KR20110042409A - Gene that increases resistance to rice stripe disease and uses thereof - Google Patents
Gene that increases resistance to rice stripe disease and uses thereof Download PDFInfo
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Abstract
Description
본 발명은 벼 줄무늬잎마름병에 대한 저항성을 증진시키는 유전자 및 이의 용도에 관한 것이다.The present invention relates to a gene for enhancing resistance to rice streaked blight and its use.
벼, 옥수수, 보리 등과 같은 화본과 작물은 인류에 식량을 공급하는 중요한 식량작물이며, 이들 작물에는 바이러스에 의해 발병하는 흑조위축병(검은줄오갈병), 오갈병 및 줄무늬잎마름병과 같은 병이 있어 경제적으로 막대한 피해를 주고 있다.Plants and crops, such as rice, corn, and barley, are important food crops that provide food to humans, and these crops are economically affected by diseases such as black atrophy disease (black string organ), ogal disease, and streaked leaf blight caused by viruses. It is doing a lot of damage.
종래의 병 저항성 품종 육성에 대한 연구는 전통적인 교배 육종에 의존하였으며, 최근의 개발된 몇몇 저항성 품종은 수직 저항성을 이용한 저항성이므로 저항성이 쉽게 무너지는 문제점이 있었다.Previous studies on breeding disease-resistant varieties depended on traditional breeding breeding, and some of the recently developed varieties of resistance-resistant varieties have a problem that resistance is easily broken because of resistance using vertical resistance.
따라서, 최근에 식물 병에 저항성을 나타내는 새로운 유전자를 탐색, 분리 및 분석하고, 그 유전자를 이용하여 형질전환된 작물을 개발하는 분자 육종 분야에 대한 연구가 활발히 진행되고 있다. 이러한 분자 육종법에 의한 병 저항성을 갖는 형질전환 식물체를 이용하는 경우에, 병에 대한 저항성으로 인한 생산량 증가뿐만 아니라, 농약의 사용량을 절감하고, 인건비 절약 등을 통한 생산성 향상 및 안전한 농산물 공급을 가능케 하기 때문에 파급 효과는 크다고 할 것이다.Therefore, researches on the field of molecular breeding have recently been actively conducted to search for, isolate and analyze new genes showing resistance to plant diseases and to develop transformed crops using the genes. In the case of using a transgenic plant having disease resistance by such a molecular breeding method, not only can the production increase due to resistance to disease, but also the use of pesticides can be reduced, productivity can be improved and labor supply can be secured through labor cost reduction, etc. The ripple effect is great.
한편, 한국등록특허 제10-0803393호에는 벼에서 OsLRP 유전자를 이용한 병 저항성을 증진시키는 방법이 개시되어 있으나, 본 발명에서와 같이 벼 줄무늬잎마름병에 대해 저항성을 증진시키는 방법에 관한 내용은 없다.On the other hand, Korean Patent No. 10-0803393 discloses a method of improving disease resistance using the OsLRP gene in rice, but there is no information on how to improve resistance to rice streaked leaves as in the present invention.
본 발명은 상기와 같은 요구에 의해 안출된 것으로서, 본 발명은 벼(Oryza sativa) 유래의 LRP(leucine rich repeat protein) 유전자인 OsLRP를 포함하는 재조합 벡터를 도입시켜 제조한 형질전환된 벼 식물체를 제공하여, 최근 들어 기후온난화로 인하여 만연된 벼 줄무늬잎마름병에 저항성이 증진된 식물체를 제공함으로써 농작물의 생산성 향상을 위한 효과적인 방법을 제공하고자 한다. The present invention has been made in accordance with the above requirements, the present invention provides a transformed rice plant prepared by introducing a recombinant vector containing OsLRP , a leucine rich repeat protein (LRP) gene derived from rice ( Oryza sativa ) In order to provide an effective method for improving productivity of crops by providing plants with increased resistance to rice streaked blight caused by recent climate warming.
상기 과제를 해결하기 위해, 본 발명은 벼 유래의 OsLRP(Oryza sativa leucine rich repeat protein) 유전자를 포함하는 재조합 벡터로 형질전환된 벼 줄무늬잎마름병에 대한 저항성이 증진된 식물체 및 이의 종자를 제공한다.In order to solve the above problems, the present invention is OsLRP ( Oryza derived from rice) sativa leucine rich repeat protein) Provided is a plant and its seeds enhanced resistance to rice streaked blight transformed with a recombinant vector comprising the gene.
본 발명은 또한, 상기 OsLRP 유전자를 이용하여 벼 줄무늬잎마름병에 대한 저항성이 증진된 벼 식물체의 제조 방법을 제공한다.The invention also, the OsLRP Provided is a method for producing rice plants having improved resistance to rice streaked blight by using a gene.
본 발명은 또한, 상기 OsLRP 유전자를 이용하여 벼 줄무늬잎마름병에 대한 저항성을 증진시키는 방법을 제공한다.The invention also, the OsLRP It provides a method of using the gene to improve resistance to rice streaked blight.
본 발명은 또한, 상기 OsLRP 유전자를 포함하는, 식물체의 벼 줄무늬잎마름병에 대한 저항성 증진용 조성물을 제공한다.The invention also, the OsLRP It provides a composition for enhancing resistance to plant rice stripe leaf blight comprising a gene.
본 발명은 또한, 상기 OsLRP 유전자를 이용하여 식물체의 생산성을 향상시키는 방법을 제공한다.The invention also, the OsLRP Provides a method of improving the productivity of the plant using the gene.
본 발명은 OsLRP 유전자를 식물 형질전환용 발현벡터에 조합하여 전이함으로써 효과적으로 식물체의 벼 줄무늬잎마름병에 대한 저항성을 증진시킬 수 있다. 따라서 기후온난화, 동계 작물 재배 등으로 인한 매개충인 벼멸구의 월동으로 최근 3년간 벼 줄무늬잎마름병이 창궐하고 있는 요즘에 살충제의 절감 효과뿐만 아니라 경제 작물 등의 수확량 증대 등에 크게 기여할 수 있을 것으로 기대된다. OsLRP invention By transferring genes in combination with expression vectors for plant transformation, it is possible to effectively enhance the resistance of plants to rice streaked blight. Therefore, it is expected to contribute greatly to the reduction of insecticides as well as to increasing the yield of economic crops in the recent three years of rice stripe blight caused by wintering of the rice hopper which is a mediator due to climate warming and winter crop cultivation.
본 발명의 목적을 달성하기 위하여, 본 발명은 벼 유래의 OsLRP(Oryza sativa leucine rich repeat protein) 유전자를 포함하는 재조합 벡터로 형질전환된 벼 줄무늬잎마름병에 대한 저항성이 증진된 식물체를 제공한다.In order to achieve the object of the present invention, the present invention is a rice-derived OsLRP ( Oryza sativa leucine rich repeat protein) Provided is a plant having improved resistance to rice streaked blight transformed with a recombinant vector containing a gene.
상기 OsLRP 유전자는 바람직하게는 서열번호 1로 표시되는 염기서열로 이루어질 수 있다. 또한, 상기 염기 서열의 변이체가 본 발명의 범위 내에 포함된다. 변이체는 염기 서열은 변화되지만, 서열번호 1의 염기 서열과 유사한 기능적 특성을 갖는 염기 서열이다. 구체적으로, OsLRP 유전자는 서열번호 1의 염기 서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. OsLRP The gene may be preferably composed of the nucleotide sequence represented by SEQ ID NO: 1. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, OsLRP The gene may comprise a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1.
폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
본 발명의 OsLRP 유전자를 포함하는 재조합 벡터는 바람직하게는 식물 발현 벡터이며, 식물 발현 벡터는 프로모터 및 터미네이터를 포함하는데, 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. "구성적(constitutive) 프로모터"는 대부분의 환경 조건 및 발달 상태 또는 세포 분화하에서 활성이 있는 프로모터이다. 형질전환체의 선택이 각종 단계에서 각종 조직에 의해서 이루어질 수 있기 때문에 구성적 프로모터가 본 발명에서 바람직할 수 있다. 따라서 구성적 프로모터는 선택 가능성을 제한하지 않는다. OsLRP of the present invention The recombinant vector comprising the gene is preferably a plant expression vector, the plant expression vector including a promoter and terminator, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an 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 conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the choice.
터미네이터는 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 알고 있다. 그러므로 터미네이터의 사용은 본 발명의 내용에서 매우 바람직하다.Terminators may use conventional terminators, such as nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium) tumefaciens ), but the terminator of the octopine gene (Octopine) gene, but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. The use of terminators is therefore highly desirable in the context of the present invention.
상기 식물체는 바람직하게는 벼이다.The plant is preferably rice.
본 발명은 또한, 상기 식물체의 종자를 제공한다. 바람직하게는, 상기 종자는 벼의 종자이다.The present invention also provides seed of the plant. Preferably, said seed is a seed of rice.
본 발명은 또한, 벼 유래의 OsLRP 유전자를 포함하는 재조합 벡터로 식물 세 포를 형질전환하는 단계; 및The present invention also relates to OsLRP derived from rice Transforming the plant cell with the recombinant vector containing the gene; And
상기 형질전환된 식물 세포로부터 형질전환 식물을 재분화하는 단계를 포함하는 벼 줄무늬잎마름병에 대한 저항성이 증진된 벼 식물체의 제조 방법을 제공한다.It provides a method for producing a rice plant improved resistance to rice streaked blight comprising the step of re-differentiating the transformed plant from the transformed plant cells.
식물의 형질전환은 DNA를 식물에 전이시키는 임의의 방법을 의미한다. 그러한 형질전환 방법은 반드시 재생 및(또는) 조직 배양 기간을 가질 필요는 없다. 식물 종의 형질전환은 이제는 쌍자엽 식물뿐만 아니라 단자엽 식물 양자를 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), 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) 입자 충격법(Klein T.M. et al., 1987, Nature 327, 70), 식물의 침윤 또는 성숙 화분 또는 소포자의 형질전환에 의한 아그로박테리움 투머파시엔스 매개된 유전자 전이에서 (비완전성) 바이러스에 의한 감염 등으로부터 적당하게 선택될 수 있다. 본 발명에 따른 바람직한 방법은 아그로박테리움 매개된 DNA 전달을 포함한다. 특히 바람직한 것은 EP A 120 516호 및 미국 특허 제4,940,838호에 기재된 바와 같은 소위 이원 벡터 기술을 이용하는 것이다.Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74), electroporation of protoplasts (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102 ), Microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), particle bombardment methods (DNA or RNA-coated) of various plant elements (Klein TM et al., 1987, Nature 327, 70), infection with (incomplete) virus in agrobacterium tumerfaciens mediated gene transfer by invasion of plants or transformation of mature pollen or vesicles, and the like. have. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.
식물의 형질전환에 이용되는 "식물 세포"는 어떤 식물 세포도 된다. 식물 세 포는 배양 세포, 배양 조직, 배양 기관 또는 전체 식물, 바람직하게는 배양 세포, 배양 조직 또는 배양 기관 및 더욱 바람직하게는 배양 세포의 어떤 형태도 된다. 바람직하게는, 상기 식물체는 벼이다."Plant cell" used for transformation of a plant may be any plant cell. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells. Preferably, the plant is rice.
"식물 조직"은 분화된 또는 미분화된 식물의 조직, 예를 들면 이에 한정되진 않으나, 뿌리, 줄기, 잎, 꽃가루, 종자, 암 조직 및 배양에 이용되는 다양한 형태의 세포들, 즉 단일 세포, 원형질체(protoplast), 싹 및 캘러스 조직을 포함한다. 식물 조직은 인 플란타(in planta)이거나 기관 배양, 조직 배양 또는 세포 배양 상태일 수 있다."Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. Plant tissue is planted in planta ) or in an organ culture, tissue culture or cell culture.
본 발명의 방법은 본 발명에 따른 재조합 벡터로 식물 세포를 형질전환하는 단계를 포함하는데, 상기 형질전환은 아그로박테리움 튜머파시엔스(Agrobacterium tumefaciens)에 의해 매개될 수 있다. 또한, 본 발명의 방법은 상기 형질전환된 식물 세포로부터 형질전환 식물을 재분화하는 단계를 포함한다. 형질전환 식물 세포로부터 형질전환 식물을 재분화하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다.The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, wherein the transformation can be mediated by Agrobacterium tumefaciens . The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.
본 발명은 또한, 벼 유래의 OsLRP 유전자를 포함하는 재조합 벡터로 식물 세포를 형질전환시켜 OsLRP 유전자를 과발현하는 단계를 포함하는 벼 줄무늬잎마름병에 대한 저항성을 증진시키는 방법을 제공한다. 하기 실시예에서 알 수 있는 바와 같이, OsLRP 유전자로 형질전환된 벼는 벼 줄무늬잎마름병 증상을 보여주지 않았다. 식물체의 형질전환 방법, 형질전환에 이용되는 재조합 벡터 및 숙주 세포로 이용될 수 있는 식물은 전술한 바와 같다.The present invention also relates to OsLRP derived from rice OsLRP by transforming plant cells with a recombinant vector containing the gene It provides a method for enhancing the resistance to rice streaked leaf blight comprising overexpressing a gene. As can be seen in the examples below, OsLRP Genetically transformed rice showed no symptoms of rice streaked blight. Plant transformation methods, recombinant vectors used for transformation and plants that can be used as host cells are as described above.
본 발명은 또한, 벼 유래의 OsLRP 유전자를 포함하는, 식물체의 벼 줄무늬잎마름병에 대한 저항성 증진용 조성물을 제공한다. 본 발명의 조성물에서, 상기 OsLRP 유전자는 바람직하게는 서열번호 1로 표시되는 염기서열로 이루어질 수 있다. 본 발명의 식물체의 벼 줄무늬잎마름병에 저항성 증진용 조성물은 유효 성분으로서 벼 유래의 OsLRP 유전자를 포함하며, 상기 유전자 OsLRP를 식물체에 형질전환시킴으로써 식물체의 벼 줄무늬잎마름병에 저항성을 증진시킬 수 있는 것이다.The present invention also relates to OsLRP derived from rice It provides a composition for enhancing resistance to plant rice stripe leaf blight comprising a gene. In the composition of the present invention, the OsLRP gene may be preferably composed of a nucleotide sequence represented by SEQ ID NO: 1. The composition for enhancing resistance to rice streaked leaf blight of the plant of the present invention is OsLRP derived from rice as an active ingredient. It includes a gene, and the gene OsLRP by transforming the plant is to improve resistance to rice streaked leaf blight of the plant.
본 발명은 또한, 벼 유래의 OsLRP 유전자를 포함하는 재조합 벡터로 식물 세포를 형질전환시켜 OsLRP 유전자를 과발현하는 단계를 포함하는 식물체의 생산성을 향상시키는 방법을 제공한다. 본 발명의 OsLRP 유전자의 식물체에서의 발현은 벼 줄무늬잎마름병에 대해 저항성을 갖게 하므로, OsLRP 유전자로 형질전환된 식물체는 대조군에 비해 식물체의 생산성이 향상될 수 있는 것이다.The present invention also relates to OsLRP derived from rice OsLRP by transforming plant cells with a recombinant vector containing the gene It provides a method for improving the productivity of a plant comprising overexpressing a gene. OsLRP of the present invention Expression of the gene in the plant makes it resistant to rice streaked blight, OsLRP Plants transformed with the gene is to improve the productivity of the plant compared to the control.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
실시예Example 1: One: OsLRPOsLRP 형질전환체에서 In transformants OsLRPOsLRP 유전자의 발현분석 Gene expression analysis
OsLRP 형질전환체의 제작과정은 선행 특허 (대한민국특허 제10-0803393호)에서 상세히 기재한 바 있다. OsLRP 형질전환체에서 OsLRP 유전자 발현을 확인하였다. 온실에서 키운 OsLRP 형질전환 벼의 잎을 채취하여 Trizol (Invitrogen Co.)을 이용하여 총 RNA를 분리하였다. 올리고 dT 프라이머(Oligo dT primer)를 이용하여 역전사(reverse transcription) 반응을 수행하고 OsLRP (AF364178) 특이 5' 프라이 머 A (5'-CTGGTCATCTGGTGCCTGAGCTTG-3': 서열번호 2)와 3' 프라이머 B (5'-GCAGTTGGTGTCATATACAGCCAG-3': 서열번호 3)로, 또한 OsActin (XM469569)은 5' 프라이머 C (5'-TCCATCTTGGCATCTCTCAG-3': 서열번호 4)와 3' 프라이머 D (5'-GTACCCGCATCAGGCATCTG-3': 서열번호 5)로 PCR(polymerase chain reaction)을 55℃ 어닐링(annealing) 온도로 30회 수행하여 OsLRP 유전자의 발현을 확인하였고 그 결과를 도 1에 나타내었다. 도 1에서 보는 바와 같이, OsLRP는 대조구에 비해 과량 발현되고 있음을 알 수 있었다.The manufacturing process of the OsLRP transformant has been described in detail in the prior patent (Korean Patent No. 10-0803393). OsLRP trait was confirmed OsLRP gene expression in the switch body. OsLRP- transformed rice leaves grown in greenhouses were harvested and total RNA was isolated using Trizol (Invitrogen Co.). Reverse transcription was performed using an oligo dT primer and OsLRP (AF364178) specific 5 'primer A (5'-CTGGTCATCTGGTGCCTGAGCTTG-3': SEQ ID NO: 2) and 3 'primer B (5 '-GCAGTTGGTGTCATATACAGCCAG-3': SEQ ID NO: 3), and OsActin (XM469569) also has 5 'primer C (5'-TCCATCTTGGCATCTCTCAG-3': SEQ ID NO: 4) and 3 'primer D (5'-GTACCCGCATCAGGCATCTG-3': PCR (polymerase chain reaction) was carried out 30 times at 55 ° C. annealing temperature in SEQ ID NO: 5 to confirm the expression of OsLRP gene, and the results are shown in FIG. 1. As shown in Figure 1, OsLRP was found to be overexpressed compared to the control.
실시예Example 2: 2: OsLRPOsLRP 형질전환체의 병 저항성 유도 여부 확인 Determine whether transformants induce disease resistance
OsLRP 형질전환체에서 병 저항성 반응이 유도되는지 여부를 확인하기 위해, 온실에서 키운 OsLRP 형질전환 벼의 잎을 채취하여 Trizol (Invitrogen Co.)을 이용하여 총 RNA를 분리하였다. 올리고 dT 프라이머를 이용하여 역전사(reverse transcription) 반응을 병 저항성 유도 마커 유전자인 OsPR10a (D38170) 특이 5' 프라이머 E (5'-GCTACAGGCATCAGTGGTCA-3': 서열번호 6)와 3' 프라이머 F (5'-GACTCAAACGCCACGAGAAT-3': 서열번호 7)로 또한 OsActin으로 PCR(polymerase chain reaction)을 55℃ 어닐링(annealing) 온도로 30회 수행하여 OsPR10a 유전자의 발현을 확인하였고 그 결과를 도 2에 나타내었다. 도 2에서 보는 바와 같이, OsPR10a는 대조구에 비해 유도 발현되고 있음을 확인할 수 있었다. To determine whether the disease resistance response is induced in OsLRP transformants, leaves of OsLRP transformed rice grown in greenhouses were harvested, and total RNA was isolated using Trizol (Invitrogen Co.). Reverse transcription was performed using oligo dT primers to identify disease resistance induction marker gene OsPR10a (D38170) specific 5 'primer E (5'-GCTACAGGCATCAGTGGTCA-3': SEQ ID NO: 6) and 3 'primer F (5'- Polymerase chain reaction (PCR) was performed 30 times at 55 ° C. annealing temperature with GACTCAAACGCCACGAGAAT-3 ′: SEQ ID NO: 7) and OsActin to confirm the expression of OsPR10a gene. The results are shown in FIG. 2. As shown in Figure 2, OsPR10a was confirmed that the induced expression compared to the control.
실시예Example 3: 3: OsLRPOsLRP 형질전환체의 벼 줄무늬잎마름병 저항성 증진효과 검정 Transformant tested the effect of increasing the resistance to rice streaked leaf blight
배양실에서 벼를 발아시켜 온실에 형질전환 벼를 파종하였다. 온실에서 벼 줄무늬바이러스가 감염된 벼로 인해 애멸구의 보독충률이 높아 온실에 많은 벼들이 벼 줄무늬잎마름병 증상을 보였다. 도 3에서 보여주는 바와 같이, 대조구는 심한 벼 줄무늬잎마름병 증상을 보여주었고, OsLRP 형질전환체는 병징이 보이지 않고 건전한 잎을 유지하고 있음을 알 수 있다.Rice was germinated in the culture room and transformed rice was sown in the greenhouse. Rices infected with rice streak virus in greenhouses have high rates of supplementation of larvae. As shown in FIG. 3, the control group showed severe rice streaked blight symptoms, and the OsLRP transformants showed no symptoms and maintained healthy leaves.
실시예Example 4: 4: OsLRPOsLRP 형질전환체와 Transformants 대조구의Control 이병체에서In this pathogen 벼 줄무늬바이러스 검출 Rice Stripe Virus Detection
온실에서 벼 줄무늬바이러스가 감염된 벼로 인해 애멸구의 보독충률이 높아 온실에 많은 벼들이 벼 줄무늬잎마름병 증상을 보였다. 이 증상이 벼 줄무늬바이러스에 의한 것인지를 확인하기 위해, 벼 줄무늬바이러스의 RNA3의 캡시드 단백질(capsid protein) 특이 5' 프라이머 G (5'-AGCCACTCTAGCTGATTTG-3': 서열번호 8)와 3'프라이머 H (5'-TGGTTATTTGGAAGGAGTG-3': 서열번호 9)로 또한 OsActin으로 PCR(polymerase chain reaction)을 55℃ 어닐링(annealing) 온도로 30회 수행하여 RSV-CP 유전자의 발현을 확인하였고 그 결과를 도 4에 나타내었다. 도 4를 보면 대조구에서는 바이러스가 대량으로 검출되었으나 OsLRP 형질전환체에서는 바이러스가 검출되지 않았다.Rices infected with rice streak virus in greenhouses have high rates of supplementation of larvae. To determine whether this symptom is caused by rice striated virus, the capsid protein specific 5 'primer G (5'-AGCCACTCTAGCTGATTTG-3': SEQ ID NO: 8) and 3 'primer H ( 5'-TGGTTATTTGGAAGGAGTG-3 ': SEQ ID NO: 9) and polymerase chain reaction (PCR) with OsActin were performed 30 times at 55 ° C. annealing temperature to confirm the expression of RSV-CP gene. Indicated. 4, a large amount of virus was detected in the control, but no virus was detected in the OsLRP transformant.
도 1은 OsLRP 형질전환체에서 OsLRP 유전자의 발현을 분석한 결과이다. 1 is a result of analyzing the expression of OsLRP gene in OsLRP transformants.
도 2는 OsLRP 형질전환체의 병 저항성 유도 여부를 확인한 결과이다. Figure 2 is a result confirming the induction of disease resistance of the OsLRP transformant.
도 3은 OsLRP 형질전환체의 줄무늬잎마름병 저항성 증진효과를 확인한 결과이다. Figure 3 is a result confirming the resistance effect of streaked leaf blight resistance of OsLRP transformants.
도 4는 OsLRP 형질전환체와 대조구의 이병체에서 벼 줄무늬바이러스를 검출한 결과이다. Figure 4 shows the results of detecting the rice streak virus in the pathogen of OsLRP transformants and control.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Gene that increases resistance to rice stripe disease and uses thereof <130> PN09189 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 992 <212> DNA <213> Oryza sativa <400> 1 ttgctgctgc tactactctt cttccccctt cctcgtgttc ctcctcttcg cctcgtctct 60 ctgccctgcc tcgtgcgcgt ccagatccgg gagggatggg ggcgggggcg ctgggggtgg 120 tggcgatggt ggcggcggcg gtggtggtgg cgatggcggg ggcgaactcc gagggcgacg 180 cgctgtcggc gctgcggcgg agcctcaggg accccggcgg ggtgctgcag agctgggacc 240 ccacgctcgt caacccctgc acctggttcc acgtcacctg cgaccgcgac aaccgcgtca 300 cgcgcctcca tcttgggaat ttgaacttat ctggtcatct ggtgcctgag cttggaaagt 360 tggatcatct gcaatatctg gagctataca agaataatat tcaaggaacg atcccatcgg 420 aacttggtaa tttgaagaat cttataagct tggacctgta caagaacaac atttctggga 480 ctatacctcc aacacttggg aaattaacgt cccttgtatt cttgcggctc aatggcaatc 540 gcttgactgg gccaatccca agggaactgg ccggaatatc tagtcttaaa gttgttgatg 600 tttcaagcaa tgatctgtgt ggaacaattc ctacatcagg accatttgag cacattcccc 660 taagcaactt tgagaagaac ccacgcttgg aaggtccaga actacaaggc ctggctgtat 720 atgacaccaa ctgctagatg gaaacacgaa gaaaagaatg ggtagaaagt aatgggcgta 780 aaatgttacc gaaaaccggg tgatatagat tatgttaggt ttggacctac cactctgtaa 840 tcctccatgc atagctcttg tttgtgctgt ttgtcggcaa tgaagttgta tgttgtagca 900 gaatactttc tttctgtgaa agtaaacatg gaactgctta tgtcatgagt caaatgttat 960 tgttatcaca aaaaaaaaaa aaaaaaaaaa aa 992 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer A <400> 2 ctggtcatct ggtgcctgag cttg 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer B <400> 3 gcagttggtg tcatatacag ccag 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer C <400> 4 tccatcttgg catctctcag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer D <400> 5 gtacccgcat caggcatctg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer E <400> 6 gctacaggca tcagtggtca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F <400> 7 gactcaaacg ccacgagaat 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer G <400> 8 agccactcta gctgatttg 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer H <400> 9 tggttatttg gaaggagtg 19 <110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Gene that increases resistance to rice stripe disease and uses the <130> PN09189 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 992 <212> DNA <213> Oryza sativa <400> 1 ttgctgctgc tactactctt cttccccctt cctcgtgttc ctcctcttcg cctcgtctct 60 ctgccctgcc tcgtgcgcgt ccagatccgg gagggatggg ggcgggggcg ctgggggtgg 120 tggcgatggt ggcggcggcg gtggtggtgg cgatggcggg ggcgaactcc gagggcgacg 180 cgctgtcggc gctgcggcgg agcctcaggg accccggcgg ggtgctgcag agctgggacc 240 ccacgctcgt caacccctgc acctggttcc acgtcacctg cgaccgcgac aaccgcgtca 300 cgcgcctcca tcttgggaat ttgaacttat ctggtcatct ggtgcctgag cttggaaagt 360 tggatcatct gcaatatctg gagctataca agaataatat tcaaggaacg atcccatcgg 420 aacttggtaa tttgaagaat cttataagct tggacctgta caagaacaac atttctggga 480 ctatacctcc aacacttggg aaattaacgt cccttgtatt cttgcggctc aatggcaatc 540 gcttgactgg gccaatccca agggaactgg ccggaatatc tagtcttaaa gttgttgatg 600 tttcaagcaa tgatctgtgt ggaacaattc ctacatcagg accatttgag cacattcccc 660 taagcaactt tgagaagaac ccacgcttgg aaggtccaga actacaaggc ctggctgtat 720 atgacaccaa ctgctagatg gaaacacgaa gaaaagaatg ggtagaaagt aatgggcgta 780 aaatgttacc gaaaaccggg tgatatagat tatgttaggt ttggacctac cactctgtaa 840 tcctccatgc atagctcttg tttgtgctgt ttgtcggcaa tgaagttgta tgttgtagca 900 gaatactttc tttctgtgaa agtaaacatg gaactgctta tgtcatgagt caaatgttat 960 tgttatcaca aaaaaaaaaa aaaaaaaaaa aa 992 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer A <400> 2 ctggtcatct ggtgcctgag cttg 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer B <400> 3 gcagttggtg tcatatacag ccag 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer C <400> 4 tccatcttgg catctctcag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer D <400> 5 gtacccgcat caggcatctg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer E <400> 6 gctacaggca tcagtggtca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer F <400> 7 gactcaaacg ccacgagaat 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer G <400> 8 agccactcta gctgatttg 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer H <400> 9 tggttatttg gaaggagtg 19
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