CN106167827A - Resistance gene of rice blast Pita, Pib molecule labelling method - Google Patents
Resistance gene of rice blast Pita, Pib molecule labelling method Download PDFInfo
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Abstract
The present invention relates to resistance gene of rice blast Pita, Pib molecule labelling method, prepare including screening, the PCR primer of blast resisting seed resource, the operation such as DNA material extraction, PCR amplification and agarose gel electrophoresis labelling, rice blast resistance gene Pita, Pib are extracted and are marked.Above-mentioned labeling method utilizes PCR primer amplification technique, respectively Pita and Pib gene is expanded to respectively on YL155/YL87, YL183/YL87, Pibdom F/Pibdom R and Lys145 F/Lys145 R amplified fragments, then gel imaging instrument is used to be analyzed confirming, finally being expanded in which fragment clearly distinguishing Pita and Pib gene, process is simple, success rate is high.
Description
Technical field
The present invention relates to rice blast Prevention Technique field, be specifically related to resistance gene of rice blast Pita, Pib and divide
Sub-labeling method.
Background technology
Rice blast (Magnuprothe grisea. Invisible element, Pyriculariagrisea) is that Oryza sativa L. is topmost
One of disease.Rice blast is also known as rice blast, because phase of causing harm, position difference are divided into Seedling pestilence, leaf pestilence, fringe pestilence, the joint class such as pestilence, grain pestilence
Type, wherein with leaf pestilence harm maximum.Rice blast is distributed widely in the countries and regions of rice cropping, the most all causes serious damage
Lose.In order to reduce the Rice Yield Loss Caused that pest and disease damage causes, the measure that the many employings of people comprehensively prevent, topmost technology has two
Kind: one is to utilize to constantly update the chemical pesticide regenerated；Two is the breeding selecting to have resistance to most disease and pest.The former not only cost
Higher and pollute environment, poison human body, be unfavorable for the sustainable development of modern agriculture.Therefore the resistance improveing rice varieties becomes
One of important goal of rice breeding worker.
Middle 1960s, Japan takes the lead in having carried out the research work of rice varieties blast resisting gene analysis, mirror
Determine 14 genes on the resistance locus of initial 8, and establish the identification system of a set of blast resistant gene analysis
(JDCs, Japanse differential cultivars), subsequently, International Rice Research Institute and Deng Chan rice state of China are the most gradually
Carry out the systematic Study of Rice Resistance To Rice Blast heredity.By in August, 2013, the most at least report 68 blast resisting positions
Point totally 83 major gene resistances.Be distributed in these gene clusters except on the 3rd extrachromosomal all rice chromosomes (2 recessiveness,
Other is dominant), wherein, Pb1, Pia, Pib, Pid2, Pid3, Pik, Pik-h/Pi54, Pik-m, Pik-p, Pish, Pita,
23 genes such as Pita, Piz-t, Pi1, Pi2, Pi5, Pi9, pi21, Pi25, Pi36, Pi37, Pi56, PiCO39 are by success
(Pi2, Pi9, Piz-t are all the multiple alleles on Piz site to clone；Pi1, Pik-h/Pi54, Pik-m, Pik-p are all Pik
Multiple alleles on site；Pid3 Yu Pi25 equipotential；Pia Yu PiCO39 equipotential).Develop large quantities of and blast resisting in recent years
The closely linked molecular marker of gene, more particularly comes from the special of resistant gene (such as Pita and Pib) sequence exploitation own
Property molecular marker (functional label), for quick, accurately, systematically identify rice varieties blast resisting genotype, and efficiently open
Exhibition blast resistant gene pyramiding breeding brings new opportunity.
Research shows, rice blast resistance gene Pita is positioned at the centromere near zone of Oryza sativa L. the 12nd chromosome.Pita base
Because comprising 2 exons and the intron of 1 1463bp, encode a length of 928 amino acid whose cytoplasma membrane receptor eggs
In vain, owing to occurring in that a single nucleotide polymorphism (SNP), i.e. codon GCT becomes TCT, and the 918th amino acid sites is by resisting
Sick alanine variation is susceptible serine, and the change of this site amino acids is for resistance protein and nontoxic gene product identification
Whether having conclusive impact, main effect Rice blast resistance Pi ta gene shows high-caliber rice blast resistance in a lot of rice district of China.
Pib gene belongs to " NBS-LRR " class disease-resistant gene, include 4 introns (164bp, 810bp, 1340bp,
308bp), full-length cDNA by the 5' untranslated region (UTR, untranslated regions) of 306bp, the ORF of 3753bp and
The 3' untranslated regions of 229bp etc. form.Pib encodes a protein product being made up of 1251 aminoacid, and this product comprises one
Individual nucleotide binding site (nucleotide binding site, NBS) and 17 leucine-rich repeat (leucine-
Rich repeats, LRRs), wherein, there are kinases 1a, 2 and 3a domain units in N-terminal NBS district, has 8 in the middle part of LRRs
The cysteine residues (Wang et al., 1999) of cluster.Pib gene can because of the change of environmental condition induction regulating controlling, such as temperature
The change of the conditions such as degree, illumination all will affect the expression (Wang et al., 1999) of this gene.Pib gene mapping is in Oryza sativa L.
2 chromosome long arm proximal end regions, and RFLP labelling RZ123, C379, C2782B etc. chain (Miyamoto et al., 1996；
Monna et al.,1997).Pib gene mapping on Oryza sativa L. the 2nd chromosome between RFLP labelling S1916 and G7030, heredity
Distance respectively 0.015cM and 0.045cM, and with RFLP labelling G7010, G7021 and G7023 be divided into from (Wang et al.,
1999).Corresponding to the position (5'-3') of Japanese fine Sequencing chromatogram at interval (the Rice Genome of 35109965-35109117
Annotation Project:TIGR version6).Resistance gene of rice blast pib is from kind " BL1 ", big to Japan
Most Pyricularia grisea Races have resistance, and bacterial strain ZB13 and ZC15 of China is also showed disease resistance response.Utilize blast resistant gene
The molecular marker R. concomitans that the susceptible gene order of Pib its own sequence and equipotential thereof is set up, can provide from rice germplasm effectively
Source is selected blast resistant gene pib quickly and accurately, and can select containing blast resisting base in segregating generation colony
The individual plant that isozygotys because of pib.
Main school Rice blast resistance Pi ta gene and Pib show high-caliber rice blast resistance in a lot of rice district of China, by extensively
It is applied to rice breeding and the production of China.The Wang Cailin chief of academy of agricultural sciences of Jiangsu Province cereal crops institute is to Jiangsu part round-grained rice
Rice has carried out blast resisting Pita and Pib genescreen, finds a lot of excellent japonica rice such as military fortune round-grained rice 21 in Jiangsu, military fortune round-grained rice 7
Number, peaceful 7007, they include Pita and Pib gene.Institute of Crop Science, Chinese Academy of Agricultural Science time gram to China
Main cultivated rice cultivars carries out blast resisting Pita and Pib gene test, result show special Xian account for 25, the Xian such as Milyang 46, survey 64-7
Rice varieties carries Pita and Pib gene.Therefore, existing seed resource carries out Pita and Pib genescreen work to field
Between breeding there is important directive function.
Summary of the invention
The invention discloses a kind of resistance gene of rice blast Pita, Pib molecule labelling method, utilize PCR primer to expand
Increasing technology, respectively Pita and Pib gene is expanded to respectively YL155/YL87, YL183/YL87, Pibdom F/Pibdom R and
On Lys145F/Lys145R amplified fragments, gel imaging instrument is then used to be analyzed confirming, clearly to distinguish Pita and Pib
Which fragment gene is finally expanded in, and process is simple, success rate is high.
For reaching above-mentioned purpose, the technical solution used in the present invention is: resistance gene of rice blast Pita, Pib molecule
Labeling method, comprises the following steps:
(1) screening of blast resisting seed resource, by japonica rice, non-glutinous rice, long-grained nonglutinous rice, Japonica Hybrid, hybridization non-glutinous rice, hybridization Xian
The each kind of rice, sterile line and restorer respectively takes the tender leaf of equal number, in order to obtain PCR primer；The gene of described PCR primer
Obtained by NCBI network address http://www.ncbi.nlm.nih.gov；
(2) PCR primer that beneficially step (1) obtains is respectively YL155/YL87, and clip size is 1042bp；YL183/
YL87, clip size is 1042bp；Pibdom F/Pibdom R, clip size is 365bp；Lys145F/Lys145R, fragment
Size is 803bp.
(3) DNA material extraction, takes fresh tissues of plants addition liquid nitrogen and is fully ground, add buffer FP1 sum
RNase, vortex shakes, and room temperature places 10min；
Add buffer FP2, the most fully mix, vortex concussion 1min；
Centrifugal 5min, is transferred to supernatant in new centrifuge tube；
By supernatant recentrifuge 5min, supernatant is transferred in new centrifuge tube；
In supernatant, add the isopropanol of 0.7 times of volume, fully mix, now there will be cotton-shaped genomic DNA；
Centrifugal 2min, abandons supernatant, retains precipitation；
Add 600 μ L 70% ethanol, vortex concussion 5sec, centrifugal 2min, abandon supernatant；This step is repeated once；
Uncapping inversion, room temperature dries the ethanol of remnants；
Add appropriate elution buffer TE, tepidarium dissolving DNA, obtain DNA solution.
(4) DNA solution that step (3) obtains is carried out PCR amplification operation.
(5) amplified production in step (4) is carried out agarose gel electrophoresis labelling.
Improving further as the present invention, the speed of described step (3) mesoscale eddies concussion is 500～3000r/min, excellent
Elect 1200r/min as.
Improving further as the present invention, in described step (3), the rotating speed of centrifugal process is 10,000r/min～15,
000r/min。
Improving further as the present invention, in described step (3), water temperature during tepidarium dissolving DNA is 45 DEG C～75
℃。
Due to the utilization of technique scheme, the present invention compared with prior art has the advantage that
The matrix illumination system scanning driving device of the present invention program, utilizes PCR primer amplification technique, respectively by Pita and
Pib gene is expanded to YL155/YL87, YL183/YL87, Pibdom F/Pibdom R and Lys145F/Lys145R amplification respectively
In fragment, gel imaging instrument is then used to be analyzed confirming, clearly to distinguish which Pita and Pib gene is finally expanded to
In fragment, process is simple, success rate is high.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail.
The size of crop breeding effect is largely determined by grasps the quantity of germ plasm resource and to its character table
Show and the depth of investigation of genetic development.Variety source is formed during long-term natural selection and artificial selection, and they are taken
With various genes, it is breed breeding and biological theory research indispensable stock source.Screening is with true
The original material of ordered goods breeding, is also the element task of crop breeding.Can neatly, the original material of selection and use rightly
Material, the breadth and depth worked by Crop Germplasm Resources is restricted.Rice blast resistance be rice varieties authorization in one important
Index, therefore the screening to company's blast resisting seed resource is significant.
Embodiment is as described below:
1, chosen material
The seed resources such as conventional japonica rice, long-grained nonglutinous rice, Japonica Hybrid, Japonica Hybrid, restorer, sterile line, it is contemplated that can detect
About 400 parts, each kind provides about 7 tender leafs (7 strain sample mixing sampling), and concrete kind and quantity by breeding portion and are correlated with
Departmental staff is supplied to laboratory after determining.
2, the design of primers of functional label
Gene order obtains related gene sequence by NCBI network address (http://www.ncbi.nlm.nih.gov).Ginseng
Primer (see table 1) is designed according to pertinent literature and primer5.0 primer-design software.Primer is by Shanghai biological engineering company limited
Synthesis.
Table 1. PCR primer
3, DNA of plants is extracted
DNA extraction uses fast-type plant genome DNA to extract test kit (sky root, Beijing).
DNA extraction step is as follows:
(1) process material: take fresh tissues of plants 100mg, add liquid nitrogen and be fully ground, add 400 μ L buffer FP1 and
The RNase of 6 μ L, vortex concussion 1min, room temperature places 10min；
(2) add 130 μ L buffer FP2, the most fully mix, vortex concussion 1min；
(3) 12,000r/min is centrifuged 5min, is transferred in new centrifuge tube by supernatant；
(4) by supernatant again 12,000r/min is centrifuged 5min, is transferred in new centrifuge tube by supernatant；
(5) in supernatant, add the isopropanol of 0.7 times of volume, fully mix, now there will be cotton-shaped genomic DNA.
12,000r/min are centrifuged 2min, abandon supernatant, retain precipitation；
(6) adding 600 μ L 70% ethanol, vortex concussion 5sec, 12,000r/min are centrifuged 2min, abandon supernatant.This step
Suddenly it is repeated once；
(7) uncapping inversion, room temperature dries the ethanol of remnants；
(8) appropriate elution buffer TE is added, 65 DEG C of water-bath dissolving DNAs, finally give DNA solution.
4, the PCR amplification of purpose fragment
Use PrimeSTAR HS DNA Polymerase (TakaRa, China) with the DNA of not homophyletic for template respectively
Expand.Reaction uses BIO-RAD PCR instrument device, reaction system and program (table 2, table 3).
Table 2 PCR reaction system
| React added solution | Addition |
| 5×PrimerSTAR buffer(Mg2+ plus) | 5μL |
| DNTP Mixture (each 2.5mM) | 2μL |
| Primer F | 0.5μL |
| Primer R | 0.5μL |
| Template DNA (50ng/ μ L) | 2μL |
| PrimerSTAR HS DNA polymerase(2.5U/μL) | 0.25μL |
| ddH2O | 14.75μL |
| Reaction cumulative volume | 25μL |
Table 3 PCR reaction condition
5, amplified production analysis
PCR primer with 1.5% agarose gel electrophoresis, analysis result under gel imaging instrument.According to gene sequencing,
The pcr amplification product of 4 pairs of primers will have following genotype: 1. can amplify 1042bp fragment with YL155/YL87, use simultaneously
YL183/YL87 can not expand the kind of product and contains Pita gene；2. can not amplify target product with YL155/YL87 (to contain
Faint and the non-specific band that can not repeat), but the kind of 1042bp fragment can be amplified with YL183/YL87 and not contain
Pita and containing its allele pita.3. can amplify 365bp fragment with Pibdom F/Pibdom R, use simultaneously
Lys145F/Lys145R can not expand the kind of product and contains Pib gene；4. can not amplify with Pibdom F/Pibdom R
Target product, but can with Y Lys145F/Lys145R amplify the kind of 803bp fragment do not contain Pib and containing its equipotential
Gene pib.
6, blast resisting conventional japonica rice Breeding Scheme (selecting breeding base, two, big Fenghe Sanya):
(selection of A and B kind needs to select suitable kind according to practical situation, has with reference to the explanation of preface part material
Branch's conventional japonica rice and long-grained nonglutinous rice all with Pita and Pib base blast resisting because of.)
In the F5 generation of breed breeding, starts the aid mark choosing selecting excellent individual plant to carry out rice blast Pita and Pib gene
Selecting experiment, experimental technique is carried out with reference to the method in the screening of above-mentioned (one) company blast resisting seed resource.Screen
Strain field to be carried out blast resisting qualification (authentication method is with reference to rice blast qualification program), therefrom filters out blast resisting
The conventional japonica rice of good quality and high output.
Below it is only the concrete exemplary applications of the present invention, protection scope of the present invention is not constituted any limitation.All employings
The technical scheme that equivalents or equivalence are replaced and formed, within the scope of all falling within rights protection of the present invention.
Claims (4)
1. resistance gene of rice blast Pita, Pib molecule labelling method, comprises the following steps:
(1) screening of blast resisting seed resource, by japonica rice, non-glutinous rice, long-grained nonglutinous rice, Japonica Hybrid, hybridization non-glutinous rice, indica Hybrid Rice, no
Educating is kind each with the restorer tender leaf that respectively takes equal number, in order to obtain PCR primer；The gene of described PCR primer passes through
NCBI network address http://www.ncbi.nlm.nih.gov obtains；
(2) PCR primer utilizing step (1) to obtain is respectively YL155/YL87, and clip size is 1042bp；YL183/YL87,
Clip size is 1042bp；Pibdom F/ Pibdom R, clip size is 365bp；Lys145 F/Lys145 R, fragment is big
Little for 803bp；
(3) DNA material extraction, takes fresh tissues of plants addition liquid nitrogen and is fully ground, add the RNase of buffer FP1 sum, vortex
Concussion, room temperature places 10 min；
Adding buffer FP2, the most fully mix, vortex shakes 1 min；
Centrifugal 5 min, are transferred to supernatant in new centrifuge tube；
By supernatant recentrifuge 5 min, supernatant is transferred in new centrifuge tube；
In supernatant, add the isopropanol of 0.7 times of volume, fully mix, now there will be cotton-shaped genomic DNA；
Centrifugal 2 min, abandon supernatant, retain precipitation；
Adding 600 L 70% ethanol, vortex shakes 5 sec, centrifugal 2 min, abandons supernatant；This step is repeated once；
Uncapping inversion, room temperature dries the ethanol of remnants；
Add appropriate elution buffer TE, tepidarium dissolving DNA, obtain DNA solution；
(4) DNA solution that step (3) obtains is carried out PCR amplification operation；
(5) amplified production in step (4) is carried out agarose gel electrophoresis labelling.
Resistance gene of rice blast Pita, Pib molecule labelling method the most according to claim 1, its special type is: institute
The speed stating the concussion of step (3) mesoscale eddies is 500～3000r/min, preferably 1200r/min.
Resistance gene of rice blast Pita, Pib molecule labelling method the most according to claim 1, its special type is: institute
Stating the rotating speed of centrifugal process in step (3) is 10,000r/min～15,000 r/min.
Resistance gene of rice blast Pita, Pib molecule labelling method the most according to claim 1, its special type is: institute
Water temperature when stating tepidarium dissolving DNA in step (3) is 45 DEG C～75 DEG C.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107988410A (en) * | 2017-12-21 | 2018-05-04 | 辽宁省盐碱地利用研究所 | Identify molecular labeling, identification method and the application of rice blast resistant gene Pita |
| CN108018371A (en) * | 2017-12-21 | 2018-05-11 | 辽宁省盐碱地利用研究所 | Identify molecular labeling, identification method and the application of Rice Resistance To Rice Blast character |
| CN108753937A (en) * | 2018-06-28 | 2018-11-06 | 扬州大学 | A kind of detection method of blast resistant gene expression and its application |
| CN113981122A (en) * | 2021-09-18 | 2022-01-28 | 华南农业大学 | A set of compatible and accurate identification, excavation and cloning technology system for rice blast Pita disease-resistant gene family alleles |
| CN116987813A (en) * | 2022-11-02 | 2023-11-03 | 江苏省农业科学院 | Rice blast multiple disease-resistant gene combination pita+Pi5+Piz-t and application thereof |
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| CN102577925A (en) * | 2012-02-17 | 2012-07-18 | 江苏省农业科学院 | Breeding method for breeding rice variety resistant to rice panicle blast in Jiangsu province |
| CN105830912A (en) * | 2016-05-10 | 2016-08-10 | 江苏丘陵地区镇江农业科学研究所 | Breeding method for rice with high yield and durable neck blast resistance |
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| WO2002034927A2 (en) * | 2000-10-20 | 2002-05-02 | Wisconsin Alumni Research Foundation | Plant genes that confer resistance to strains of magnaporthe grisea having avr1 co39 cultivar specificity gene |
| CN102577925A (en) * | 2012-02-17 | 2012-07-18 | 江苏省农业科学院 | Breeding method for breeding rice variety resistant to rice panicle blast in Jiangsu province |
| CN105830912A (en) * | 2016-05-10 | 2016-08-10 | 江苏丘陵地区镇江农业科学研究所 | Breeding method for rice with high yield and durable neck blast resistance |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988410A (en) * | 2017-12-21 | 2018-05-04 | 辽宁省盐碱地利用研究所 | Identify molecular labeling, identification method and the application of rice blast resistant gene Pita |
| CN108018371A (en) * | 2017-12-21 | 2018-05-11 | 辽宁省盐碱地利用研究所 | Identify molecular labeling, identification method and the application of Rice Resistance To Rice Blast character |
| CN108753937A (en) * | 2018-06-28 | 2018-11-06 | 扬州大学 | A kind of detection method of blast resistant gene expression and its application |
| CN113981122A (en) * | 2021-09-18 | 2022-01-28 | 华南农业大学 | A set of compatible and accurate identification, excavation and cloning technology system for rice blast Pita disease-resistant gene family alleles |
| CN116987813A (en) * | 2022-11-02 | 2023-11-03 | 江苏省农业科学院 | Rice blast multiple disease-resistant gene combination pita+Pi5+Piz-t and application thereof |
| CN116987813B (en) * | 2022-11-02 | 2024-02-13 | 江苏省农业科学院 | Rice blast multiple resistance gene combination Pita+Pi5+Piz-t and its application |
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