CN102660777A - Method for constructing suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs - Google Patents
Method for constructing suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs Download PDFInfo
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Abstract
The invention discloses a method for constructing a suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs. The method includes steps of: sample processing, total ribonucleic acid (RNA) extraction, mRNA purification, suppression subtractive hybridization and polymerase chain reaction (PCR) amplification of testing parts and driven elements, PCR products and polarization mode dispersion 18-T (pMD18-T) carrier connection restraining, connection product conversion, monoclonal storing and the like. The SSH library of oryza rufipogon threatened by bacterial blight germs constructed by the method is high in sensitivity, even length of insertion elements of the library is 450bp, a large number of difference expressed sequence tags (ESTs) of the oryza rufipogon threatened by the bacterial blight germs can be obtained fast in one step, partial sequence information is provided for separating overall-length cDNA sequence of clone bacterial blight resisting related genes, simultaneously important theoretical basis for improvement of biology is provided and new bacterial blight resisting related genes can be obtained.
Description
Technical field
The present invention relates to the plant gene engineering technology field, be specifically related on the common wild-rice blade, inoculate artificially bacterial leaf spot pathogenic bacteria and cause the stress-inducing condition, make up the common wild-rice SSH library that bacterial leaf-blight is coerced.
Background technology
Previously (the inhibition difference subtracts hybridization supression subtravtive hybridization to SSH, and SSH) application of library on plant is a lot.Like the difference expression gene of separating plant different developmental phases, or the genes involved of separating plant flower development, leaf aging and root development (Kim JY et al, Biochimicaet Biophysica Acta, 1489:389-392,1999; Kim JY et al, Molecular Cell, 9:392-397,1999; Hinderhofer K and Zentgraf U, Planta, 213:469-473,2001).In recent years, the research of the gene differential expression of SSH technology under various stress conditions is also more and more, as sick worm coerce, (Luo Meng, Acta Genetica Sinica, 29:535-530,2002 such as subzero treatment, water stress, radiation are coerced; Bahn SC et al, Biochimicaet Biophysica Acta, 1522:134-137,2001, Happe T and Kaminski A, Europen Journal of Biochemistry, 269:1022-1032,2002; Savenstrand H et al, Plant and Cell Physiology, 43:402-410.).More than differential gene expression is many under SSH technical study environment stress or the inductive condition to use in research, has found that also a lot of participation specified conditions induce down the gene of specifically expressing, still utilize so far SSH technical study common wild-rice (
Oryza rufipogonGriff.) gene differential expression does not also appear in the newspapers under bacterial leaf spot pathogenic bacteria is coerced, and does not have relevant patent yet.The present invention sets up the construction process in the common wild-rice SSH library that a kind of bacterial leaf spot pathogenic bacteria coerces and the verification method of difference expression gene, to reach the new gene of efficient excavation high resistance to hoja blanca.
Summary of the invention
The technical problem that the present invention will solve is to overcome prior art to lack and to utilize SSH technical study common wild-rice to coerce the defective and the deficiency of gene differential expression down at bacterial leaf spot pathogenic bacteria, its objective is the construction process that the common wild-rice SSH library that a kind of bacterial leaf spot pathogenic bacteria that can induce resisting bacterial leaf-blight genes involved differential expression in the common wild-rice blade effectively coerces is provided.
The common wild-rice SSH library constructing method that bacterial leaf spot pathogenic bacteria provided by the present invention is coerced may further comprise the steps:
(1) bacterium that connects of common wild-rice blade material is handled
1. with the bacterial leaf-blight bacteria strain, line on the NA culture medium flat plate, cultivate 2-3 d for 28 ℃ and carry out activation, wash lawn with sterile distilled water after the activation, wheat flange protractor turbidimetry is mixed with 6 * 10
8Ml
-1Bacterium liquid;
2. inoculate the common wild-rice blade of bacterial leaf spot pathogenic bacteria in 26-28 ℃ of greenhouse with the scissors that has picked bacterium liquid from the leaf-cutting inoculation method that blade tip deducts 2-3 cm; Each bacterial strain connects 3 piers; 30 leaves, contrast replaces bacterium liquid inoculation common wild-rice blade as contrast with sterilized water;
3. connect behind the bacterium every at a distance from 24 h, respectively at 24 h, 48 h, 72 h, 96 h, 120 h, the sampling of 144 h equivalent, the control group of butt joint sterilized water also simultaneously respectively equivalent take a sample;
The common wild-rice blade that 4. will connect 6 stages that 24 h behind the bacterium, 48 h, 72 h, 96 h, 120 h, 144 h collect mixes; As test side (Tester); The blade in 6 stages of contrast sterilized water inoculation mixes too, as driven element (Driver);
(2) extraction of total RNA
1. get test side (Tester) common wild-rice blade and driven element (Driver) common wild-rice blade respectively, place mortar to add liquid nitrogen respectively and grind fast, per 100 mg samples add 1ml TRIzol extracting solution, the vortex 15 s mixings that vibrate;
2. above-mentioned sample is placed 5 min at 15-30 ℃, add 200 μ l chloroforms, every use 1 ml TRIzol adds 200 μ l chloroforms, builds pipe racks, and vortex 15 s that vibrate place 3 min for 15-30 ℃;
3. 4 ℃, centrifugal 15 min of 12000 rpm, this moment, solution was divided into three layers: xanchromatic is an organic phase, and what middle layer and upper strata were colourless is water, and mainly there is aqueous phase in RNA, is transferred to water in another new no RNase centrifuge tube;
4. add isopyknic Virahol to the aqueous phase that obtains, mixing is placed 20-30 min for 15-30 ℃.4 ℃, centrifugal 10 min of 12000 rpm abandon supernatant;
5. the volume(tric)fraction that adds the preparation of DEPC water is 75% ethanolic soln washing precipitation, and the volume(tric)fraction that every use 1 ml TRIzol uses 1ml DEPC water to prepare is 75% ethanolic soln;
6. 4 ℃, centrifugal 3 min of 5000 rpm.With the careful sucking-off supernatant liquid of rifle head, keep deposition.15-30 ℃ is dried 2-3 min, and adding 30-100 μ l does not have the RNase distilled water, fully dissolve RNA after, preserve or get into next step test for-70 ℃;
(3) purifying of mRNA
1. 5 mg Oligo (dT) Cellulose add 0.5 ml1 * Binding Buffer, mixing, and 15-30 ℃, centrifugal 2 min of 2000 g remove supernatant;
2. total RNA0.3mg, less than 500 μ l volumes use DEPC-ddH
2O supplies, and adds isopyknic 2 * Binding Buffer, mixing;
3. the mixed solution of step (3) in 2. joined 1. abundant mixing among Oligo (dT) Cellulose of step (3), 65 ℃ of water-bath 5 min, or put upside down mixing; Place 30 min for 15-30 ℃, centrifugal 3 min of 2000 g abandon supernatant;
4. in mRNA-Oligo (dT) Cellulose mix, add 0.5 ml, 1 * Binding Buffer, mixing; 15-30 ℃, centrifugal 3 min of 2000 g abandon supernatant, repeat this step once;
5. add 0.5 ml Wash Buffer, mixing; 15-30 ℃, centrifugal 3 min of 2000 g abandon supernatant, repeat this step once;
6. add 65 ℃ of RNA Elution of 200 μ l, 15-30 ℃, centrifugal 3 min of 4000 g collect supernatant, repeat this step 1-2 time;
7. the LAP that adds isopyknic Virahol and 1.6%, abundant mixing, 4 ℃, centrifugal 20 min of 12000 g abandon supernatant.The washing with alcohol that adds 700 μ l 70%, 4 ℃, centrifugal 5 min of 12000 g;
8. dry deposition, 30-50 μ l DEPC-ddH
2The O dissolving, 1% agarose gel electrophoresis detects;
(4) the inhibition difference of test side (Tester) cDNA and driven element (Driver) cDNA subtracts hybridization
1. utilize ThermoScript II respectively will the side of test (Tester) mRNA reverse transcription becomes test side (Tester) and driven element (Driver) the second chain cDNA with driven element (Driver) mRNA;
2. in test side (Tester) and driven element (Driver) the second chain cDNA reaction tubes, add respectively
RsaThe I enzyme, 37 ℃ of enzymes are cut 1.5 h;
3.
RsaTest side after the I enzyme is cut (Tester) cDNA is connected for 16 ℃ with joint and spends the night;
4. test side (Tester) cDNA that has connected joint with
RsaThe driven element that the I enzyme is cut (Driver) cDNA differs from and subtracts hybridization, adds 200 μ l dilution buffer dilution hybridization solution after hybridization is accomplished, and places-20 ℃ to preserve or carry out the next round test.
(5) formation in the bacterial leaf spot pathogenic bacteria common wild-rice SSH library of coercing
1. with the hybridization solution of last round of dilution as template, carry out the pcr amplification first time, and then with the second time PCR reaction solution carry out second pcr amplification of taking turns as template, concrete reaction system and response procedures are following:
PCR for the first time: reaction system: 10 * PCR reaction buffer, 2.5 μ l; DNTP Mix (10 μ M) 0.5 μ l; PCR Primer (10 μ M) 1 μ l; 50 * Advantage cDNA Polymerase Mix, 0.5 μ l, the hybridization solution template of 1 μ l dilution, distilled water less than 25 μ l; PCR response procedures: 75 ℃ of 5 min; 94 ℃ of 30 sec, 66 ℃ of 30 sec, 72 ℃ of 90 sec, 27 circulations;
PCR for the second time: reaction system: get 1 μ l for the first time the PCR reaction solution as template; 10 * PCR reaction buffer, 2.5 μ l; DNTP Mix (10 mM) 0.5 μ l, Neasted Primer 1 (10 μ M) 1 μ l, Neasted Primer 2 (10 μ M) 1 μ l; 50 * Advantage cDNA Polymerase Mix, 0.5 μ l, distilled water supplies 25; Response procedures: 94 ℃ of 30 sec, 68 ℃ of 30 sec, 72 ℃ of 90 sec, 27 circulations;
2. the PCR product that the second time, pcr amplification obtained is connected with the pMD18-T carrier, 16 ℃ of connections are spent the night;
3. will connect product through transformed into escherichia coli DH5 α competent cell, and coat on the LB flat board that has added AMP, IPTG, X-gal, 37 ℃ of incubated overnight carry out indigo plant and screen day shift;
4. picking white mono-clonal is preserved from the above-mentioned flat board, promptly is built into the common wild-rice SSH library that bacterial leaf spot pathogenic bacteria of the present invention is coerced, totally 1295 clones, 1269 hickies wherein, 26 locus coeruleus.
The used template of the present invention is two; One is to connect bacterial leaf spot pathogenic bacteria to handle the every common wild-rice blade at a distance from 6 stages that 24 h, 48 h, 72 h, 96 h, 120 h, 144 h collect in back; After mixing, extract total RNA, the cDNA that reverse transcription becomes is as test side (Tester); Another is to replace the inoculation of bacterium liquid with water, and without connecing the blade that bacterial leaf spot pathogenic bacteria is handled, the cDNA that total RNA reverse transcription of extraction becomes is as driven element (Driver).The library that the present invention makes up differs from as driven element (Driver) as test side (Tester) and the common wild-rice blade that do not connect the bacterial leaf spot pathogenic bacteria processing with the common wild-rice blade material that connects bacterial leaf spot pathogenic bacteria and handle and subtracts hybridization; Basically eliminate the non-specific expressing gene after coerced by bacterial leaf spot pathogenic bacteria, and enrichment receive the difference expression gene of bacterial leaf spot pathogenic bacteria stress-inducing.
The invention has the beneficial effects as follows:
A. can obtain the differential expression ESTs that a large amount of common wild-rices receive the bacterial leaf spot pathogenic bacteria stress-inducing once, fast; For the separating clone disease-resistant related gene provides ESTs sequence information foundation; The differential expression ESTs sequence that is obtained provides partial sequence information for the separating clone full length cDNA sequence, for biological improvement etc. the important theory foundation is provided simultaneously; Can obtain new resisting bacterial leaf-blight genes involved, can cultivate disease-resistant varieties through the transgenic method rice transformation behind the separating clone.
B. this library constructing method is highly sensitive, has passed through the inrichment of equalization and target fragment, can guarantee that the cDNA that receives bacterial leaf spot pathogenic bacteria to coerce the low abundance expression in back can be detected; This library has 1295 clones; 34 positive colonies of random choose all have fragment to insert; Insert clip size between 200-900bp, mean length is at 450bp, from library clone number and the insertion fragment distribution range that obtains at present; The constructed SSH library of the present invention is a better inhibited subtracted library, lays a good foundation for from common wild-rice, excavating and clone the resisting bacterial leaf-blight genes involved.
C. the constructed SSH library of the present invention can be used for studying common wild-rice resisting bacterial leaf-blight mechanism principle etc.Also can aspect molecular biology research, be used for gene functional research such as protein modified state, the Western marking, immunosorption, immunoprecipitation etc.Aspect RESEARCH ON CELL-BIOLOGY, be used for the resisting bacterial leaf-blight Study on Mechanism, research cell proliferation, differentiation, apoptosis, cell signaling process etc.
Description of drawings
Fig. 1 is the insertion fragment detected result in the constructed bacterial leaf spot pathogenic bacteria of the present invention common wild-rice SSH library of coercing, M:DNA Marker; 1-34: the part positive colony of picking at random.
Fig. 2 is the RT-PCR proof diagram from the differential expression ESTs that is numbered OR68 and two ESTs sequences of OR5 of the common wild-rice SSH library random choose that the constructed bacterial leaf spot pathogenic bacteria of the present invention is coerced, M:2000 DNAMarker; 1,3 does not connect bacterium handles blade; 2,4 connect bacterium handles blade, 1-2:OR68; 3-4:OR5.
Fig. 3 is the RT-PCR proof diagram from the differential expression ESTs that is numbered OR7, OR70, three ESTs sequences of OR143 of the common wild-rice SSH library random choose that the constructed bacterial leaf spot pathogenic bacteria of the present invention is coerced, M:2000 DNAMarker; 1,3,5 do not connect bacterium handles blade; 2,4,6 connect bacterium handles blade, 1-2:OR7; 3-4:OR70; 5-6:OR143.
Embodiment
Below in conjunction with embodiment the present invention is done further description, but be not construed as limiting the invention.Bacterial leaf spot pathogenic bacteria C1 bacterial strain that embodiment is used and bacterial leaf spot pathogenic bacteria Y8 bacterial strain are to utilize the pathogenic general character that causes bacterial leaf-blight of bacterial leaf spot pathogenic bacteria to come infected plant, and the various bacterial leaf spot pathogenic bacterias that therefore have virulence can both reach effect of the present invention.Bacterial leaf spot pathogenic bacteria C1 bacterial strain is the typical pathogenic strains of China, and bacterial leaf spot pathogenic bacteria Y8 is that Chinese yunnan is economized typical pathogenic strains, and the purpose that adopts two kinds of inoculation is to guarantee to connect the bacterium success, also can adopt a kind of inoculation.Reagent used among following each embodiment all can be bought from the biochemical reagents shop.
The common wild-rice SSH library constructing method that embodiment 1 bacterial leaf spot pathogenic bacteria is coerced may further comprise the steps:
(1) bacterium that connects of common wild-rice blade material is handled
1. with bacterial leaf spot pathogenic bacteria C1 bacterial strain and bacterial leaf spot pathogenic bacteria Y8 bacterial strain, line on the NA culture medium flat plate, cultivate 2-3 d for 28 ℃ and carry out activation, wash lawn with sterile distilled water after the activation, wheat flange protractor turbidimetry is mixed with 6 * 10
8Ml
-1Bacterium liquid;
2. after afternoon 15:00; Inoculate the common wild-rice blade of bacterial leaf spot pathogenic bacteria in the greenhouse with the scissors that has picked bacterium liquid from the leaf-cutting inoculation method that blade tip deducts 2-3 cm; Each bacterial strain connects 3 piers; 30 leaves, contrast promptly replace bacterium liquid inoculation common wild-rice blade with sterilized water, inoculate as contrast with sterilized water;
3. connect behind the bacterium every at a distance from 24 h, i.e. the 24th h, 48 h, 72 h, 96 h, 120 h, the sampling of 144 h equivalent, the control group of butt joint sterilized water also simultaneously equivalent take a sample, put into the liquid nitrogen quick-frozen to sample immediately after the sampling, be stored in-70 ℃ of refrigerators subsequent use;
The common wild-rice blade that 4. will connect 6 stages that 24 h behind the bacterium, 48 h, 72 h, 96 h, 120 h, 144 h collect mixes; As test side (Tester); Contrast is that the blade in 6 stages of sterilized water inoculation mixes too, as driven element (Driver)
(2) extraction of total RNA
1. get Tester common wild-rice blade and Driver common wild-rice blade respectively, place mortar to add liquid nitrogen respectively and grind fast, per 100 mg samples add 1ml TRIzol extracting solution, the vibration mixing;
2. above-mentioned sample is placed 5 min at 18～22 ℃, the nucleic acid-protein mixture is separated fully.Add 200 μ l chloroforms, every use 1 ml TRIzol adds 200 μ l chloroforms, builds pipe racks, and thermal agitation 15 s place 3 min for 22 ℃;
3. 4 ℃, centrifugal 15 min of 12000 rpm, this moment, solution was divided into three layers: the xanchromatic organic phase, mainly there are aqueous phase in the water that middle layer and upper strata are colourless, RNA, are transferred to water (about 500 μ l) in another new no RNase centrifuge tube;
4. add isopyknic Virahol to the aqueous phase that obtains, mixing is placed 20-30 min for 18～22 ℃.4 ℃, centrifugal 10 min of 12000 rpm abandon supernatant;
5. add 75% ethanol (preparation of DEPC water) washing precipitation, every use 1 ml TRIzol precipitates with the 1ml75% washing with alcohol;
6. 4 ℃, centrifugal 3 min of 5000 rpm.With the careful sucking-off supernatant liquid of rifle head, keep deposition.18～22 ℃ are dried 2-3 min, and adding 30-100 μ l does not have RNase, fully dissolve RNA after, preserve or get into next step test for-70 ℃.
(3) purifying of mRNA
1. 5 mg Oligo (dT) Cellulose add 0.5 ml1 * Binding Buffer, mixing, and 18～22 ℃, centrifugal 2 min of 2000 g remove supernatant;
2. total RNA0.3mg, less than 500 μ l volumes use DEPC-ddH
2O supplies, and adds isopyknic 2 * Binding Buffer, gently mixing;
3. the mixed solution of step (3) in 2. joined 1. abundant mixing among Oligo (dT) Cellulose of step (3), 65 ℃ of water-bath 5 min, or put upside down abundant mixing gently; Place 30 min for 18～22 ℃, centrifugal 3 min of 2000 g abandon supernatant;
4. in mRNA-Oligo (dT) Cellulose mix, add 0.5 ml, 1 * Binding Buffer, mixing.Room temperature, centrifugal 3 min of 2000 g abandon supernatant, repeat this step once;
5. add 0.5 ml Wash Buffer, mixing.18～22 ℃, centrifugal 3 min of 2000 g abandon supernatant, repeat this step once;
6. add 65 ℃ of RNA Elution of 200 μ l, 18～22 ℃, centrifugal 3 min of 4000 g collect supernatant, repeat this step 1-2 time;
7. the LAP that adds isopyknic Virahol and 1.6%, abundant mixing, 4 ℃, centrifugal 20 min of 12000 g abandon supernatant; The washing with alcohol that adds 700 μ l 70%, 4 ℃, centrifugal 5 min of 12000 g;
8. dry deposition, 30-50 μ l DEPC-ddH
2The O dissolving, 1% agarose gel electrophoresis detects
(4) the inhibition difference of Tester cDNA and Driver cDNA subtracts hybridization
1. reverse transcription becomes Tester and the Driver second chain cDNA with Driver mRNA with Tester mRNA respectively to utilize ThermoScript II;
2. in Tester and the Driver second chain cDNA reaction tubes, add respectively
RsaThe I enzyme, 37 ℃ of enzymes are cut 1.5 h;
3.
RsaTester cDNA after the I enzyme is cut is connected for 16 ℃ with joint and spends the night;
4. the Tester cDNA that has connected joint with
RsaThe Driver cDNA that the I enzyme is cut differs from and subtracts hybridization, adds 200 μ l dilution buffer dilution hybridization solution after hybridization is accomplished, and places-20 ℃ to preserve or carry out the next round test.
(5) formation in the bacterial leaf spot pathogenic bacteria common wild-rice SSH library of coercing
1. with the hybridization solution of last round of dilution as template, carry out the pcr amplification first time, and then with the second time PCR reaction solution carry out second pcr amplification of taking turns as template, concrete reaction system and response procedures are following:
PCR for the first time: reaction system: 10 * PCR reaction buffer, 2.5 μ l; DNTP Mix (10 μ M) 0.5 μ l; PCR Primer (10 μ M) 1 μ l; 50 * Advantage cDNA Polymerase Mix, 0.5 μ l, the hybridization solution template of 1 μ l dilution, distilled water less than 25 μ l; PCR response procedures: 75 ℃ of 5 min; 94 ℃ of 30 sec, 66 ℃ of 30 sec, 72 ℃ of 90 sec, 27 circulations.
PCR for the second time: reaction system: get 1 μ l for the first time the PCR reaction solution as template; 10 * PCR reaction buffer, 2.5 μ l; DNTP Mix (10 mM) 0.5 μ l, Neasted Primer 1 (10 μ M) 1 μ l, Neasted Primer 2 (10 μ M) 1 μ l; 50 * Advantage cDNA Polymerase Mix, 0.5 μ l, distilled water supplies 25; Response procedures: 94 ℃ of 30 sec, 68 ℃ of 30 sec, 72 ℃ of 90 sec, 27 circulations.
2. the PCR product that the second time, pcr amplification obtained is connected with the pMD18-T carrier, 16 ℃ of connections are spent the night;
3. will connect product through transformed into escherichia coli DH5 α competent cell, and coat on the LB flat board that has added AMP, IPTG, X-gal, 37 ℃ of incubated overnight carry out indigo plant and screen day shift;
4. picking white mono-clonal is preserved from the above-mentioned flat board, promptly is built into the common wild-rice SSH library that bacterial leaf spot pathogenic bacteria of the present invention is coerced, totally 1295 clones, 1269 hickies wherein, 26 locus coeruleus.
Insert the detection of cDNA clip size in the common wild-rice SSH library that the constructed bacterial leaf spot pathogenic bacteria of embodiment 2 the present invention is coerced
(1) 34 positive colonies of random choose from the common wild-rice SSH library that builds are inoculated in respectively and contain in the antibiotic LB liquid nutrient medium of AMP, and 28 ℃, 180 rpm shake overnight cultures;
(2) from 34 positive colony bacterium liquid, use the alkaline lysis method of extracting plasmid respectively;
(3) plasmid that extracts with step (2) is set up the PCR reaction system: 10 * PCR damping fluid, 5.0 μ l, MgCl as template
23 μ l, dNTP 4 μ l, Nested PCR primers F 1 μ l, Nested PCR primer R 1 μ l, Taq polysaccharase 0.5 μ l, plasmid template 1 μ l, sterilized water supply 50 μ l reaction volumes;
(4) PCR response procedures: 94 ℃ of preparatory sex change 5 min, 94 ℃ of sex change 35 s then, 55 ℃ of annealing 30 s, 72 ℃ are extended 1 min, totally 35 circulations, last 72 ℃ are extended 10min.
(5) after the PCR reaction finishes, carry out the detection of PCR product with 1% agarose gel electrophoresis.
Detected result is as shown in Figure 1, and 34 positive colonies of random choose all have fragment to insert, and inserts clip size between 200-900bp, and mean length is at 450bp.From the library clone number and insertion fragment distribution range that obtain at present, the constructed SSH library of the present invention is a better inhibited subtracted library, lays a good foundation for from common wild-rice, excavating and clone the resisting bacterial leaf-blight genes involved.
The application in the common wild-rice SSH library that embodiment 3 embodiment 1 constructed bacterial leaf spot pathogenic bacteria is coerced
Its step is following:
(1) whole mono-clonals are inoculated in respectively from the constructed SSH library of embodiment 1 contained in the antibiotic LB liquid nutrient medium of AMP, and 28 ℃, 180 rpm shake overnight cultures;
(2) mail bacterium liquid to China big genome company and check order, sequencing result adopts DNAStar software to remove carrier, and ContigExpress software carries out sequence assembly, removes Tumor-necrosis factor glycoproteins;
(3) with the nonredundancy sequence at NCBI (http://www.ncbi.nlm.nih.gov); American National bioinformation center) albumen database and nucleic acid database in compare respectively, and criterion is with reference to the EST of paddy rice, Arabidopis thaliana research, with blastx as a result consistence greater than 40%; Score value is greater than 80; Consistence is greater than 50% as a result for blastn, and score value is divided into according to screening and functional annotation greater than 80, and analytical results is sorted out and summed up; The blastx comparison has 341 ESTs sequences and known functional protein homology; 104 ESTs sequences and unknown function albumen homology are arranged, have 49 ESTs sequences in albumen database, not find homologous protein simultaneously, have no albumen to be complementary with it.Blastn comparison has the ESTs of 417 sequences and known function to have higher homology, the ESTs homology of 77 sequences and unknown function.
(4) the bioinformatic analysis result of basis (3) step; 5 ESTs sequences of random choose; Corresponding clone's numbering is respectively OR5, OR7, OR68, OR70 and OR143 in the SSH library; Use Primer5.0 software to design primer respectively; With the common wild-rice leaf cDNA of having inoculated bacterial leaf spot pathogenic bacteria with do not connect leaf cDNA that bacterial leaf spot pathogenic bacteria handles as template, 5 ESTs sequences of picked at random are carried out the RT-PCR checking respectively, detecting it, to belong to the inducible expression gene that bacterial leaf spot pathogenic bacteria coerces still be the constitutive expression gene;
(5) the RT-PCR reaction system is following: MgCl
22 μ l, 10 * PCR damping fluid, 2.5 μ l, dNTP 2 μ l, each 1 μ l of upstream and downstream primer, Taq polysaccharase 0.5 μ l, cDNA template 1 μ l, sterilized water supply 25 μ l reaction volumes;
(6) the RT-PCR response procedures is following: 94 ℃ of sex change 5 min, and 94 ℃ of 35 s then, annealing (it is fixed that temperature press primer) 30 s, 72 ℃ are extended 1 min, totally 35 circulations, last 72 ℃ of extension 10min.5 pairs of RT-PCR primers, PCR are divided into 5 groups to carry out.
The detected result of 5 ESTs sequences of picked at random is: wherein the ESTs of clone numbering OR5 and OR68 in not inoculating the common wild-rice blade that bacterial leaf spot pathogenic bacteria handles with inoculated its purpose band (shown in Figure 2) that all can increase in the bacterial leaf spot pathogenic bacteria processing common wild-rice blade; Show that these two genes are the constitutive expression genes in the common wild-rice, but not the inducible expression gene.3 ESTs that are numbered OR7, OR70 and OR143 from clone in the library are through the RT-PCT checking; These 3 genes its purpose band that in not inoculating the common wild-rice that bacterial leaf spot pathogenic bacteria handles, all can not increase; Come out (shown in Figure 3) can only increase in the common wild-rice of having inoculated bacterial leaf spot pathogenic bacteria; Show that thus these 3 genes belong to the inducible expression gene that bacterial leaf spot pathogenic bacteria is coerced in common wild-rice, but not the constitutive expression gene, infer that these 3 genes have participated in disease-resistant responsing reaction in the disease-resistant process of common wild-rice; Infer that further it is a common wild-rice resisting bacterial leaf-blight genes involved, even be disease-resistant gene.Through comparing at NCBI, the homology of these 5 ESTs and known function gene is all higher, and OR5 has 92% homology with the Pikm2-TS gene that contains the NBS-LRR structural domain, and the nucleotide sequence of OR5 is shown in SEQ ID NO:1 in the sequence table; OR7 and phosphoinositide 3-kinase (phosphatidylinositol 3-kinase) have 81% homology, and the nucleotide sequence of OR7 is shown in SEQ ID NO:2 in the sequence table; The homology of OR68 and somatic embryo protein kinase 1 (somatic embryogensis protein kinase 1) is 98%, and the nucleotide sequence of OR68 is shown in SEQ ID NO:3 in the sequence table; The homology of OR70 and abc transport albumen (ABC transporter family protein) gene is 98%, the nucleotide sequence of OR70 is shown in SEQ ID NO:4 in the sequence table; The homology of OR143 and glycine dehydrogenase (glycine dehydrogenase) is 96%, and the nucleotide sequence of OR143 is shown in SEQ ID NO:5 in the sequence table; Therefore we tentatively think these 5 ESTs genes encodings of from the library, obtaining is respectively the pairing albumen of homologous genes with it.
This application shows that the constructed library of the inventive method can obtain the differential expression ESTs that a large amount of common wild-rices receive the bacterial leaf spot pathogenic bacteria stress-inducing once, fast; For the separating clone disease-resistant related gene provides ESTs sequence information foundation; The differential expression est sequence information that is obtained helps separating clone cDNA full length sequence, for biological improvement etc. the important theory foundation is provided simultaneously; Can obtain new resisting bacterial leaf-blight genes involved, can cultivate disease-resistant varieties through the transgenic method rice transformation behind the separating clone.Can be used for studying common wild-rice resisting bacterial leaf-blight mechanism principle etc.Also can aspect molecular biology research, be used for gene functional research such as protein modified state, the Western marking, immunosorption, immunoprecipitation etc.Aspect RESEARCH ON CELL-BIOLOGY, be used for the resisting bacterial leaf-blight Study on Mechanism, research cell proliferation, differentiation, apoptosis, cell signaling process etc.
< 110>KUNMING INST OF BOTANY CAS
< 120>construction process in the bacterial leaf spot pathogenic bacteria common wild-rice SSH library of coercing
<130> -
<160> 5
<170> PatentIn?version?3.3
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gtactggcgc?aagtagtcaa?taataccaat?gacaagctct?ttcttctcct?cgtcgattcc 60
aacaagtagt?gagtaatcca?tgacatccgc?tgtcgcaaga?aaagaagtgt?cattccaaac 120
ggctctttcc?aatcttcgtt?ttgccttgct?ccctaaaaat?attggctttg?tatgcaatgc 180
ctctaggagg?tttgaatcca?agaggacttt?actctcactg?gatgtatagc?gtgagcgcaa 240
tgaacccttt?agatcataga?ctctagatat?cttcctttca?aagaaaagat?tctccatcac 300
catgaggtcc?atctttactt?ccctaccacc?ttttaagccc?ttgatattaa?cctgatatac 360
tcctacgatt?ttggccaggc?aagttggact?acctgaagtc?agggattcca?tcaaatatct 420
gaagt 425
<210> 3
<211> 278
<212> DNA
< 213>common wild-rice (Oryza rufipogon Griff.)
<220>
<221> misc_feature
<222> (41)..(41)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (110)..(110)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (150)..(150)
<223> n?is?a,?c,?g,?or?t
<400> 3
aaaagaaacg?aaagacaaaa?tcacaaagca?tccaaccttt?nctcgacggc?ttcctcgccc 60
ttgtttgtgt?atatgacaga?cagatcgata?gctggcaaat?ctggagggan?aaatggttta 120
cttgtaaccc?tccccggtgc?ctcggtaatn?ttcaagtagg?ctctgcactt?ggttgtcgtt 180
agaaatgcat?ggccgggcgg?aattgcaaca?cattgttgtg?gcggctctgc?gtctgtaatg 240
gctgaacaac?tgatgcctct?ctctgctgta?ttgtacgt 278
<210> 4
<211> 474
<212> DNA
< 213>common wild-rice (Oryza rufipogon Griff.)
<220>
<221> misc_feature
<222> (109)..(109)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (134)..(134)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (156)..(156)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (237)..(237)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (272)..(272)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (306)..(306)
<223> n?is?a,?c,?g,?or?t
<220>
<221> misc_feature
<222> (425)..(425)
<223> n?is?a,?c,?g,?or?t
<400> 4
tccctctctc?tggcctcaag?attcctttca?aggtaataat?tgtaatctcc?ttgatagtcc 60
tgaatagtct?gatctttcac?ttcaataact?ctgttaacaa?tttgctttnc?gaaataccgg 120
tcatgagaaa?ctgntataac?agtgcctgtg?tattcngaaa?ttgcctcctc?aagcatttct 180
ttcgatggaa?tatcaaggtg?atttgttggt?tcatccaata?tcaataaagt?agatggngtc 240
accaagaact?tgcagaaggc?aagcctcgcc?tnctctccac?cacttaagaa?ttgaaccttt 300
ctatcnaaca?tatcatccct?aaagttgcaa?cgaccaagga?gacctttgat?atcatcaatt 360
ttccaatcct?ctgctgcttc?tgctacagta?tccagcacag?ttttctccaa?atcaagtgct 420
tcagnctgat?tttgctcgaa?ataattaggc?aatacattat?gctccccaag?aagt 474
<210> 5
<211> 643
<212> DNA
< 213>common wild-rice (Oryza rufipogon Griff.)
<400> 5
acggcttagt?ccatgagtcg?ctcatcagaa?gttggggcga?gtgaggagca?ctcttgagga 60
cgttgttgtt?cacatctgct?ttgccacttt?ctatctctgc?aatttcttcc?ctgattgaga 120
ttagagcatc?acggaaccta?tctagttcgg?ccttgctttc?actttcagtt?ggttcaatca 180
taagtgtgcc?tggaacaggc?catgacatag?taggtgcatg?gaatccatag?tccatcaaac 240
gctttgcaac?atcctctggc?tctataccag?cagtcgtcct?aaatcctctt?aaatcaataa 300
tgaactcatg?ggcaacagtt?ccattgactc?cgcggaaaag?aactgggcag?tgtttctcca 360
gacgctttgt?catgtagttt?gcattcaaga?ttgcaatctt?tgaagcgtca?gtgagaccct 420
tagaacccat?catggctatg?tatgtgtagg?agattggtag?aatcaacgct?gatccccatg 480
gggcagcaga?aatggtacct?gcccggtctg?tgattctaag?gcaccttttg?atgacctgat 540
tattgatggg?ctgttcacag?agatcctaca?gaggacgtcg?tcagagctaa?atgagatcca 600
gttcttcgag?gatggatcct?ggcactccat?caagccacag?agg 643
Claims (1)
1. the bacterial leaf spot pathogenic bacteria common wild-rice SSH library constructing method of coercing may further comprise the steps:
(1) bacterium that connects of common wild-rice blade material is handled
1. with the bacterial leaf-blight bacteria strain, line on the NA culture medium flat plate, cultivate 2-3 d for 28 ℃ and carry out activation, wash lawn with sterile distilled water after the activation, wheat flange protractor turbidimetry is mixed with 6 * 10
8Ml
-1Bacterium liquid;
2. inoculate the common wild-rice blade of bacterial leaf spot pathogenic bacteria in 26-28 ℃ of greenhouse with the scissors that has picked bacterium liquid from the leaf-cutting inoculation method that blade tip deducts 2-3 cm; Each bacterial strain connects 3 piers; 30 leaves, contrast replaces bacterium liquid inoculation common wild-rice blade as contrast with sterilized water;
3. connect behind the bacterium every at a distance from 24 h, respectively at 24 h, 48 h, 72 h, 96 h, 120 h, the sampling of 144 h equivalent, the control group of butt joint sterilized water also simultaneously respectively equivalent take a sample;
The common wild-rice blade that 4. will connect 6 stages that 24 h behind the bacterium, 48 h, 72 h, 96 h, 120 h, 144 h collect mixes, and as test side, the blade in 6 stages of contrast sterilized water inoculation mixes too, as driven element;
(2) extraction of total RNA
1. get test side's common wild-rice blade and driven element common wild-rice blade respectively, place mortar to add liquid nitrogen respectively and grind fast, per 100 mg samples add 1ml TRIzol extracting solution, the vortex 15 s mixings that vibrate;
2. place 5 min at 15-30 ℃, add 200 μ l chloroforms, every use 1 ml TRIzol adds 200 μ l chloroforms, builds pipe racks, and vortex 15 s that vibrate place 3 min for 15-30 ℃;
3. 4 ℃, centrifugal 15 min of 12000 rpm, this moment, solution was divided into three layers: xanchromatic is an organic phase, and what middle layer and upper strata were colourless is water, and mainly there is aqueous phase in RNA, is transferred to water in another new no RNase centrifuge tube;
4. add isopyknic Virahol to the aqueous phase that obtains, mixing is placed 20-30 min for 15-30 ℃; 4 ℃, centrifugal 10 min of 12000 rpm abandon supernatant;
5. the volume(tric)fraction that adds the preparation of DEPC water is 75% ethanolic soln washing precipitation, and the volume(tric)fraction that every use 1 ml TRIzol uses 1ml DEPC water to prepare is 75% ethanolic soln;
6. 4 ℃, centrifugal 3 min of 5000 rpm; With the careful sucking-off supernatant liquid of rifle head, keep deposition; 15-30 ℃ is dried 2-3 min, and adding 30-100 μ l does not have the RNase distilled water, fully dissolve RNA after, preserve or get into next step test for-70 ℃;
(3) purifying of mRNA
1. 5 mg Oligo (dT) Cellulose add 0.5 ml1 * Binding Buffer, mixing, and 15-30 ℃, centrifugal 2 min of 2000 g remove supernatant;
2. total RNA 0.3 mg, supplying with DEPC-ddH2O of less than 500 μ l volumes adds isopyknic 2 * Binding Buffer, mixing;
3. the mixed solution of step (3) in 2. joined 1. abundant mixing among Oligo (dT) Cellulose of step (3), 65 ℃ of water-bath 5 min, or put upside down mixing; Place 30 min for 15-30 ℃, or put upside down mixing gently, 18～22 ℃, centrifugal 3 min of 2000 g abandon supernatant;
4. in mRNA-Oligo (dT) Cellulose mix, add 0.5 ml, 1 * Binding Buffer, mixing; 15-30 ℃, centrifugal 3 min of 2000 g abandon supernatant, repeat this step once;
5. add 0.5 ml Wash Buffer, mixing; 15-30 ℃, centrifugal 3 min of 2000 g abandon supernatant, repeat this step once;
6. add 65 ℃ of RNA Elution of 200 μ l, 15-30 ℃, centrifugal 3 min of 4000 g collect supernatant, repeat this step 1-2 time;
7. the LAP that adds isopyknic Virahol and 1.6%, abundant mixing, 4 ℃, centrifugal 20 min of 12000 g abandon supernatant;
The washing with alcohol that adds 700 μ l 70%, 4 ℃, centrifugal 5 min of 12000 g;
8. dry deposition, 30-50 μ l DEPC-ddH
2The O dissolving, 1% agarose gel electrophoresis detects
(4) the inhibition difference of test side cDNA and driven element cDNA subtracts hybridization
1. utilize ThermoScript II respectively will the side of test mRNA reverse transcription becomes test side r and the driven element second chain cDNA with driven element mRNA;
2. in test side and the driven element second chain cDNA reaction tubes, add respectively
RsaThe I enzyme, 37 ℃ of enzymes are cut 1.5 h;
3.
RsaTest side cDNA after the I enzyme is cut is connected for 16 ℃ with joint and spends the night;
4. the test side cDNA that has connected joint with
RsaThe driven element cDNA that the I enzyme is cut differs from and subtracts hybridization, adds 200 μ l dilution buffer dilution hybridization solution after hybridization is accomplished, and places-20 ℃ to preserve or carry out the next round test;
(5) formation in the bacterial leaf spot pathogenic bacteria common wild-rice SSH library of coercing
1. with the hybridization solution of last round of dilution as template, carry out the pcr amplification first time, and then with the second time PCR reaction solution carry out second pcr amplification of taking turns as template, concrete reaction system and response procedures are following:
PCR for the first time: reaction system: 10 * PCR reaction buffer, 2.5 μ l; DNTP Mix (10 μ M) 0.5 μ l; PCR Primer (10 μ M) 1 μ l; 50 * Advantage cDNA Polymerase Mix, 0.5 μ l, the hybridization solution template of 1 μ l dilution, distilled water less than 25 μ l; PCR response procedures: 75 ℃ of 5 min; 94 ℃ of 30 sec, 66 ℃ of 30 sec, 72 ℃ of 90 sec, 27 circulations;
PCR for the second time: reaction system: get 1 μ l for the first time the PCR reaction solution as template; 10 * PCR reaction buffer, 2.5 μ l; DNTP Mix (10 mM) 0.5 μ l, Neasted Primer 1 (10 μ M) 1 μ l, Neasted Primer 2 (10 μ M) 1 μ l; 50 * Advantage cDNA Polymerase Mix, 0.5 μ l, distilled water supplies 25; Response procedures: 94 ℃ of 30 sec, 68 ℃ of 30 sec, 72 ℃ of 90 sec, 27 circulations;
2. the PCR product that the second time, pcr amplification obtained is connected with the pMD18-T carrier, 16 ℃ of connections are spent the night;
3. will connect product through transformed into escherichia coli DH5 α competent cell, and coat on the LB flat board that has added AMP, IPTG, X-gal, 37 ℃ of incubated overnight carry out indigo plant and screen day shift;
4. picking white mono-clonal is preserved from the above-mentioned flat board, promptly is built into the common wild-rice SSH library that described bacterial leaf spot pathogenic bacteria is coerced.
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| CN103614473A (en) * | 2013-11-27 | 2014-03-05 | 华南农业大学 | Preparation method and applications of cDNA chip for detecting drug resistance level of bactrocera dorsalis resistant to trichlorphon and beta-cypermethrin |
| CN103725746A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Coli group and colibacillus testing medium |
| CN104004753A (en) * | 2014-05-15 | 2014-08-27 | 福建农林大学 | Method for separating and regulating peanut embryonic development gene |
| CN106244600A (en) * | 2016-09-21 | 2016-12-21 | 云南省农业科学院生物技术与种质资源研究所 | The oryza officinalis Oo92082 gene of leaf spot bacteria abduction delivering and specific amplification primer thereof |
| CN107475780A (en) * | 2017-09-25 | 2017-12-15 | 中国计量大学 | Structure, the authentication method in Pomacea canaliculata SSH libraries under a kind of low temperature stress |
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| CN103725746A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Coli group and colibacillus testing medium |
| CN103614473A (en) * | 2013-11-27 | 2014-03-05 | 华南农业大学 | Preparation method and applications of cDNA chip for detecting drug resistance level of bactrocera dorsalis resistant to trichlorphon and beta-cypermethrin |
| CN103614473B (en) * | 2013-11-27 | 2015-01-21 | 华南农业大学 | Preparation method and applications of cDNA chip for detecting drug resistance level of bactrocera dorsalis resistant to trichlorphon and beta-cypermethrin |
| CN104004753A (en) * | 2014-05-15 | 2014-08-27 | 福建农林大学 | Method for separating and regulating peanut embryonic development gene |
| CN106244600A (en) * | 2016-09-21 | 2016-12-21 | 云南省农业科学院生物技术与种质资源研究所 | The oryza officinalis Oo92082 gene of leaf spot bacteria abduction delivering and specific amplification primer thereof |
| CN107475780A (en) * | 2017-09-25 | 2017-12-15 | 中国计量大学 | Structure, the authentication method in Pomacea canaliculata SSH libraries under a kind of low temperature stress |
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