydration stress. However, after 15 minutes of drought stress treatment, CaFAF1- overexpressing Arabidopsis plants showed lower leaf temperatures compared to wild-type plants.
상기 결과들은 CaFAF1이 ABA-매개성 기공폐쇄 및 가뭄 반응 마커 유전자의 발현을 조절하여 애기장대 식물의 가뭄 스트레스에 대한 저항성을 음성 조절 (negative regulation)한다는 것을 시사한다.The above results suggest that CaFAF1 negatively regulates the resistance to drought stress in Arabidopsis plants by regulating ABA-mediated stomatal closure and the expression of drought response marker genes.
실시예 5. Example 5. CaFAF1-CaFAF1- 침묵 고추 식물의 염분 스트레스 내성 감소 확인Confirmation of reduced salt stress tolerance in silent pepper plants
가뭄 및 염분 스트레스에 대한 식물의 반응 매커니즘은 대부분 중복된다. 상기 실시예에서 CaFAF1이 가뭄 스트레스 내성의 음성 조절인자임을 확인하였으므로, CaFAF1이 염분 스트레스에 대한 식물 반응에도 관여하는지 확인하였다. Plant response mechanisms to drought and salt stress largely overlap. Since CaFAF1 was confirmed to be a negative regulator of drought stress tolerance in the above example, it was confirmed whether CaFAF1 is also involved in plant response to salt stress.
먼저, 4주령의 대조군 및 CaFAF1-침묵 고추 식물을 소금 용액에서 3일간 배양하여 염분 스트레스를 가한 후 표현형을 관찰했다. 도 24에 나타낸 바와 같이, 고염 환경에서 대조군에 비해 CaFAF1-침묵 고추 식물이 더 많이 시든 것으로 나타났다. First, 4-week-old control and CaFAF1- silenced pepper plants were cultured in salt solution for 3 days and their phenotypes were observed after applying salt stress. As shown in Figure 24, more CaFAF1- silenced pepper plants wilted compared to the control group in a high-salt environment.
분자 수준 및 생리적 수준에서 가뭄 스트레스 내성에 대한 CaFAF1의 기능을 구체적으로 살펴보기 위해, CaFAF1-침묵에 따른 스트레스 관련 유전자 CaDREB1, CaOSR1, CaP5CS, 및 CaRAB18 유전자의 발현 변화를 확인했다. 도 25에 나타낸 바와 같이, 고염 스트레스를 처리하였을 때 상기 유전자들의 발현은 대조군에 비해 CaFAF1-침묵 고추에서 감소되어 있었다.To specifically examine the function of CaFAF1 in drought stress tolerance at the molecular and physiological levels, changes in the expression of stress-related genes CaDREB1 , CaOSR1 , CaP5CS , and CaRAB18 genes following CaFAF1- silencing were confirmed. As shown in Figure 25, when treated with high salt stress, the expression of the above genes was reduced in CaFAF1- silenced peppers compared to the control group.
염분 스트레스는 전해질 누출 (electrolyte leakage) 및 지질 과산화 (lipid peroxidation)을 증가시키는 것으로 알려져 있다. 따라서 CaFAF1 유전자 침묵이 상기 두 가지 현상을 유도하는지 확인하기 위해, 4주령의 대조군 및 CaFAF1-침묵 고추 식물을 200 mM의 NaCl 용액에서 20시간 배양한 후 전해질 누출 정도 및 지질 과산화 정도를 확인했다. 먼저 전해질 누출을 비교한 결과, 고염 스트레스를 가했을 때 두 식물 계통 모두에서 전해질 누출이 증가하긴 했으나, 대조군 식물에 비해 CaFAF1-침묵 고추 식물에서 전해질 누출이 더욱 증가한 것을 확인할 수 있었다 (도 26). 또한, 지질 과산화에 의한 생성물인 말론알데히드 (malondialdehyde, MDA) 함량 역시 대조군에 비해 CaFAF1-침묵 고추 식물에서 더욱 증가한 것으로 나타났다 (도 27). Salt stress is known to increase electrolyte leakage and lipid peroxidation. Therefore, to confirm whether CaFAF1 gene silencing induces the above two phenomena, 4-week-old control and CaFAF1- silenced pepper plants were cultured in a 200 mM NaCl solution for 20 hours, and then the degree of electrolyte leakage and lipid peroxidation were checked. First, as a result of comparing electrolyte leakage, it was confirmed that electrolyte leakage increased in both plant lines when high salt stress was applied, but electrolyte leakage increased more in CaFAF1- silenced pepper plants compared to control plants (FIG. 26). In addition, the content of malondialdehyde (MDA), a product of lipid peroxidation, was also found to be more increased in CaFAF1- silenced pepper plants compared to the control group (FIG. 27).
또한, 염분 스트레스는 프롤린 (proline) 생합성에 관여하는 유전자들의 발현을 촉진하여 프롤린 축적을 유도하는 것으로 알려져 있다. CaFAF1-침묵 고추 식물에서 CaP5CS 유전자의 발현이 증가한 사실을 기초로, 대조군 및 CaFAF1-침묵 고추 식물에 염분 스트레스를 20시간 동안 가한 후, 두 식물에서 프롤린 함량을 비교했다. 그 결과, 도 28에 나타낸 바와 같이, 소금 스트레스에 의해 두 식물 모두에서 프롤린 함량이 증가하였으나, 대조군에 비해 CaFAF1-침묵 고추에서 더욱 증가한 것을 확인할 수 있었다. In addition, salt stress is known to induce proline accumulation by promoting the expression of genes involved in proline biosynthesis. Based on the fact that the expression of the CaP5CS gene was increased in CaFAF1- silenced pepper plants, control and CaFAF1- silenced pepper plants were subjected to salt stress for 20 hours and the proline content in the two plants was compared. As a result, as shown in Figure 28, the proline content increased in both plants due to salt stress, but it was confirmed that the proline content increased further in the CaFAF1- silenced pepper compared to the control group.
상기 결과들은 CaFAF1의 높은 발현은 스트레스 관련 유전자의 발현 및 세포막 안정성을 조절함으로써 염분 스트레스에 대한 식물체의 내성을 향상시킨다는 것을 시사한다.The above results suggest that high expression of CaFAF1 improves plant tolerance to salt stress by regulating the expression of stress-related genes and cell membrane stability.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다. The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.
<110> CHUNG-ANG UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> CaFAF1 genes and Method for improving the resistance to the drought and salt stress using CaFAF1 in plants <130> MP21-114 <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> CaFAF1_amino acid sequence <400> 1 Met Ser Ala Ala Asn Ser Met Ser Ile Asn Asn Phe Asn Ser Thr Leu 1 5 10 15 Lys Met Glu Lys Glu Ala Thr Thr Gln Ser Ile Pro Leu Pro Pro Thr 20 25 30 Lys Met Gly Ile Val Lys Gln Gly Ile Val Ser Ile Leu Gly Ser Asp 35 40 45 Ser Asp Lys Arg Asn Lys Ala Ala Ala Ala Val Ser Ile Arg Arg Thr 50 55 60 Leu Ser Ala Asp Met Ser Ser Lys Lys Trp Leu Ser Gln Asn Gly Phe 65 70 75 80 Thr Pro Ile Lys Lys Ile Ala Ser Ser Lys Glu Leu Asp Val Leu Gly 85 90 95 Gln Asp Asp Val Trp Lys Ser Ile Gln Asn Gly Thr Asn Lys Ala Glu 100 105 110 Pro Thr Ser Leu Asp Val Trp Ser Ser Ile Leu Thr Gln Lys Lys Gln 115 120 125 Glu Glu Ser Ser Thr Leu Pro Pro Pro Tyr Val His Pro Leu Leu Lys 130 135 140 Lys Ser Ser Ser Ser Leu Thr Glu Lys Ser Leu Glu Ile Cys Thr Glu 145 150 155 160 Ser Leu Gly Ser Glu Thr Gly Ser Glu Ile Ser Leu Ser Ser Tyr Pro 165 170 175 Pro Ser Asp Thr Glu Glu Asp Lys Asp Asp His Pro Gln Lys Gln Gln 180 185 190 Gln Glu Glu Lys Gln Glu Leu Ser Phe Gln Ser Phe Glu Glu Phe Pro 195 200 205 Val Ile Lys Phe Asn His Asn Lys Lys Met Ser Ser Pro Pro Lys Ser 210 215 220 Phe Pro Pro Pro Ile Ser Ser Leu Ser Ala Glu Asp Asn Lys Pro Ser 225 230 235 240 Val His Met Gln Ser Arg Arg Gln Asn Gly Arg Leu Ile Val Glu Ala 245 250 255 Val Ser Val Pro Pro Gln Asn His Phe Arg Ser Arg Arg Val Glu Gly 260 265 270 Arg Leu Val Leu Thr Leu Ile Thr Thr Ser Ser Glu Gly Ile His Gln 275 280 285 Gln Val Ala Glu Phe Glu Gln Ala Phe Asp Asp Phe Gln Glu Val Asp 290 295 300 His Asn Tyr Asp Asp Glu Val Glu Asp Lys Gly Asn Gly Thr Lys Gly 305 310 315 320 Met Gln Ile Val Leu Glu Gln Lys Pro Arg Leu Ser Asn Gly Arg Ile 325 330 335 Asn Val Asn Thr Thr Thr Leu Met Met Lys Gln Leu Met Gly Gly Leu 340 345 350 Glu Lys Lys Asn His His Ile Thr Trp Pro Lys Lys Phe Asn Lys Ser 355 360 365 Ile Lys Leu Ile Glu Glu Glu Asn Glu Ile Thr Pro Ile Pro Gln Ser 370 375 380 Leu Pro Gln Ile Ser Gln Leu Ile Thr Ser Pro Asn Pro Pro Glu Ala 385 390 395 400 Ser Ser Phe Asn Ala Cys Asp Tyr Phe Trp Lys Lys Asn Pro Thr Val 405 410 415 Thr Thr Ile Val Asn Ser Tyr Asn Asp Lys Ser Thr Thr Lys Lys Ala 420 425 430 Ser Tyr Glu Gln Gln Asp Leu Val Leu Met Arg Gly Asn Lys Gly Asp 435 440 445 Ile Asn Tyr Leu Val Pro Leu Leu Lys Gly Cys Lys Glu Gln Arg Arg 450 455 460 Ser Leu Leu Ile Trp Glu Pro Tyr Cys Ile Ala Thr Ser 465 470 475 <210> 2 <211> 1434 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_nucleotide sequence <400> 2 atgtcagcag ctaattcaat gagcatcaac aacttcaact caacattgaa gatggaaaag 60 gaggcaacaa cacaaagtat cccactaccc cccactaaga tggggatagt gaaacaagga 120 atagtgtcca ttcttggatc agactctgac aaaagaaaca aggctgctgc agctgtttca 180 attaggagaa ccctttcagc tgatatgtca tccaagaaat ggctatcaca aaatggtttc 240 acccctatca agaagattgc ttcctcaaaa gaacttgatg ttcttggaca agatgatgtt 300 tggaaatcaa tccaaaatgg tactaacaaa gctgagccaa catcacttga tgtttggagc 360 tcaattttaa cacaaaagaa acaagaggag tcttccacac ttccacctcc ttatgtccac 420 cctctcttga aaaaatcatc tagctcttta actgaaaaaa gtcttgaaat ttgtaccgaa 480 agtcttggat cagaaactgg ttctgagatt agcctctctt cttaccctcc ttctgacact 540 gaggaagaca aagatgatca tcctcaaaaa caacaacaag aagaaaaaca agagttgtcc 600 tttcaatcgt ttgaggaatt tccagttatt aagtttaacc acaacaaaaa aatgtcatct 660 cctcccaagt ctttccctcc accaatttct tccttgtctg cagaagacaa caagccctct 720 gttcatatgc aatctcgcag acagaatggt aggttgattg ttgaagctgt atctgttcct 780 cctcagaacc attttcgctc tcggcgcgtt gaaggtcgcc ttgttctcac cttaatcacc 840 acttcatcag agggcattca tcaacaagtg gcagagttcg agcaggcctt cgacgatttt 900 caagaagttg atcacaacta tgatgatgaa gtggaggata aaggaaatgg gacaaaaggg 960 atgcaaattg tgcttgagca aaaaccaaga ttgtcaaatg ggaggataaa tgtgaacaca 1020 acaacgttga tgatgaagca gctcatggga ggtctagaga aaaagaatca tcatataaca 1080 tggcctaaaa agttcaacaa atcaatcaaa ttgattgaag aggagaatga aatcacccca 1140 attcctcaat cacttccaca aatctctcaa ctcatcacat caccaaatcc acctgaagca 1200 tcttctttca atgcctgtga ctatttctgg aagaaaaatc caactgtgac gaccattgtc 1260 aatagctaca atgacaaaag tacaacaaag aaagcatcat atgaacagca agatttggtc 1320 ctaatgaggg gaaacaaagg ggacatcaat tatttggtgc ctttgttgaa aggatgcaag 1380 gaacaaagga ggtctctact catttgggag ccatattgca ttgccacctc ctaa 1434 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_cloning primer_FW <400> 3 atgactcttc tcactcctcc tccga 25 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_cloning primer_RV <400> 4 ttaggaggtg gcaatgcaat atg 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_cloning primer_w/o stop codon <400> 5 ggaggtggca atgcaatatg 20 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_RT-PCR primer_FW <400> 6 atcatcatat aacatggcct aaaaa 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_RT-PCR primer_RV <400> 7 ttcatatgat gctttctttg ttgta 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaACT1_RT-PCR primer_FW <400> 8 gacgtgacct aactgataac ctgat 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaACT1_RT-PCR primer_RV <400> 9 ctctcagcac caatggtaat aactt 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaNCED3_RT-PCR primer_FW <400> 10 ttaaggatct taagcgtggt tatgt 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaNCED3_RT-PCR primer_RV <400> 11 agattagttc aagaacgtga attgg 25 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaOSR1_RT-PCR primer_FW <400> 12 atggaggcac aactgcaccg tc 22 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaOSR1_RT-PCR primer_RV <400> 13 ggcccaccat gaacttctgc ac 22 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CaRAB18_RT-PCR primer_FW <400> 14 atgtcgcact acgagaacca atatag 26 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CaRAB18_RT-PCR primer_RV <400> 15 atcatcctca gagctgctgg agc 23 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaDREB1_RT-PCR primer_FW <400> 16 ggaatttcaa aagaatcatc tagca 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaDREB1_RT-PCR primer_RV <400> 17 agctagaaga gtactgcatc caaaa 25 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CaP5CS_RT-PCR primer_FW <400> 18 acaagattta gtgatgggtt ccgc 24 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CaP5CS_RT-PCR primer_RV <400> 19 gctgaaacac ttgtaatccc aggata 26 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13720_RT-PCR primer_FW <400> 20 gaacagagaa aatatgaacc atttg 25 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13720_RT-PCR primer_RV <400> 21 tgatctctct ctttgctatt tgatt 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13730_RT-PCR primer_FW <400> 22 gttcttgcat ccaatagtac ttctc 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13730_RT-PCR primer_RV <400> 23 gccaaaaaga acaaaaatat aacaa 25 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA11g16000_RT-PCR primer_FW <400> 24 attcaatgct tatgagtact tttgg 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA11g16000_RT-PCR primer_RV <400> 25 gagaggaaaa tcatattggt aatga 25 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AtActin8_RT-PCR primer_FW <400> 26 caactatgtt ctcaggtatt gcaga 25 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AtActin8_RT-PCR primer_RV <400> 27 gtcatggaaa cgatgtctct ttagt 25 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NCED3_RT-PCR primer_FW <400> 28 acatggaaat cggagttaca gatag 25 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NCED3_RT-PCR primer_RV <400> 29 agaaacaaca aacaagaaac agagc 25 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RAB18_RT-PCR primer_FW <400> 30 ggaagaaggg aataacacaa aagat 25 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RAB18_RT-PCR primer_RV <400> 31 gcgttacaaa ccctcattat tttta 25 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RD29B_RT-PCR primer_FW <400> 32 gttgaagagt ctccacaatc acttg 25 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RD29B_RT-PCR primer_RV <400> 33 atacaaatcc ccaaactgaa taaca 25 <210> 34 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> DREB2A_RT-PCR primer_FW <400> 34 ctacaaagcc tcaactacgg aatac 25 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> DREB2A_RT-PCR primer_RV <400> 35 aaactcggat agagaatcaa cagtc 25 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Xbal-CaFAF1_VIGS primer_FW <400> 36 tctagaaaca tcatcttgtc caagaacat 29 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Xhol-CaFAF1_VIGS primer_RV <400> 37 ctcgagtgtc agcagctaat tcaatg 26 <110> CHUNG-ANG UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> CaFAF1 genes and Method for improving the resistance to the drought and salt stress using CaFAF1 in plants <130> MP21-114 <160> 37 <170> KoPatentIn 3.0 <210> 1 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> CaFAF1_amino acid sequence <400> 1 Met Ser Ala Ala Asn Ser Met Ser Ile Asn Asn Phe Asn Ser Thr Leu 1 5 10 15 Lys Met Glu Lys Glu Ala Thr Thr Gln Ser Ile Pro Leu Pro Pro Thr 20 25 30 Lys Met Gly Ile Val Lys Gln Gly Ile Val Ser Ile Leu Gly Ser Asp 35 40 45 Ser Asp Lys Arg Asn Lys Ala Ala Ala Ala Val Ser Ile Arg Arg Thr 50 55 60 Leu Ser Ala Asp Met Ser Ser Lys Lys Trp Leu Ser Gln Asn Gly Phe 65 70 75 80 Thr Pro Ile Lys Lys Ile Ala Ser Ser Lys Glu Leu Asp Val Leu Gly 85 90 95 Gln Asp Asp Val Trp Lys Ser Ile Gln Asn Gly Thr Asn Lys Ala Glu 100 105 110 Pro Thr Ser Leu Asp Val Trp Ser Ser Ile Leu Thr Gln Lys Lys Gln 115 120 125 Glu Glu Ser Ser Thr Leu Pro Pro Pro Tyr Val His Pro Leu Leu Lys 130 135 140 Lys Ser Ser Ser Ser Leu Thr Glu Lys Ser Leu Glu Ile Cys Thr Glu 145 150 155 160 Ser Leu Gly Ser Glu Thr Gly Ser Glu Ile Ser Leu Ser Ser Tyr Pro 165 170 175 Pro Ser Asp Thr Glu Glu Asp Lys Asp Asp His Pro Gln Lys Gln Gln 180 185 190 Gln Glu Glu Lys Gln Glu Leu Ser Phe Gln Ser Phe Glu Glu Phe Pro 195 200 205 Val Ile Lys Phe Asn His Asn Lys Lys Met Ser Ser Pro Pro Lys Ser 210 215 220 Phe Pro Pro Pro Ile Ser Ser Leu Ser Ala Glu Asp Asn Lys Pro Ser 225 230 235 240 Val His Met Gln Ser Arg Arg Gln Asn Gly Arg Leu Ile Val Glu Ala 245 250 255 Val Ser Val Pro Pro Gln Asn His Phe Arg Ser Arg Arg Val Glu Gly 260 265 270 Arg Leu Val Leu Thr Leu Ile Thr Thr Ser Ser Glu Gly Ile His Gln 275 280 285 Gln Val Ala Glu Phe Glu Gln Ala Phe Asp Asp Phe Gln Glu Val Asp 290 295 300 His Asn Tyr Asp Asp Glu Val Glu Asp Lys Gly Asn Gly Thr Lys Gly 305 310 315 320 Met Gln Ile Val Leu Glu Gln Lys Pro Arg Leu Ser Asn Gly Arg Ile 325 330 335 Asn Val Asn Thr Thr Thr Leu Met Met Lys Gln Leu Met Gly Gly Leu 340 345 350 Glu Lys Lys Asn His His Ile Thr Trp Pro Lys Lys Phe Asn Lys Ser 355 360 365 Ile Lys Leu Ile Glu Glu Glu Asn Glu Ile Thr Pro Ile Pro Gln Ser 370 375 380 Leu Pro Gln Ile Ser Gln Leu Ile Thr Ser Pro Asn Pro Pro Glu Ala 385 390 395 400 Ser Ser Phe Asn Ala Cys Asp Tyr Phe Trp Lys Lys Asn Pro Thr Val 405 410 415 Thr Thr Ile Val Asn Ser Tyr Asn Asp Lys Ser Thr Thr Lys Lys Ala 420 425 430 Ser Tyr Glu Gln Gln Asp Leu Val Leu Met Arg Gly Asn Lys Gly Asp 435 440 445 Ile Asn Tyr Leu Val Pro Leu Leu Lys Gly Cys Lys Glu Gln Arg Arg 450 455 460 Ser Leu Leu Ile Trp Glu Pro Tyr Cys Ile Ala Thr Ser 465 470 475 <210> 2 <211> 1434 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_nucleotide sequence <400> 2 atgtcagcag ctaattcaat gagcatcaac aacttcaact caacattgaa gatggaaaag 60 gaggcaacaa cacaaagtat cccactaccc cccactaaga tggggatagt gaaacaagga 120 atagtgtcca ttcttggatc agactctgac aaaagaaaca aggctgctgc agctgtttca 180 attaggagaa ccctttcagc tgatatgtca tccaagaaat ggctatcaca aaatggtttc 240 acccctatca agaagatgc ttcctcaaaa gaacttgatg ttcttggaca agatgatgtt 300 tggaaatcaa tccaaaatgg tactaacaaa gctgagccaa catcacttga tgtttggagc 360 tcaattttaa cacaaaagaa acaagaggag tcttccacac ttccacctcc ttatgtccac 420 cctctcttga aaaaatcatc tagctcttta actgaaaaaaa gtcttgaaat ttgtaccgaa 480 agtcttggat cagaaactgg ttctgagatt agcctctctt cttaccctcc ttctgacact 540 gaggaagaca aagatgatca tcctcaaaaa caacaacaag aagaaaaaca agagttgtcc 600 tttcaatcgt ttgaggaatt tccagttatt aagtttaacc acaacaaaaaa aatgtcatct 660 cctcccaagt ctttccctcc accaatttct tccttgtctg cagaagacaa caagccctct 720 gttcatatgc aatctcgcag acagaatggt aggttgattg ttgaagctgt atctgttcct 780 cctcagaacc attttcgctc tcggcgcgtt gaaggtcgcc ttgttctcac cttaatcacc 840 acttcatcag agggcattca tcaacaagtg gcagagttcg agcaggcctt cgacgatttt 900 caagaagttg atcacaacta tgatgatgaa gtggaggata aaggaaatgg gacaaaaggg 960 atgcaaattg tgcttgagca aaaaccaaga ttgtcaaatg ggaggataaa tgtgaacaca 1020 acaacgttga tgatgaagca gctcatggga ggtctagaga aaaagaatca tcatataaca 1080 tggcctaaaa agttcaacaa atcaatcaaa ttgattgaag aggagaatga aatcacccca 1140 attcctcaat cacttccaca aatctctcaa ctcatcacat caccaaatcc acctgaagca 1200 tcttctttca atgcctgtga ctatttctgg aagaaaaatc caactgtgac gaccattgtc 1260 aatagctaca atgacaaaag tacaacaaag aaagcatcat atgaacagca agatttggtc 1320 ctaatgaggg gaaacaaagg ggacatcaat tatttggtgc ctttgttgaa aggatgcaag 1380 gaacaaagga ggtctctact catttgggag ccatattgca ttgccacctc ctaa 1434 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_cloning primer_FW <400> 3 atgactcttc tcactcctcc tccga 25 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_cloning primer_RV <400> 4 ttaggaggtg gcaatgcaat atg 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_cloning primer_w/o stop codon <400> 5 ggaggtggca atgcaatatg 20 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_RT-PCR primer_FW <400> 6 atcatcatat aacatggcct aaaaa 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaFAF1_RT-PCR primer_RV <400> 7 ttcatatgat gctttctttg ttgta 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaACT1_RT-PCR primer_FW <400> 8 gacgtgacct aactgataac ctgat 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaACT1_RT-PCR primer_RV <400> 9 ctctcagcac caatggtaat aactt 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaNCED3_RT-PCR primer_FW <400> 10 ttaaggatct taagcgtggt tatgt 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaNCED3_RT-PCR primer_RV <400> 11 agattagttc aagaacgtga attgg 25 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaOSR1_RT-PCR primer_FW <400> 12 atggaggcac aactgcaccg tc 22 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaOSR1_RT-PCR primer_RV <400> 13 ggccccaccat gaacttctgc ac 22 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CaRAB18_RT-PCR primer_FW <400> 14 atgtcgcact acgagaacca atatag 26 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CaRAB18_RT-PCR primer_RV <400> 15 atcatcctca gagctgctgg agc 23 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaDREB1_RT-PCR primer_FW <400> 16 ggaatttcaa aagaatcatc tagca 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaDREB1_RT-PCR primer_RV <400> 17 agctagaaga gtactgcatc caaaa 25 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CaP5CS_RT-PCR primer_FW <400> 18 acaagattta gtgatgggtt ccgc 24 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CaP5CS_RT-PCR primer_RV <400> 19 gctgaaacac ttgtaatccc aggata 26 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13720_RT-PCR primer_FW <400> 20 gaacagagaa aatatgaacc atttg 25 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13720_RT-PCR primer_RV <400> 21 tgatctctct ctttgctatt tgatt 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13730_RT-PCR primer_FW <400> 22 gttcttgcat ccaatagtac ttctc 25 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA01g13730_RT-PCR primer_RV <400> 23 gccaaaaaga acaaaaatat aacaa 25 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA11g16000_RT-PCR primer_FW <400> 24 attcaatgct tatgagtact tttgg 25 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CA11g16000_RT-PCR primer_RV <400> 25 gagaggaaaa tcatattggt aatga 25 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AtActin8_RT-PCR primer_FW <400> 26 caactatgtt ctcaggtatt gcaga 25 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AtActin8_RT-PCR primer_RV <400> 27 gtcatggaaaa cgatgtctct ttagt 25 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NCED3_RT-PCR primer_FW <400> 28 acatggaaat cggagttaca gatag 25 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NCED3_RT-PCR primer_RV <400> 29 agaaaacaaca aacaagaaac agagc 25 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RAB18_RT-PCR primer_FW <400>30 ggaagaaggg aataacacaa aagat 25 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RAB18_RT-PCR primer_RV <400> 31 gcgttacaaa ccctcattat tttta 25 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RD29B_RT-PCR primer_FW <400> 32 gttgaagagt ctccacaatc acttg 25 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RD29B_RT-PCR primer_RV <400> 33 atacaaatcc ccaaactgaa taaca 25 <210> 34 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> DREB2A_RT-PCR primer_FW <400> 34 ctacaaagcc tcaactacgg aatac 25 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> DREB2A_RT-PCR primer_RV <400> 35 aaactcggat agagaatcaa cagtc 25 <210> 36 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Xbal-CaFAF1_VIGS primer_FW <400> 36 tctagaaaca tcatcttgtc caagaacat 29 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Xhol-CaFAF1_VIGS primer_RV <400> 37 ctcgagtgtc agcagctaat tcaatg 26
Claims (15)
상기 발현 억제제는 CaFAF1 유전자의 서열, 상기 CaFAF1 유전자의 서열에 상보적인 서열, 또는 상기 CaFAF1 유전자 서열의 단편에 대한 mRNA에 상보적으로 결합하여 발현을 억제하는 siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), 리보자임 (ribozyme), DNAzyme, PNA (peptide nucleic acids), 및 안티센스 뉴클레오티드 중 어느 하나인 것인, 식물체의 건조 스트레스 저항성 증진용 조성물.
A composition for improving dry stress resistance in plants, comprising as an active ingredient an expression inhibitor of the CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) protein consisting of the amino acid sequence of SEQ ID NO: 1,
The expression inhibitor is siRNA (small interference RNA), shRNA (short hairpin) that binds complementary to the sequence of the CaFAF1 gene, a sequence complementary to the sequence of the CaFAF1 gene, or mRNA for a fragment of the CaFAF1 gene sequence to inhibit expression. A composition for enhancing dry stress resistance in plants, which is any one of RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids), and antisense nucleotide.
상기 CaFAF1 단백질은 서열번호 2의 염기서열로 이루어진 CaFAF1 유전자로 암호화되는 것인, 식물체의 건조 스트레스 저항성 증진용 조성물.
According to paragraph 1,
A composition for enhancing dry stress resistance in plants, wherein the CaFAF1 protein is encoded by the CaFAF1 gene consisting of the base sequence of SEQ ID NO: 2.
상기 CaFAF1 유전자는 고추로부터 분리된 것인, 식물체의 건조 스트레스 저항성 증진용 조성물.
According to paragraph 3,
A composition for enhancing dry stress resistance in plants, wherein the CaFAF1 gene is isolated from pepper.
A method for enhancing drought stress resistance in plants, comprising the step of inhibiting the expression of CaFAF1 protein consisting of the amino acid sequence of SEQ ID NO: 1 by treating the composition of claim 1.
A transgenic plant with improved drought stress resistance by the method of claim 8.
상기 형질전환 식물체는 고추인 것을 특징으로 하는, 형질전환 식물체.
According to clause 9,
A transgenic plant, characterized in that the transgenic plant is a pepper.
A plant containing at least one selected from the group consisting of a CaFAF1 ( Capsicum annuum FANTASTIC FOUR 1) protein consisting of the amino acid sequence of SEQ ID NO: 1, a CaFAF1 gene encoding the CaFAF1 protein, and a recombinant vector containing the CaFAF1 gene as an active ingredient. Composition for improving salt stress resistance.
(S1) 서열번호 1의 아미노산 서열로 이루어진 CaFAF1 단백질을 암호화하는 유전자를 식물체에 형질전환 하는 단계; 및
(S2) 상기 형질전환된 식물체에서 CaFAF1 단백질을 과발현시키는 단계.
A method for enhancing salt stress resistance in plants, comprising the following steps:
(S1) Transforming a plant with a gene encoding the CaFAF1 protein consisting of the amino acid sequence of SEQ ID NO: 1; and
(S2) Step of overexpressing CaFAF1 protein in the transformed plant.
A transgenic plant with improved salt stress resistance by the method of claim 14.
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