CN103184285B - Identification method of rice bacterial leaf blight resistance - Google Patents
Identification method of rice bacterial leaf blight resistance Download PDFInfo
- Publication number
- CN103184285B CN103184285B CN201310091649.4A CN201310091649A CN103184285B CN 103184285 B CN103184285 B CN 103184285B CN 201310091649 A CN201310091649 A CN 201310091649A CN 103184285 B CN103184285 B CN 103184285B
- Authority
- CN
- China
- Prior art keywords
- paddy rice
- mirna1425
- measured
- bacterial leaf
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 89
- 235000009566 rice Nutrition 0.000 title claims abstract description 89
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 35
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 88
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 55
- 108091070501 miRNA Proteins 0.000 claims abstract description 26
- 230000014509 gene expression Effects 0.000 claims abstract description 22
- 239000002679 microRNA Substances 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 239000000523 sample Substances 0.000 claims description 13
- 241000233866 Fungi Species 0.000 claims description 11
- 238000011081 inoculation Methods 0.000 claims description 11
- 201000010099 disease Diseases 0.000 claims description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 8
- 239000007791 liquid phase Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 6
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000010359 gene isolation Methods 0.000 claims description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 238000013518 transcription Methods 0.000 abstract 2
- 230000035897 transcription Effects 0.000 abstract 2
- 230000030279 gene silencing Effects 0.000 abstract 1
- 238000012226 gene silencing method Methods 0.000 abstract 1
- 239000000700 radioactive tracer Substances 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 15
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 239000003550 marker Substances 0.000 description 5
- 229920002401 polyacrylamide Polymers 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241001272684 Xanthomonas campestris pv. oryzae Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000001965 potato dextrose agar Substances 0.000 description 2
- PKZCRWFNSBIBEW-UHFFFAOYSA-N 2-n,2-n,2-trimethylpropane-1,2-diamine Chemical compound CN(C)C(C)(C)CN PKZCRWFNSBIBEW-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000004160 Ammonium persulphate Substances 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 1
- 102100029075 Exonuclease 1 Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 1
- 235000019395 ammonium persulphate Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 244000037666 field crops Species 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-M glutaminate Chemical compound [O-]C(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-M 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
Abstract
The invention discloses an identification method of rice bacterial leaf blight resistance and an application of miRNA1425 genetic locus, and belongs to the field of biotechnology. The identification method comprises the following steps: inoculating paddy rice to be detected and infected paddy rice; separating miRNA genes of the paddy rice to be detected and the infected paddy rice; detecting the expressions of the miRNA genes; and determining whether the paddy rice to be detected is resistant to bacterial leaf blight. The invention further discloses the application of the miRNA1425 genetic locus in identification of rice bacterial leaf bright resistance. The invention uses the transcription of the miRNA1425 gene locus to identify whether paddy rice is resistant to bacterial leaf blight, and achieves success. The transcription of the miRNA1425 gene locus provided by the invention is detection based on expression level, so as to avoid misjudgment caused by bacterial leaf blight resistance gene silencing; and the identification method provided by the invention does not use the conventional DNA linkage tracer method, so as to avoid misjudgment caused by untightness of DNA linkage, improve the detection accuracy and avoid unnecessary production loss.
Description
Technical field
The present invention relates to biological technical field, particularly the authentication method of the bacterial leaf spot resistance of a kind of paddy rice.
Background technology
Paddy rice is the topmost food crop of China, and bacterial leaf-blight is one of paddy rice two large Major Diseases, belongs to worldwide disease, and with Rice In Asian Cultivated Rice, district attaches most importance to.Bacterial leaf-blight sickness rate is high, infection is fast, and morbidity rice field underproduction 20-30%, even can reach 50%.
Cultivating and utilizing disease-resistant variety is to solve that the grain drop in production that bacterial leaf-blight causes is most economical, the most effective means, therefore, need to identify cultivated Bacterial Blight Resistance in Rice.At present, the method of identifying bacterial blight of rice adopts DNA (Deoxyribonucleic acid conventionally, thymus nucleic acid) molecule marker, utilize the principle of DNA linked marker, identify that whether paddy rice is with bacterial leaf spot resistance gene, compare with the conventional genetic marker of tradition application, molecule marker has and is not subject to season, environmental restraint, whether does not have expression and genomic dna variation and the advantage such as abundant thereof.
In realizing process of the present invention, contriver finds that prior art at least exists following problem:
Even if exist resistant gene also not represent that this resistant gene has activity in ill plant, if this resistant gene silence, when producing, still can show as so susceptible, interference experiment personnel's judgement, if directly put in production, can bring loss to grain-production.
Summary of the invention
In order to solve, utilize DNA molecular marker to identify the whether not accurate enough shortcoming of resisting bacterial leaf-blight of paddy rice, the embodiment of the present invention provides the authentication method of the bacterial leaf spot resistance of a kind of paddy rice.Described technical scheme is as follows:
On the one hand, the invention provides the authentication method of the bacterial leaf spot resistance of a kind of paddy rice, comprise the following steps:
Inoculate paddy rice to be measured and susceptible paddy rice;
MiRNA gene in separated described paddy rice to be measured and described susceptible paddy rice;
Detect the expression of the miRNA1425 gene locus in described miRNA gene;
According to the detection of expression result of described miRNA1425 gene locus, judge whether resisting bacterial leaf-blight of described plant to be measured.
Particularly, the sequence of described miRNA1425 gene locus is as shown in SEQ ID NO:1 in sequence table.
Particularly, bacterial leaf spot fungus strain P6 is inoculated on described paddy rice to be measured and described susceptible paddy rice.
Further, the position of described inoculation is the blade of described paddy rice to be measured and described susceptible paddy rice.
Particularly, 5-10 minute after inoculation, separated described miRNA gene.
Further, adopt the separated described miRNA gene of miRNA gene isolation test kit.
Particularly, adopt liquid phase Northern hybrid method to detect the expression of described miRNA1425 gene locus.
Further, the sequence of the probe that described liquid phase Northern hybrid method is used is as shown in SEQ IDNO:2 in sequence table, and 5 ' end of described probe is marked with fluorescein FITC.
Particularly, judge described plant to be measured whether resisting bacterial leaf-blight method as: if the expression of the expression of the miRNA1425 gene locus of described susceptible paddy rice and the miRNA1425 gene locus of described paddy rice to be measured equate, described paddy rice to be measured is susceptible; If the expression of the miRNA1425 gene locus of described susceptible paddy rice is obviously greater than the expression of the miRNA1425 gene locus of described paddy rice to be measured, described paddy disease-resistant to be measured.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: miRNA gene and common rna gene are different, its sequence is shorter, be not easy degraded, therefore, at human diseases, obtained application in as cancer detection, but utilize miRNA gene test and diagnosis of plant disease example seldom, mainly because the research of Mirnas of plant gene lags behind the research of mankind miRNA gene relatively, by the paddy rice miRNA gene of great amount of samples and the research of bacterial leaf spot resistance relation, contriver find the expression of miRNA1425 gene and bacterial leaf spot resistance closely related, thus, propose first miRNA1425 gene and there is the ability that detects paddy rice bacterial leaf spot resistance, the present invention detects the expression of miRNA1425 gene locus in paddy rice to be measured and susceptible paddy rice, by the comparison of result, identify whether resisting bacterial leaf-blight of paddy rice to be measured, the present invention utilizes miRNA1425 gene locus to identify whether paddy rice has bacterial leaf spot resistance, and obtained success, transcribing of miRNA1425 gene locus provided by the invention is the detection being based upon on expression level, avoided the erroneous judgement being caused by the resistant gene silence of bacterial leaf-blight, the present invention does not utilize conventional DNA linked marker method, avoided the erroneous judgement closely not causing because DNA is chain, improved the accuracy detecting, avoided unnecessary production loss.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the electrophoretogram that the embodiment of the present invention provides.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.Reagent used in the present invention is commercial reagent.
Embodiment
The present invention passes through the probe bulk crossing of the miRNA gene of paddy rice and the good miRNA1425 gene of mark, and by non-sex change polyacrylamide detected through gel electrophoresis result, and the result of paddy rice to be measured and susceptible paddy rice is compared, and then judging whether resisting bacterial leaf-blight of paddy rice to be measured, its specific experiment process is as follows.
Susceptible paddy rice: choosing growth normal and surface does not have obvious disease and pest, the rice paddy seed of the not resisting bacterial leaf-blight that label is 9311.
Paddy rice to be measured: the rice paddy seed of choosing label and be 9311 resisting bacterial leaf-blight.
Wherein, in the rice paddy seed of resisting bacterial leaf-blight, there is resistant gene Xa23.
The pre-treatment of seed: be 70% by concentration alcohol-pickled 2 minutes, with deionized water, wash 2 times again, soaked overnight in the water of 30 ℃, at the temperature of 30 ℃, seed is carried out to vernalization, after bud is longer, be seeded in rice seedling bed, young plants management is identical with common cultivation custom, when rice shoot grows to 4-5 blade, to transplant to large Tanaka, field management is identical with common cultivation custom.
Bacterial leaf spot fungus strain is cultivated: select paddy rice is had to strong pathogenic bacterial leaf spot fungus strain P6(Xanthomonasoryzae pv.oryzae Philippine race6), this bacterial leaf spot fungus strain P6 derives from Inst. of Genetics and Development Biology, CAS, by bacterial leaf spot fungus strain P6, in culture temperature, be under the condition of 30 ℃, in potato dextrose agar (Potato-Dextrose-Agar, PSA) on substratum, cultivate, the composition of substratum is as follows: the sucrose that mass volume ratio is 1%, mass volume ratio is 1% peptone, mass volume ratio is the agar that 0.1% glutaminate and mass volume ratio are 1.5%.Incubation time is 2-5 days, by bacterial leaf spot fungus strain P6 be cultured to bacterium that naked eyes can observe dense till.
Bacterial leaf spot bacteria concentration is measured: before the blade of Inoculated Rice, bacterial leaf spot fungus strain P6 is dissolved in the water, (U.S. Quawell company produces to utilize spectrophotometer, model is Q5000) measure the extinction OD600 value at 600nm place, and bacterium liquid is diluted to OD600 value is 0.5, now the concentration of bacterium liquid is approximately 5 * 10
8cfu/ml left and right, for the blade of Inoculated Rice.
Bacterial leaf spot fungus strain inoculation: before paddy rice to be measured and susceptible Rice Heading, inoculate respectively bacterial leaf spot fungus strain P6, inoculation method is leaf-cutting inoculation, particularly, scissors is immersed to 1-2 second in the bacterium liquid that OD600 value is 0.5 bacterial leaf spot fungus strain P6, use again the scissors that speckles with bacterium liquid from cutting off blade apart from vane tip 1-2cm, now, count inoculation 0 hour, at inoculation 5-10 minute, get respectively each 0.5 gram of left and right of blade of paddy rice to be measured and susceptible paddy rice inoculation position, be respectively charged in the centrifuge tube that volume is 1.5ml, and drop in liquid nitrogen and preserve immediately, and paddy rice to be measured and susceptible paddy rice are got at least respectively 3 parts of sample materials and repeat experiment.
The extraction of miRNA gene: (miRNA gene isolation test kit is commercially available to utilize miRNA gene isolation test kit, article No.: R6727, production company: Omega, this test kit specifically comprises: RNA centrifugal column, genomic dna is removed centrifugal column, collection tube, MCL lysis buffer, XD binding buffer liquid, RNA elutriant II, DEPC water) the miRNA gene in separated paddy rice to be measured and susceptible rice leaf, concrete operation method is as follows: the blade that takes out respectively paddy rice to be measured and susceptible paddy rice from liquefied ammonia, and two groups of blades are placed in respectively to two mortars, in mortar, pour liquid nitrogen into immediately, and fully grind, respectively get the centrifuge tube that the ground powder of about 100mg is placed in 1.5ml, the lysate that adds 700 μ L, vortex 30 seconds is to mix sample, 55 ℃ are incubated 30 minutes, under the room temperature condition that is 12000 * g at centrifugal force centrifugal 5 minutes, supernatant liquor is transferred in centrifugal column centrifugal, for removing the DNA of genome, centrifugal 2 minutes of 12000 * g room temperature, and the liquid rotating of outflow is moved in the centrifuge tube of a new 1.5mL, in liquid, add the dehydrated alcohol of 1.1 times of this liquid volumes vortex to mix for 20 seconds, liquid rotating is moved in RNA centrifugal column to 12000 * g room temperature centrifugal 1 minute, abandon centrifugate, add ethanol that 500 μ L concentration are 96-100% in RNA centrifugal column, 12000 * g room temperature is centrifugal 1 minute again, abandon centrifugate, add 500 μ L XD binding buffer liquid in RNA centrifugal column, centrifugal 1 minute of 12000 * g room temperature, abandon centrifugate, add 750 μ L RNA elutriant II in RNA centrifugal column, centrifugal 1 minute of 12000 * g room temperature, abandon centrifugate, add again 750 μ L RNA elutriant II in RNA centrifugal column, centrifugal 1 minute of 12000 * g room temperature, abandon centrifugate.RNA centrifugal column is being greater than under the top speed of 12000 * g, centrifugal 2 minutes of room temperature, in RNA centrifugal column, add 30-50 μ L DEPC water, under room temperature, place 5 minutes, be greater than under the top speed of 12000 * g room temperature, centrifugal 1 minute, the liquid of centrifugal acquisition is miRNA cdna solution, and this solution is stored in to-70 ℃.
MiRNA gene quantification: utilize spectrophotometer (U.S. Quawell company produces, and model is Q5000) to measure the concentration of the miRNA gene obtaining.
The sequence of miRNA1425 gene locus: 5 '-uaggauucaauccuugcugcu-3 '.
The hybridization probe of miRNA1425 gene locus: the DNA probe of design miRNA1425 gene locus is 5 '-agcagcaaggattgaatccta-3 ', wherein, 5 ' end of this probe is marked with fluorescein FITC.This probe mark is completed by Shanghai Invitrigen company with synthetic.
Preparing hybrid damping fluid: its composition is as follows: the sodium phosphate buffer of 30mM pH8.0,0.3M NaCl solution and 10mM EDTA, after preparing, the DEPC(diethyl pyrocarbonate that is 0.1% by concentration; Diethylpyrocarbonate) process after 12 hours for 37 ℃, 120 ℃ of sterilizings 20 minutes are stand-by.
Liquid phase Northern hybridization: with two PCR pipes, get respectively the paddy rice to be measured of 5 μ g and the miRNA solution of the blade of susceptible paddy rice, the hybridization probe of the miRNA1425 gene locus of the hybridization buffer of 5 μ l and 1ul10pM/ul, mix, make liquid phase Northern hybridization solution, 42 ℃ of insulations, add the exonuclease I(You NEB company of 5ul2000U/ml to produce after 1 hour, commodity article No. is M0303) digest 2 hours.Wherein, adopt liquid phase Northern hybrid method, 5 ' the end at probe has a FITC mark, this is marked at can be luminous under uv irradiating, so, after electrophoresis completes, be placed directly in the lower irradiation of ultraviolet ray and can see band, the step that does not need these troubles of radioautograph, does not need 3 ' end to carry out mark yet, and its method is simple.In addition, those have not been fallen by dnase digestion by the probe in hybridization in liquid, do not affect last result.
Concentration is the preparation of 6% non-sex change polyacrylamide gel: the non-sex change polyacrylamide gel mother liquor that the concentration of getting 20mL ACT:Bis=29:1 is 30% (production of Google bio tech ltd, Wuhan), (Shanghai Di Shen Bioisystech Co., Ltd produces to add concentration and be the tbe buffer liquid of 1 times, article No. is B1610733) 80mL, be mixed with concentration and be 6% non-sex change polyacrylamide gelating soln, the non-sex change polyacrylamide gelating soln 16mL that to get the concentration for preparing be 6%, add ammonium persulphate that 16 μ L concentration are 10% and the N of 160 μ L, N, N', N'-Tetramethyl Ethylene Diamine (TEMED), the concentration >99.0% of TEMED wherein, after above-mentioned solution is mixed, encapsulating among offset plate, (produced by Beijing Liu Yi plant and instrument factory by glue-pouring device rapidly, model is DYCZ-24DN), and room temperature can be used after placing 2 hours.
Electrophoresis detection: by above-mentioned liquid phase Northern hybridization solution add 2ul10 * electrophoretic buffer (U.S. Lifetechnology company produces, commodity article No. is AM8556), utilizing concentration is 6% non-sex change polyacrylamide gel electrophoresis 30 minutes under the voltage of 100V, under ultraviolet ray, observe and take a picture, the results are shown in accompanying drawing 1.
As shown in Figure 1, in figure, the band in left side is susceptible paddy rice, the band on right side is paddy rice to be measured, wherein, the strip width in left side is obviously greater than the width of the band on right side, so the expression of the miRNA1425 gene locus of susceptible paddy rice is greater than the expression of the miRNA1425 gene locus of paddy rice to be measured, paddy disease-resistant to be measured.
Press the susceptible paddy rice of aforesaid method inoculation P6 bacterial leaf spot bacterium and the continued growth in land for growing field crops of paddy rice to be measured after 15 days, measure withered blade length, result shows that the withered blade length of susceptible paddy rice is greater than respectively 10cm, and the withered blade length of paddy rice to be measured is less than respectively 1cm, the susceptible paddy rice that shows plantation shows as susceptible, the paddy rice to be measured of plantation detects as disease-resistant, consistent with experimental result.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. an authentication method for the bacterial leaf spot resistance of paddy rice, is characterized in that, said method comprising the steps of:
Inoculate paddy rice to be measured and susceptible paddy rice;
MiRNA gene in separated described paddy rice to be measured and described susceptible paddy rice;
Detect the expression of the miRNA1425 gene locus in described miRNA gene;
According to the detection of expression result of described miRNA1425 gene locus, judge whether resisting bacterial leaf-blight of described plant to be measured.
2. method according to claim 1, is characterized in that, the sequence of described miRNA1425 gene locus is as shown in SEQ ID NO:1 in sequence table.
3. method according to claim 2, is characterized in that, bacterial leaf spot fungus strain P6 is inoculated on described paddy rice to be measured and described susceptible paddy rice.
4. method according to claim 3, is characterized in that, the position of described inoculation is the blade of described paddy rice to be measured and described susceptible paddy rice.
5. method according to claim 2, is characterized in that, 5-10 minute after inoculation, separated described miRNA gene.
6. method according to claim 5, is characterized in that, adopts the separated described miRNA gene of miRNA gene isolation test kit.
7. method according to claim 2, is characterized in that, adopts liquid phase Northern hybrid method to detect the expression of described miRNA1425 gene locus.
8. method according to claim 7, is characterized in that, the sequence of the probe that described liquid phase Northern hybrid method is used is as shown in SEQ ID NO:2 in sequence table, and 5 ' end of described probe is marked with fluorescein FITC.
9. method according to claim 2, it is characterized in that, judge described plant to be measured whether resisting bacterial leaf-blight method as: if the expression of the expression of the miRNA1425 gene locus of described susceptible paddy rice and the miRNA1425 gene locus of described paddy rice to be measured equate, described paddy rice to be measured is susceptible; If the expression of the miRNA1425 gene locus of described susceptible paddy rice is greater than the expression of the miRNA1425 gene locus of described paddy rice to be measured, described paddy disease-resistant to be measured.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310091649.4A CN103184285B (en) | 2013-03-21 | 2013-03-21 | Identification method of rice bacterial leaf blight resistance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310091649.4A CN103184285B (en) | 2013-03-21 | 2013-03-21 | Identification method of rice bacterial leaf blight resistance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103184285A CN103184285A (en) | 2013-07-03 |
| CN103184285B true CN103184285B (en) | 2014-08-06 |
Family
ID=48675731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310091649.4A Expired - Fee Related CN103184285B (en) | 2013-03-21 | 2013-03-21 | Identification method of rice bacterial leaf blight resistance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103184285B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104141007B (en) * | 2014-06-30 | 2016-09-28 | 江汉大学 | The method of miRNA393a gene forecast bacterial blight of rice |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1155308C (en) * | 2000-06-05 | 2004-06-30 | 华中农业大学 | Breading method for anti-rice bacterial leaf-blight breed |
| CN100381465C (en) * | 2005-01-19 | 2008-04-16 | 中国科学院遗传与发育生物学研究所 | Bacterial leaf spot resistance related protein and its coding gene and uses |
-
2013
- 2013-03-21 CN CN201310091649.4A patent/CN103184285B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103184285A (en) | 2013-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103184286B (en) | Identification method of rice bacterial leaf blight resistance | |
| CN104531750A (en) | Method for labelling ascochyta citrullina by adopting green fluorescent protein (GFP) | |
| CN101974651B (en) | Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian | |
| CN103060322B (en) | Fusarium oxysporum bitter gourd specialized molecular marker and application thereof | |
| CN103255222B (en) | Primers and method for identifying greenhouse trialeurodes vaporariorum and bemisia tabaci by utilizing mitochondria SCAR (sequence characterized amplified regions) marker | |
| CN103866038B (en) | For detecting tobacco to the N gene-specific primer of TMV resistance to, detection method and test kit | |
| CN102399888A (en) | Molecular identification method for cochliobolus miyabeanus pathogens | |
| CN101200769B (en) | Detection method for discriminating medicinal lucid ganoderma by using special gene sequence | |
| CN101086015B (en) | Mushroom 45 bacteria molecular specific mark and its obtaining method and uses | |
| CN103184285B (en) | Identification method of rice bacterial leaf blight resistance | |
| CN111321242A (en) | Rapid molecular detection method and application of rubber tree anthracnose pathogen Siamese anthrax | |
| CN103184288B (en) | Identification method of rice bacterial leaf blight resistance and application of miRNA1318 genetic locus | |
| CN110606877A (en) | Transcription factor for improving wheat rust-resistant varieties and screening method thereof | |
| CN102154123A (en) | Dioscorea nipponica Makino Fusarium sp. and application thereof | |
| CN104212896B (en) | The Molecular Identification primer of a kind of Tobacco Angular Leaf Spot Disease bacterium and authentication method | |
| CN109504686A (en) | Tomato SlCaM6 gene is improving the application in low-temperature resistance | |
| CN105018612A (en) | Quantitative detection method of garlic virus | |
| CN103184287B (en) | The authentication method of the bacterial leaf spot resistance of paddy rice and the application of miRNA172a | |
| CN104673787A (en) | Molecular marker, detection method and application of hypsizigus marmoreus strain SIEF2632 | |
| CN101812445B (en) | Kit for quickly extracting paddy DNA | |
| CN101397587A (en) | Method for preparing live bacteria internal standard based on gene substitution technique | |
| CN101086017B (en) | Mushroom 507 bacteria molecular specific mark and its obtaining method and uses | |
| CN101988126B (en) | Nucleic acid composition for detecting Datura ferox and application thereof | |
| CN100389209C (en) | Primer for detecting seed purity and its method | |
| CN110055267A (en) | A kind of rice cytosolic kinase-encoding gene OsRLCK5 and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140806 |