CN102604974B - Rice blast resistance gene Piym2 and application thereof - Google Patents
Rice blast resistance gene Piym2 and application thereof Download PDFInfo
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Abstract
The invention discloses a rice blast resistance gene Piym2 and application thereof, and provides the nucleotide sequence of the rice blast resistance gene Piym2 of paddy rice and the amino acid sequence of a protein coded by the rice blast resistance gene Piym2, and the gene is a member belonging to the CC-NBS-LRR resistance gene family. The invention also provides primers for amplifying the rice blast resistance gene Piym2 of paddy rice and the application of the gene in transforming paddy rice or other plants to breed disease-resistant varieties. When being transferred into diseased plants, the gene can help to produce new disease-resistant plants. In particular, hybridization and transformation methods can be used for adding a plurality of disease-resistant genes into plants, while the problem of linkage drag in the genome, which appears in the conventional breeding technique, cannot occur, moreover, the breeding time can be shortened, and the variety-breeding efficiency can be increased.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of resistance gene of rice blast
piym2separating clone and application.
Background technology
Paddy rice is one of most important food crop in the world, and whole world population over half be take rice as main food.Rice blast is one of topmost disease of paddy rice, and the annual paddy underproduction caused because of rice blast in the whole world reaches 10%～30%, when serious, reaches 30%～50%, and even No kernels or seeds are gathered, as in a year of scarcity.Area occurring throughout the year 3,800,000 mu of left and right at China's rice blast, causes the paddy loss of several hundred million kilograms, the grain security that is directly threatening country.
Cultivate and utilize disease-resistant variety to be acknowledged as most economical effective and eco-friendly rice blast prophylactico-therapeutic measures always.But, due to the Population Structure of Magnaporthe grisea complexity, its pathogenic being easy to is morphed in addition, some the potential or rapid breedings new pathogenic microspecies that produce, a disease-resistant variety is often promoted 3-5 susceptibleization is just occurred.Extensively excavating disease-resistant resource, identify and clone's disease-resistant gene, cultivate wide spectrum, permanent disease-resistant new variety, is the task of top priority of the sick breeding research work of current rice anti-rice blast.
Along with molecular biological fast development, at least existing more than 70 rice anti-rice blast major gene located by molecule so far, lay respectively on 11 rice chromosomes except the 3rd karyomit(e), wherein more than 2/3, be positioned at the 6th, 11 and 12 karyomit(e)s, and cluster distributes.Aspect the rice blast resistance gene clone, existing 18 resistant genes of formal report,
pib, Pita, Pi9,
pi2, Piz-t,
pi-d2,
pi-d3,
pi36,
pi37,
pik,
pik-m,
pik-p,
pi5, Pia,
pit,
pish,
pi21, Pb1.Remove
pid2the serine/threonine acceptor class kinases of encoding,
pi21the albumen of a proline rich of coding represents outside a kind of new resistant gene type that remaining 16 gene of having cloned all belongs to NBS-LRR (Nucleotide binding site-Leucine rich repeat) gene family separately.
pib(Wang et al., 1999) are first rice blast resistance genes be cloned, and the clone, from Japanese japonica rice variety TohokuIL9, is positioned at the chromosomal long-armed end of paddy rice the 2nd.It includes 4 introns, and full-length cDNA is comprised of 1 5' non-translational region (UTR), the open reading frame (ORF) of 1 and 1 3' non-translational region etc.
pibthe protein product be comprised of 1251 amino acid of encoding, belong to typical NBS-LRR class disease-resistant gene.The variation of envrionment conditions (as temperature, illumination etc.) can affect
pibexpression.
pita(Bryan et al., 2000) clone, from Japanese japonica rice variety Yashiro-Mochi, is positioned at paddy rice the 12nd zone, the nearly kinetochore of karyomit(e).The intron that it comprises 2 exons and 1, the plasma membrane receptor protein of encoding and being comprised of 928 amino acid, comprise NBS structural domain and rich leucine structural domain (LRD).
pi-tathe product of anti-sense site coding only has an amino acid whose difference, and the L-Ala of 918 becomes Serine.
pi-tathe mediation resistance may be the result of Pi-ta albumen and AVR-Pita interactions between protein, but needs one be divided into it from single dominant gene
ptr (t)participation (Jia et al., 2008).
pi9(Qu et al., 2006),
pi2with
piz-t(Zhou et al., 2006) all be positioned at the nearly centric region of Short arm of chromosome 6, all belong to the NBS-LRR genoid, but their 2 introns lay respectively at before the NBS district and the LRR district after, and hold unlike the N that the intron of other NBS-LRR genes is positioned at the kinases 2 in NBS district.
pi 2with
pi z-tthe height homology, both only have eight amino acid differences, are distributed in 3 conservative LRR structural areas, have shown that the LRR structural area is relevant with the microspecies specialization of gene.
pi-d2(Chen et al., 2006) and
pi-d3all clone from China's rice variety ground paddy (Shang et al., 2009).
pi-d2be positioned at the nearly centric region of Short arm of chromosome 6, the serine/threonine protein receptor kinase of encoding, its anti-sense difference is caused by single base mutation, and the 441st amino acid sports methionine(Met) (M) by Isoleucine (I).
pi-d3be positioned at the long-armed nearly centric region of the 6th karyomit(e), contain 2 exons, the albumen formed by 923 amino acid of encoding, product contains NBS-LRR structural domain and MHD motif.Its anti-sense difference is also because a single base mutation causes, and the 2208th of the LRR structural domain of disease-resistant variety the C of Nucleotide place sports T in susceptible variety, causes the premature termination of proteins encoded.
pi36(Zhai et al., 2011) clone, from rice variety Q61, be positioned on the 8th the short arm of a chromosome.Its 1056 amino acid of encoding is single copy in genome, the constructive expression, and only there is an amino acid whose difference (Asp-Ser) in albumen anti-, sense site coding on the 590th amino acid whose position.
pi37(Zhai et al., 2011) clone from japonica rice variety St. No.1, be positioned at the 1st karyomit(e) long-armed on.Its 1290 amino acid of encoding, be also constitutive expression, and the acquisition of its resistance causes because displacement occurs for the 239th (alanine mutation is α-amino-isovaleric acid) and the 247th amino acids (methionine(Met) sports Isoleucine).
pik(Zhai et al., 2011),
pik-m(Ashikawa et al., 2008) and
pik-p(Yuan et al., 2011) are
pik3 allelotrope on site, be positioned at the long-armed proximal end of the 11st karyomit(e) zone.The resistance of these 3 genes provides by 2 closely linked NBS-LRR class complementary gene.
pik2 complementary gene coding albumen with
pik-mwith
pik-pthe corresponding coded albumen of allelotrope has very high homology.
pi5(Lee
et al., 2009) and
pia(Okuyama et al., 2011) also all comprise 2 NBS-LRR genoids.
pi5be positioned at the 9th nearly kinetochore of karyomit(e) short, its 2 NBS-LRR genoids 1025 and 1063 amino acid of encoding respectively, one expressed by pathogenic bacterium inducing, another constructive expression.
piabe positioned at the 11st the short arm of a chromosome, although its 2 adjacent opposite directions of NBS-LRR genoid.
pit(Hayashi and Yoshida, 2009) clone, from Japanese japonica rice variety K59, is positioned at the 1st karyomit(e) proximal end zone.With susceptible
pit (Npb)different, disease-resistant
pit (K59)promotor in inserted a long retrotransposon, simultaneously at NBS and LRR structural domain, 2 amino acid substitutions are respectively arranged.Retrotransposon activity
pitthe expression of gene, right
pitrecover the resistance function and play vital effect.
pish(Takahashi et al., 2010) clone from Japanese japonica rice variety new No. 2, be positioned at the 1st karyomit(e) long-armed on.It utilizes retrotransposon insertion mutation method clone, with
pi37amino acid consistence large 98%.
pi21(Fukuoka et al., 2009) are first recessive blast resistant genes be cloned, and are the main effect QTLs be positioned on the 4th karyomit(e).With susceptible variety, compare, disease-resistant variety Owarihatamochi's
pi21the disappearance of 21bp and 48bp is arranged respectively in gene.Susceptible allelotrope
pi21the albumen of a proline rich formed by 266 amino acid of coding, the negative regulator disease resistance.
pb1(Hayashi et al, 2010) are genes with strain resistance and fringe pest resistance, from rice variety Modan, are arranged in long-armed upper one of the 11st karyomit(e) and take the tandem repetitive sequence that 60-kb is unit.
pb1gene does not comprise the P-loop structure in the NBS zone, and degeneration has also occurred some other motif.Containing
pb1kind in,
pb1the transcriptional level of gene increases with developmental process, and its functionalization repeats to settle in the encoding sequence upstream promoter sequence to activate " being sunk into sleep " by genome
pb1result.
The clone of rice blast resistance gene and functional analysis thereof, provide certain information for people understand the rice anti-rice blast molecule mechanism.At first, in 18 genes of having cloned, have 16 to belong to NBS-LRR genoid family, have reason to believe, in paddy rice, the blast resistant gene overwhelming majority should be the NBS-LRR genoid; Secondly, most disease-resistant gene clusters distribute, and bunch between gene homology very high, implying paddy rice and resistant gene in pathogenic bacteria coevolution process likely by copying, recombinate, the modes such as insertion and base mutation produce different resistant genes; In addition, confirmed " gene pairs gene " hypothesis that the nontoxic gene of resistant gene and pathogenic bacteria is identified mutually on molecular level, with respect to the tachytely of nontoxic gene, paddy rice need to constantly produce new resistant gene.
The separation of rice blast resistance gene and acquisition, for the blast resisting transgenic breeding provides GENE SOURCES.In the rice blast resistance gene of having cloned, except the partial resistance gene
pi21with
pb1outward, other genes all show complete resistance, and its microspecies specialization is strong, and resistance is easy to lose; As for
pi21with
pb1although, possess some feature of durable resistance, a little less than resistance level, in rice blast time of being very popular, be difficult to really play a role.Because the field Pyricularia grisea Race is numerous and speed of mutation is fast, in order to tackle better the harm of rice blast, clone more blast resistant gene and just seem particularly important.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of rice blast resistance gene and application thereof, described gene is proceeded to susceptible plant, contribute to produce new disease-resistant plants, particularly can be with hybridizing and method for transformation cumulative a plurality of disease-resistant genes in plant, and can not produce in traditional breeding technology Linkage drag problem in the genome of following appearance, and can shorten breeding time, improve breed breeding efficiency.
The purpose of this invention is to provide a kind of resistance gene of rice blast, its nucleotide sequence is as shown in SEQ ID NO.1.A kind of NBS-LRR of this DNA sequence encoding proteinoid, its aminoacid sequence is as listed as SEQ ID NO. 2, and structure is as shown in Figure 8.
Thereby another object of the present invention is to provide the coded protein of above-mentioned blast resistant gene, its aminoacid sequence is as shown in SEQ ID NO.2.
Another object of the present invention is to provide the Auele Specific Primer pair for the above-mentioned blast resistant gene that increases, and its forward primer is as shown in SEQ ID NO.3, and reverse primer is as shown in SEQ ID NO.4.
Another object of the present invention is to provide the carrier that contains above-mentioned blast resistant gene.
What the present invention separated, cloned
piym2resistant gene coding NBS-LRR albumen.This kind of albumen comprises two main structural domain: NBS and LRR zone, wherein the NBS structural domain contains conservative kinases 1a(kinase 1a): kinase 1a:GGAGKS, kinases 2(kinase 2): LLYLDDV and kinases 3a(kinase 3a): GSRVLVTSRQ.And the C-end of this albumen is 27 typical LRR repetitions, its leucine content is 16.4%.
The present invention comprise equally by
piym2the primary structure of resistant gene partly connects the suitable formed mosaic gene of adjusting sequence, and comprises the plant of this gene and the seed of this kind of plant in genome.This gene can be natural or chimeric.For example, will comprise the promotor of the fragment of this gene and a constitutive expression as the Cauliflower flower also connections such as 35S promoter of virus, or the promotor that the promotor specific expressed with tissue/developmental stage or envrionment conditions are induced is connected.Wherein, tissue and developmental stage comprise leaf, fruit, seed and flower etc., and the envrionment conditions bag expands attack, anaerobic condition and the light etc. of pathogenic bacteria.
Rice blast resistance gene provided by the invention
piym2there is important using value.By described
piym2coding sequence is connected to plant vector, imports to paddy rice or other plant cell, can obtain the transgenosis disease-resistant variety of expressing said gene, thereby is applied to produce.Gene constructed in plant conversion carrier under the present invention, can do suitable modification to affiliated gene or its regulating and controlling sequence, also can be before its transcription initiation codon by other promotor, replace original in mover, thereby widen and strengthen the resistance to pathogenic bacteria.
The present invention has following significantly beneficial effect:
piym2deriving from Yunnan Province of China japonica rice variety YMG, is the local variety of a wide spectrum, durable resistance.Clone Origin, in the rice blast resistance gene of YMG, can be widely used in the breeding of anti-rice blast rice.
Will
piym2gene proceeds to susceptible plant, contributes to produce new disease-resistant plants.Particularly can use hybridization and method for transformation cumulative a plurality of disease-resistant genes in plant, and can not produce in traditional breeding technology Linkage drag problem in the genome of following appearance, and can shorten breeding time, improve breed breeding efficiency; The clone of disease-resistant gene be solve in traditional breeding method plant between transgenosis have the basis of obstacle.
The accompanying drawing explanation
Fig. 1 is resistance gene of rice blast
piym2heredity and physical map
Fig. 2 is
piym2the purpose fragment agarose gel electrophoresis figure that primer amplifies from anti-sense parent DNA
Wherein: M1:15kb DNA molecular amount mark; M2:2kb DNA molecular amount mark
Fig. 3 is complementary carrier pCAMBIA1305.1 structure iron
Fig. 4 is over-express vector pCUbi1390 structure iron
Fig. 5 is resistance gene of rice blast
piym2genome complementation transforms T
1the blast resistance identification figure of plant
Wherein, 1:YMG; 2:Fujisaka5; 3-14 means that respectively 12 take the transgenosis individual plant that Fujisaka5 is background.R represents disease-resistant, and S represents susceptible.A. each T
1the rice blast resistance phenotype of individual plant; B. each T
1the hpt marker gene type of individual plant, the PCR reaction system is as follows: 10 μ l 2 * PCR Buffer for KOD FX, 4 μ l dNTPs (2mM each), 1 μ l primer (10 μ M, F+R), 0.4 μ l KOD FX enzyme (1U/ μ l, TOYOBO), 1 μ l DNA profiling (100ng/ μ l), add ddH
2o to 20 μ l; Amplification reaction condition: 94 ℃ of 2 min; 98 ℃ of 10s → 55 ℃ 30s → 68 ℃ of 50s, → 68 ℃ of 5 min of 35 circulations, 16 ℃ of preservations.
Fig. 6 is resistance gene of rice blast
piym2cross to express and transform T
1the blast resistance identification figure of plant
Wherein, 1:YMG; 2:Fujisaka5; 3-12 means that respectively 10 take the transgenosis individual plant that Fujisaka5 is background.R represents disease-resistant, and S represents susceptible.A. each T
1the rice blast resistance phenotype of individual plant; B. each T
1the hpt marker gene type of individual plant, PCR reaction system and amplification reaction condition are with Fig. 5 B.
Fig. 7 is resistance gene of rice blast
piym2the structure iron of gene;
Fig. 8 is resistance gene of rice blast
piym2the aminoacid sequence structure iron of coding;
Wherein, 2 CC(coil-coil that the N end is inferred for composition with single underlined letter) structural domain; 3 conservative regions of the letter shift NBS that Intermediate Gray list underscore overstriking are tilted; 590-1288 amino acid of the C-end of this albumen is that 27 LRR repeat.
Embodiment
The experimental technique used in following embodiment if no special instructions, be ordinary method (for example: the rugged adopted people in mountain, high slope nur are write. rice blast and breeding for disease resistance (Ling Zhongzhuan, Sun Changqi translate). agriculture press, 1990; Lu Tiegang etc. write. molecular genetics. and Higher Education Publishing House, 2008).In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
embodiment 1: resistance gene of rice blast
piym2genetic analysis and Primary Location
In order to excavate and identify the rice blast new resistance gene, to not carrying the susceptible japonica rice local variety Lijiang xintuanheigu (Lijiangxintuanheigu of any main effect blast resistant gene, be called for short LTH) and Yunnan local japonica rice variety wool paddy (Yangmaogu, be called for short YMG) the preparing hybrid combination, adopt F
1, F
2and F
3colony conducts in-depth research the rice blast resistance heredity of YMG, identifies and has located
piym1with
piym2two Major resistance genes.Wherein,
piym2to the performance of the rice blast fungi isolates CH43 from China's Indica Rice Region resistance, be positioned in paddy rice the 1st karyomit(e) long-armed upper (Fig. 1).
embodiment 2: rice blast resistance gene
piym2fine Mapping and candidate gene prediction
The present invention utilizes map based cloning and bioinformatics method clone resistant gene
piym2.For this gene of Fine Mapping, utilize from LTH/YMG F
2that in colony, separates shows 1990 of extremely susceptible individual plants to the rice blast strain identification, and shows 1060 of extremely disease-resistant individual plants, by using linked marker and F
3the checking of offspring's phenotype, will
piym2be positioned between mark CAPS2 and dCAPS5.In the fine reference sequences of Japan, this interval is 78.8 Kb, comprises 10 candidate genes, wherein 4 genes (
os01g57270,
os01g57280,
os01g57310with
pish) be and the gene of disease-resistant relevant NBS-LRR structure, all be gathered in clone B1100D10 upper, cross over the interval of 52.0Kb.Order-checking is found, in YMG, this interval is approximately 43 Kb, utilize FGENESH software (http://linux1.softberry.com/berry. phtml) to carry out the predictive genes discovery, this 43 Kb sequence contains 7 genes, and wherein 2 is NBS-LRR genoid (Fig. 1).Determine that by following experiment second NBS-LRR genoid (candidate gene) is target gene
piym2.
embodiment 3: rice blast resistance gene
piym2the separation of candidate gene and the structure of carrier
In order to isolate
piym2, according to the sequence obtained, design the Auele Specific Primer of candidate gene by primer-design software, by high-fidelity KOD enzyme (TOYOBO), by PCR method, candidate gene is separated; Be connected to after pcr amplification product is purified on pCAMBIA1305.1 and pCUbi1390 carrier, and proceed to intestinal bacteria Trans10(TransGen Biotech) the middle preservation.The positive plasmid that the connection product is cut checking through enzyme is sent to the rich Deco skill Development Co., Ltd's order-checking advanced in years in Beijing (Biomed).
One, pcr amplification
According to the sequence obtained, design the Auele Specific Primer of candidate gene by primer-design software, wherein primer sequence is respectively:
1305F:
CCATGATTACGAATTCTTTCATTAGGTGCACCCACCCACAAG (SEQ ID NO.3)
1305R:
TACCGAGCTCGAATTCTGAAGATGGGCGATGGGAGTCATGT (SEQ ID NO.4)
1390F:
TTACTTCTGCACTAGGTACCGTGTTATACAATGGCGGAGGTGGTGCTA (SEQ ID NO.5)
1390R:
TCCGTCGACCTGCAGGTACCAAGCACAATTCATCTGAATTCCTTCCAGCG (SEQ ID NO.6）
Take the DNA of disease-resistant parent YMG by high-fidelity KOD enzyme by PCR method is template, utilizes primer 1390F (SEQ ID NO.5) and 1390R (SEQ ID NO.6) amplification
piym2coding region fragment 3867bp, utilize 1305F (SEQ ID NO.3) and 1305R (SEQ ID NO.4) amplification
piym2full-length gene group fragment 7006bp (SEQ ID NO.1).The PCR reaction system is as follows: 2 * PCR Buffer for KOD FX, 25 μ l, 10 μ l dNTPs (2mM each), 2 μ l primer (10pmol, F+R), 1 μ l KOD FX enzyme (1U/ μ l, TOYOBO), 2 μ l DNA profilings (100ng/ μ l), add ddH
2o to 50 μ l.Amplification reaction condition: 94 ℃ of 2 min; 98 ℃ of 10 s → 60 ℃ 30s → 68 ℃ of 10 min, 35 circulations; 68 ℃ of 5 min, 16 ℃ of preservations.
Two, PCR product and plasmid retrieve and purification
The PCR product is electrophoresis on 1.5% sepharose, and electrophoretic buffer is 1 * TAE, when electrophoresis indicator tetrabromophenol sulfonphthalein migrates to (160V, 20min) can the DNA isolation fragment time, powered-down.Record result on the gel image analysis instrument, electrophorogram as shown in Figure 2.Cut the purpose band under ultraviolet lamp, utilize sepharose to reclaim test kit (day root biotech company) and reclaim, utilize test kit (the rich Deco skill Development Co., Ltd that steps in Beijing) to extract plasmid, use
ecoRIenzyme is cut the pCAMBIA1305.1 carrier, uses
kpnIafter enzyme is cut the pCUbi1390 carrier, recovery is standby.
Three, the connection of candidate gene transforms
Utilize Clonetech In-fusion
pCR Cloning system (TaKaRa) is to after purifying
piym2full-length gene group fragment (7006bp) and coding region fragment (3867bp, refer in SEQ ID NO.1 the 1598th to 5464) respectively restructuring to the pCAMBIA1305.1 carrier
ecoRIrestriction enzyme site, and the pCUbi1390 carrier
kpnIrestriction enzyme site, and transform in intestinal bacteria Trans10 (TransGen Biotech).The carrier schematic diagram as shown in Figure 3 and Figure 4.
The recombining reaction program:
Linear plasmid (100-200 ng/ μ l) 3 μ l
The PCR product of purifying (100-200 ng/ μ l) 1 μ l
5×in-fusion HD Enzyme Premix 1 μl
Response procedures: 50 ° of C 60 min, 4 ° of C 30min
Four, enzyme is cut checking and order-checking
Positive colony plasmid after conversion is respectively through restriction enzyme
ecoRIor
kpnIafter enzyme is cut checking, the positive plasmid in connection is sent to the rich Deco skill Development Co., Ltd's order-checking advanced in years in Beijing (Biomed).
embodiment 4: rice blast resistance gene
piym2genetic complementation test and the Resistance Identification of transformant
To, through the positive colony plasmid of sequence verification, contain
piym2the complementary carrier pCAMBIA1305.1 of full-length gene group and containing
piym2the over-express vector pCUbi1390 of coding region, import respectively in agrobacterium strains EHA105 by electric shock, and transform susceptible rice varieties Fujisaka5.Utilize Totomycin mark HygF(GGAGCATATACGCCCGGAGT, SEQ ID NO.7) and HygR(CTGCCCGCTGTCTAC AACC, SEQ ID NO.8) detect the T that turns complementary carrier and over-express vector
0in generation, filter out transgenic positive plant transplanting.Contain the phase at rice tillering, utilize the rice blast strain identification to carry out the injection inoculation evaluation, found that the T that turns complementary carrier at 33
0in plant, 29 strain performance disease resistance responses are arranged, 4 strains show susceptible reaction; At 48 T that turn over expression vector
0in plant, 21 strain performance resistance disease resistance responses are arranged, 27 performance susceptible reactions (table 1).To T
1strain is carried out the spray inoculation evaluation, finds all by susceptible T
0the T that individual plant is derivative
1strain all show isozygoty susceptible, and by disease-resistant T
0the T that individual plant is derivative
1strain shows disease-resistant or anti-sense and separates (table 1).These results show,
piym2transgenosis can complementary pair for the resistance of examination rice blast diseased plant.Fig. 5 and Fig. 6 provide part T
1for plant leaf scab figure, wherein, Rice Resistance To Rice Blast is measured by the classification of 0-5 level: the 0-3 level is disease-resistant R, and 4-5 is susceptible S.
Above result also illustrates,
piym2transgenosis can genetic stability, can directly utilize
piym2gene transformation paddy rice susceptible variety, obtain the anti-rice blast rice new germ plasm that contains this gene, also can utilize sexual hybridization to shift in the resistance transformant
piym2gene is improved other japonica rice rice varieties, can also utilize sexual hybridization that this gene is directly applied to rice breeding.
Table 1
piym2the genetic complementation test analytical results of gene
embodiment 5:
piym2gene with
piym2the structure of resistance protein
piym2full length cDNA sequence be 5088bp, contain a 3867bp(and refer in SEQ ID NO.1 the 1598th to 5464) coding region (open reading frame), 5 ' and 3 ' non-translational region is respectively 717bp(and refers in SEQ ID NO.1 the 881st to 1597) and 504bp(refer in SEQ ID NO.1 the 5465th to 5502, the 5611st to the 6076th).By icp gene group DNA and cDNA sequence, the 3 ' non-translational region of finding gene contains a 108bp(and refers in SEQ ID NO.1 the 5503rd to 5610) intron (Fig. 7)
piym2protein polypeptide formed by 1288 amino-acid residues of genes encoding, molecular weight is 146 KD, and iso-electric point is 6.24, and aminoacid sequence is as shown in SEQ ID NO.2.
piym2albumen belongs to NBS-LRR albumen, and its structure as shown in Figure 8.Utilize COIL to the analysis showed that the 51-88 of this protein polypeptide and 124-152 amino acid are 2 CC(coil-coil) structural domain.Conservative kinase 1a(GGAGKS in the NBS structural domain) be positioned at 220-225 amino acid of this polypeptide; Kinase 2(LLYLDDV) be positioned at 295-301 amino acid of this polypeptide; Kinase 3a(GSRVLVTSRQ) be positioned at 325-364 amino acid of this polypeptide.And 590-1288 amino acid of the C-end of this albumen is 27 LRR repetitions, its leucine content is 16.4%.
Claims (7)
1. a resistance gene of rice blast, its nucleotide sequence is as shown in SEQ ID NO.1.
2. the cDNA of gene claimed in claim 1.
3. the protein of genes encoding claimed in claim 1, it is characterized in that: its aminoacid sequence is as shown in SEQ ID NO.2.
4. the expression cassette that contains gene claimed in claim 1 or cDNA claimed in claim 2.
5. the expression vector that contains gene claimed in claim 1 or cDNA claimed in claim 2.
6. a method for preparing the transgenic paddy rice of blast resisting, is characterized in that, utilizes the described gene of claim 1 or cDNA rice transformation claimed in claim 2, selects the plant that resistance strengthens in transfer-gen plant.
7. obtain the primer pair of gene claimed in claim 1, its forward primer is as shown in SEQ ID NO.3, and reverse primer is as shown in SEQ ID NO.4.
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| CN104293800A (en) * | 2014-09-11 | 2015-01-21 | 南京大学 | Rice blast resistance gene RMg40 and application thereof |
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| "水稻稻瘟病抗性基因研究进展";谢培 等;《湖南农业科学》;20111231(第5期);第79-81,82页 * |
| "稻瘟病抗病基因Pia的抗性分析及精细定位";曾晓珊 等;《中国科学》;20111231;第41卷(第1期);第70-77页 * |
| 曾晓珊 等."稻瘟病抗病基因Pia的抗性分析及精细定位".《中国科学》.2011,第41卷(第1期),第70-77页. |
| 谢培 等."水稻稻瘟病抗性基因研究进展".《湖南农业科学》.2011,(第5期),第79-81,82页. |
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