KR20230045886A - dsRNA targeting HC-pro middle region for inhibiting PepMoV replication and uses thereof - Google Patents
dsRNA targeting HC-pro middle region for inhibiting PepMoV replication and uses thereof Download PDFInfo
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Abstract
Description
본 발명은 PepMoV (Pepper Mottle Virus)의 증식을 억제하는 HC-pro (helper component proteinase) 중앙 부위 표적 dsRNA 및 이의 용도에 관한 것이다.The present invention relates to dsRNA targeting the central region of HC-pro (helper component proteinase) that inhibits the proliferation of Pepper Mottle Virus (PepMoV) and uses thereof.
RNAi (RNA interference) 기술을 기반으로, 표적 RNA의 상동성 있는 RNA를 발현시킨 식물은 바이러스 방제 측면에서 효과적이고 영속적인 장점을 가지고 있다. 하지만, 이러한 형질전환 방법은 시간이 오래 걸리고, 고비용이며, 작물마다 효과적인 형질전환 방법이 다르고, GMO (Genetically modified organism)로 인한 국가적 규제 등의 단점을 가지고 있다. 이러한 문제점을 극복하기 위해서 RNAi 기작을 응용하여 바이러스 dsRNA를 식물에 직접 처리하는 방법이 대두되고 있다. dsRNA는 식물체에 주입된 후 성숙과정을 거쳐 다양한 siRNA (small interfering RNA)를 형성하고, siRNA는 Argonaute 단백질에 로딩되어 표적 mRNA와 상보적으로 결합 및 절단하여 표적 유전자의 발현을 조절한다.Based on RNAi (RNA interference) technology, plants expressing RNA homologous to the target RNA have an effective and durable advantage in terms of virus control. However, these transformation methods have disadvantages such as time consuming, high cost, different effective transformation methods for each crop, and national regulations due to genetically modified organisms (GMOs). In order to overcome these problems, a method of directly processing viral dsRNA into plants by applying the RNAi mechanism has emerged. After dsRNA is injected into plants, it undergoes a maturation process to form various small interfering RNAs (siRNAs). The siRNAs are loaded into Argonaute proteins and complementarily bind to and cleave target mRNAs to regulate the expression of target genes.
PepMoV (Pepper Mottle Virus)의 유전자를 표적으로 하는 dsRNA를 외래 합성 후 식물에 처리했을 때, 바이러스의 증식이 현저히 감소된다는 것은 보고된 바 없다.It has not been reported that the proliferation of the virus is significantly reduced when dsRNA targeting the gene of PepMoV (Pepper Mottle Virus) is exogenously synthesized and then treated with plants.
한편, 한국공개특허 제2011-0051539호에는 'PepMoV에 대한 내성이 증진된 고추의 형질전환체 및 그 제조방법'이 개시되어 있고, 한국공개특허 제2015-0140510호에는 '담배나방 방제용 dsRNA, 이를 포함하는 살충제 조성물 및 방제방법'이 개시되어 있으나, 본 발명의 'PepMoV의 증식을 억제하는 HC-pro 중앙 부위 표적 dsRNA 및 이의 용도'에 대해서는 기재된 바가 없다.On the other hand, Korean Patent Publication No. 2011-0051539 discloses 'a transformant of pepper with improved resistance to PepMoV and a manufacturing method thereof', and Korean Patent Publication No. 2015-0140510 discloses 'dsRNA for controlling tobacco moth, An insecticide composition and control method comprising the same are disclosed, but nothing is described about the ' HC-pro central region targeting dsRNA that inhibits the growth of PepMoV and its use' of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 PepMoV의 HC-pro 유전자의 중앙 부위(419 내지 949번째 염기)를 표적으로 하는 dsRNA를 제작하고 이를 모델 식물에 전처리하고 바이러스의 증식 여부를 확인한 결과, HC-pro 유전자의 중앙 부위 중에서 5' 부위를 표적으로 하는 dsRNA 처리에 의해 PepMoV의 증식이 현저히 억제되는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs, and the present inventors prepared dsRNA targeting the central region (bases 419 to 949) of the HC-pro gene of PepMoV, pretreated it in a model plant, and tested whether or not the virus proliferated. As a result of confirming, the present invention was completed by confirming that the proliferation of PepMoV was significantly suppressed by dsRNA treatment targeting the 5' region of the central region of the HC-pro gene.
상기 과제를 해결하기 위해, 본 발명은 서열번호 1의 염기서열로 표시되며, PepMoV (Pepper mottle virus)의 HC-pro (helper component proteinase) 유전자의 발현을 억제하는, PepMoV 증식억제용 dsRNA (double-stranded RNA)를 제공한다.In order to solve the above problems, the present invention is represented by the nucleotide sequence of SEQ ID NO: 1 and suppresses the expression of the HC-pro (helper component proteinase) gene of PepMoV (Pepper mottle virus), dsRNA (double- stranded RNA).
또한, 본 발명은 상기 dsRNA를 유효성분으로 포함하는 PepMoV 방제용 조성물을 제공한다.In addition, the present invention provides a composition for controlling PepMoV comprising the dsRNA as an active ingredient.
또한, 본 발명은 상기 조성물을 식물, 식물의 종자 또는 재배지에 처리하는 단계를 포함하는 PepMoV의 방제 방법을 제공한다.In addition, the present invention provides a method for controlling PepMoV comprising the step of treating plants, plant seeds or plantings with the composition.
또한, 본 발명은 상기 dsRNA를 포함하는 재조합 벡터를 제공한다.In addition, the present invention provides a recombinant vector containing the dsRNA.
본 발명은 HC-pro 유전자의 중앙 부위를 표적으로 하는 dsRNA를 유효성분으로 하는 바이러스 억제제로서, 외래 합성 dsRNA가 바이러스 방제를 위한 제제로 응용될 수 있는 가능성을 제시하였다. 또한, 본 발명의 dsRNA는 화학작물보호제 대체재로서 dsRNA를 활용하기 위한 기반 연구 및 혁신적인 Mode Of Action(MOA) 작물보호제 개발 등을 위한 농생명공학에 적용될 수 있을 것이다.The present invention is a virus inhibitor containing dsRNA targeting the central region of the HC-pro gene as an active ingredient, suggesting the possibility that exogenous synthetic dsRNA can be applied as a virus control agent. In addition, the dsRNA of the present invention can be applied to agricultural biotechnology for research on the use of dsRNA as a substitute for chemical crop protection agents and development of innovative Mode Of Action (MOA) crop protection agents.
도 1은 PepMoV의 HC-pro 유전자의 중앙 부위(HC-pro_middle)를 표적으로 하는 dsRNA의 표적 위치 및 서열을 보여준다. 노란색 부분은 다른 dsRNA와 중첩(overlap)되는 부위를 나타낸다.
도 2는 담배 식물에서 HC-pro 유전자의 중앙 부위 표적 dsRNA 처리에 따른 PepMoV의 증식 억제 효과를 qPCR로 확인한 결과(A) 및 UV 조건에서 관찰한 사진(B)이다. 도 2A는 바이러스 처리 8일 후의 결과로, **는 p < 0.01, ***는 p < 0.001을 의미한다. 또한, dsRNA_Hc-pro는 HC-pro 유전자의 중앙 부위 전체를 표적으로 하는 dsRNA를 의미하며, dsRNA_Hc-pro #1은 HC-pro 유전자의 중앙 부위에서 5'을 표적으로 하는 dsRNA를 의미하며, dsRNA_Hc-pro #2는 HC-pro 유전자의 중앙 부위에서 middle을 표적으로 하는 dsRNA를 의미하고, dsRNA_Hc-pro #3은 HC-pro 유전자의 중앙 부위에서 3'을 표적으로 하는 dsRNA를 의미한다.Figure 1 shows the target location and sequence of dsRNA targeting the central region (HC-pro_middle) of the HC-pro gene of PepMoV. The yellow part represents the overlapping region with other dsRNAs.
FIG. 2 is a result (A) and a photo (B) observed under UV conditions confirming the growth inhibitory effect of PepMoV by qPCR according to treatment with dsRNA targeting the central region of the HC-pro gene in tobacco plants. Figure 2A shows the results after 8 days of virus treatment, ** means p < 0.01, *** means p < 0.001. In addition, dsRNA_Hc-pro refers to a dsRNA targeting the entire central region of the HC-pro gene, dsRNA_Hc-
본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1의 염기서열로 표시되며, PepMoV (Pepper mottle virus)의 HC-pro (helper component proteinase) 유전자의 발현을 억제하는, PepMoV 증식억제용 dsRNA (double-stranded RNA)를 제공한다.In order to achieve the object of the present invention, the present invention is represented by the nucleotide sequence of SEQ ID NO: 1 and suppresses the expression of the HC-pro (helper component proteinase) gene of PepMoV (Pepper mottle virus), PepMoV proliferation inhibitory dsRNA ( double-stranded RNA).
본 발명에 따른 dsRNA는 세포내에서 RNA 간섭(RNAi)에 의해 작용한다. RNA 간섭은 식물을 포함한 진핵생물에서 폭넓게 존재하는 전사 후 유전자 발현 조절 기작이다. 세포에 도입된 dsRNA는 RNAse Ⅲ 효소인 Dicer에 의해 처리되어, siRNA (small interference RNA)의 형태로 Argonaute 단백질과 리보뉴클레오복합체를 형성하여 표적 부위에 상보적 결합을 통해 표적 유전자를 절단하거나, 단백질 합성을 억제한다. 이로 인해 식물 세포에 도입된 바이러스 유전자를 표적으로 한 dsRNA에 유도된 RNA 간섭은 식물에 감염된 바이러스의 표적 유전자 발현 억제를 통하여 바이러스의 증식을 직접적으로 감소시키는 방식으로 작용한다.The dsRNA according to the present invention acts by RNA interference (RNAi) in cells. RNA interference is a post-transcriptional gene expression regulatory mechanism widely present in eukaryotes including plants. The dsRNA introduced into cells is processed by Dicer, an RNAse Ⅲ enzyme, and forms a ribonucleocomplex with Argonaute protein in the form of siRNA (small interference RNA) to cleave the target gene through complementary binding to the target site, or protein inhibit synthesis. As a result, RNA interference induced by dsRNA targeting viral genes introduced into plant cells directly reduces the proliferation of viruses through suppression of target gene expression of viruses infected in plants.
본 발명에 따른 PepMoV 증식억제용 dsRNA는 서열번호 4의 염기서열로 이루어진 PepMoV의 HC-pro 유전자 중앙 부위를 표적으로 하는 dsRNA로, 서열번호 1의 염기서열로 이루어진 것일 수 있다.The dsRNA for inhibiting PepMoV proliferation according to the present invention is a dsRNA targeting the central region of the HC-pro gene of PepMoV composed of the nucleotide sequence of SEQ ID NO: 4, and may be composed of the nucleotide sequence of SEQ ID NO: 1.
본 발명의 일 구현 예에 있어서, 상기 서열번호 1의 염기서열로 이루어진 dsRNA는 HC-pro 유전자 중앙 부위의 5' 부위를 표적으로 하며(도 1 참고), 서열번호 2의 염기서열로 이루어진 HC-pro 유전자 중앙 부위의 middle 부위 표적 dsRNA 및 서열번호 3의 염기서열로 이루어진 HC-pro 유전자 중앙 부위의 3' 부위를 표적으로 하는 dsRNA에 비해 PepMoV 증식억제 효과가 우수한 것이 특징이다.In one embodiment of the present invention, the dsRNA consisting of the nucleotide sequence of SEQ ID NO: 1 targets the 5' region of the central region of the HC-pro gene (see FIG. 1), and the HC -pro gene consisting of the nucleotide sequence of SEQ ID NO: 2 Compared to the dsRNA targeting the middle region of the pro gene and the dsRNA targeting the 3' region of the central region of the HC-pro gene consisting of the nucleotide sequence of SEQ ID NO: 3, PepMoV is characterized by superior proliferation inhibitory effect.
본 발명에 사용된 dsRNA 또는 "이중가닥 RNA" 는 센스 및 안티센스가 결합된 것으로, 실질적으로 상보적인 2개의 핵산 가닥을 포함하는 이중체 구조를 갖는 리보핵산 분자의 복합체를 지칭한다. 이러한 이중가닥 RNA는 세포내로 도입시 단일가닥으로 분리되어, siRNA의 형태로 표적 전사 RNA에 결합하여, 이로부터 단백질이 합성되는 것을 방해한다. 따라서, 이중가닥을 형성할 수 있을 정도의 상보성을 가지는 한, 센스 및 안티센스 두 가닥이 완전히 상보적일 필요는 없고, 이런 의미에서 한 가닥의 서열만으로도, 다른 가닥의 서열은 적절하게 결정될 수 있을 것이다. 용어 "상보적"은, 두 가닥의 올리고뉴클레오티드 또는 폴리뉴클레오티드가 특정 조건 하에 혼성화하여 이중체(duplex) 구조를 형성할 수 있는 능력을 지칭한다. 본 발명에서는 dsRNA가 세포에 도입되어 본 발명에 따른 목적을 달성하는 한 다양한 상보성이 사용될 수 있다. 예를 들면, 두 가닥이 완전히 상보적이거나, 또는 실질적으로 상보적일 수 있다. 일 구현 예에선, 최대 4개, 3개 또는 2개 또는 1개의 미스매치된 염기 쌍을 포함할 수 있다.As used herein, dsRNA or “double-stranded RNA” refers to a complex of ribonucleic acid molecules having a duplex structure comprising two substantially complementary nucleic acid strands, in which sense and antisense are combined. When introduced into cells, these double-stranded RNAs are separated into single strands, bind to target transcriptional RNAs in the form of siRNAs, and prevent protein synthesis therefrom. Therefore, as long as they have sufficient complementarity to form a double strand, the two sense and antisense strands do not need to be completely complementary, and in this sense, the sequence of the other strand can be appropriately determined with only the sequence of one strand. The term "complementary" refers to the ability of two strands of an oligonucleotide or polynucleotide to hybridize under certain conditions to form a duplex structure. In the present invention, various complementarity can be used as long as dsRNA is introduced into cells to achieve the purpose according to the present invention. For example, the two strands may be completely complementary or substantially complementary. In one embodiment, it may contain up to 4, 3 or 2 or 1 mismatched base pairs.
본 발명의 상기 서열번호 1의 염기서열은 dsRNA에서 센스(sense) 가닥의 염기서열을 의미한다.The nucleotide sequence of SEQ ID NO: 1 of the present invention refers to the nucleotide sequence of the sense strand in dsRNA.
또한, 본 발명에 따른 dsRNA는 다수의 뉴클레오티드에서의 실질적인 변형 및 당해 분야에 공지된 모든 다양한 유형의 화학적 변형을 포함할 수 있다. 또한, 상기 dsRNA는 naked 형태의 dsRNA일 수 있고, dsRNA의 안정성 또는 표적 세포 내로의 흡수를 증진시킬 수 있는 다양한 물질 등으로 변형된 형태일 수 있다.In addition, dsRNAs according to the present invention may include substantial modifications at multiple nucleotides and all of the various types of chemical modifications known in the art. In addition, the dsRNA may be a naked dsRNA, or may be a modified form with various materials capable of enhancing dsRNA stability or uptake into target cells.
본 발명은 또한, 상기 dsRNA를 유효성분으로 포함하는 PepMoV 방제용 조성물을 제공한다.The present invention also provides a composition for controlling PepMoV comprising the dsRNA as an active ingredient.
본 발명에 따른 PepMoV 방제용 조성물에 있어서, 상기 dsRNA는 서열번호 1의 염기서열로 표시되며, PepMoV의 HC-pro 유전자의 발현을 억제하는 dsRNA이다.In the composition for controlling PepMoV according to the present invention, the dsRNA is represented by the nucleotide sequence of SEQ ID NO: 1 and is a dsRNA that suppresses the expression of the HC-pro gene of PepMoV.
본 발명에 따른 PepMoV 방제용 조성물은 예를 들어, 직접 분사가능한 용액, 분말 및 현탁액의 형태 또는 고농축 수성, 유성 또는 다른 현탁액, 분산액, 에멀젼, 유성 분산액, 페이스트, 분진, 흩뿌림 물질 또는 과립제로 제조할 수 있으나, 이에 제한되지 않는다.The composition for controlling PepMoV according to the present invention is prepared, for example, in the form of directly sprayable solutions, powders and suspensions or as highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, scattering materials or granules. It can, but is not limited thereto.
본 발명의 PepMoV 방제용 조성물은 다양한 형태로 제제화할 수 있다. 상기 제제는 예를 들어 용매 및/또는 담체를 첨가함으로써 제조될 수 있다. 종종, 비활성 첨가제 및 표면-활성 물질, 예를 들어 유화제 또는 분산제를 제제에 혼합한다. 적합한 표면-활성 물질은 방향족 술폰산(예를 들어 리그노술폰산, 페놀-술폰산, 나프탈렌- 및 디부틸나프탈렌술폰산), 지방산, 알킬- 및 알킬아릴술포네이트, 알킬 라우릴 에테르, 지방 알코올 술페이트의 알칼리 금속, 알카라인 토금속, 암모늄염, 술페이트화 헥사-, 헵타- 및 옥타데칸올, 지방 알코올 글리콜 에테르의 염, 술포네이트 나프탈렌 및 이의 유도체, 포름알데히드의 축합물, 나프탈렌 또는 나프탈렌술폰산, 페놀 및 포름알데히드의 축합물, 폴리옥시에틸렌옥틸 페놀 에테르, 에톡실화 이소옥틸-, 옥틸- 또는 노닐페놀, 알킬페닐 또는 트리부틸페닐 폴리글리콜 에테르, 알킬아릴폴리에테르 알코올, 이소트리데실 알코올, 지방 알코올/에틸렌 옥사이드 축합물, 에톡실화 피마자유, 폴리옥시에틸렌 알킬에테르 또는 폴리옥시프로필렌, 라우릴 알코올 폴리글리콜 에테르 아세테이트, 소르비톨 에스테르, 리그닌-술파이트 폐액 또는 메틸셀룰로오스일 수 있으나, 이에 제한되지는 않는다.The composition for controlling PepMoV of the present invention can be formulated in various forms. The formulation can be prepared, for example, by adding a solvent and/or a carrier. Often, inactive additives and surface-active substances such as emulsifiers or dispersants are mixed into the formulation. Suitable surface-active substances are aromatic sulfonic acids (eg lignosulfonic acids, phenol-sulfonic acids, naphthalene- and dibutylnaphthalenesulfonic acids), fatty acids, alkyl- and alkylarylsulfonates, alkyl lauryl ethers, alkalis of fatty alcohol sulfates. Metals, alkaline earth metals, ammonium salts, sulfated hexa-, hepta- and octadecanols, salts of fatty alcohol glycol ethers, sulfonates naphthalene and its derivatives, condensates of formaldehyde, naphthalene or naphthalenesulfonic acids, phenols and formaldehyde Condensates, polyoxyethyleneoctyl phenol ethers, ethoxylated isooctyl-, octyl- or nonylphenols, alkylphenyl or tributylphenyl polyglycol ethers, alkylarylpolyether alcohols, isotridecyl alcohols, fatty alcohol/ethylene oxide condensates , ethoxylated castor oil, polyoxyethylene alkyl ether or polyoxypropylene, lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin-sulfite waste liquid or methylcellulose, but is not limited thereto.
적합한 고형 담체 물질은 원칙적으로, 모두 다공성이고, 농업적으로 허용가능한 담체, 예를 들어 광물토류(예컨대 실리카, 실리카 겔, 실리케이트, 활석, 고령토, 석회암, 석회, 초크, 보울, 황토, 점토류, 백운석, 규조토류, 황산칼슘, 황산 마그네슘, 산화마그네슘, 분쇄 합성물질), 비료(예컨대 황산암모늄, 인산암모늄, 질산암모늄, 우레아), 식물성 제품(예컨대 곡물 가루, 나무 껍질 가루, 목분(wood meal) 및 견과 껍질 가루) 또는 셀룰로오스 분말일 수 있으나, 이에 제한되지는 않는다. 또한, 상기 고형 담체는 1종류 또는 2종류 이상을 혼합하여 사용할 수도 있다.Suitable solid carrier materials are, in principle, all porous, agriculturally acceptable carriers, for example mineral earths (such as silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, boulders, loess, clays, Dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetics), fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), vegetable products (e.g. grain flour, bark meal, wood meal) and nut shell powder) or cellulose powder, but is not limited thereto. In addition, one type or a mixture of two or more types may be used for the above solid carrier.
본 발명은 또한, 상기 조성물을 식물, 식물의 종자 또는 재배지에 처리하는 단계를 포함하는 PepMoV의 방제 방법을 제공한다.The present invention also provides a method for controlling PepMoV comprising the step of treating plants, plant seeds or plantings with the composition.
본 발명의 PepMoV 방제 방법에 있어서, 상기 조성물은 서열번호 1의 염기서열로 표시되며, PepMoV의 HC-pro 유전자의 발현을 억제하는 dsRNA를 유효성분으로 함유하는 조성물이다.In the PepMoV control method of the present invention, the composition is represented by the nucleotide sequence of SEQ ID NO: 1 and contains dsRNA that inhibits the expression of the HC-pro gene of PepMoV as an active ingredient.
또한, 상기 처리는 PepMoV를 방제하기 위해 dsRNA의 유효량을 포함하는 조성물을 물로 균일하게 희석한 후 동력살포기와 같은 적절한 살포장치를 이용하여 식물에 직접 살포하거나, dsRNA의 유효량을 포함하는 조성물을 식물이나 식물의 종자에 침지하거나 관주, 즉, 분무하여 수행할 수 있다. 침지하는 방법의 경우, 조성물을 식물 주변의 토양에 붓거나 또는 종자를 조성물에 담가둘 수 있다. 본 발명의 방법에 적용될 수 있는 식물은 특별히 제한되지 않는다.In addition, in order to control PepMoV, the composition containing an effective amount of dsRNA is uniformly diluted with water and then directly sprayed on plants using an appropriate spraying device such as a power sprayer, or a composition containing an effective amount of dsRNA is applied to plants or plants. This can be done by dipping or drenching, i.e. spraying, the seeds of the plant. For the immersion method, the composition can be poured into the soil around the plants or the seeds can be immersed in the composition. Plants that can be applied to the method of the present invention are not particularly limited.
본 발명의 '유효량'은 재배 식물 상에서 바이러스를 방제하는 데 충분하거나, 처리된 식물에 실질적인 손상을 초래하지 않는 조성물의 양을 의미한다. 이러한 양은 넓은 범위 내에서 달라질 수 있고, 다양한 인자, 처리된 재배 식물의 구체적 종류 및 상태, 서식 장소, 또는 기후 조건 등에 따라 좌우된다.An 'effective amount' in the present invention means an amount of the composition that is sufficient to control viruses on cultivated plants or does not cause substantial damage to the treated plants. This amount can vary within wide limits and depends on various factors, such as the specific type and condition of the treated cultivated plant, locus of habitat, or climatic conditions.
본 발명은 또한, 상기 서열번호 1의 염기서열로 표시되는 dsRNA를 포함하는 재조합 벡터를 제공한다.The present invention also provides a recombinant vector containing the dsRNA represented by the nucleotide sequence of SEQ ID NO: 1.
본 발명에 따른 dsRNA는 센스 및 안티센스 방향을 동시에 발현하는 바이너리 형질전환용 벡터(Daisuke Miki와 Ko Shimamoto, Plant Cell Physiol. 45(4):490-495, 2004) 등에 게이트웨이 시스템(Gateway system; Invitrogen, USA) 방법으로 클로닝되어 사용될 수 있다. 이러한 벡터에는 다양한 프로모터, 예를 들면, 과발현을 위한 프로모터, 상시 발현을 위한 프로모터, 스트레스 조건에서 발현되는 유도성 프로모터, 목적 외래 서열을 식물생육 기간 중 일정 시기에 발현되도록 하는 프로모터, 식물조직의 특정 부분에만 발현하게 하는 프로모터 또는 목적 외래 서열의 발현을 높이기 위해 인핸서가 결합된 하이브리드 프로모터를 포함할 수 있다.The dsRNA according to the present invention can be used in a binary transformation vector (Daisuke Miki and Ko Shimamoto, Plant Cell Physiol. 45(4):490-495, 2004) that simultaneously expresses sense and antisense directions, etc. USA) can be cloned and used. Such vectors include various promoters, such as promoters for overexpression, promoters for constitutive expression, inducible promoters expressed under stress conditions, promoters for expressing target foreign sequences at certain times during plant growth, and plant tissue-specific promoters. A hybrid promoter to which an enhancer is linked may be included to increase the expression of a promoter or a foreign sequence of interest to be expressed only in a part thereof.
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 examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.
재료 및 방법Materials and Methods
1. dsRNA의 합성1. Synthesis of dsRNA
Thermofisher의 RNAi Megascript RNAi kit를 사용하여 dsRNA를 합성하였다. 각 dsRNA의 길이가 250bp가 넘지 않도록 하며 HC-pro 유전자의 중앙 부위(419 내지 949번째 염기) 내 모든 서열이 포함되도록 각 dsRNA 간에 일정 길이가 중첩(overlap) 되도록 디자인하였다(도 1).dsRNA was synthesized using Thermofisher's RNAi Megascript RNAi kit. The length of each dsRNA did not exceed 250 bp, and each dsRNA was designed to overlap with a certain length so that all sequences in the central region (bases 419 to 949) of the HC-pro gene were included (Fig. 1).
2. 모델식물에의 dsRNA 주입2. Injection of dsRNA into model plants
이전의 연구에서 dsRNA를 전처리할 경우, 바이러스 증식 억제 효과가 가장 높다는 결과를 얻었다. 이에 본 발명에서는, 바이러스 처리 2일 전에 25 ng/㎕ 농도의 dsRNA를 인필트레이션(infiltration) 기법으로 3주령의 담배(Nicotiana benthamiana) 식물체의 3개 잎에 100 ㎍ 씩 각각 주입하였다. 2일 후 바이러스(PepMoV isolation 134)를 아그로박테리움 형질전환 방법으로 식물체에 발현시켰다. 간단하게, pSNU1 vector에 assemble된 GFP 발현 PepMoV isolate 134 (NCBI accession no. EU586123)를 실험에 사용하였다 (Phu et al., J Virol Methods. 2019 Mar;265:26-34). 해당 벡터를 전기천공법으로 아그로박테리움 튜메파시엔스(Agrobacterium tumefaciens) GV3101 균주에 도입시키고, 상기 아그로박테리움 균주를 OD600 값이 0.5가 되도록 배양한 후, 상기 배양액 4㎖을 dsRNA를 처리한 식물체에 처리하였다.In a previous study, it was found that pretreatment with dsRNA had the highest effect of inhibiting viral growth. Accordingly, in the present invention, 100 μg each of dsRNA at a concentration of 25 ng/μl was injected into three leaves of a 3-week-old tobacco ( Nicotiana benthamiana ) plant by
바이러스 처리 시점 0 dpi (day post inoculation), 5 dpi 및 8 dpi에 GFP 발현 수준을 보기 위해 UV 하에서 사진을 촬영하였다.Photographs were taken under UV to see the GFP expression level at 0 dpi (day post inoculation), 5 dpi, and 8 dpi of virus treatment.
3. qPCR (quantitative Polymerase chain reaction)3. qPCR (quantitative polymerase chain reaction)
dsRNA에 의한 PepMoV 증식 억제 효과를 확인하기 위해, 실험 기간 동안 얻은 샘플로 총 RNA를 추출한 후 DNase 처리하고, 역전사 반응을 통해 cDNA로 합성한 뒤 qPCR을 진행하여 GFP의 상대적인 발현 수준을 측정하였다.To confirm the inhibitory effect of PepMoV proliferation by dsRNA, total RNA was extracted from samples obtained during the experiment, treated with DNase, synthesized into cDNA through reverse transcription, and qPCR was performed to measure the relative expression level of GFP .
(reference)L23
(reference)
실시예 1. PepMoV의 Example 1. PepMoV HC-proHC-pro 유전자 중앙 부위를 표적으로 하는 dsRNA의 바이러스 증식 억제능 분석 Analysis of dsRNA targeting the central region of the gene to inhibit viral growth
본 발명자는 PepMoV의 HC-pro 유전자의 중앙 부위(서열번호 4)를 다시 5', middle, 및 3'의 세 부위로 구분하여 이들 각각을 표적으로 하는 dsRNA를 제작하였다(도 1). 상기 각 dsRNA의 PepMoV 증식 억제능을 분석하기 위해, dsRNA를 전처리한 후 PepMoV를 식물체에 처리하고 바이러스 게놈 내 클로닝된 GFP의 발현 수준을 분석하였다.The present inventors divided the central region (SEQ ID NO: 4) of the HC-pro gene of PepMoV into three regions, 5', middle, and 3', and prepared dsRNA targeting each of them (FIG. 1). In order to analyze the ability of each dsRNA to inhibit PepMoV growth, plants were treated with PepMoV after pretreatment with dsRNA, and the expression level of GFP cloned in the virus genome was analyzed.
그 결과, 바이러스만 단독 처리한 조건(PepMov)과 비교하여 HC-pro 유전자 표적 dsRNA를 전처리하고 바이러스를 처리한 실험군에서는 GFP의 상대적 발현 수준이 현저히 감소되어, PepMoV의 증식이 억제되고 있음을 알 수 있었다(도 2A). 특히, HC-pro 유전자의 중앙 부위 중에서 5' 표적 dsRNA 처리군에서 3' 또는 middle 부위 표적 dsRNA 처리군보다 증식 억제 효과가 보다 우수하게 확인되었고, 이는 바이러스 처리 후 5일 및 8일째의 GFP 형광 발현에서도 동일하게 나타났다(도 2B). 상기 결과는 본 발명에 따른 dsRNA가 PepMoV 방제에 효과적으로 사용될 수 있음을 시사하였다.As a result, compared to the virus-only treatment condition (PepMov), the relative expression level of GFP was significantly reduced in the experimental group pre-treated with HC-pro gene-targeting dsRNA and virus-treated, indicating that PepMoV proliferation was inhibited. (Fig. 2A). In particular, among the central region of the HC-pro gene, the 5' target dsRNA treatment group showed a better proliferation inhibitory effect than the 3' or middle region target dsRNA treatment group, which was confirmed by the GFP fluorescence expression on the 5th and 8th days after virus treatment. The same appeared in (Fig. 2B). The above results suggested that the dsRNA according to the present invention can be effectively used for controlling PepMoV.
<110> Seoul National University R&DB Foundation <120> dsRNA targeting HC-pro middle region for inhibiting PepMoV replication and uses thereof <130> PN21315 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 210 <212> RNA <213> Artificial Sequence <220> <223> dsRNA HC-pro_mid_5' <400> 1 gaaagggaac gagaauacaa gugaggaaug gucgacugcu cgacaacauu uaaaggagcu 60 ggugagauuu cagaagaaua gaacugauaa uauaaagaaa ggugacuugg caucauucag 120 aaauaagcuu ucugcucgug cacaguacaa uuuguauuua ucaugcgaua aucagcuuga 180 caagaaugcu aguuuucuau ggggucagcg 210 <210> 2 <211> 209 <212> RNA <213> Artificial Sequence <220> <223> dsRNA HC-pro_mid_mid <400> 2 ucaugcgaua aucagcuuga caagaaugcu aguuuucuau ggggucagcg agaauaccau 60 gcacgucggu uuuuccuaaa cuucuuucaa caaauagacc caucaaaagg uuauuugucg 120 uaugaagauc ggaccauacc aaaugguucu cgaaaguuag cuauaggcaa cuuaauuguu 180 ccacucgauu uagcugaauu ccgaaaacg 209 <210> 3 <211> 210 <212> RNA <213> Artificial Sequence <220> <223> dsRNA HC-pro_mid_3' <400> 3 cuauaggcaa cuuaauuguu ccacucgauu uagcugaauu ccgaaaacgc augaagggca 60 ucgacacuca gcaaccacca auugguaagu acuguacaag ccaauuggau gggaauuuug 120 uguauccgug cugcugcacg acgcuugaug auggccaacc aauucgauca gcuguuuacg 180 caccgacuaa gaaacauuua guuguuggua 210 <210> 4 <211> 530 <212> DNA <213> Pepper mottle virus <400> 4 gaaagggaac gagaatacaa gtgaggaatg gtcgactgct cgacaacatt taaaggagct 60 ggtgagattt cagaagaata gaactgataa tataaagaaa ggtgacttgg catcattcag 120 aaataagctt tctgctcgtg cacagtacaa tttgtattta tcatgcgata atcagcttga 180 caagaatgct agttttctat ggggtcagcg agaataccat gcacgtcggt ttttcctaaa 240 cttctttcaa caaatagacc catcaaaagg ttatttgtcg tatgaagatc ggaccatacc 300 aaatggttct cgaaagttag ctataggcaa cttaattgtt ccactcgatt tagctgaatt 360 ccgaaaacgc atgaagggca tcgacactca gcaaccacca attggtaagt actgtacaag 420 ccaattggat gggaattttg tgtatccgtg ctgctgcacg acgcttgatg atggccaacc 480 aattcgatca gctgtttacg caccgactaa gaaacattta gttgttggta 530 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggacgacggc aactacaaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttgtactcca gcttgtgccc 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aaggatgccg tgaagaagat gt 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gcatcgtagt caggagtcaa cc 22 <110> Seoul National University R&DB Foundation <120> dsRNA targeting HC-pro middle region for inhibiting PepMoV replication and uses <130> PN21315 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 210 <212> RNA <213> artificial sequence <220> <223> dsRNA HC-pro_mid_5' <400> 1 gaaagggaac gagaauacaa gugagggaaug gucgacugcu cgacaacauu uaaaggagcu 60 ggugagauuu cagaagaaua gaacugauaa uauaaagaaa ggugacuugg caucauucag 120 aaauaagcuu ucugcucgug cacaguacaa uuuguauuua ucaugcgaua aucagcuuga 180 caagaaugcu aguuuucuau ggggucagcg 210 <210> 2 <211> 209 <212> RNA <213> artificial sequence <220> <223> dsRNA HC-pro_mid_mid <400> 2 ucaugcgaua aucagcuuga caagaaugcu aguuuucuau ggggucagcg agaauaccau60 gcacgucggu uuuuccuaaa cuucuuucaa caaauagacc caucaaaagg uuauuugucg 120 uaugaagauc ggaccauacc aaaugguucu cgaaaguuag cuauaggcaa cuuaauuguu 180 ccacucgauu uagcugaauu ccgaaaacg 209 <210> 3 <211> 210 <212> RNA <213> artificial sequence <220> <223> dsRNA HC-pro_mid_3' <400> 3 cuauaggcaa cuuaauuguu ccacucgauu uagcugaauu ccgaaaacgc augaagggca 60 ucgacacuca gcaaccacca auugguaagu acuguacaag ccaauuggau gggaauuuug 120 uguauccgug cugcugcacg acgcuugaug auggccaacc aauucgauca gcuguuuacg 180 caccgacuaa gaaacauuua guuguuggua 210 <210> 4 <211> 530 <212> DNA <213> pepper mottle virus <400> 4 gaaagggaac gagaatacaa gtgaggaatg gtcgactgct cgacaacatt taaaggagct 60 ggtgagattt cagaagaata gaactgataa tataaagaaa ggtgacttgg catcattcag 120 aaataagctt tctgctcgtg cacagtacaa tttgtattta tcatgcgata atcagcttga 180 caagaatgct agttttctat ggggtcagcg agaataccat gcacgtcggt ttttcctaaa 240 cttctttcaa caaatagacc catcaaaagg ttatttgtcg tatgaagatc ggaccatacc 300 aaatggttct cgaaagttag ctataggcaa cttaattgtt ccactcgatt tagctgaatt 360 ccgaaaacgc atgaagggca tcgacactca gcaaccacca attggtaagt actgtacaag 420 ccaattggat gggaattttg tgtatccgtg ctgctgcacg acgcttgatg atggccaacc 480 aattcgatca gctgtttacg caccgactaa gaaacattta gttgttggta 530 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 5 ggacgacggc aactacaaga 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 6 ttgtactcca gcttgtgccc 20 <210> 7 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 7 aaggatgccg tgaagaagat gt 22 <210> 8 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 8 gcatcgtagt caggagtcaa cc 22
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| KR20150140510A (en) * | 2014-06-05 | 2015-12-16 | 안동대학교 산학협력단 | dsRNA for the control of Helicoverpa assulta, insecticide composition and control method comprising it |
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| KR20150140510A (en) * | 2014-06-05 | 2015-12-16 | 안동대학교 산학협력단 | dsRNA for the control of Helicoverpa assulta, insecticide composition and control method comprising it |
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