CN109280082B - A kind of rice blast resistance gene Pi57 and its coding albumen and application - Google Patents

Abstract

The present invention discloses a kind of rice blast resistance gene Pi57 and its coding albumen and application.The nucleotide sequence of the rice blast resistance gene Pi57 is as shown in SEQ ID NO:2.Its amino acid sequence for encoding albumen is as shown in SEQ ID NO:3.Gene Pi57 of the present invention averagely reaches 91% or more from the anti-spectrum of 322 Pyricularia oryzae bacterial strains of Cambodia, Laos, Burma, Thailand, Vietnam and China to acquisition, has the characteristics that wide spectrum blast resisting, is the outstanding anti-source of breeding for disease resistance；The seed analysis of disease-resistant plant and its offspring that gene Pi57 is obtained by the genetic transformation of gene shows that gene Pi57 can stablize and is hereditary to offspring.

Description

A kind of rice blast resistance gene Pi57 and its coding albumen and application

Technical field

The present invention relates to field of plant genetic project technology, and in particular to rice blast resistance gene and its coding and are answered at albumen With.

Background technique

Crop disease is the Main Factors for causing crop yield loss.With the continuous growth of world population, Crop Improvement The resistance level of disease is to increase grain yield, Ensuring Food Safety seems especially important.Rice is the most important grain in the world One of food crop, the whole world have the population of more than half using rice as staple food.Caused by fungi (Magnaporthe oryzae) Rice blast be that world's rice production district causes harm one of disease the most serious, the whole world is every year because of the damage caused by rice blast harm 7,000,000,000 dollars were quenched, morbidity field yield causes the loss of 10-30%, and serious field even has no harvest.It was verified that breeding It is to control that the disease is the most economical, efficient and environmentally friendly measure in Rice Production with resistant rice cultivars are promoted, and it is disease-resistant Excavation, research and the utilization of gene are effective basis for carrying out breeding for disease resistance and core.

By extensive genetic analysis, is identified from the cultivated rice of Oryza and wild rice at present, positions more than 100 a rice blasts Sick resistant gene^[1].Since first rice blast resistance gene successful clone in 1999,26 rice have at least been cloned at present Seasonal febrile diseases resistant gene^[1-3].It is removed in these genes cloned by the topology discovery for encoding albumen to these disease-resistant genes Pid-2 gene, which encodes a receptor protein kinase and pi21 gene one, has heavy metal binding domain and Pro-rich structural domain Agnoprotein outside, other genes cloned encode have nucleotide binding site and leucine repetitive structure domain (NBS- LRR albumen)^[4], NBS-LRR albumen can be further divided into albumen of the albumen n end with TIR structural domain or CC structural domain again, that is, have The albumen of TIR-NBS-LRR or CC-NBS-LRR structural domain.Further study showed that the NBS-LRR of plant disease resistance genes coding Direct or indirect interaction can occur for the albumen that can directly encode with pathogen nontoxic gene, and then cause the resistance of host anti- It answers.As the albumen that rice blast resistance gene Pita is encoded in rice is directly encoded with avirulence gene of rice blast AvrPita Albumin A vrPita interaction, so as to cause Resistant reaction；And the NBS-LRR albumen of the disease-resistant gene RPM1 coding in arabidopsis is It identifying the host albumen RIN4 modified by pathogen, and then causes the Resistant reaction of host, such identification is indirect identification, Rather than the direct interaction of ill-resistant protein and pathogen nontoxic gene.Constantly being cloned for disease-resistant gene, especially has different knots Anti-disease mechanism further to further investigate rice is found and realizes disease-resistant persistent way by the clone of structure type disease-resistant gene Diameter, to realize that the sustainable use of resistant rice cultivars lays the foundation.

[1]Ashikani S,Rafili MY,Shabanimofrad M,Ghasemzadeh A,Ravanfar SA, Latif MA(2016)Molecular progress on the mapping and cloning of functional genes for blast resistance in rice(Oryza sativa L.):current status and future considerations.Crit Rev Biotechnol 36:353-367

[2]Ballini E,Morel JB,Droc G,Price A,Courtois B,Notteghem JL,Tharreau D(2008)A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance.Mol Plant Microbe Interact 21:859–868

[3]Deng Y,Zhai K,Xie Z,Yang D,Zhu X,Liu J,Wang X,Qin P,Yang Y,Zhang G,Li Q,Zhang J,Wu S,Milazzo J,Mao B,Wang E,Xie H,Tharreau D,He Z.Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance.Science,2017,355:962-965.DOI:10.1126/science.aai8898

[4]Dangl JL and Jones JD.Plant pathogens and integrated defence Responses to infection.Nature, 2001,411:826-833

Summary of the invention

It is existing to solve the burden of the gene linkage as caused by genetic linkage in conventional cross-breeding of the existing technology Rice blast resistance gene in most gene to there is anti-spectrum narrow, and be difficult in conventional breeding to obtain disease resistance it is good, its Its comprehensive agronomy character again excellent new varieties the technical issues of and overcome the problems, such as that resistance is easy to lose, the present invention provides one kind Rice blast resistance gene Pi57 and its coding albumen and application.

Technical scheme is as follows:

1. a kind of rice blast resistance gene Pi57, nucleotide sequence is as shown in SEQ ID NO:2.

2. the coding albumen of rice blast resistance gene Pi57 described in technical solution 1, amino acid sequence such as SEQ ID Shown in NO:3.

3. the primers F 57-SalI-Fw and primers F 57- of rice blast resistance gene Pi57 described in amplification technique scheme 1 The base sequence of AscI-Rv, the primers F 57-SalI-Fw as shown in SEQ ID NO:7, the primers F 57-AscI-Rv's Base sequence is as shown in SEQ ID NO:8.

4. the preparation method of rice blast resistance gene Pi57 described in technical solution 1: with resistant donor rice introgression line IL- The genomic DNA of E1454 is template, and primers F 57-SalI-Fw and primers F 57-AscI-Rv described in technical solution 3 are carried out PCR amplification obtains rice blast resistance gene Pi57.

5. a kind of rice blast resistance gene Pi57 answering in Molecular Breeding for Blast Resistance described in technical solution 1 With.

6. rice blast resistance gene Pi57 is transferred in the rice of sense rice blast according to application described in technical solution 5, Obtain the rice with rice blast resistance.

Compared with prior art, innovative point of the invention and the utility model has the advantages that

Gene Pi57 of the present invention is to 322 Pyricularia oryzaes acquired from Cambodia, Laos, Burma, Thailand, Vietnam and China The anti-spectrum of bacterial strain averagely reaches 91% or more, has the characteristics that wide spectrum blast resisting, is the outstanding anti-source of breeding for disease resistance.Gene Pi57 is from long male wild rice, since it is big with the genome difference of cultivated rice, according to the reference sequence of known rice genome Column are difficult to obtain the sequence of targeting regions.The method that the present invention passes through genomic walking (genome walking), successfully obtains The unknown DNA sequence dna of targeting regions in rice introgression line IL-E1454 is laid a good foundation for subsequent clone.

It is transferred in susceptible rice varieties using rice blast resistance gene Pi57 provided by the invention, can produce new resist Sick rice varieties；Meanwhile after cloning disease-resistant gene, it is difficult that the utilization of gene can break in traditional breeding way gene between different plant species The problem of with transformation, blast resistant gene Pi57 provided by the invention can be transferred into rice with transformation technology, pass through base The conversion of cause, the period needed for both can greatly shortening breeding, while can avoid target gene present in traditional breeding technology The problem of being brought with the Linkage drag of other bad character genes of control.Disease-resistant plant that the present invention is obtained by genetic transformation and Gene Pi57 stabilization can be hereditary to offspring by way of conventional sexual hybridization by the seed of its offspring.Gene Pi57 has wide Compose blast resisting the characteristics of, using the gene be bred as disease-resistant varieties can large area planted.

It is the base sequence of the promoter of gene Pi57 shown in SEQ ID NO:1 in sequence table.

It is the nucleotide sequence of gene Pi57 shown in SEQ ID NO:2 in sequence table.

It is the amino acid sequence of the coding albumen of gene Pi57 shown in SEQ ID NO:3 in sequence table, it includes 3 masters The structural domain wanted: transmembrane domain, ARM (armadillo repeats) repetitive structure domain and Pro-rich structural domain, wherein Transmembrane domain is (29-191aa)；ARM repetitive structure domain (207-811aa)；Pro-rich structural domain (817-839aa).

It is the cDNA nucleotide sequence of gene Pi57 shown in SEQ ID NO:4 in sequence table.

It is the primer of the cDNA nucleotide sequence of amplification gene Pi57 shown in SEQ ID NO:5 in sequence table The base sequence of Pi57cDNA-Fw.

It is the primer of the cDNA nucleotide sequence of amplification gene Pi57 shown in SEQ ID NO:6 in sequence table The base sequence of Pi57cDNA-Rv.

It is the base sequence of the primers F 57-SalI-Fw of amplification gene Pi57 shown in SEQ ID NO:7 in sequence table.

It is the base sequence of the primers F 57-AscI-Rv of amplification gene Pi57 shown in SEQ ID NO:8 in sequence table.

It is the base sequence for expanding hygromycin gene Molecular Identification primer hptFw shown in SEQ ID NO:9 in sequence table.

It is the base sequence for expanding hygromycin gene Molecular Identification primer hptRv shown in SEQ ID NO:10 in sequence table Column.

It is the base sequence of amplification gene Pi57 Molecular Identification primer Pi57SPFw shown in SEQ ID NO:11 in sequence table Column.

It is the base sequence of amplification gene Pi57 Molecular Identification primer Pi57SPRv shown in SEQ ID NO:12 in sequence table Column.

Detailed description of the invention

Fig. 1: resistance gene of rice blast Pi57 Strategies For The Cloning figure.

Fig. 2: Transgenic Rice Plants identify electrophoretogram: wherein swimming lane M is molecular weight marker DL2000, and swimming lane 1-17 is to turn Change the different T of pCAMBIAAscI-Pi57-3 plasmid₀It is that template carries out selection markers hygromycin for transgenic plant genomic DNA The amplification PCR product of gene (hpt), swimming lane B are clear water control, and swimming lane N is the DNA cloning of transformation receptor kind as a result, swimming lane P For with the amplification of the pCAMBIAAscI-Pi57-3 plasmid positive control containing gene Pi57.

Fig. 3: transgenic positive transformed plant, transgenosis susceptible receptor kind Yunnan are inoculated with using Pyricularia oryzae HN09-1C-7 Excellent No. 1 of round-grained rice and blast resisting donor rice introgression line IL-E1454 response diagram.The 17th article of blade of 1- is transgenosis T₀In generation, is different Plant, DJY1 are the blades of the susceptible receptor kind Dianjingyou No.1 of transgenosis, and IL-E1454 is the resistant donor containing gene Pi57 The blade of rice introgression line IL-E1454.

Fig. 4: transgenosis T is inoculated with using Pyricularia oryzae HN09-1C-7₁For the coseparation analysis figure of plant.M is molecular weight mark Remember DL2000,1 converts kind Dianjingyou No.1 for susceptible receptor, and 2 be blast resistant gene Pi57 donor rice introgression line IL- E1454,3-6 are the T of transgenosis Pi57₁For disease plant, 7-12 is transgenosis Pi57T₁For disease-resistant plant.

Specific embodiment

According to gene cloning and the implementation strategy of functional verification, the present invention is made further using attached drawing and implementation process It is described below, embodiments of the present invention are not limited thereto, unless otherwise specified, technological means employed in implementation process is Conventional means well-known to those skilled in the art.By implementing, complete the separation clone of gene Pi57, functional character and point Son identifies that the gene Pi57 cloned in the present invention can be assembled with genetic transformation carrier appropriate, passes through the strategy of transgenosis Genetic transformation is carried out, the transgenic line for obtaining susceptible rice varieties after transgenosis Pi57 has wide spectrum blast resisting Characteristic.

For the clone for implementing gene Pi57, early period has been carried out following experimental study (referring to Dong et in this laboratory al.,2017.Fine mapping of Pi57(t)conferring broad spectrum resistance against Magnaporthe orzyae in introgression line IL-E1454derived from Oryza longistaminata.PLOS ONE,12(10):e0186201.https://doi.org/10.1371/ Journal.pone.o186201):

(1) anti-spectral property of the rice blast resistance gene Pi57 to rice blast

In order to which for gene Pi57 to the resistance level of rice blast, laboratory utilizes source in clear rice introgression line IL-E1454 In 322 Pyricularia oryzae strain inoculated IL-E1454 of China, Cambodia, Laos, Burma, Thailand and Vietnam, wherein 44 are come From Cambodia, 30 come from Laos, and 25 come from Chinese (table from Vietnam, 176 from Thailand, 28 from Burma, 19 1).Anti- spectrum measurement result shows that gene Pi57 spectrum anti-to the bacterial strain of China reaches 90.91%, and spectrum anti-to the bacterial strain of Cambodia reaches To 95.45%, spectrum anti-to the bacterial strain of Laos and Burma reaches 100%, and spectrum anti-to the bacterial strain of Thailand reaches 83.33%, to Vietnam Bacterial strain it is anti-spectrum reach 78.57%；91% or more is averagely reached to the anti-spectrum of 322 parts of bacterial strains of 6 country's acquisitions.From these mirror Fixed result can be seen that the characteristic that gene Pi57 has wide spectrum blast resisting, can be used as the anti-source of breeding for disease resistance at these Place is utilized (Dong et al., 2017).

The anti-spectrum analysis of 1 rice blast resistance gene Pi57 of table

Remarks: a indicates the bacterial strain number of analysis；B indicates specific gene to the fastness frequency of the Pyricularia oryzae bacterial strain measured (%)

(2) Strategies For The Cloning of blast resistant gene Pi57

Early period utilizes 39445 F from IL-E1454/RD23 and IL-E1454/ Lijiang xintuanheigu₂For group of individuals At target group, this research department completes the positioning of blast resistant gene Pi57 early period, which is located at the dyeing of rice the 12nd The nearly centromere region of body galianconism.Using the label with gene close linkage, construct using the genome of OryzasativaLcv.Nipponbare as reference sequences Gene Pi57 electronics physical map (Fig. 1).Then it is the design primer referred to the genome of OryzasativaLcv.Nipponbare and combines gene The method that group step moves (genome walking), obtains the region gene Pi57 in resistant donor introgression line IL-E1454 Sequence information, and carry out gene forecast analysis (applicant not yet announce IL-E1454 material targeting regions DNA sequence dna letter Breath), then, according to acquired sequence information, the present invention has carried out following work:

Biomaterial rice introgression line IL-E1454 and Pyricularia oryzae HN09-1C-7 involved in embodiment are in non-patent Document: Dong et al., 2017.Fine mapping of Pi57 (t) conferring broad spectrum resistance against Magnaporthe orzyae in introgression line IL-E1454derived from Oryza longistaminata.PLOS ONE,12(10):e0186201.https://doi.org/10.1371/ Journal.pone.o186201) open, applicant has preservation, and applicant's guarantee is from present patent application day to the public in 20 years It provides.Applicant contact address: Yunnan Province, Panlong District Beijing Road, Kunming 2238, Yunnan Agriculatural Academy's agricultural environment money Source research institute, postcode: 650205.

Each reagents such as pCAMBIA1300AscI carrier, rice varieties Dianjingyou No.1 involved in embodiment be it is commercially available, Pyricularia oryzae HN09-1C-7 bacterial strain is unit preservation.

Embodiment 1 determines blast resistant gene Pi57 by candidate gene approach

One, gene Pi57 candidate gene approach

Using softberry platform (http://www.softberry.com), the IL-E1454 obtained to seminar exists The sequence of targeting regions carries out the annotation analysis of targeting regions gene, finds to contain the candidate gene of 3 predictions in targeting regions (Pi57-1 (LOC_os12g18690), Pi57-2 (LOC_os12g18710) and Pi57-3 (LOC_os12g18729).According to base Because of the sequence information of annotation, design primer and using RT-PCR analysis shows, 3 candidate genes only have Pi57-3 gene expression, Other 2 genes (Pi57-1 and Pi57-2) are not expressed, thus the candidate gene of gene Pi57 is determined as Pi57-3.

The acquisition of the full length cDNA sequence of two, gene Pi57

Using Trizol reagent, total serum IgE is extracted from the spire of rice introgression line IL-E1454, and with Powerscript II Transcriptase (Takara company) reverse transcription synthesizes the first chain cDNA.With primer Pi57cDNA-Fw, (its base sequence is such as Shown in SEQ ID NO:5) and primer Pi57cDNA-Rv (its base sequence is as shown in SEQ ID NO:6), by PCR amplification, obtain CDNA sequence, that is, gene Pi57 full length cDNA sequence of gene Pi57-3 is obtained, the cDNA sequence of gene Pi57 such as SEQ ID NO: It is 3804bp shown in 4.Its pcr amplification reaction condition is as follows: 50 μ l of overall reaction system, contains 10 5 × PrimeStar of μ l Buffer, 1 μ l high-fidelity Taq enzyme PrimeStar GXL DNA Polymerase, 4 μ l dNTP (2.5 μM of each), 2 μ l Pi57cDNA-Fw (10 μM), 2 μ l Pi57cDNA-Rv (10 μM), 2 μ l cDNA templates, and double aqua sterilisas that steam are supplied to 50 μ l. PCR reaction condition be 98 DEG C 10 seconds, 68 DEG C 4 minutes, altogether carry out 32 circulation.

The sequence of three, gene Pi57 is analyzed

Genomic dna sequence comparative analysis using cDNA and the Pi57 gene of the gene Pi57 of acquisition shows Pi57's Genome sequence contains 3 intrones, wherein first exon is 1~1136bp；Second exon be the 1465th~ 1725bp；Third exon is 4393~6319bp；4th exon is 6637-7116bp.In cDNA sequence, The code area of gene Pi57 be 1159bp~3726bp, coding region sequence length be 2568bps, gene Pi57 encode one by The albumen of 855 amino acid composition.

The genetic transformation complementation confirmatory experiment of 2 blast resistant gene Pi57 of embodiment

One, the amplification and acquisition of gene Pi57

Using the genomic DNA of disease-resistant donor parents rice introgression line IL-E1454 as template, primers F 57-SalI-Fw is utilized (its base sequence is as shown in SEQ ID NO:7) and primers F 57-AscI-Rv (its base sequence is as shown in SEQ ID NO:8) expand Increase the sequence of the promoter (its nucleotide sequence such as SEQ ID NO:1) comprising gene Pi57 itself, obtains the complete of gene Pi57 Long sequence.Pcr amplification reaction condition is as follows: 50 μ l of overall reaction system, contains 10 μ 5 × PrimeStar of l Buffer, 1 μ l high Fidelity Taq enzyme PrimeStar GXL DNA Polymerase, 4 μ l dNTP (2.5 μM of each), 2 μ l Pi57-SalI-Fw (10 μM), the 2 μ l of genomic DNA of 2 μ l Pi57-AscI-Rv (10 μM), IL-E1454 supply double aqua sterilisas that steam to 50 μ l. PCR reaction condition be 98 DEG C 10 seconds, 68 DEG C of 12min, altogether carry out 32 circulation.The nucleotide sequence of gene Pi57 such as SEQ ID Shown in NO:2.

The genetic transformation of 3 gene Pi57 of embodiment

One, the building of gene Pi57 genetic transformation carrier and genetic transformation

(1) preparation of cloning vector and gene Pi57 amplified production: using restriction enzyme Sal I in two 1.5ml Centrifuge tube in respectively digestion pCAMBIA1300AscI carrier and amplification obtain gene Pi57 product (by described in embodiment 2 expansion Increase gene Pi57): 2 μ l of Sal I enzyme cutting buffering liquid (NEB3.1), restriction enzyme Sal I 1 μ l, pCAMBIA1300AscI 5 μ l of 5 μ l of carrier or gene Pi57 segment, and distilled water is added and supplies 20 μ l reaction solutions respectively；In 37 DEG C of digestion 30min, then Carry out the purifying of segment respectively using QIAquick Gel Extraction Kit；PCAMBIA1300AscI carrier and gene Pi57 product after purification is again Digestion, two groups of endonuclease reactions are carried out with restriction enzyme A sc I respectively are as follows: 2 μ l of enzyme cutting buffering liquid (Cutsmart), 1 AscI μ l, 10 μ l of 10 μ l of pCAMBIA1300AscI carrier or gene Pi57 segment after purification, supply 20 μ l with distilled water；37℃ Digestion 30min；Product after purifying two groups of endonuclease reactions again obtains Sal I and Asc I double digestion respectively The DNA of pCAMBIA1300AscI carrier and gene Pi57, and carry out quantifying for DNA concentration.

(2) connection of target fragment and carrier connection reaction and clone: is carried out using T4 ligase: connection buffer (10 × T4ligation buffer) 2 μ l, the 1 μ l (10ng) of pCAMBIA1300AscI carrier after double digestion, the gene after double digestion 4 μ l (50ng) of DNA fragmentation, the 1 μ l of T4 ligase of Pi57, distilled water supply 20 μ l；It is connected overnight at 10 DEG C；It is heavy with dehydrated alcohol Shallow lake connection product, and Electroporation obtains positive colony (pCAMBIA1300AscI-Pi57-3) simultaneously into Escherichia coli DH10B Sequencing analysis is carried out, gene Pi57 sequence is as shown in SEQ ID NO:2.

(3) acquisition of the genetic transformation of Rice Callus and transgenic plant

Using Agrobacterium-mediated genetic transformation method (Hiei Y, Ohta S, Komari T, et al.Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence Analysis of the boundaries of the T-DNA.The Plant Journal, 1994,6:271-282) carry out The genetic transformation of gene is tested: by the pCAMBIA1300AscI-Pi57 Cloning Transformation of acquisition into agrobacterium strains EHA105, And the rice varieties Dianjingyou No.1 of sense rice blast is further infected with Agrobacterium, it is obtained by resistant calli screening, differentiation Transgenic plant.

(4) Molecular Identification of transgenic plant and disease-resistant phenotypic evaluation

The transgenic plant of acquisition utilizes the hygromycin B gene sequence carried on genetic transformation carrier pCAMBIA1300AscI Column primer is identified, carries out PCR amplification identification (pcr amplification reaction system by the DNA that amplification rotaring gene plant blade extracts It is same as Example 2 with response procedures).Genetic transformation obtains 17 plants of transgenic plants, 17 plants of T of identified acquisition altogether₀In generation, turns The all positives of gene plant, electrophorogram are shown in Fig. 2, and Molecular Identification is respectively as follows: hptFw (its base sequence such as SEQ with primer Shown in ID NO:9) and hptRv (base sequence is as shown in SEQ ID NO:10).

17 plants of transgenic plants, inoculation are inoculated with using the Pyricularia oryzae HN09-1C-7 to cause a disease to receptor kind Dianjingyou No.1 As a result and the reaction of control is as shown in Figure 3.1st-the 17 leaf is transgenic plant, and DJY1 is transformation receptor susceptible variety The blade of Dianjingyou No.1, IL-E1454 are the reaction of the donor of Pi57 containing blast resistant gene rice introgression line IL-E1454.Inoculation T₀Reaction result discovery, 17 plants of transgenic plants have 16 plants of performances disease-resistant.

By T₀After transgenic plant breeding selfing, T is obtained₁For seed, and to the T of acquisition₁It is isolated for group Analysis experiment, to the T of transgenosis₁Molecule and phenotypic evaluation analysis are carried out for plant.Molecular Identification primer Pi57SPFw (its alkali Basic sequence is as shown in SEQ ID NO:11) and Pi57SPRv (its base sequence is as shown in SEQ ID NO:12) progress, such as Fig. 4 institute Show.The result shows that the T containing transgenosis Pi57₁It is disease-resistant for plant performance, and the performance of the plant without transgenosis Pi57 is susceptible, Show the characteristic isolated.Show that gene Pi57 can be transferred in susceptible variety by the method for conversion, and passes through breeding Choice, obtain homozygous disease-resistant plant, be applied to paddy disease-resistant breeding, the rice varieties of breeding blast resisting, to solve water Rice produces the problem of upper rice varieties are endangered by rice blast.

Sequence table

<110>Yunnan Prov Academy Of Agricultural Sciences, Agricultural Research Institute Of Environmental Resources

<120>a kind of rice blast resistance gene Pi57 and its coding albumen and application

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1955

<212> DNA

<213>rice introgression line IL-E1454 (Oryza sativa)

<400> 1

ataggagagg aagagagagg ctggacagag ggagtactta cctgcgcatg ccgcatgcac 60

gatggggatg agtaccggat tttttgtttg ttaatatata tgggtccacc gatttaatag 120

aaaatcaatg gttaaaatat atgggtccac cgatttaacg gaaaatcaat agctagatat 180

ttattagatt ttccatgatt ttctatattt tattagagtg ccacgtggcg actcaagagt 240

gtttatagga tgacacatgg cggtttagga gcatttgtag gaagtttaat ggacttttag 300

tatataataa tagataatag aagatttatt agagtgccac gtggcgactc aagagtgttt 360

ataggatgac acatggcggt ttaggagcat ttgtaggaag tttaatggac ttttagtata 420

taataataga tagattttga ataagatagc agtaatatag tatatcacta tatatacgtt 480

attggttcat aggtatgcac cattaaatta aatctacaat gtccaaaaat aacaagtcat 540

attggcgctt tgcatggcaa tcgagaatta ttaaaaatca tgaaaaacta gtgatatgga 600

ttatatcatc atttgaagat gcataaaatt tggggtccat tttattcagc ggttaggaca 660

gtacgggaat aacctaaagt attcggagta tagggcatgc gattttgttt ctctttaatt 720

cctatttaaa acaaatggta catgggtgtt tcatactaag tttggttaat tagggaacac 780

aaatcatact aagcttatat atcagaattc atgcaatgaa agtatacgat ataaaaataa 840

gaattaacca aacaaaatta tactttaatt agtccgtggt gacggaccat ttacaaatga 900

tccacaccta cttattcaga tctctttttt tttttcaaaa aagagatgtg atgtgaaaaa 960

aagaaaggcg cagaagtaaa tgtaagtaga atatatattt aaatatatcc taaaatctat 1020

aaatacatct atagtataaa agaaattttg tgaccgaaag ttttaaaaaa taagatatgc 1080

taatattcaa aaaagatata cacatagtac aaaatgatag tctaatagta atcaactcta 1140

ttattgcgac acaatatttt ctcataaatt taagattgca tggttccaag taatattagt 1200

ttagaattca tatgtttcaa gggtagatca aagagtttca taaccaatat actgatataa 1260

tgttcaaagc aaccccaaac taattattga caaaaacaaa tttagaagag agtcaaagaa 1320

tttcaagaaa aagtaaccca cacataaaac atccaagata aaaattcaaa caacatcaaa 1380

taaagatgcc ggcgcatatg cgcgggctac aacttcctag tttcgtaaaa aaagttggac 1440

aagatttatc cgtagatacc gctcttgagc aatggtgtcg ggatcgtgat tcgtgaaccg 1500

tgtggactgg ggtttgcatc ctgttccgta tacaccagat ttttttttcc tgatgatcac 1560

atccccactt gcctgcatcg cttttgtggc ctctccatag aatggttaat tcacggtaaa 1620

aaacgatttt tttatgacac caccctttta tttttactgg cggccatgac aaaacaccgc 1680

ttgtaaaatt atacggaaag attcaaagtc acgaacctcc agcgaacggc ctcttaaaga 1740

agccagcaaa tagcaatatt tttacaagcg gacctcttaa gaagctacta gcgaaaatcc 1800

attgtttacc agcagacttc ttaagaggcc gtgagcaaaa aaaaaccaat tttctctgtc 1860

ggctgctaag aggtccacca acaaaaaaaa aaacatcatt gcactaaaaa ataattgaac 1920

acaataaaaa ccaccaccta ccgtgccacc ttgcc 1955

<210> 2

<211> 7116

<212> DNA

<213>rice introgression line IL-E1454 (Oryza sativa)

<220>

<221> exon

<222> (1)..(1136)

<223>first exons

<220>

<221> exon

<222> (1465)..(1725)

<223>second exons

<220>

<221> exon

<222> (4393)..(6319)

<223>third exon

<220>

<221> exon

<222> (6637)..(7116)

<223>the 4th exons

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accaccactt ccctctctcc tcctcatcct catcttcata tctagataga cctcatcttc 60

tccagccacc accgcctccc tccccaccgg tggtggcggc gggaagacag cggctcggct 120

gagcccggcg ggtagggcaa cgatttagta tgggaggagc agcggcgact tctctacacg 180

acggcgttcg gaggcggatc tagcagcggc gactccatcc agcggtggat ccgccgcctc 240

ctagtcctag ctctctctcc cccgctcctc cctcctcccc ttcccccgcc gccaccaccc 300

accccctctc cctctctcag aactggtgcg cggggagcgg tggaggctgc ggcggcgaag 360

gctggggggg ctcggcagcg gcggcgacgg tgagaccggt agcagtgatg gtggagggga 420

cggatccgat ggcggtggtt ctcgtcagcc agttcaagga tgacgacagc ggcggctgcg 480

gcaacgatgg acaggctctg ggcggctcct ccaccctcct cccctctctc atatccaacg 540

actcggggtg gtaggacgtg gccgacgcat ccggcggcgg agctcgggta tcaatccggc 600

atgttttttt ttctccaaaa tcaaattttc ctggtggtcg ggaaacggat tttcaatttt 660

tttttacgct cgaaaatctt ttttcgctgg cggtcaacgt aacttaaccg cttgtgaaaa 720

caagctttaa agtttcgcta gtgaagattg ttattttcac tagccttttg tttgcgggcg 780

gctgacagtg ccaccagcga aaaattcttt tgctagtagt gattgctaga tggtcagcag 840

agaacttgtg gagtatatga ctatcaagta tcaaccaaca gtacacttca gcagaaagag 900

gatggttcta gctctccggt ggtttaatat tgaaacgtac agcccaccca ccaccacccc 960

agtcgtcccc agctaagcgc tcccttgctt gcctcagtag acgagctgcc aactagtagc 1020

actccagcaa gcagcgggtc agcggagtcc ggagggcgca cgtgcgccgc agcgaggtag 1080

cagcgtggcc aggcggcggc tggctggcca gccgggttcg gcaagcggct tagcaggtaa 1140

ttttctatca catcgttcta attttaatca aactactaat tttagcatga actaaacaca 1200

cccttggttg cgactttggg agtcgatata taggccttgc tcgtagttcg ttgattttgg 1260

ggttaaactg gcgaggaact ttacttggat accacctggg agttgatgtg attttagtgt 1320

ttagtacagt ttagagttta ttacctaccg gtttcttggt gcctggcgca cactgagcat 1380

gcaaatgcag tgagagccag tatcatctaa gacggctgga cctgaaatat tttaccatgt 1440

atatatcaaa ctcgacttga tcaggtgttt agcggaggaa ggatcgatgt ccggcgccgg 1500

acagagccgt ggtcatcgcc ttggattaca cattgattcg gattggccag aggtcttgtt 1560

gatcaatgac tatgcggtgt tcatggggta cctgtcgatg gttgtcaccg ggacggggtt 1620

ccttgtgctc acttggtcca ccgtcatcct cctcggtgga ttcgtctcca tgctatccaa 1680

caaggacttc tggagtctca cggtgatcac gctcgttcaa acaaggtgag ctatctatag 1740

ccatctcaac cagacttata tttatttcgc gttcccaaca ttaaccagtt gagccaagca 1800

gttggagttt cgcattgcta gctagctact agtttactgt cagatctagc tttcaacact 1860

tcagtgttgc gaactgaata tgctgcaaaa cgttgcaact ccagagtggc tttatatact 1920

acattgatat gaaaatattc aagtcccata tttgcaaaat cgactatttt taaaatcaat 1980

tgttttctcc ccctttaaag aagcatgcat tttttatatt tctctcccct ttgacaatga 2040

tttcatcaag aattttaaaa gagaaaatat aaccaataaa gcataactat catcatttca 2100

tgattgtcac atgtaataat cacttctttt cttaattttt tatttttctg aatttttttt 2160

gagagctttg aaatgtacct aaaacaattc atggtcatat gttgcaagac aaatcagata 2220

aattcatcaa ctagttaaca agcatgcatt aagtataaac atgaatcaaa catattacaa 2280

ttaagctcaa atcaataata aagattaatg atttataatg caacatattt acaagtacat 2340

atgttgaaaa aaaatcccta ctggattatc actctttctc aaattaactt catcgcaaaa 2400

ataaccaaga gaattttccg caagtattac aaattttggt actcattttg acattgaata 2460

taaaatatat atctcaatta tttcgagtat gttcttgttg ttaagactgt ttctaacgat 2520

tcggtactca ttattttcct cactgcaaat acgtctttat cgtcaagact gtttccaagg 2580

attatgtatt catttttttt tctctcatca taagcaactt tttattctct tgcgacattt 2640

ggaacaaaca agaagcaaat tgaggaataa caatccaata agcataaatc attcaaactc 2700

ggcacttgct acaacattgc atatttacat aaatcaagta aaagaattag tgtcatgcat 2760

catctcactt aaaaagcaat atccaagcca catgaaaagg ctaaaatgca tacaagctaa 2820

catataatat acctcaatta ttttggaagc atgcacaaga atatactaga ggtatatacc 2880

aaagttcttc ttttctcatt tgttctttat ttttcagatt tagagcaatg catggatata 2940

gacatgaatg caatgaaaaa gaacaaactc ccctcaaata aacatccaat tctccgcgaa 3000

gaaaaatcaa gaagaaatca aaaaaatttg aaaaagacat ccaattgcca atgatatcac 3060

gaaggttctt atgaattgac ataaatccac ttgaattacc aaaactacca cttcaaacac 3120

tagcatgaaa tgctaataaa cgaagaagta aaacaaagtt atgtcaactc aaaagtacat 3180

ttatgttatc ttgaaaagca atcaccgtct ccacattgaa cgatcatggg gagtatgccg 3240

ccaaaataca tacgaacatg aagatgtcga ggccccgtgt tggggttagt cataagaaat 3300

tttagaatcc aacactaagt cacatgacaa atcttaacta attaaaagat tcatattttt 3360

cctttcatca cctcaatttt cgtccgtccc ttgcttcgtt cgcaagatgc gataactagg 3420

actctgcttc tttcgttcct catccgattg tatttaggta gggcaatcgg agtgggaaat 3480

ccgccgcacg gccttgccgg gaagaagtaa ggaattgggg aagagagaag cggagtccta 3540

gctatgtaaa ccatatgcat gcgatatgga aagcttcacc atatttatgc gatatggaaa 3600

ccttcaccat acgcacacaa caccatatgt atgcgatatg gaaaccttca cctccacgtg 3660

cattgttctc tggcctgagc atcctgtatg atatccttga ccatccggac ctcaacttct 3720

tccaagccta gtcccgatcc atcgccagct atactctcat agtcttaagc aacacataca 3780

catgaataac aaagaaactc catatccgaa tacaagttaa caccaacaat atgactcaca 3840

tcagcataca atagtatcac atacgaaaac cgactccctt ctggaatcac ccggtctgat 3900

cggtgcctcc tgcccggtct gacaggcacc caatgctgac cgcattgttt accatccgaa 3960

aatactatcc accgtatttt caacaacact taccgttgac aattgttcat caccaaaatt 4020

aatcatcata tgattctaca acagtcagat acagtcagat ataggcatgt tcggagagat 4080

gccatactag aattgaaatg tcaaattttg gaagagaaaa aaagcaatag agttaataaa 4140

aaagaaaaag aaagggctac tctctgcatt catcctttca tggcaaaatt aattttacac 4200

gatcgccaaa agtaaatttg atcactaatt aatgtgaaat tactttgaca cgatggcatg 4260

tatcgaatct tattttacca ttgtgtgcat ccctaacaaa aaaaaaaacg tacttactct 4320

ttccactatc gataaatcag tccttttaac cggaaaattc atccttccaa acaaaaaaaa 4380

aatctgaaac aggatattcg atgtttttct gaatggaaaa gtaagccaca ttgggtactc 4440

attgaagcgc ttatgcaagg ccgcacgctt catcgcgttg ccccataacc ataagaaggt 4500

tgggttcagg ggtgctgttc gagtgcttgt gttcaccatc gtcttgtgtc cgctgttcct 4560

gctctacatg tttgggctct tcgtttcccc ctggatttcg ctgtggcgtc taatccagca 4620

ggattacggc gtgacggccg gagacagcag cagcaaggca cacctgcagc ctgcgctggt 4680

ggttctctac tccctggccc tgttccaggg cgtcctcttc tactacaggg ccatctctgc 4740

ttgggaagaa cagaagctag tgaaagatgt ggccgacaaa tacatgtttg atacagtgtc 4800

acgcagttca gtttcggatt atttacatga gatcaaggtg ggatgtgaga atgacccgtc 4860

ctttgccaga gggaggaacc tgatcacata cgccgtcaag ctgatggaat ccacatcacc 4920

ggatgggtac ctttcaggtg cacggattct tgacacactc atcaagttta atagggatgc 4980

tgatgcatcg gggagcgaat tcccggggca gagtatgcag atttacaata tgattggatc 5040

cgcaacctcc agtcccatac tccacaactt agttcagatg ctggattcca agagtgcata 5100

tgatggagag atcaggttgc gcgccgcgag gattgtggat cactttgctg gtgagatccg 5160

tttggacaag atcctacaag ggatccggtg tgtatcttcc ttgcttgaac tcgagcagaa 5220

attatttcag gattcttctg aagaagagga tgatcaccag atttctgtca aagaaaagga 5280

ttattacccc aaagactatg aacagatgca acttataggc atgcagatcc ttttaaacct 5340

ctcctatgac aagaacaact tgtccctcat gagcaacaca gatgatccgg ccttgatcaa 5400

caagattgtg gcacttataa cgtccaaggg atcacttcac aaaaaagaac ataacgaatg 5460

gtcccgtatg gcagagctcg gcgtgaagat actaagccga tttatgcgat ttatgtatgg 5520

ccctacaaaa tcaaacaata ttctgtggca tgaaatatca acaagcagca aagcaatcgg 5580

caccttggag agcattcttg agtgtgacca atgtgactct gtgctgaaga aacacgccat 5640

aaggattctc agaaggatat tcatggatac atcttcagct atgggtgaag gagacagaga 5700

aaggttcatc ggctccttga tggacatgtc tcttcacaac agtaatggtg actttcaaaa 5760

tctggcaggt gtagatctgg cgctcaagaa acaagggtta agcattctca aagaaatata 5820

cttgaatcca tcttcgatta tggacgaagg agacagagaa aggttcatcg gctccctgat 5880

ggacatgttt cttgacaaca gtaagggcga ctttggaaat ctgccaggtg aagatctgga 5940

cctcaagaaa caagagttaa gcattctcaa agaaatatgc atggatccat cttcgtttat 6000

gggcgaaagt gacagagaaa aattcatcgg catcctgatg gacatgtttc ttcacaacag 6060

caagggcgac ttgtttgaaa aactagcagg tgatgatctg gtgcagatat gtagaagaag 6120

tggaagcagt gccgccatca tattgaagaa atatggtcat gatattgtta attgtattgc 6180

tgatactcgt tcaagcgtgt acagcagcat gcacagaaaa attgcagcaa agatcctcaa 6240

tcatctgtgt agtccctact ccactgacga ggaacatctt cagaatctga aggaggccat 6300

cattgatttg atacccaagg tacgttagtc gctgtcattt atacttccta tgtcaacaaa 6360

caattttttt catgataatg aaatacataa ctctggcagc ctctacattg tcagatgcac 6420

gaaaccatag ctcattaaat agtttggcaa cctctacatt cgcagaaatc catgggtaac 6480

aaacttgttt aaagcaaaac tgaagcacaa cttgtatcat taaaatttca tccatgctta 6540

ctgaatatcc atcagagtag tctccaaaag atgggacact gcttttatat gcatcctatt 6600

tatatcggaa tgaccgaaag cactgtaatg ttgcaggtgc taagagaagc acttggttgg 6660

gggttgacag gaaaagagat acaaggagtg gcagtttcag gccttgaagg tacccaagat 6720

gatgactgga aattgcagga agcattggcg tccctctgtg caaccgtgtt taacagaatt 6780

gtcagcaagg atgcagattt gactgctcgg ttcaacaaca tcgccgctgg tatctgcgac 6840

caggcagcga agcctcgcgt gaccttcgca gacctcatca aagaagctgt gaaagttcat 6900

cgcatggaat ttaagaaacc agaaccacca aaaccacctg ccggcccggc actttacgag 6960

tttatgcctg cgtactaccc cccgccccac tttatgtatt tgagcgagga agatcccaac 7020

gcgtgttgta tctcttgata actccctaat ttgtaatgca aaccgaggat gaaaaaaata 7080

tatatgccta tacggtactg ctccatttcc aaataa 7116

<210> 3

<211> 855

<212> PRT

<213>rice introgression line IL-E1454 (Oryza sativa)

<220>

<221> TRANSMEM

<222> (29)..(191)

<223>transmembrane domain

<220>

<221> DOMAIN

<222> (207)..(811)

<223>ARM repetitive structure domain

<220>

<221> DOMAIN

<222> (817)..(839)

<223>Pro-rich structural domain

<400> 3

Met Ser Gly Ala Gly Gln Ser Arg Gly His Arg Leu Gly Leu His Ile

1 5 10 15

Asp Ser Asp Trp Pro Glu Val Leu Leu Ile Asn Asp Tyr Ala Val Phe

20 25 30

Met Gly Tyr Leu Ser Met Val Val Thr Gly Thr Gly Phe Leu Val Leu

35 40 45

Thr Trp Ser Thr Val Ile Leu Leu Gly Gly Phe Val Ser Met Leu Ser

50 55 60

Asn Lys Asp Phe Trp Ser Leu Thr Val Ile Thr Leu Val Gln Thr Arg

65 70 75 80

Ile Phe Asp Val Phe Leu Asn Gly Lys Val Ser His Ile Gly Tyr Ser

85 90 95

Leu Lys Arg Leu Cys Lys Ala Ala Arg Phe Ile Ala Leu Pro His Asn

100 105 110

His Lys Lys Val Gly Phe Arg Gly Ala Val Arg Val Leu Val Phe Thr

115 120 125

Ile Val Leu Cys Pro Leu Phe Leu Leu Tyr Met Phe Gly Leu Phe Val

130 135 140

Ser Pro Trp Ile Ser Leu Trp Arg Leu Ile Gln Gln Asp Tyr Gly Val

145 150 155 160

Thr Ala Gly Asp Ser Ser Ser Lys Ala His Leu Gln Pro Ala Leu Val

165 170 175

Val Leu Tyr Ser Leu Ala Leu Phe Gln Gly Val Leu Phe Tyr Tyr Arg

180 185 190

Ala Ile Ser Ala Trp Glu Glu Gln Lys Leu Val Lys Asp Val Ala Asp

195 200 205

Lys Tyr Met Phe Asp Thr Val Ser Arg Ser Ser Val Ser Asp Tyr Leu

210 215 220

His Glu Ile Lys Val Gly Cys Glu Asn Asp Pro Ser Phe Ala Arg Gly

225 230 235 240

Arg Asn Leu Ile Thr Tyr Ala Val Lys Leu Met Glu Ser Thr Ser Pro

245 250 255

Asp Gly Tyr Leu Ser Gly Ala Arg Ile Leu Asp Thr Leu Ile Lys Phe

260 265 270

Asn Arg Asp Ala Asp Ala Ser Gly Ser Glu Phe Pro Gly Gln Ser Met

275 280 285

Gln Ile Tyr Asn Met Ile Gly Ser Ala Thr Ser Ser Pro Ile Leu His

290 295 300

Asn Leu Val Gln Met Leu Asp Ser Lys Ser Ala Tyr Asp Gly Glu Ile

305 310 315 320

Arg Leu Arg Ala Ala Arg Ile Val Asp His Phe Ala Gly Glu Ile Arg

325 330 335

Leu Asp Lys Ile Leu Gln Gly Ile Arg Cys Val Ser Ser Leu Leu Glu

340 345 350

Leu Glu Gln Lys Leu Phe Gln Asp Ser Ser Glu Glu Glu Asp Asp His

355 360 365

Gln Ile Ser Val Lys Glu Lys Asp Tyr Tyr Pro Lys Asp Tyr Glu Gln

370 375 380

Met Gln Leu Ile Gly Met Gln Ile Leu Leu Asn Leu Ser Tyr Asp Lys

385 390 395 400

Asn Asn Leu Ser Leu Met Ser Asn Thr Asp Asp Pro Ala Leu Ile Asn

405 410 415

Lys Ile Val Ala Leu Ile Thr Ser Lys Gly Ser Leu His Lys Lys Glu

420 425 430

His Asn Glu Trp Ser Arg Met Ala Glu Leu Gly Val Lys Ile Leu Ser

435 440 445

Arg Phe Met Arg Phe Met Tyr Gly Pro Thr Lys Ser Asn Asn Ile Leu

450 455 460

Trp His Glu Ile Ser Thr Ser Ser Lys Ala Ile Gly Thr Leu Glu Ser

465 470 475 480

Ile Leu Glu Cys Asp Gln Cys Asp Ser Val Leu Lys Lys His Ala Ile

485 490 495

Arg Ile Leu Arg Arg Ile Phe Met Asp Thr Ser Ser Ala Met Gly Glu

500 505 510

Gly Asp Arg Glu Arg Phe Ile Gly Ser Leu Met Asp Met Ser Leu His

515 520 525

Asn Ser Asn Gly Asp Phe Gln Asn Leu Ala Gly Val Asp Leu Ala Leu

530 535 540

Lys Lys Gln Gly Leu Ser Ile Leu Lys Glu Ile Tyr Leu Asn Pro Ser

545 550 555 560

Ser Ile Met Asp Glu Gly Asp Arg Glu Arg Phe Ile Gly Ser Leu Met

565 570 575

Asp Met Phe Leu Asp Asn Ser Lys Gly Asp Phe Gly Asn Leu Pro Gly

580 585 590

Glu Asp Leu Asp Leu Lys Lys Gln Glu Leu Ser Ile Leu Lys Glu Ile

595 600 605

Cys Met Asp Pro Ser Ser Phe Met Gly Glu Ser Asp Arg Glu Lys Phe

610 615 620

Ile Gly Ile Leu Met Asp Met Phe Leu His Asn Ser Lys Gly Asp Leu

625 630 635 640

Phe Glu Lys Leu Ala Gly Asp Asp Leu Val Gln Ile Cys Arg Arg Ser

645 650 655

Gly Ser Ser Ala Ala Ile Ile Leu Lys Lys Tyr Gly His Asp Ile Val

660 665 670

Asn Cys Ile Ala Asp Thr Arg Ser Ser Val Tyr Ser Ser Met His Arg

675 680 685

Lys Ile Ala Ala Lys Ile Leu Asn His Leu Cys Ser Pro Tyr Ser Thr

690 695 700

Asp Glu Glu His Leu Gln Asn Leu Lys Glu Ala Ile Ile Asp Leu Ile

705 710 715 720

Pro Lys Val Leu Arg Glu Ala Leu Gly Trp Gly Leu Thr Gly Lys Glu

725 730 735

Ile Gln Gly Val Ala Val Ser Gly Leu Glu Gly Thr Gln Asp Asp Asp

740 745 750

Trp Lys Leu Gln Glu Ala Leu Ala Ser Leu Cys Ala Thr Val Phe Asn

755 760 765

Arg Ile Val Ser Lys Asp Ala Asp Leu Thr Ala Arg Phe Asn Asn Ile

770 775 780

Ala Ala Gly Ile Cys Asp Gln Ala Ala Lys Pro Arg Val Thr Phe Ala

785 790 795 800

Asp Leu Ile Lys Glu Ala Val Lys Val His Arg Met Glu Phe Lys Lys

805 810 815

Pro Glu Pro Pro Lys Pro Pro Ala Gly Pro Ala Leu Tyr Glu Phe Met

820 825 830

Pro Ala Tyr Tyr Pro Pro Pro His Phe Met Tyr Leu Ser Glu Glu Asp

835 840 845

Pro Asn Ala Cys Cys Ile Ser

850 855

<210> 4

<211> 3804

<212> DNA

<213>rice introgression line IL-E1454 (Oryza sativa)

<220>

<221> 5’UTR

<222> (1)..(1158)

<223>5 ' non-translational regions

<220>

<221> CDS

<222> (1159)..(3726)

<223>gene coding region

<220>

<221> 3’UTR

<222> (3727)..(3804)

<223>3 ' non-translational regions

<400> 4

accaccactt ccctctctcc tcctcatcct catcttcata tctagataga cctcatcttc 60

tccagccacc accgcctccc tccccaccgg tggtggcggc gggaagacag cggctcggct 120

gagcccggcg ggtagggcaa cgatttagta tgggaggagc agcggcgact tctctacacg 180

acggcgttcg gaggcggatc tagcagcggc gactccatcc agcggtggat ccgccgcctc 240

ctagtcctag ctctctctcc cccgctcctc cctcctcccc ttcccccgcc gccaccaccc 300

accccctctc cctctctcag aactggtgcg cggggagcgg tggaggctgc ggcggcgaag 360

gctggggggg ctcggcagcg gcggcgacgg tgagaccggt agcagtgatg gtggagggga 420

cggatccgat ggcggtggtt ctcgtcagcc agttcaagga tgacgacagc ggcggctgcg 480

gcaacgatgg acaggctctg ggcggctcct ccaccctcct cccctctctc atatccaacg 540

actcggggtg gtaggacgtg gccgacgcat ccggcggcgg agctcgggta tcaatccggc 600

atgttttttt ttctccaaaa tcaaattttc ctggtggtcg ggaaacggat tttcaatttt 660

tttttacgct cgaaaatctt ttttcgctgg cggtcaacgt aacttaaccg cttgtgaaaa 720

caagctttaa agtttcgcta gtgaagattg ttattttcac tagccttttg tttgcgggcg 780

gctgacagtg ccaccagcga aaaattcttt tgctagtagt gattgctaga tggtcagcag 840

agaacttgtg gagtatatga ctatcaagta tcaaccaaca gtacacttca gcagaaagag 900

gatggttcta gctctccggt ggtttaatat tgaaacgtac agcccaccca ccaccacccc 960

agtcgtcccc agctaagcgc tcccttgctt gcctcagtag acgagctgcc aactagtagc 1020

actccagcaa gcagcgggtc agcggagtcc ggagggcgca cgtgcgccgc agcgaggtag 1080

cagcgtggcc aggcggcggc tggctggcca gccgggttcg gcaagcggct tagcaggtgt 1140

ttagcggagg aaggatcg atg tcc ggc gcc gga cag agc cgt ggt cat cgc 1191

Met Ser Gly Ala Gly Gln Ser Arg Gly His Arg

1 5 10

ctt gga tta cac att gat tcg gat tgg cca gag gtc ttg ttg atc aat 1239

Leu Gly Leu His Ile Asp Ser Asp Trp Pro Glu Val Leu Leu Ile Asn

15 20 25

gac tat gcg gtg ttc atg ggg tac ctg tcg atg gtt gtc acc ggg acg 1287

Asp Tyr Ala Val Phe Met Gly Tyr Leu Ser Met Val Val Thr Gly Thr

30 35 40

ggg ttc ctt gtg ctc act tgg tcc acc gtc atc ctc ctc ggt gga ttc 1335

Gly Phe Leu Val Leu Thr Trp Ser Thr Val Ile Leu Leu Gly Gly Phe

45 50 55

gtc tcc atg cta tcc aac aag gac ttc tgg agt ctc acg gtg atc acg 1383

Val Ser Met Leu Ser Asn Lys Asp Phe Trp Ser Leu Thr Val Ile Thr

60 65 70 75

ctc gtt caa aca agg ata ttc gat gtt ttt ctg aat gga aaa gta agc 1431

Leu Val Gln Thr Arg Ile Phe Asp Val Phe Leu Asn Gly Lys Val Ser

80 85 90

cac att ggg tac tca ttg aag cgc tta tgc aag gcc gca cgc ttc atc 1479

His Ile Gly Tyr Ser Leu Lys Arg Leu Cys Lys Ala Ala Arg Phe Ile

95 100 105

gcg ttg ccc cat aac cat aag aag gtt ggg ttc agg ggt gct gtt cga 1527

Ala Leu Pro His Asn His Lys Lys Val Gly Phe Arg Gly Ala Val Arg

110 115 120

gtg ctt gtg ttc acc atc gtc ttg tgt ccg ctg ttc ctg ctc tac atg 1575

Val Leu Val Phe Thr Ile Val Leu Cys Pro Leu Phe Leu Leu Tyr Met

125 130 135

ttt ggg ctc ttc gtt tcc ccc tgg att tcg ctg tgg cgt cta atc cag 1623

Phe Gly Leu Phe Val Ser Pro Trp Ile Ser Leu Trp Arg Leu Ile Gln

140 145 150 155

cag gat tac ggc gtg acg gcc gga gac agc agc agc aag gca cac ctg 1671

Gln Asp Tyr Gly Val Thr Ala Gly Asp Ser Ser Ser Lys Ala His Leu

160 165 170

cag cct gcg ctg gtg gtt ctc tac tcc ctg gcc ctg ttc cag ggc gtc 1719

Gln Pro Ala Leu Val Val Leu Tyr Ser Leu Ala Leu Phe Gln Gly Val

175 180 185

ctc ttc tac tac agg gcc atc tct gct tgg gaa gaa cag aag cta gtg 1767

Leu Phe Tyr Tyr Arg Ala Ile Ser Ala Trp Glu Glu Gln Lys Leu Val

190 195 200

aaa gat gtg gcc gac aaa tac atg ttt gat aca gtg tca cgc agt tca 1815

Lys Asp Val Ala Asp Lys Tyr Met Phe Asp Thr Val Ser Arg Ser Ser

205 210 215

gtt tcg gat tat tta cat gag atc aag gtg gga tgt gag aat gac ccg 1863

Val Ser Asp Tyr Leu His Glu Ile Lys Val Gly Cys Glu Asn Asp Pro

220 225 230 235

tcc ttt gcc aga ggg agg aac ctg atc aca tac gcc gtc aag ctg atg 1911

Ser Phe Ala Arg Gly Arg Asn Leu Ile Thr Tyr Ala Val Lys Leu Met

240 245 250

gaa tcc aca tca ccg gat ggg tac ctt tca ggt gca cgg att ctt gac 1959

Glu Ser Thr Ser Pro Asp Gly Tyr Leu Ser Gly Ala Arg Ile Leu Asp

255 260 265

aca ctc atc aag ttt aat agg gat gct gat gca tcg ggg agc gaa ttc 2007

Thr Leu Ile Lys Phe Asn Arg Asp Ala Asp Ala Ser Gly Ser Glu Phe

270 275 280

ccg ggg cag agt atg cag att tac aat atg att gga tcc gca acc tcc 2055

Pro Gly Gln Ser Met Gln Ile Tyr Asn Met Ile Gly Ser Ala Thr Ser

285 290 295

agt ccc ata ctc cac aac tta gtt cag atg ctg gat tcc aag agt gca 2103

Ser Pro Ile Leu His Asn Leu Val Gln Met Leu Asp Ser Lys Ser Ala

300 305 310 315

tat gat gga gag atc agg ttg cgc gcc gcg agg att gtg gat cac ttt 2151

Tyr Asp Gly Glu Ile Arg Leu Arg Ala Ala Arg Ile Val Asp His Phe

320 325 330

gct ggt gag atc cgt ttg gac aag atc cta caa ggg atc cgg tgt gta 2199

Ala Gly Glu Ile Arg Leu Asp Lys Ile Leu Gln Gly Ile Arg Cys Val

335 340 345

tct tcc ttg ctt gaa ctc gag cag aaa tta ttt cag gat tct tct gaa 2247

Ser Ser Leu Leu Glu Leu Glu Gln Lys Leu Phe Gln Asp Ser Ser Glu

350 355 360

gaa gag gat gat cac cag att tct gtc aaa gaa aag gat tat tac ccc 2295

Glu Glu Asp Asp His Gln Ile Ser Val Lys Glu Lys Asp Tyr Tyr Pro

365 370 375

aaa gac tat gaa cag atg caa ctt ata ggc atg cag atc ctt tta aac 2343

Lys Asp Tyr Glu Gln Met Gln Leu Ile Gly Met Gln Ile Leu Leu Asn

380 385 390 395

ctc tcc tat gac aag aac aac ttg tcc ctc atg agc aac aca gat gat 2391

Leu Ser Tyr Asp Lys Asn Asn Leu Ser Leu Met Ser Asn Thr Asp Asp

400 405 410

ccg gcc ttg atc aac aag att gtg gca ctt ata acg tcc aag gga tca 2439

Pro Ala Leu Ile Asn Lys Ile Val Ala Leu Ile Thr Ser Lys Gly Ser

415 420 425

ctt cac aaa aaa gaa cat aac gaa tgg tcc cgt atg gca gag ctc ggc 2487

Leu His Lys Lys Glu His Asn Glu Trp Ser Arg Met Ala Glu Leu Gly

430 435 440

gtg aag ata cta agc cga ttt atg cga ttt atg tat ggc cct aca aaa 2535

Val Lys Ile Leu Ser Arg Phe Met Arg Phe Met Tyr Gly Pro Thr Lys

445 450 455

tca aac aat att ctg tgg cat gaa ata tca aca agc agc aaa gca atc 2583

Ser Asn Asn Ile Leu Trp His Glu Ile Ser Thr Ser Ser Lys Ala Ile

460 465 470 475

ggc acc ttg gag agc att ctt gag tgt gac caa tgt gac tct gtg ctg 2631

Gly Thr Leu Glu Ser Ile Leu Glu Cys Asp Gln Cys Asp Ser Val Leu

480 485 490

aag aaa cac gcc ata agg att ctc aga agg ata ttc atg gat aca tct 2679

Lys Lys His Ala Ile Arg Ile Leu Arg Arg Ile Phe Met Asp Thr Ser

495 500 505

tca gct atg ggt gaa gga gac aga gaa agg ttc atc ggc tcc ttg atg 2727

Ser Ala Met Gly Glu Gly Asp Arg Glu Arg Phe Ile Gly Ser Leu Met

510 515 520

gac atg tct ctt cac aac agt aat ggt gac ttt caa aat ctg gca ggt 2775

Asp Met Ser Leu His Asn Ser Asn Gly Asp Phe Gln Asn Leu Ala Gly

525 530 535

gta gat ctg gcg ctc aag aaa caa ggg tta agc att ctc aaa gaa ata 2823

Val Asp Leu Ala Leu Lys Lys Gln Gly Leu Ser Ile Leu Lys Glu Ile

540 545 550 555

tac ttg aat cca tct tcg att atg gac gaa gga gac aga gaa agg ttc 2871

Tyr Leu Asn Pro Ser Ser Ile Met Asp Glu Gly Asp Arg Glu Arg Phe

560 565 570

atc ggc tcc ctg atg gac atg ttt ctt gac aac agt aag ggc gac ttt 2919

Ile Gly Ser Leu Met Asp Met Phe Leu Asp Asn Ser Lys Gly Asp Phe

575 580 585

gga aat ctg cca ggt gaa gat ctg gac ctc aag aaa caa gag tta agc 2967

Gly Asn Leu Pro Gly Glu Asp Leu Asp Leu Lys Lys Gln Glu Leu Ser

590 595 600

att ctc aaa gaa ata tgc atg gat cca tct tcg ttt atg ggc gaa agt 3015

Ile Leu Lys Glu Ile Cys Met Asp Pro Ser Ser Phe Met Gly Glu Ser

605 610 615

gac aga gaa aaa ttc atc ggc atc ctg atg gac atg ttt ctt cac aac 3063

Asp Arg Glu Lys Phe Ile Gly Ile Leu Met Asp Met Phe Leu His Asn

620 625 630 635

agc aag ggc gac ttg ttt gaa aaa cta gca ggt gat gat ctg gtg cag 3111

Ser Lys Gly Asp Leu Phe Glu Lys Leu Ala Gly Asp Asp Leu Val Gln

640 645 650

ata tgt aga aga agt gga agc agt gcc gcc atc ata ttg aag aaa tat 3159

Ile Cys Arg Arg Ser Gly Ser Ser Ala Ala Ile Ile Leu Lys Lys Tyr

655 660 665

ggt cat gat att gtt aat tgt att gct gat act cgt tca agc gtg tac 3207

Gly His Asp Ile Val Asn Cys Ile Ala Asp Thr Arg Ser Ser Val Tyr

670 675 680

agc agc atg cac aga aaa att gca gca aag atc ctc aat cat ctg tgt 3255

Ser Ser Met His Arg Lys Ile Ala Ala Lys Ile Leu Asn His Leu Cys

685 690 695

agt ccc tac tcc act gac gag gaa cat ctt cag aat ctg aag gag gcc 3303

Ser Pro Tyr Ser Thr Asp Glu Glu His Leu Gln Asn Leu Lys Glu Ala

700 705 710 715

atc att gat ttg ata ccc aag gtg cta aga gaa gca ctt ggt tgg ggg 3351

Ile Ile Asp Leu Ile Pro Lys Val Leu Arg Glu Ala Leu Gly Trp Gly

720 725 730

ttg aca gga aaa gag ata caa gga gtg gca gtt tca ggc ctt gaa ggt 3399

Leu Thr Gly Lys Glu Ile Gln Gly Val Ala Val Ser Gly Leu Glu Gly

735 740 745

acc caa gat gat gac tgg aaa ttg cag gaa gca ttg gcg tcc ctc tgt 3447

Thr Gln Asp Asp Asp Trp Lys Leu Gln Glu Ala Leu Ala Ser Leu Cys

750 755 760

gca acc gtg ttt aac aga att gtc agc aag gat gca gat ttg act gct 3495

Ala Thr Val Phe Asn Arg Ile Val Ser Lys Asp Ala Asp Leu Thr Ala

765 770 775

cgg ttc aac aac atc gcc gct ggt atc tgc gac cag gca gcg aag cct 3543

Arg Phe Asn Asn Ile Ala Ala Gly Ile Cys Asp Gln Ala Ala Lys Pro

780 785 790 795

cgc gtg acc ttc gca gac ctc atc aaa gaa gct gtg aaa gtt cat cgc 3591

Arg Val Thr Phe Ala Asp Leu Ile Lys Glu Ala Val Lys Val His Arg

800 805 810

atg gaa ttt aag aaa cca gaa cca cca aaa cca cct gcc ggc ccg gca 3639

Met Glu Phe Lys Lys Pro Glu Pro Pro Lys Pro Pro Ala Gly Pro Ala

815 820 825

ctt tac gag ttt atg cct gcg tac tac ccc ccg ccc cac ttt atg tat 3687

Leu Tyr Glu Phe Met Pro Ala Tyr Tyr Pro Pro Pro His Phe Met Tyr

830 835 840

ttg agc gag gaa gat ccc aac gcg tgt tgt atc tct tga taactcccta 3736

Leu Ser Glu Glu Asp Pro Asn Ala Cys Cys Ile Ser

845 850 855

atttgtaatg caaaccgagg atgaaaaaaa tatatatgcc tatacggtac tgctccattt 3796

ccaaataa 3804

<210> 5

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

accaccactt ccctctctcc tc 22

<210> 6

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ggaaatggag cagtaccgta taggc 25

<210> 7

<211> 37

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

acgcgtcgac ataggagagg aagagagagg ctggaca 37

<210> 8

<211> 37

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ttggcgcgcc ccaaacttgc aggaaactag tagaaac 37

<210> 9

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

acactacatg gcgtgatttc at 22

<210> 10

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

tccactatcg gcgagtactt ct 22

<210> 11

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

agagtgcata tgatggagag at 22

<210> 12

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

tctggtgatc atcctcttct tc 22

Claims (5)

1. a kind of rice blast resistance gene Pi57, nucleotide sequence is as shown in SEQ ID NO:2.

2. the coding albumen of rice blast resistance gene Pi57 described in claim 1, amino acid sequence such as SEQ ID NO:3 institute Show.

3. the preparation method of rice blast resistance gene Pi57 described in claim 1, it is characterised in that: with resistant donor rice infiltration Enter be IL-E1454 genomic DNA be template, carry out PCR amplification with primers F 57-SalI-Fw and primers F 57-AscI-Rv and obtain To rice blast resistance gene Pi57, the base sequence of the primers F 57-SalI-Fw is as shown in SEQ ID NO:7, the primer The base sequence of F57-AscI-Rv is as shown in SEQ ID NO:8.

4. a kind of application of the rice blast resistance gene Pi57 described in claim 1 in Molecular Breeding for Blast Resistance.

5. application according to claim 4, it is characterised in that: rice blast resistance gene Pi57 is transferred to sense rice blast In rice, the rice with rice blast resistance is obtained.