CN103898209B - The detection method of avirulence gene of rice blast AVR-pik-B - Google Patents
The detection method of avirulence gene of rice blast AVR-pik-B Download PDFInfo
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Abstract
The invention belongs to biology field, be specifically related to a kind of detection method of avirulence gene of rice blast.Described method from Pyricularia oryzae STb gene, amplifies one section of nucleotide sequence with primer pair SEQ ID NO:1 and SEQ ID NO:2, use NlaIII enzymic digestion again, obtain the DNA fragmentation of length-specific, the detection that whether there is nontoxic gene AVR-pik-B in Pyricularia oryzae can be applied to, to determine the nontoxic gene type entrained by Pyricularia oryzae, thus carry out rice varieties layout and disease-resistant variety seed selection according to this result.
Description
Technical field
The invention belongs to biology field, be specifically related to utilize the nontoxic gene in primer special and corresponding enzyme butt case detection Pyricularia oryzae.
Background technology
The rice blast caused by fungi Magnaporthe oryzae is the primary disease in world's Rice Production.Mutual work between Pyricularia oryzae and paddy rice host meets " gene-for-gene theory " of Flor, and even Pyricularia oryzae has nontoxic gene, and rice varieties carries corresponding disease-resistant gene, and paddy rice will produce resistance to Pyricularia oryzae.Nontoxic gene is class important pathogen thing gene a---important member for exciton that can change host cell state during host and pathogen are done mutually.Each dominant resistant gene in host plant, the nontoxic gene that corresponding existence one is dominant in pathogenic fungi; The disease-resistant gene product of host carries out identification with the nontoxic gene product of direct or indirect mode to pathogenic bacteria and starts defense response, and then produces the resistance to this pathogenic bacteria.Only have in host plant and there is corresponding disease-resistant gene, and in germ, there is corresponding nontoxic gene, plant just can show disease resistance, then the non-compatible reaction of pathogenic bacteria and host plant is caused, impel local cells generation hypersensitive cell downright bad, to stop the expansion of pathogenic micro-organism in plant body [Liu JL et al., 2010].The nontoxic gene of qualification Pyricularia oryzae, contributing to people selects the rice varieties with corresponding disease-resistant gene to carry out rational deployment according to the nontoxic gene type of different areas epidemic link, simultaneously breeding man can carry out cultivating and promoting by the rice varieties that utilizes the seed selection of molecule marker supplementary means to have corresponding disease-resistant gene of the nontoxic gene entrained by the Major Epidemic bacterial strain of field, thus reduces the threat that the use of agricultural chemicals and rice blast causes Rice Production.
Rice blast resistance gene Pik is positioned at paddy rice 11 chromosome long arm, has stronger, stable resistance [Fjellstrom et al., 2004] to many Chinese rice blast microspecies, this gene has 4 allelotrope, be respectively pik-p, pik-m, pik-s and pik-h.Avirulence gene of rice blast AVR-pik was cloned in 2009 [YoshidaK et al., 2009], known 5 kinds of allelic gene types at present, be respectively AVR-pik-A, AVR-pik-B, AVR-pik-C, AVR-pik-D, AVR-pik-E, the wherein type that finds at first at the beginning of being this gene evolution of AVR-pik-D, can be pik/pik-p/pik-m identified, and other several types be this gene with the saltant type [Hiroyuki Kanzaki et al., 2012] that occurs successively in rice varieties disease-resistant gene common evolutionary.The scope that these nontoxic genes can be the identification of pik disease-resistant allelotrope is smaller relative to AVR-pik-D type, as AVR-pik-B only can and disease-resistant gene pikm and pikp between there is non-affinity and react (not falling ill), therefore the such allelotrope of precise Identification, in order to detect the specificity of Field epidemic rice blast fungi isolates, the disease-resistant gene of people entrained by rice varieties can be contributed to and carry out rational deployment, alleviate the harm of rice blast.
Summary of the invention
For the nontoxic gene entrained by precise Identification Pyricularia oryzae, and then seed selection and popularization have the rice varieties of corresponding blast resistant gene targetedly, alleviate the harm of rice blast, the invention provides a kind of method for detecting avirulence gene of rice blast AVR-pik-B, may be used for the detection of nontoxic gene entrained by Pyricularia oryzae, think that Variety distribution and disease-resistant variety seed selection provide reference.
The invention provides following solution:
For detecting the primer pair DNA whether avirulence gene of rice blast AVR-pik-B exists, as shown in SEQ ID NO:1 and SEQ IDNO:2.
The present invention further provides primer pair shown in SEQ ID NO:1 and SEQ ID NO:2 and detect the purposes on avirulence gene of rice blast AVR-pik-B.
A kind of method detecting avirulence gene of rice blast AVR-pik-B and whether exist, with sequence shown in SEQ ID NO:1 and SEQ ID NO:2 for primer, increase DNA to be detected, if amplify the specific fragment of 503bp size and amplicon is containing nontoxic gene AVR-pik-B containing, for example sequence shown in SEQ ID NO:3.
A kind of differentiation avirulence gene of rice blast AVR-pik-B and other allelic method, with sequence shown in SEQ ID NO:1 and SEQ ID NO:2 for primer, pcr amplification DNA to be detected, obtain the amplified production of 503bp, further with NIaIII enzymic digestion, if PCR primer can not be NlaIII enzymic digestion, then there is nontoxic gene AVR-pik-B in DNA to be detected; If PCR primer enzyme can obtain 367bp and 136bp two DNA fragmentations after cutting, then show allelotrope AVR-pik-A, AVR-pik-C, AVR-pik-D or AVR-pik-E of there is avirulence gene of rice blast AVR-pik-B in DNA to be detected.
Above-mentioned primer and enzymatic cleavage methods are identified the DNA that preferred rice blast hypha extracts, and detect whether carry avirulence gene of rice blast AVR-pik-B, easy to detect, accuracy rate is high, can be Breeding of Blast Resistant Rice and Variety distribution provides reference.
Accompanying drawing explanation
Fig. 1 is with Pyricularia oryzae DNA for template, with sequence shown in SEQ ID NO:1 and ISEQ ID NO:2 for the gel electrophoresis figure of primer extension product after NlaIII enzymic digestion;
Wherein, M:600bp molecular weight marker (TIANGEN MD101), 200ng; Fragment length is respectively: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp.
A：AVR-pik-A；B：AVR-pik-C；C：AVR-pik-D；D：AVR-pik-B：E：AVR-pik-E
CK: for PCR primer enzyme cut before electrophoretic band; EC: for PCR primer enzyme cut after electrophoretic band.
Obtain 367bp and 136bp two DNA fragmentations after the amplified production NIaIII enzymic digestion of wherein AVR-pik-A, AVR-pik-C, AVR-pik-D and AVR-pik-E, and the amplified production of AVR-pik-B is be still 503bp after NIaIII enzymic digestion.
Fig. 2 is with 6 strain Pyricularia oryzae DNA of field separation for template, with sequence shown in SEQ ID NO:1 and SEQ ID NO:2 for the gel electrophoresis figure of primer extension product after NlaIII enzymic digestion;
Wherein, M:600bp molecular weight marker (TIANGEN MD101), 200ng; Fragment length is respectively: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp.
1,2,3,4,5,6 are respectively the electrophoresis result of different strains amplified production after NlaIII enzymic digestion, visible 1,2,3, No. 5 bacterial strain PCR primer enzymes are still 1 fragment being about 500bp after cutting, and the PCR primer enzyme of No. 4 and No. 6 bacterial strains obtains two bar segment after cutting, and sequence length is about 370bp and 130bp.
Fig. 3 is the inoculated identification result of Pyricularia oryzae on 4 pik and allelotrope Monogenic lines thereof through qualification tool nontoxic gene AVRpik-B.
The Resistant expression of several kind to this bacterial strain is respectively: pikm and pikp shows high resistance, sense in piks performance, and pik performance is susceptible, the high sense of Lijiang xintuanheigu performance.
Fig. 4 is with 6 strain Pyricularia oryzae DNA of field separation for template, the gel electrophoresis figure being primer extension product with sequence shown in SEQ ID NO:1 and SEQ ID NO:2;
Wherein, M:600bp molecular weight marker (TIANGEN MD101), 200ng; Fragment length is respectively: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp.
1,2,3,4,5,6 electrophoresis result being respectively different strains amplified production.
Embodiment
Embodiment 1:DNA extraction, pcr amplification and electrophoretic analysis
One, Pyricularia oryzae filament is cultivated
The appropriate rice blast hypha of picking, is inoculated in 200ml liquid nutrient medium (containing KH in often liter of liquid nutrient medium
2pO
40.5g, K
2hPO
40.5g, MgSO
40.5g, CaCl
20.1g, glucose 20g and yeast extract paste 5g) in, 25 DEG C and 120r/m concussion are cultivated centrifugal 10 minutes of 6d, 5000r/m and are collected mycelium, are placed on sterilizing filter paper and place 36h at 37 DEG C of baking ovens, be stored in after oven dry-20 DEG C for subsequent use.
Two, DNA extraction (CTAB method):
Take 0.1g oven dry mycelium to grind in liquid nitrogen, (note: do not make sample melt) after liquid nitrogen vaporization is complete, proceed to rapidly containing 65 DEG C of preheated extracting solution (1M Tris pH8.0,100mM; 5M NaCl, 1.0M; 0.5M EDTA, 20mM; 10%CTAB, 2.0%) in the centrifuge tube of 400 μ L.Shake up gently.
60 ~ 65 DEG C of water-baths 30 ~ 60 minutes, within every 10 minutes, shake is evenly gently.
Add isopyknic chloroform/primary isoamyl alcohol (24:1), gentle shake, makes into milk sap.
Under room temperature (temperature is not less than 15 DEG C), the centrifugal 5min of 12000r/min, gets supernatant liquor.
Carefully extract with macropore Tip head the dehydrated alcohol (or isopyknic primary isoamyl alcohol) that supernatant liquor 200 μ L adds 2 times of volumes of precooling, place more than 20 minutes under the condition of-20 DEG C, centrifugal 5 minutes of 5000r/min, collecting precipitation.
70% washing with alcohol precipitation 1 ~ 2 time.
Open pipe lid, put clean place and dry, milky DNA precipitation is transparent.
100 μ LTE (Tris-Hcl10mM, PH8.0; EDTA1mM, PH8.0) dissolution precipitation, and be diluted to 100 μ g/mL ,-20 DEG C save backup.
Three, pcr amplification:
Pcr amplification reaction carries out in PCR amplification instrument.
1. reaction system is as follows:
2 × Taq PCR Master Mix (Ai Delai PC0902) 10 μ L, each 0.5 μ L of primer of 10 μMs, the template DNA 1 μ L of 100 μ g/mL, ddH
2o8 μ L.
3.PCR response procedures is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 30s, 35 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.
Four, PCR primer detects and purifying
Get 4 μ LPCR products, electrophoresis in 1% sepharose, through goldview dyeing, application gel imaging system log.
PCR primer purification kit (TIANGEN M1419) is utilized to carry out purifying to product.
Five, enzyme cuts test
Endonuclease reaction system (20 μ L): 10 × NEB4 damping fluid 2 μ L, purified pcr product≤1 μ g, 100 × BSA0.2 μ L, restriction endonuclease 0.5 μ L, sterilizing ddH
2o complements to 20 μ L
Enzyme tangent condition: 37 DEG C of incubation 3.5h.
Six, enzyme cut after product electrophoresis detection
Get 4 μ L digestion products, electrophoresis in 1% sepharose, through goldview dyeing, application gel imaging system log.
Embodiment 2: the detection of nontoxic gene AVR-pik-B in Pyricularia oryzae
One, the Pyricularia oryzae gathered from field is through separation and purification, cultivate Pyricularia oryzae according to the method in embodiment 1 and extract mycelia DNA, carry out pcr amplification again, electrophoresis detection amplification in 1% sepharose, as seen from Figure 2, with the DNA of 6 strain Pyricularia oryzaes for template, with sequence shown in SEQ IDNO:1 and SEQ ID NO:2 for primer amplification all can obtain the sequence of about 500bp.
Two, carry out PCR primer purifying according to the method for embodiment 1 and enzyme is cut, digestion products gets 4 μ L, electrophoresis in 1% sepharose, through goldview dyeing, and application gel imaging system log (Fig. 3).As seen from Figure 3,1,2,3, the PCR primer enzyme of No. 5 bacterial strains is still 1 fragment being about 500bp after cutting, and the PCR primer enzyme of No. 4 and No. 6 bacterial strains obtains two bar segment after cutting, and sequence length is about 370bp and 130bp, shows 1,2,3, No. 5 bacterial strains carry nontoxic gene AVR-pik-B.
Three, the pcr amplification product of 6 Pyricularia oryzaes of the first step acquisition send the order-checking of calm and peaceful company of Beijing Sino-U.S., result shows 1,2,3, No. 5 bacterial strain PCR primer sequences are consistent with sequence shown in SEQ ID NO:3, and proving that it carries further is nontoxic gene AVR-pik-B really.
Embodiment 3: carry the rice blast fungi isolates of nontoxic gene AVR-pik-B and the mutual work between different paddy rice Monogenic lines
Transplanted by the Pyricularia oryzae carrying nontoxic gene AVR-pik-B on rolled oats tomato juice agar, cultivate 5 ~ 7d, treat that mycelia is covered with under 25 ~ 27 DEG C of conditions, after wiping aerial hyphae with the cotton swab of sterilizing, moisturizing is cultivated, and Pyricularia oryzae can large volume production spore.When the rice seedling of 4 anti-rice blast rice Monogenic lines (pik, pikp, pikm, piks) and Lijiang xintuanheigu (without disease-resistant gene contrast) grows to 5 leaf 1 heart, each bacterial strain conidium of numerous for above-mentioned expansion cultivation is used 120ml sterile water wash respectively, filtering with double gauze loads in triangular flask, preparation spore suspension (concentration is have about 20 spores under 120 powered microscope fields), isolates spray inoculation respectively.24h after inoculation, moisturizing on the brandreth being overlying on above vinyl disc with plastic film, covered with sunshade net prevents direct sunlight on the plastic film.7-10d investigation after inoculation, pathogenic response type and microspecies are determined to evaluate by national physiological races of rice blast fungus Combined Trials group (1980) unified standard.
Investigation Recording criteria:
0 grade anosis ... high resistance (HR)
1 grade of brown point only having a needle point size ... anti-(R)
2 grades of slightly large brown points ... anti-(R)
The little scab of grey that 3 grades of circles are slightly large, edge brown, lesion diameter 1 ~ 2mm ... in anti-(MR)
4 grades of typical fusiform scabs, long 1 ~ 2cm, is confined between two master pulses usually.To cause harm area less than 2% ... middle sense (MS)
5 grades of typical scabs, area 3% ~ 10% of causing harm ... middle sense (MS)
6 grades of typical scabs, area 11% ~ 25% of causing harm ... sense (S)
7 grades of typical scabs, area 26%o ~ 50% of causing harm ... sense (S)
8 grades of typical scabs, area 51% ~ 75% of causing harm ... high sense (HS)
9 grades of whole blades are withered ... high sense (HS)
As seen from Figure 4, without scab on rear 7d, pikp and pikm two the Monogenic lines blades of inoculation, to the Pyricularia oryzae performance high resistance of carrying virus gene AVR-pik-B; Piks Monogenic lines having diameter be greater than the scab of about 2mm, is middle sense to this strains expressed; And Monogenic lines pik and without the Lijiang xintuanheigu blade of any blast resistant gene all occurring diameter is greater than the fusiformis scab of 2cm, and area of causing harm is comparatively large, shows as sense and high sense respectively to rice blast.This inoculation test result shows that the Pyricularia oryzae of tool nontoxic gene AVR-pik-B can be the rice varieties carrying pikm and pikp disease-resistant gene and identified, the parent of these two disease-resistant genes of tool can be selected when prompting breeding man carries out breed breeding to carry out the resistance improvement of kind, also the rice varieties carrying pikm and pikp disease-resistant gene can be selected during variety popularization to carry out rational deployment, avoid the generation of rice blast.
Claims (1)
1. distinguish avirulence gene of rice blast AVR-pik-B and other allelic method for one kind, with sequence shown in SEQ ID NO:1 and SEQ ID NO:2 for primer, pcr amplification DNA to be detected, obtain the amplified production of 503bp, further with NlaIII enzymic digestion, if PCR primer can not be NlaIII enzymic digestion, then there is nontoxic gene AVR-pik-B in DNA to be detected; If PCR primer enzyme can obtain 367bp and 136bp two DNA fragmentations after cutting, then show allelotrope AVR-pik-A, AVR-pik-C, AVR-pik-D or AVR-pik-E of there is avirulence gene of rice blast AVR-pik-B in DNA to be detected.
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| CN107904324A (en) * | 2017-12-30 | 2018-04-13 | 武汉大学 | The codominant marker system of avirulence gene of rice blast AVR Pik and its primer and application |
| CN117344048A (en) * | 2023-09-28 | 2024-01-05 | 江苏省农业科学院 | RPA-LFD primer probe group for detecting avirulence gene Avr-Pik, kit and application thereof |
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Non-Patent Citations (3)
| Title |
|---|
| Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions;Hiroyuki Kanzaki;《The Plant Journal》;20121231;第72卷;894–907 * |
| Association Genetics Reveals Three Novel Avirulence Genes from the Rice Blast Fungal Pathogen Magnaporthe oryzae;Kentaro Yoshida 等;《The Plant Cell》;20090531;第21卷;1573-1591 * |
| 辽宁省稻瘟病菌无毒基因型鉴定及分析;王世维 等;《中国农业科学》;20141231;第47卷(第3期);462-472 * |
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