KR20000012621A - Vectors having the Genes of Superoxide Dismutase and Ascorbate Peroxidase, and Transformed Plants by the Vector - Google Patents

Vectors having the Genes of Superoxide Dismutase and Ascorbate Peroxidase, and Transformed Plants by the Vector Download PDF

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KR20000012621A
KR20000012621A KR1019990057774A KR19990057774A KR20000012621A KR 20000012621 A KR20000012621 A KR 20000012621A KR 1019990057774 A KR1019990057774 A KR 1019990057774A KR 19990057774 A KR19990057774 A KR 19990057774A KR 20000012621 A KR20000012621 A KR 20000012621A
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plants
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superoxide dismutase
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곽상수
권석윤
이행순
랜디알렌
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복성해
생명공학연구소
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    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

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Abstract

PURPOSE: The aim is to provide transformed plant having resistance of environmental stress by using vector containing superoxide dismutase(SOD) and as corbate peroxidase(APX). CONSTITUTION: For practicing example of preparing vector, cDNA of MnSOD and APX is prepared and inserted in between 35S promoter and 35S terminator of cauliflower mosaic virus(pCGN1578). It is cut with HindIII to produce about 1.7kb-1.5kb of DNA fragments. pGPTV-Bar is used as transforming vector for double transformation. GUS gene of pGPTV-Bar is removed by using EcoR1 and SmaI and the fragment containing SOD and APX is inserted into the vector. Plasmid DNA of the prepared vector is introduced to Agrobaterium tumefaciens(LBA4404).

Description

슈퍼옥사이드 디스뮤타제 및 아스코르베이트 퍼옥시다제 유전자를 동시에 함유하는 벡터 및 그에 의해 형질전환된 식물체{Vectors having the Genes of Superoxide Dismutase and Ascorbate Peroxidase, and Transformed Plants by the Vector}Vectors having the Genes of Superoxide Dismutase and Ascorbate Peroxidase, and Transformed Plants by the Vector} Simultaneously Containing Superoxide Dismutase and Ascorbate Peroxidase Genes

본 발명은 슈퍼옥사이드 디스뮤타제(superoxide dismutase, SOD)와 아스코르베이트 퍼옥시다제(ascorbate peroxidase, APX)를 이용한 환경스트레스 내성식물의 개발에 관한 것으로, 상세하게는 완두에서 분리한 슈퍼옥사이드 디스뮤타제의 cDNA와 아스코르베이트 퍼옥시다제의 cDNA를 식물체의 엽록체에서 과발현되게 조작한 식물형질전환 벡터, 상기 벡터로 형질전환되어 슈퍼옥사이드 디스뮤타제와 아스코르베이트 퍼옥시다제가 과발현됨으로써 산화적인 스트레스(oxidative stress)를 야기하는 파라킷(paraquat, methyl viologen)과 중금속 카드늄에 대한 내성이 있는, 산화적인 스트레스에 저항성을 가진 식물체에 관한 것이다.The present invention relates to the development of environmental stress resistant plants using superoxide dismutase (SOD) and ascorbate peroxidase (APX), specifically, superoxide dismux isolated from peas. Oxidative stress due to overexpression of superoxide dismutase and ascorbate peroxidase by transforming the cDNA of the other agent and the cDNA of ascorbate peroxidase into the plant transformation vector overengineered in the chloroplasts of the plant. It is about plants that are resistant to oxidative stress, which is resistant to paraquat (methyl viologen) and heavy metal cadmium causing oxidative stress.

슈퍼옥사이드 디스뮤타제는 슈퍼옥사이드 음이온 래디칼(O2 -.)을 과산화수소 (H2O2)로 전환시키는 효소로서, 포함하고 있는 보족금속원소에 따라 CuZnSOD, MnSOD 및 FeSOD로 구분된다. 이들은 종류에 따라 세포내에서의 존재위치가 달라, CuZnSOD는 세포질과 엽록체에, MnSOD는 미토콘드리아에, FeSOD는 엽록체에 각각 존재한다.Superoxide dismutase is an enzyme that converts superoxide anion radicals (O 2- . ) To hydrogen peroxide (H 2 O 2 ), and is classified into CuZnSOD, MnSOD and FeSOD depending on the containing metal element. Depending on the type, they exist in the cell, and CuZnSOD is present in the cytoplasm and chloroplasts, MnSOD is present in the mitochondria, and FeSOD is present in the chloroplasts.

아스코르베이트 퍼옥시다제는 아스코르베이트를 전자공여체로 사용하여 H2O2를 물로 전환시키는 효소로서 식물체와 곤충에서 발견되며, 식물체에서는 세포질, 엽록체의 스트로마 및 틸라코이드막에 존재하는 것으로 밝혀져 있다.Ascorbate peroxidase is an enzyme that converts H 2 O 2 into water using ascorbate as an electron donor. It is found in plants and insects, and it has been found in plants to be present in the cytoplasm, chloroplast stromal and thylakoid membranes.

식물체의 엽록체에서는 비교적 높은 농도의 산소가 상존하며, 빛에너지를 이용하여 물을 분해하는 과정에서 발생되는 전자의 에너지를 활용하기 위한 전자전달계가 존재하고 있어 다양한 산화적 스트레스에 민감하게 반응한다. 따라서 엽록체의 항산화능력을 높일 수 있다면 환경스트레스 조건에서도 식물의 생산성을 유지시킬 수 있을 것으로 기대된다.In the chloroplasts of plants, relatively high concentrations of oxygen are present and electron transfer systems exist to utilize the energy of electrons generated in the process of decomposing water by using light energy, which is sensitive to various oxidative stresses. Therefore, if the antioxidant capacity of the chloroplast can be increased, it is expected that the productivity of the plant can be maintained even under environmental stress conditions.

항산화효소, 특히 슈퍼옥사이드 디스뮤타제 또는 아스코르베이트 퍼옥시다제를 이용한 형질전환 식물체를 개발하고 산화스트레스에 대한 저항성을 조사한 보고(Free Rad. Biol. Med. 23: 473-479)가 있으나 엽록체에서 슈퍼옥사이드 음이온 래디칼과 과산화수소를 효과적으로 제거하는데 기여하는 SOD와 APX를 동시에 발현시킨 보고는 없다. CuZnSOD, MnSOD 및 FeSOD를 도입한 형질전환 식물체는 파라킷에 의한 산화적 스트레스와 광저해(photoinhibition)에 저항성을 가지며, APX를 도입한 형질전환 식물체도 산화적 스트레스 및 광저해에 대한 저항성을 가지는 것으로 보고되고 있다.There have been reports of the development of transgenic plants using antioxidant enzymes, particularly superoxide dismutase or ascorbate peroxidase, and their resistance to oxidative stress (Free Rad. Biol. Med. 23: 473-479). There are no reports of simultaneous expression of SOD and APX, which contribute to the effective removal of superoxide anion radicals and hydrogen peroxide. Transgenic plants with CuZnSOD, MnSOD and FeSOD are resistant to oxidative stress and photoinhibition by parakites, and transgenic plants with APX also have resistance to oxidative stress and photoinhibition. Is being reported.

생체 내에 존재하는 슈퍼옥사이드 음이온 래디칼과 과산화수소는 Harber-Weiss반응에 의해 가장 강한 활성 산소종인 수산화래디칼(·OH)을 생성한다. SOD와 퍼옥시다제는 이러한 활성산소종의 제거와 수산화래디칼의 예방에 관여하는 항산화효소이다. 따라서 SOD와 APX를 엽록체에 동시에 발현시키는 것은 SOD 또는 APX의 단독발현의 효과보다 더욱 효율적일 것으로 기대된다.Superoxide anion radicals and hydrogen peroxide present in the living body produce the strongest active radicals (· OH) by the Harber-Weiss reaction. SOD and peroxidase are antioxidant enzymes involved in the removal of these reactive oxygen species and the prevention of hydroxyl radicals. Therefore, simultaneous expression of SOD and APX in chloroplasts is expected to be more efficient than the effect of SOD or APX alone expression.

본 발명의 목적은 식물체에 환경스트레스에 대한 저항성을 부여하기 위해 슈퍼옥사이드 디스뮤타제 및 아스코르베이트 퍼옥시다제를 엽록체에 동시 발현시킨 식물체를 개발하고자 하는 것이다.An object of the present invention is to develop a plant in which superoxide dismutase and ascorbate peroxidase are co-expressed in chloroplasts in order to give the plant resistance to environmental stress.

도 1은 본 발명에 의한 형질전환 식물체 및 비 형질전환 식물체에서 SOD 동위효소의 native gel 분석사진 및 활성도 그래프.1 is a native gel analysis and activity graph of SOD isozymes in transgenic plants and non-transgenic plants according to the present invention.

도 2는 본 발명에 의한 형질전환 식물체 및 비 형질전환 식물체에서 APX 동위효소의 native gel 분석사진 및 활성도 그래프.Figure 2 is a native gel analysis and activity graph of APX isoenzyme in transgenic plants and non-transgenic plants according to the present invention.

도 3은 담배 잎 절편세포의 파라킷에 의한 스트레스 내성을 조사한 그래프.Figure 3 is a graph of stress resistance by parakitet of tobacco leaf section cells.

도 4는 담배 식물체의 파라킷에 의한 스트레스 내성을 조사한 그래프.Figure 4 is a graph examining the stress resistance by the parakitets of tobacco plants.

도 5는 파라킷 처리에 의한 형질전환식물체 잎의 내성을 보여주는 사진.Figure 5 is a photograph showing the resistance of the transgenic plant leaves by parakit treatment.

도 6은 본 발명에 의한 형질전환 식물체의 카드뮴 내성을 보여주는 그래프.Figure 6 is a graph showing the cadmium resistance of transgenic plants according to the present invention.

도 7은 본 발명에 의한 형질전환식물체의 카드늄 내성을 보여주는 사진.Figure 7 is a photograph showing the cadmium resistance of the transformed plant according to the present invention.

상기와 같은 목적을 달성하기 위하여 본 발명은 슈퍼옥사이드 디스뮤타제 및 아스코르베이트 퍼옥시다제를 엽록체에 동시 발현시킬 수 있는 벡터 및 상기 벡터로 형질전환되어 산화적 스트레스에 저항성을 나타내는 형질전환 식물체를 제공한다.In order to achieve the above object, the present invention provides a vector capable of co-expressing superoxide dismutase and ascorbate peroxidase on chloroplasts and transformed plants transformed with the vector and resistant to oxidative stress. to provide.

본 발명에서 상기 슈퍼옥사이드 디스뮤타제 및 아스코르베이트 퍼옥시다제는 완두에서 얻었으며, 형질전환의 대상이 되는 식물체는 담배로 결정하였다.In the present invention, the superoxide dismutase and ascorbate peroxidase were obtained from peas, and the plant to be transformed was determined to be tobacco.

먼저, 환경스트레스에 대한 저항성 담배를 개발하기 위하여, 완두에서 분리한 슈퍼옥사이드 디스뮤타제(CuZnSOD 및 MnSOD)의 유전자 및 아스코르베이트 퍼옥시다제 (APX)의 유전자를 조작하여 합성된 단백질이 엽록체로 이동하도록 식물 형질전환벡터를 제작한다.First, in order to develop tobacco resistant to environmental stress, a protein synthesized by manipulating the genes of superoxide dismutases (CuZnSOD and MnSOD) and ascorbate peroxidase (APX) isolated from peas was converted into chloroplasts. Construct plant transformation vectors to move.

형질전환 식물체를 제작하는 순서는 ① 카나마이신에 저항성을 갖고 있는 담배에 슈퍼옥사이드 디스뮤타제(CuZnSOD, MnSOD)를 형질전환시키고 이어서 상기 형질전환 담배에 바스타를 선별표지로 하여 아스코르베이트 퍼옥시다제를 재도입하거나, 반대로 ② 카나마이신에 대한 저항성을 가진 담배에 아스코르베이트 퍼옥시다제를 먼저 형질전환 시킨 뒤 바스타를 선별표지로 사용하여 슈퍼옥사이드 디스뮤타제를 재도입한다. 이러한 방식에 의해 이중형질전환 담배 식물체가 제작된다.The procedure for producing a transgenic plant is to transform superoxide dismutase (CuZnSOD, MnSOD) into tobacco which is resistant to kanamycin, and then ascorbate peroxidase is selected by using a bacter as a marker. Reintroduce or reverse ② Ascorbate peroxidase to tobacco with resistance to kanamycin, and then superoxide dismutase is reintroduced using Vaster as a screening label. In this way a bitransformed tobacco plant is produced.

이렇게 제직된 단일 또는 이중 형질전환 담배 식물체를 Native gel 분석하면 도입된 CuZnSOD 및 MnSOD의 가 나타나며, 본 발명에서 제작한 이중형질전환체는 대조식물보다 SOD 비활성도(units/mg protein)가 수 배 증가한다.Native gel analysis of the weaved single or double transgenic tobacco plants resulted in the introduction of CuZnSOD and MnSOD, and the double transformants prepared in the present invention had several times more SOD activity (units / mg protein) than the control plants. do.

본 발명에 의한 이중 형질전환 식물체의 잎 절편을 파라킷이 함유된 용액에 저리하는 경우 용액의 전기전도도는 비 형질전환 식물체보다 47%에서 82%가 감소한 값을 나타내는 바, 이는 SOD 및 APX가 동시에 과발현되어 산화 스트레스 환경에 내성을 나타낸 결과이다. 또한 식물체에 다양한 농도의 파라킷을 처리하는 경우, SOD 및 APX가 이중발현하는 식물체의 잎의 가시적인 손상이 비형질전환 식물체의 손상보다 매우 적게 나타나므로 본 발명에 의한 이중 형질전환 식물체가 산화적 스트레스에 보다 저항성이 있는 것을 알 수 있다.When leaf fragments of a double transgenic plant according to the present invention are stored in a solution containing a parakit, the electrical conductivity of the solution is 47% to 82% lower than that of the non-transgenic plant, which means that SOD and APX simultaneously Overexpression results in resistance to oxidative stress environments. In addition, when the plants are treated with various concentrations of parakites, since the visible damage of the leaves of the plants dual expression of SOD and APX appears much less than that of the non-transformed plants, the double transgenic plants according to the present invention are oxidative. It can be seen that it is more resistant to stress.

나아가, 본 발명에서 개발된 형질전환 식물체는 중금속 카드늄(cadmium)에 내성이 있는 것으로 밝혀져, 엽록체에 과발현된 SOD와 APX는 중금속에 의해 유도되는 산화적 스트레스를 경감시키는 효과가 있음을 알 수 있다.Furthermore, the transgenic plants developed in the present invention were found to be resistant to heavy metal cadmium, and SOD and APX overexpressed in the chloroplasts have an effect of reducing oxidative stress induced by heavy metals.

이하, 본 발명을 하기 실시예에 의거하여 좀더 상세하게 설명한다. 하기 실시예는 본 발명을 설명하기 위한 예시일 뿐, 본 발명의 범위가 이들 만으로 제한되는 것은 아니며, 이에 의해 본 발명의 기술적 사상의 범위가 변경되거나 축소되지 않는다. 또한 유전자 조작법은 정립되어 알려져 있기 때문에 하기 실시예에서는 유전자 조작을 위한 구체적인 조건이나 방법의 기재를 생략한다.Hereinafter, the present invention will be described in more detail based on the following examples. The following examples are only examples for describing the present invention, and the scope of the present invention is not limited thereto, and the scope of the technical idea of the present invention is not changed or reduced. In addition, since genetic engineering methods are known and well-known, the following example omits description of specific conditions or methods for genetic manipulation.

실시예 1 : 이중 형질전환을 위한 형질전환 벡터의 제작Example 1 Preparation of Transformation Vector for Double Transformation

합성된 단백질을 엽록체로 이동시킬 수 있는 신호(signal)를 가지고 있는 형태로 하기의 모든 형질전환용 벡터를 제작하였다.All the following transformation vectors were prepared in a form having a signal capable of transferring the synthesized protein to chloroplasts.

CuZnSOD가 엽록체에 과발현된 담배종자는 Sen Gupta 등(Plant Physiol 103:988-991, 1993)에 의해 개발된 것을, MnSOD가 엽록체에 과발현된 담배종자는 Schake(MS thesis, Texas Tech University, 1995)가 개발한 것을 각각 사용하였다. APX가 엽록체에 과발현된 담배종자는 Webb과 Allen(Plant Physiol Suppl 108:64, 1995)이 개발한 것을 사용하였다. 형질전환에 사용되는 MnSOD와 APX는 모두 완두에서 분리한 것으로 상기 MnSOD 및 APX 과발현 담배의 개발에 사용한 것과 동일하다.Tobacco seeds overexpressing CuZnSOD on chloroplasts were developed by Sen Gupta et al. (Plant Physiol 103: 988-991, 1993). Tobacco seeds overexpressing MnSOD on chloroplasts were described by Schake (MS thesis, Texas Tech University, 1995). Each developed was used. Tobacco seeds overexpressed APX in chloroplasts were those developed by Webb and Allen (Plant Physiol Suppl 108: 64, 1995). MnSOD and APX used for transformation are all isolated from pea and are the same as those used for the development of MnSOD and APX overexpressing tobacco.

먼저 MnSOD와 APX의 cDNA를 제작하여 pCGN1578에 콜리플라워 모자이크 바이러스의 35S 프로모터 및 35S 터미네이터 사이에 삽입하였다. 이를 HindIII로 절단하여 프로모터에서 터미네이터에 이르는 전장 각각 약 1.7kb 및 1.5kb인 DNA 절편을 얻었다.First, cDNAs of MnSOD and APX were prepared and inserted into pCGN1578 between the 35S promoter and 35S terminator of cauliflower mosaic virus. This was digested with HindIII to obtain DNA fragments of about 1.7 kb and 1.5 kb, respectively, from the promoter to the terminator.

이중 형질전환을 위해 사용한 형질전환용 벡터는 바스타를 선별표지로 한 pGPTV-Bar를 사용하였다. pGTPV-Bar의 GUS 유전자를 EcoRI 및 SmaI으로 제거하고, 앞서 얻은 아스코르베이트 퍼옥시다제 및 슈퍼옥사이드 디스뮤타제를 포함하는 절편을 삽입하였다. 이렇게 제작된 벡터의 플라스미드 DNA를 아그로박테리움 (Agrobacterium tumefaciens LBA4404)에 도입하였다.As a transforming vector used for double transformation, pGPTV-Bar was used as a selection label of Vasta. The GUS gene of pGTPV-Bar was removed with EcoRI and SmaI, and a fragment containing the ascorbate peroxidase and superoxide dismutase obtained above was inserted. The plasmid DNA of the thus prepared vector was introduced into Agrobacterium (Agrobacterium tumefaciens LBA4404).

실시예 2 : 식물체의 형질전환Example 2 Transformation of Plants

실시예 1에서 제작한 형질전환용 벡터(를 함유하는 상기 아그로박테리움)를 이용하여 CuZnSOD가 발현하는 식물체에는 아스코르베이트 퍼옥시다제가, APX가 발현되는 식물체에는 MnSOD가 각각 도입되도록 형질전환하였다.Ascorbate peroxidase was expressed in plants expressing CuZnSOD and MnSOD was introduced in plants expressing APX using the transformation vector prepared in Example 1 (containing the Agrobacterium). .

CuZnSOD 및 APX가 발현되는 식물체는 카나마이신에 대한 저항성을 가지고 있기 때문에 2mg/L의 바이알라포스(biolaphos)를 포함하는 배지에서 선별하면서 형빌전환식물체를 유도하였다.Since plants expressing CuZnSOD and APX have resistance to kanamycin, the transformed plants were induced by selection in a medium containing 2 mg / L of biolaphos.

CuZnSOD 및 APX가 동시에 발현되는 식물체는 CA로, APX 및 MnSOD가 동시에 발현되는 식물체는 AM로 각각 명명하였다. 또한 CA 식물체에 AM을 수분시켜 얻은 것을 C/A 식물체, AM에 CA를 수분시켜 얻은 A/C 식물체로 명명하였는데, 이들 2대째 식물체는 두 종류의 SOD(CuZnSOD 및 MnSOD)와 APX가 동시에 엽록체에서 발현되는 식물체들이다.Plants in which CuZnSOD and APX are simultaneously expressed are named CA, and plants in which APX and MnSOD are simultaneously expressed are named AM. The A / C plants obtained by polluting AM in CA plants were named C / A plants, and the A / C plants obtained by polluting CA in AM. These two generations of plants have two types of SODs (CuZnSOD and MnSOD) and APX simultaneously in chloroplasts. Plants that are expressed.

실시예 3 : 질전환식물체에서 SOD 및 APX의 활성분석Example 3 Analysis of SOD and APX Activity in Transgenic Plants

실시예 2에서 제작한 형질전환 식물체에서 SOD 및 APX의 활성을 확인하기 위해 담배잎 절편을 재료로 이용하여 각 효소의 native gel분석과 활성을 조사하였다. SOD의 native gel 분석은 Bowler 등의 방법(EMBO J 10: 1723-1732, 1991)을, 효소활성은 McCord와 Fridovich의 방법(J Biol Chem 244: 6049-6055, 1969)을 적용하였다. APX의 native gel 분석은 Mittler와 Zilinskas의 방법(Plant J 5: 397-405, 1994)을, 효소활성은 Nakano와 Asada의 방법(Plant Cell Physiol 22: 867-880, 1981)을 이용하였다.In order to confirm the activity of SOD and APX in the transformed plant prepared in Example 2, the native gel analysis and activity of each enzyme were investigated using tobacco leaf slices as a material. For native gel analysis of SOD, Bowler et al. (EMBO J 10: 1723-1732, 1991) and McCord and Fridovich's method (J Biol Chem 244: 6049-6055, 1969) were applied. Native gel analysis of APX was performed by Mittler and Zilinskas (Plant J 5: 397-405, 1994), and enzymatic activity by Nakano and Asada (Plant Cell Physiol 22: 867-880, 1981).

도 1은 도입된 SOD를 확인하기 위한 native gel 분석사진과 효소활성을 조사하여 그래프로 나타낸 것이다. 본 발명에서 제작한 CA, AM, C/A 및 A/C 식물체에서 CuZnSOD 또는 MnSOD의 밴드가 확인되었다(도 1의 A). 또한 비 형질전환 담배에 비하여 CA식물체는 1.15배, AM식물체는 2.0배, C/A식물체는 2.4배, 그리고 A/C식물체는 2.8배의 SOD 비활성이 증가하였다(도 1의 B).1 is a graph showing the native gel assay and enzyme activity to check the introduced SOD. A band of CuZnSOD or MnSOD was identified in CA, AM, C / A and A / C plants produced in the present invention (A of FIG. 1). In addition, compared to non-transforming tobacco, CA plants increased 1.15 times, AM plants 2.0 times, C / A plants 2.4 times, and A / C plants 2.8 times increased SOD inactivation (B in FIG. 1).

도 2는 도입된 APX를 확인하기 위한 native gel 분석사진과 효소활성을 조사하여 그래프로 나타낸 것이다. 본 발명에 의한 CA, AM, C/A 및 A/C식물체에 CuZnSOD 또는 MnSOD의 밴드가 확인되었다(도 2의 A). 또한 비형질전환 담배에 비하여 CA식물체에서 7.6배, AM식물체에서 3.9배, C/A 식물체에서 3.6배, 그리고 A/C 식물체에서 3.0배의 APX 비활성 증가가 나타났다(도 2의 B).Figure 2 shows the graph by examining the native gel assay and enzyme activity for identifying the introduced APX. The band of CuZnSOD or MnSOD was confirmed in CA, AM, C / A and A / C plants according to the present invention (A of FIG. 2). In addition, APX inactivation was increased by 7.6 times in CA plants, 3.9 times in AM plants, 3.6 times in C / A plants, and 3.0 times in A / C plants compared to non-transformed tobacco (FIG. 2B).

실시예 4 : 형질전환식물체의 산화적 스트레스에 대한 내성 조사Example 4 Investigation of Resistance to Oxidative Stress of Transgenic Plants

담배 잎 절편을 이용하여 산화적 스트레스에 대한 내성을 조사하여 도 3에 나타내었다.Tobacco leaf slices were examined for resistance to oxidative stress and shown in FIG. 3.

담배 잎 절편(지름 7mm)을 2μM 또는 5μM의 파라킷(methyl viologen)을 포함하는 0.4M sortibol 용액에 띄우고 12시간동안 암처리하여 파라킷이 조직에 흡수될 수 있도록 한 후, 24시간 및 48시간 동안 빛을 쪼여주면서 전도도계(Orion, Model 162)를 이용하여 용액의 전도도를 측정하였다. 또한 48시간 후 잎 절편과 용액을 autoclave하여 세포내의 내용물을 완전히 용액으로 유출시킨 다음 동일한 방법으로 용액의 전도도를 측정하였다. Autoclave한 후의 전도도 값과 24시간 및 48시간 처리한 후의 전도도를 비교하여 % ion leakage 값을 구하였다. 도 3의A는 2μM 파라킷을 처리한 결과로, 24시간 후 비형질전환식물체에 대한 형질전환식물체의 상대적인 이온유출정도는 CA 식물체의 경우 58%, AM 식물체의 경우 81%, C/A 식물체의 경우 52% 그리고 A/C 식물체의 경우 18%이었다. 48시간 후 CA 식물체의 경우 50%, AM 식물체의 경우 56%, C/A 식물체의 경우 37% 그리고 A/C의 경우 32%이었다. 도 3의 B는 5μM의 파라킷을 처리한 결과로, 24시간 후 비 형질전환 식물체에 대한 형질전환식물체의 상대적인 이온유출정도는 CA 식물체의 경우 97%, AM 식물체의 경우 86%, C/A 식물체의 경우 85% 그리고 A/C 식물체의 경우 42%이었다. 48시간 후 CA 식물체의 경우 96%, AM 식물체의 경우 86%, C/A 식물체의 경우 77% 그리고 A/C의 경우 59%이었다.Tobacco leaf slices (7 mm in diameter) were floated in a 0.4 M sortibol solution containing 2 μM or 5 μM parakit (methyl viologen) and dark treated for 12 hours to allow the parakit to be absorbed into the tissue, followed by 24 and 48 hours. The conductivity of the solution was measured using a conductivity meter (Orion, Model 162) while irradiating light. After 48 hours, the leaf sections and the solution were autoclaved to completely drain the contents of the cells into the solution, and the conductivity of the solution was measured in the same manner. The% ion leakage value was obtained by comparing the conductivity values after autoclave with those after 24 and 48 hours treatment. 3A shows the result of treatment of 2 μM parakit, the relative ion release rate of the transformed plants to non-transformed plants after 24 hours was 58% for CA plants, 81% for AM plants, and C / A plants. 52% for A / C plants and 18% for A / C plants. After 48 hours, they were 50% for CA plants, 56% for AM plants, 37% for C / A plants and 32% for A / C. FIG. 3B shows the results of treatment of 5 μM parakites. The relative ion release rate of the transgenic plants to non-transgenic plants after 24 hours was 97% for CA plants, 86% for AM plants, and C / A. 85% for plants and 42% for A / C plants. After 48 hours, they were 96% for CA plants, 86% for AM plants, 77% for C / A plants, and 59% for A / C.

실시예 5 : 식물체 수준에서 파라킷에 의한 산화적 스트레스에 대한 저항성 조사Example 5 Investigation of Resistance to Oxidative Stress by Parakites at the Plant Level

3-4 엽기의 식물체에 25μM, 50μM 그리고 100μM의 파라킷을 분무하고 3일 후 잎에 나타나는 내성정도와, 파라킷 처리한 식물체에 나타난 손상을 보여주는 사진을 각각 도 4와 5에 도시하였다.Figures 4 and 5 show photographs showing the resistance of the parakited plants and the degree of resistance to the leaves after spraying 25 μM, 50 μM and 100 μM parakites on 3-4 foliar plants after 3 days.

25μM의 파라킷을 처리한 경우 비 형질전환 식물체 및 SOD 또는 APX가 단독발현되는 식물체들은 잎의 3.7-33%가 손상을 받은 반면, 이중 형질전환 식물체(CA, AM, C/A, A/C 식물체)에서는 잎의 1.7-2.7%만이 손상되었다. 50μM 파라킷을 처리한 경우 비 형질전환 식물체 및 SOD 또는 APX가 단독발현되는 식물체들은 잎의 17-57%가 손상을 받은 반면 이중 형질전환 식물체에서는 잎의 5.3-10%에 손상이 나타났다. 100μM의 파라킷을 처리한 경우 비 형질전환 식물체 및 SOD 또는 APX가 단독발현되는 식물체들은 잎의 60-82%가 손상을 받은 반면 이중 형질전환 식물체에서는 잎의 21-38%에 손상이 나타났다.Non-transformed plants and plants that express SOD or APX alone when treated with 25 μM parakited damaged 3.7-33% of the leaves, whereas double transgenic plants (CA, AM, C / A, A / C). Plants only damaged 1.7-2.7% of the leaves. Treatment with 50 μM parakit showed that 17-57% of the leaves were damaged in non-transformed and SOD or APX-only plants, whereas 5.3-10% of the leaves in double transgenic plants. Non-transformed plants and plants with SOD or APX expression alone were damaged in 100 μM parakites, while in 21-38% of the leaves in double transgenic plants.

실시예 6 : 식물체 수준에서 카드뮴에 의한 산화적 스트레스에 대한 저항성 조사Example 6: Investigation of resistance to oxidative stress by cadmium at plant level

도 6은 본 발명에 의한 형질전환 식물체와 비 형질전환 식물체에 카드뮴이 첨가된 용액을 가한 후 성장도를 조사한 것이다.Figure 6 shows the growth after the addition of a solution added cadmium to the transgenic plants and non-transgenic plants according to the present invention.

도 6의 A에서 볼 수 있듯이 카드뮴이 없는 경우 양자간의 성장(식물체 길이)에는 차이가 거의 없었으나 50μM Cd이 가해진 경우 비 형질전환 식물체는 성장이 50% 정도 감소되는 반면에 본 발명에 의한 형질전환 식물체는 오히려 50% 정도의 성장증가가 관찰되었다. 50μM Cd 존재하에서 형질전환 식물체 및 비 형질전환 식물체의 생중량 및 건중량을 조사하였다(도 6의 B). 생중량은 양자간에 어떤 유의관계가 발견되지 않고 거의 유사한 정도를 나타내지만, 건조중량은 형질전환 식물체가 비 형질전환 식물체에 비해 약 2~5배에 이른다. 따라서 전체적으로 본 발명에 의한 형질전환 식물체는 카드뮴에 대하여 상당한 내성이 있음을 알 수 있다.As shown in FIG. 6A, in the absence of cadmium, there was almost no difference in the growth (plant length) between the two, but when 50 μM Cd was added, the non-transgenic plants reduced the growth by 50%, whereas the transformation according to the present invention. Plant growth was observed by 50%. The live and dry weights of the transgenic and non transgenic plants were examined in the presence of 50 μM Cd (FIG. 6B). Raw weight shows almost similar level without any significant relationship between them, but dry weight is about 2 to 5 times higher than that of non-transgenic plants. Therefore, it can be seen that the transgenic plants according to the present invention have considerable resistance to cadmium.

이상과 같이, 본 발명에서는 산화적 스트레스에 보다 저항성을 갖는 식물체를 개발하였으며, 특히 SOD와 APX를 동시에 엽록체에서 발현시킴으로써 환경스트레스에 저항성을 갖는 식물체를 개발할 수 있었다. 따라서 이러한 SOD 및 APX를 엽록체에서 동시에 발현시키는 방법은 점차 악화되고 있는 환경에서 생산성을 유지할 수 있는 식물체의 개발에 유용하게 이용될 수 있을 것으로 기대된다.As described above, in the present invention, plants having more resistance to oxidative stress were developed, and in particular, plants having resistance to environmental stress could be developed by simultaneously expressing SOD and APX in chloroplasts. Therefore, the method of simultaneously expressing the SOD and APX in the chloroplasts is expected to be useful for the development of plants that can maintain productivity in an increasingly worsening environment.

Claims (4)

슈퍼옥사이드 디스뮤타제 및 아스코르베이트 퍼옥시다제 유전자를 동시에 함유하며, 엽록체에서 발현되는 것을 특징으로 하는 벡터.A vector containing both superoxide dismutase and ascorbate peroxidase genes, wherein the vector is expressed in the chloroplast. 슈퍼옥사이드 디스뮤타제 및 아스코르베이트 퍼옥시다제 유전자를 동시에 함유하며, 엽록체에서 발현되는 것을 특징으로 하는 벡터로 형질전환되어 산화적 스트레스에 저항성을 나타내는 것을 특징으로 하는 형질전환 식물체.A transgenic plant containing both superoxide dismutase and ascorbate peroxidase genes, transformed into a vector characterized in that it is expressed in the chloroplast and exhibiting resistance to oxidative stress. 슈퍼옥사이드 디스뮤테이즈 및 아스코르베이트 퍼옥시다제 유전자를 동시에 함유하며, 엽록체에서 발현되는 벡터의 제조방법.A method for producing a vector containing both superoxide dismutase and ascorbate peroxidase genes, which is expressed in chloroplasts. 슈퍼옥사이드 디스뮤타제 및 아스코르베이트 퍼옥시다제 유전자를 동시에 함유하며, 엽록체에서 발현되는 것을 특징으로 하는 벡터로 형질전환되어 산화적 스트레스에 저항성을 나타내는 것을 특징으로 하는 형질전환 식물체의 제조방법.A method for producing a transgenic plant, characterized in that it contains a superoxide dismutase and ascorbate peroxidase gene simultaneously, and is transformed into a vector characterized by being expressed in chloroplasts to exhibit resistance to oxidative stress.
KR1019990057774A 1999-12-15 1999-12-15 Vectors having the Genes of Superoxide Dismutase and Ascorbate Peroxidase, and Transformed Plants by the Vector KR20000012621A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006054815A1 (en) * 2004-11-17 2006-05-26 Korea Research Institute Of Bioscience And Biotechnology Recombinant expression vector for production of plants having multiple stress tolerances, and method for preparing multiple stress-tolerant plants using the same
KR100704751B1 (en) * 2003-11-07 2007-04-10 한국생명공학연구원 Recombinant expression vector for production of plants having multiple stress tolerances and method for preparing multiple stress-tolerant plants using the same

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100704751B1 (en) * 2003-11-07 2007-04-10 한국생명공학연구원 Recombinant expression vector for production of plants having multiple stress tolerances and method for preparing multiple stress-tolerant plants using the same
WO2006054815A1 (en) * 2004-11-17 2006-05-26 Korea Research Institute Of Bioscience And Biotechnology Recombinant expression vector for production of plants having multiple stress tolerances, and method for preparing multiple stress-tolerant plants using the same

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