KR101024112B1 - Improving method for Plant disease resistance - Google Patents

Improving method for Plant disease resistance Download PDF

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KR101024112B1
KR101024112B1 KR1020090001958A KR20090001958A KR101024112B1 KR 101024112 B1 KR101024112 B1 KR 101024112B1 KR 1020090001958 A KR1020090001958 A KR 1020090001958A KR 20090001958 A KR20090001958 A KR 20090001958A KR 101024112 B1 KR101024112 B1 KR 101024112B1
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oshrl
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박상렬
변명옥
문석준
한세연
박수철
신동진
윤충효
배신철
황덕주
김정구
이부영
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Abstract

본 발명은 식물병 저항성을 증진시키는 방법으로서, 더욱 상세하게는 서열번호 1의 염기서열을 갖는 OsHRL(Oryza sativa HR-like lesion-inducing gene) 유전자를 포함하는 형질전환용 벡터로 식물 세포를 형질전환하여 OsHRL 유전자를 과발현시키는 단계를 포함하는 식물병 저항성을 증진시키는 방법에 관한 것이다. The present invention is a method for enhancing plant disease resistance, and more specifically, transforming plant cells with a transformation vector comprising an OsHRL (Oryza sativa HR-like lesion-inducing gene) gene having a nucleotide sequence of SEQ ID NO: 1. The present invention relates to a method for enhancing plant disease resistance comprising overexpressing an OsHRL gene.

본 발명에 따르면, 병 저항성 작물개발을 위한 분자육종소재로 이용가능하며 타 작물에도 이용가능할 것으로 기대된다. 또한, 병 저항성 품종육성으로 농약사용절감과 생산성 향상 및 안전한 농산물 공급이 가능하다는 이점이 있다. According to the present invention, it is expected to be available as a molecular breeding material for the development of disease resistant crops and also to other crops. In addition, by fostering disease-resistant varieties, there is an advantage that it is possible to reduce pesticide use, improve productivity, and provide safe agricultural products.

Description

식물병의 저항성 증진 방법{Improving method for Plant disease resistance}Improving method for plant disease resistance

본 발명은 식물병 저항성을 증진시키는 방법으로서, 더욱 상세하게는 서열번호 1의 염기서열을 갖는 OsHRL(Oryza sativa HR-like lesion-inducing gene) 유전자를 포함하는 형질전환용 벡터로 식물 세포를 형질전환하여 OsHRL 유전자를 과발현시키는 단계를 포함하는 식물병 저항성을 증진시키는 방법에 관한 것이다. The present invention is a method for enhancing plant disease resistance, and more specifically, transforming plant cells with a transformation vector comprising an OsHRL (Oryza sativa HR-like lesion-inducing gene) gene having a nucleotide sequence of SEQ ID NO: 1. The present invention relates to a method for enhancing plant disease resistance comprising overexpressing an OsHRL gene.

종래 병 저항성 품종 육성의 연구는 전통적인 교배 육종에 의존하였으며 최근까지 벼 흰잎마름병 저항성 관련 벼 Xa 유전자들에 대한 연구는 많이 되어 오고 있으나 저항성이 쉽게 무너지는 경향이 있었다.Previous studies on breeding disease-resistant varieties have relied on traditional breeding breeds. Until recently, research on rice Xa genes related to rice leaf blight resistance has been conducted, but resistance tended to collapse easily.

본 발명은 병 저항성 작물 육종을 위하여 병 저항성 유전자를 분리ㆍ 분석한 후 벼에 형질전환시켜 벼 흰잎마름병 저항성 작물개발용 유전자를 개발하고자 한다. The present invention is to develop a gene for the development of rice leaf blight resistant crops by separating and analyzing the disease resistance gene for breeding disease resistant crops and transforming the rice.

상기 과제를 해결하기 위하여 본 발명은 서열번호 1의 염기서열을 갖는 OsHRL(Oryza sativa HR-like lesion-inducing gene) 유전자를 포함하는 형질전환용 벡터로 식물 세포를 형질전환하여 OsHRL 유전자를 과발현시키는 단계를 포함하는 식물병의 저항성 증진 방법을 제공한다. In order to solve the above problems, the present invention comprises the steps of overexpressing the OsHRL gene by transforming a plant cell with a transformation vector comprising an OsHRL (Oryza sativa HR-like lesion-inducing gene) gene having a nucleotide sequence of SEQ ID NO: 1 It provides a method for enhancing resistance of plant diseases comprising a.

또한, 본 발명은 식물병 저항성을 증진시키는, 서열번호 1의 염기서열을 갖는 OsHRL(Oryza sativa HR-like lesion-inducing gene) 유전자를 포함하는 형질전환용 벡터를 제공한다.In addition, the present invention provides a transformation vector comprising an OsHRL (Oryza sativa HR-like lesion-inducing gene) gene having a nucleotide sequence of SEQ ID NO: 1, which enhances plant disease resistance.

본 발명에 따르면, 병 저항성 작물개발을 위한 분자육종소재로 이용가능하며 타 작물에도 이용가능할 것으로 기대된다. 또한, 병 저항성 품종육성으로 농약사용절감과 생산성 향상 및 안전한 농산물 공급이 가능하다는 이점이 있다. According to the present invention, it is expected to be available as a molecular breeding material for the development of disease resistant crops and also to other crops. In addition, by fostering disease-resistant varieties, there is an advantage that it is possible to reduce pesticide use, improve productivity, and provide safe agricultural products.

[[ 실시예1Example 1 ] ] YeastYeast twotwo -- hybridhybrid 에 의한 유전자 선발Gene selection by

Marker exchange mutagenesis를 통해 병원성 관련 기능을 확인한 Xoo effector인 avrBs2를 bait로 하고 Xoo 처리 3시간 후의 동진벼 cDNA library를 prey로하여 yeast two-hybrid를 실시하여 β-galactosidase 활성을 보이는 클론을 선발한 것을 도 1에 나타내었다.  A clone showing the β-galactosidase activity was selected by performing yeast two-hybrid using avrBs2, a Xoo effector whose pathogenic function was confirmed through marker exchange mutagenesis, as a bait, and a cDNA library as a prey after 3 hours of Xoo treatment. Shown in

[[ 실시예2Example 2 ] 벼 (] Rice ( OsyzaOsyza sativasativa )에서 )in RTRT -- PCRPCR 에 의해 By OsHRLOshrl 유전자 분리 Gene isolation

도 1에서 얻어진 클론들을 염기서열 분석하고 blast 조사 결과 병 저항성과 관련 있는 유전자를 분리하였으며 OsHRL로 명명하고 그 염기서열 및 아미노산을 도 2 및 서열번호 1에 나타내었다 (GenBank Accession number; FJ548850). The clones obtained in FIG. 1 were sequenced, and genes related to disease resistance were isolated from blast irradiation, named OsHRL, and the sequences and amino acids thereof are shown in FIG. 2 and SEQ ID NO: 1 (GenBank Accession number; FJ548850).

[[ 실시예3Example 3 ] ] OsHRLOshrl 유전자의 발현양상 Expression of genes

벼 종자를 2-3일간 발아시켜 파종 후 6주 된 동진벼에 PSA배지에서 2일간 키운 벼 흰잎마름병 균을 1mM MgCl2에 현탁시켜 OD600에서 1로 조정하여 가위접종 한 후 시간대별로 sampling하였다. 각각의 시료를 RNeasy Plant Mini Kit (Qiagen Co.)을 이용하여 total RNA를 분리하였다. 1㎍의 총 RNA로 reverse transcriptase를 이용하여 first strand를 만들고 OsHRL 특이 primer인 OsHRL-F (5'-ATG GGG TTC GTC TCC TTC G-3')와 OsHRL-R (5'-CTA GTT CGT CTT CGA CTT GGG A-3'), actin의 5‘ 특이 primer Actin-F (5'-GTC TGC GAT AAT GGA ACT GGT ATG GTC AAG GCT-3’) 과 3‘ 특이 primer Actin-R (5'-GTA CCC GCA TCA GGC ATC TG-3')을 이용하여 PCR을 수행하였고 그 결과는 도 3에 나타내었다. Rice seed germinated for 2-3 days, seeded rice leaf blight bacteria grown in PSA medium for 2 days in Dongjin rice 6 weeks after sowing in 1mM MgCl 2 , adjusted to 1 at OD600, and sampled by time. Each sample was isolated from total RNA using the RNeasy Plant Mini Kit (Qiagen Co.). Using 1 ㎍ of total RNA, make a first strand using reverse transcriptase and OsHRL-F (5'-ATG GGG TTC GTC TCC TTC G-3 ') OsHRL-R (5'-CTA GTT CGT CTT CGA CTT GGG A-3 '), 5' specific primer Actin-F of actin (5'-GTC TGC GAT AAT GGA ACT GGT ATG GTC AAG GCT-3 ') and 3 PCR was performed using 'specific primer Actin-R (5'-GTA CCC GCA TCA GGC ATC TG-3') and the results are shown in FIG.

도 3에서 보는 바와 같이 OsHRL 유전자는 벼 흰잎마름병원균처리에서 1시간 이후부터 발현이 유도됨을 알 수 있었다.As shown in FIG. 3, the OsHRL gene was found to be induced after 1 hour in rice leaf blight pathogen treatment.

[[ 실시예4Example 4 ] 형질전환용 운반체 작성 및 형질전환Transformation carrier creation and transformation

도 3에서 보여주는 바와 같이 OsHRL 유전자가 병원균에 의해 발현이 증진되는 효과를 보았기 때문에 기능분석을 위해 식물 형질전환용 벡터를 만들었다. 먼저, OsHRL clone으로 과발현 벡터를 작성하기 위해 5‘ primer A (5’- AA AAA GCA GGC TAC ATG GGG TTC GTC TCC TTC-3’)와 3’ primer B (5’-A GAA AGC TGG GTC CTA GTT CGT CTT CGA CTT G-3’)로 polymerase chain reaction (PCR)에 의해 증폭하였고 5‘ primer iA (5'-AA AAA GCA GGC TCC TTT GTG CAG CTC TTC ATG-3')와 3’ primer B (5’-A GAA AGC TGG GTC CTA GTT CGT CTT CGA CTT G-3’)로 polymerase chain reaction (PCR)에 의해 증폭한 후 각각 attB1 (5'-G GGG ACA AGT TTG TAC AAA AAA GCA GGC T-3') 과 attB2 (5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3')와 다시 PCR로 증폭한 후 Invitrogen의 Gateway cloning system을 이용하여 pDONOR201에 BP 반응에 의해 entry clone을 만들었다. 이 entry clone으로부터 식물발현 벡터인 pB7WG2D와 pB7GWIWG2(2)에 각각 LR 반응에 의해 식물발현용 벡터를 만들었다. 그 지도는 도 4에 나타내었다.  As shown in FIG. 3, since the expression of the OsHRL gene was seen to be enhanced by the pathogen, a plant transformation vector was made for functional analysis. First, 5 'primer A (5'- AA AAA GCA GGC TAC ATG GGG TTC GTC TCC TTC-3') and 3 'primer B (5'-A GAA AGC TGG GTC CTA GTT) Amplified by polymerase chain reaction (PCR) with CGT CTT CGA CTT G-3 ') and 5' primer iA (5'-AA AAA GCA GGC TCC TTT GTG CAG CTC TTC ATG-3 ') and 3' primer B (5 Amplified by polymerase chain reaction (PCR) with '-A GAA AGC TGG GTC CTA GTT CGT CTT CGA CTT G-3') and then attB1 (5'-G GGG ACA AGT TTG TAC AAA AAA GCA GGC T-3 ') ) And PCR were amplified again with attB2 (5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3 ') and the entry clone was made by BP reaction to pDONOR201 using Invitrogen's Gateway cloning system. Plant expression vectors were generated from the entry clones by LR reactions to pB7WG2D and pB7GWIWG2 (2), respectively. The map is shown in FIG.

제조된 OsHRL/pB7WG2D 벡터와 OsHRL-i/pB7GWIWG2(2) 벡터를 각각 아그로박테리움 투메파시엔스 LBA4404 (Agrobacterium tumefaciens LBA4404)에 electrophoration으로 도입한 다음 벼 (품종: 동진)에 형질전환하였다. Shooting 된 개체를 MSO media에 옮겨 27～29℃/light 조건에서 배양하여 뿌리가 내리고 1주일이 지나면 pot에 심어 온실에서 배양하고 1주일 후 0.3% 바스타를 spray하여 생존한 개체를 선발하였다. 그 결과 25개의 과발현 형질전환체와 200개의 발현억제 형질전환체를 얻었다.The prepared OsHRL / pB7WG2D vector and OsHRL-i / pB7GWIWG2 (2) vector were respectively used as Agrobacterium tumefaciens LBA4404 ( Agrobacterium tumefaciens LBA4404) was introduced by electrophoration and then transformed into rice (breed: Dongjin). Shooting individuals were transferred to MSO media and cultured at 27 ～ 29 ℃ / light conditions, and after 1 week, roots were planted in pots, incubated in greenhouse, and after 1 week, 0.3% batha was sprayed to select surviving individuals. As a result, 25 overexpressing transformants and 200 expression suppressing transformants were obtained.

[[ 실시예5Example 5 ] 형질전환체의 분자생물학적 특성분석] Molecular Biology Characterization of Transformant

OsHRL 과발현 및 발현억제 형질전환체에의 잎으로부터 총 RNA를 분리한 후 OsHRL 유전자를 탐침으로 하여 northern 혼성화를 실시하여 발현을 분석하였고 그 결과는 도 5에 나타내었다.    OsHRL overexpression and expression suppression Total RNA was isolated from the leaves of the transformant, and then subjected to northern hybridization using the OsHRL gene as a probe to analyze expression. The results are shown in FIG. 5.

[[ 실시예6Example 6 ] ] OsHRLOshrl 유전자 형질전환 벼의 벼 흰잎마름병 저항성 검정 Genetically Transgenic Rice Rice Leaf Blight Resistance Assay

OsHRL 유전자 과발현 및 발현 억제 형질전환체가 벼 흰잎마름병에 대해 저항성을 보이는지 알아보기 위해 PSA배지에서 2일간 키운 벼흰잎마름병균을 1mM MgCl2에 현탁시켜 OD600에서 1로 조정하여 가위로 잎의 끝을 자르는 방법에 의해 접종하였다. 접종 14일 후에 병원균이 진전되는 정도를 자로 측정하였고 그 결과는 도 6에 나타내었다.   OsHRL Gene Overexpression and Expression Inhibition To find out if the transformants are resistant to rice leaf blight, two days of PSA cultures were suspended in 1 mM MgCl2 and adjusted to 1 at OD600 to cut the leaves with scissors. Inoculated by After 14 days of inoculation, the extent of pathogen progress was measured by rule. The results are shown in FIG. 6.

도 1은 Xoo의 avrBs2와 벼 cDNA library를 이용한 Yeast two-hybrid 선발 결과이다. 1 is a result of selecting Yeast two-hybrid using avrBs2 and rice cDNA library of Xoo.

도 2는 OsHRL을 암호화하는 유전자의 염기서열 및 아미노산 서열을 나타낸다. Figure 2 shows the nucleotide sequence and amino acid sequence of the gene encoding OsHRL.

도 3은 OsHRL 유전자의 발현양상: RT-PCR결과이다. Figure 3 shows the expression pattern of the OsHRL gene: RT-PCR.

도 4는 OsHRL 형질전환용 벡터 지도이다. 4 is a vector map for OsHRL transformation.

도 5는 형질전환체에서의 OsHRL의 발현분석을 나타낸다.5 shows expression analysis of OsHRL in transformants.

도 6은 OsHRL 유전자 과발현 및 발현억제 형질전환체의 벼 흰잎마름병에 대한 저항성 검정 결과이다.Figure 6 shows the results of resistance test against rice leaf blight of OsHRL gene overexpression and expression inhibitory transformants.

<110> Republic of KOREA <120> Improving method for Plant disease <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 474 <212> DNA <213> Oryza sativa HR-like lesion-inducing gene <400> 1 atggggttcg tctccttcgc tgggagggtc ctcttcgcct ccgtcttcct cctctccgcc 60 taccaggagt ttagtgaatt tggagctgat ggtggaccag ctgcaaaggc ccttcggcct 120 aagtataacg tcttcaccaa aaatatttct gcacatttgg gagtagcagt gcctcatgtt 180 gagttgaagc acattgttgc tgctaccatt ggtctgaagg gtctgggagg tctccttttt 240 atcctgagca gttcgtttgg tgcttatctc ctgctgattt acctcgcttt tatcacacct 300 gttgtctacg acttctacaa ctacaacatg gagaagtccg aatttgtgca gctcttcatg 360 aagttcacac agaatttggc tctctttggg gcgcttcttt tcttcctggg catgaagaac 420 tccattccca agaggcaggc caagaagaag gctcccaagt cgaagacgaa ctag 474 <110> Republic of KOREA <120> Improving method for Plant disease <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 474 <212> DNA <213> Oryza sativa HR-like lesion-inducing gene <400> 1 atggggttcg tctccttcgc tgggagggtc ctcttcgcct ccgtcttcct cctctccgcc 60 taccaggagt ttagtgaatt tggagctgat ggtggaccag ctgcaaaggc ccttcggcct 120 aagtataacg tcttcaccaa aaatatttct gcacatttgg gagtagcagt gcctcatgtt 180 gagttgaagc acattgttgc tgctaccatt ggtctgaagg gtctgggagg tctccttttt 240 atcctgagca gttcgtttgg tgcttatctc ctgctgattt acctcgcttt tatcacacct 300 gttgtctacg acttctacaa ctacaacatg gagaagtccg aatttgtgca gctcttcatg 360 aagttcacac agaatttggc tctctttggg gcgcttcttt tcttcctggg catgaagaac 420 tccattccca agaggcaggc caagaagaag gctcccaagt cgaagacgaa ctag 474  

Claims (4)

서열번호 1의 염기서열을 갖는 OsHRL(Oryza sativa HR-like lesion-inducing gene) 유전자를 포함하는 형질전환용 벡터로 식물 세포를 형질전환하여 OsHRL 유전자를 과발현시키는 단계를 포함하는 식물병의 저항성 증진 방법.Method of enhancing resistance of a plant disease comprising overexpressing an OsHRL gene by transforming a plant cell with a transformation vector comprising an OsHRL (Oryza sativa HR-like lesion-inducing gene) gene having a nucleotide sequence of SEQ ID NO: 1 . 식물병 저항성을 증진시키는, 서열번호 1의 염기서열을 갖는 OsHRL(Oryza sativa HR-like lesion-inducing gene) 유전자를 포함하는 형질전환용 벡터.A transformation vector comprising an Oryza sativa HR-like lesion-inducing gene (OsHRL) gene having a nucleotide sequence of SEQ ID NO: 1, which enhances plant disease resistance. 제 1항에 있어서, The method of claim 1, 상기 식물병은 흰잎마름병인 것을 특징으로 하는 식물병의 저항성 증진 방법.The plant disease is a leaf blight disease resistance method of plant diseases, characterized in that. 제2항의 형질전환용 벡터로 벼를 형질전환시킴으로써 병 저항성이 증진된 벼 식물체.A rice plant having improved disease resistance by transforming rice with the transformation vector of claim 2.
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KR20020015179A (en) * 2000-08-21 2002-02-27 김강권 Gene encoding for a transcription factor which regulates the expression of defense related genes in rice and method for inducing the systemic acquired resistance by using this gene
KR20060031524A (en) * 2004-10-08 2006-04-12 동아대학교 산학협력단 A pathogenesis-related gene ogpr1 isolated from wild rice, the sequences of amino acid and the transgenic plant using the same
KR100803393B1 (en) 2006-09-05 2008-02-14 대한민국 A method enhancing disease resistance using oslrp gene in rice

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KR20020015179A (en) * 2000-08-21 2002-02-27 김강권 Gene encoding for a transcription factor which regulates the expression of defense related genes in rice and method for inducing the systemic acquired resistance by using this gene
KR20060031524A (en) * 2004-10-08 2006-04-12 동아대학교 산학협력단 A pathogenesis-related gene ogpr1 isolated from wild rice, the sequences of amino acid and the transgenic plant using the same
KR100803393B1 (en) 2006-09-05 2008-02-14 대한민국 A method enhancing disease resistance using oslrp gene in rice

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