CN102675434A - Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein - Google Patents
Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein Download PDFInfo
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Abstract
The invention discloses a magnaporthe oryzae isolated protein and gene thereof as well as a nucleotide sequence for applying peptide with 20 amino acids and coding a peptide segment and application of the nucleotide sequence. According to the magnaporthe oryzae isolated protein, the plant resistance can be improved, the defense reaction of the plant can be induced, and the resistance of the plant can be remarkably improved by the magnaporthe oryzae isolated protein; the magnaporthe oryzae isolated protein is low in concentration and quick in onset of action; and rice leaves are treated by using a 10muMHis-MoHrip1 solution for 3 days and then the treated rice leaves are inoculated to magnaporthe oryzae, and the inhibition rate for pathogenesis can reach 63.89 percent. The amino acid sequence of the magnaporthe oryzae isolated protein is shown as SEQIDNO.2. The invention also relates to a gene for coding the protein. The amino acid sequence of the gene is shown as SEQIDNO.1.
Description
Technical field:
The present invention relates to have more than the nucleotide sequence of 20 amino acid whose peptides, this peptide section of encoding with and use, particularly a kind ofly can improve that plant resistance to environment stress and inducing plant defensive raction come from the separating protein of rice blast fungus (Magnaporthe oryzae) and encode this proteinic nucleotide sequence and application thereof.
Background technology:
Rice blast is one of important disease of paddy rice, can cause the underproduction significantly, the underproduction 40%～50% when serious, even No kernels or seeds are gathered, as in a year of scarcity, all there is generation in each rice district, the world.Rice blast pathogen rice blast fungus (Magnaporthe oryzae) is on classification position, and asexual generation belongs to the Deuteromycotina Pyricularia Sacc., and sexual generation belongs to Ascomycotina.At present chemical prevention and seed selection kind are mainly taked in the control of Plant diseases.But they often exist the drawback that can't forgo.Last century, the plant induction of resistance received publicity along with development of biology, and exciton more receives publicity as the main factor of inducing plant resistance.Therefore from this pathogenic bacteria, seek out and have new activity, the active substance of new function has become the focus that domestic and international scientist pays close attention to.
As the rice blast fungus of the main disease of China paddy rice, the protein of its generation is a kind of important effector molecule as exciton.It transmits the exciton signal of pathogenic bacteria through the acceptor molecule of identification and effect vegetable cell surface or subcellular components; Start the defence system of plant; The local organization of inducing plant produces supersensitivity necrocytosis, and makes plant finally produce systemic acquired resistance through the systems communicate of local cells semiochemicals.
Research rice blast fungus exciton is not a lot of both at home and abroad at present.From rice blast fungus, isolated the activeconstituents that some can cause the plant disease-resistant reaction or cause the plant morbidity.As as far back as H.Kanon in 1993, M.Haga etc. just in the cell walls of rice blast fungus, be separated to a kind of gp excite son (H.Kanon, M.Haga etc., J.Pesticide Sci., 993,18:299-308).Nineteen ninety-five; The U.Schaffrath of Germany etc. is separated to the gp of an about 15.6kDa from rice blast fungus, this gp is injected rice leaf, can effectively must improve the ability (U.Schaffrath etc. of the anti-rice blast fungus of paddy rice; Physiological and Molecular Plant Pathology; 1995,46,293-307).J.Koga in 1998 etc. isolate two kinds of unsaturated fatty acids excitons (J.Koga etc., The Journal of Biological Chemistry, 1998,48,273) from rice blast fungus.
People such as domestic Agricultural University Of South China Li Yun peak isolate an about 66kDa from the rice blast fungus cell walls gp excites son, and this gp can inducing paddy rice blade px and the active rising of phenylalanine ammonia lyase.Biological activity assay result after heat, trypsinase and sodium periodate are handled shows that this proteic avtive spot is glycosyl part (Li Yunfeng etc., a Plant Pathology 2004,34 (2): 127～132).Obtained the albumen exciton of a 40kDa in the cell of rice blast fungus such as the Xu Feng of Plant Protection institute, Chinese Academy of Agricultral Sciences.This albumen can enhancement of plant growth quality (Acta Agriculturae Boreali-Sinica, 2006,21:1-5).
All documents of having published show that rice blast fungus can produce exciton really, cause that the defensive raction of plant improves the resistance of plant.Though exciton character and kind that this bacterium produces have nothing in common with each other; But their protein sequence and gene order report are seldom; So seek a kind of new protein exciton, clear and definite its sequence information is very important with the resistance that further improves plant for disclosing function.
Summary of the invention:
The purpose of this invention is to provide a kind of application that can improve rice blast fungus separating protein and gene order and this albumen and the gene thereof of plant resistance to environment stress and inducing plant defensive raction.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
Rice blast fungus of the present invention (Magnaporthe oryzae) bacterial strain has carried out preservation on February 8th, 2012 in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; Postcode: 100101, the numbering of registering on the books CGMCC No:5773, classification called after: rice blast fungus (Magnaporthe oryzae).
A kind of isolating protein, its aminoacid sequence is shown in SEQ ID NO:2.
The invention still further relates to the application of above-mentioned protein in improving plant resistance to environment stress and inducing plant defensive raction.
The application of protein of the present invention in improving plant resistance to environment stress and inducing plant defensive raction, wherein, said plant is tobacco and paddy rice.
The invention still further relates to a kind of isolating polynucleotide, its nucleotides sequence one of is classified as what follows:
(1) the proteinic polynucleotide sequence of encoding amino acid sequence such as SED ID NO:2;
(2) with (1) in polynucleotide sequence according to basepairing rule complementary polynucleotide sequence.
The isolating polynucleotide of the present invention, wherein: encoding amino acid sequence like the proteinic polynucleotide sequence of SED ID NO:2 shown in SEQ ID NO:1.
The invention still further relates to a kind of recombinant vectors, it contains above-mentioned isolating polynucleotide.
Recombinant vectors of the present invention, wherein said carrier are the recombinant vectors that between the BamH1/ site of pET-28a (+) carrier and Sal1 site, inserts above-mentioned isolating polynucleotide.
A kind of host cell of genetic engineering, it contains above-mentioned recombinant vectors.
The host cell of genetic engineering of the present invention, wherein said host cell are the intestinal bacteria Transetta (DE3) that contains above-mentioned recombinant vectors.
Protein of the present invention is meant the nutrient solution from rice blast fungus (Magnaporthe oryzae), the supernatant of nutrient solution or the protein of thalline.
Advantage of the present invention is: this kind protein can significantly improve the resistance of plant, and concentration is low rapid-action, inoculates rice blast fungus behind the 10 μ M His-MoHrip1 solution-treated rice leaf 3d, and the inhibiting rate that rice blast fungus is fallen ill can reach 63.89%.This protein provides new approach for the disease resistance that improves plant, thereby in agriculture prodn, has broad application prospects.
Description of drawings:
Fig. 1 passes through HP Q HiTrap for crude protein
TMThe color atlas of anion-exchange column, solid line are absorption value, and dotted line is a NaCl concentration.
Fig. 2 changes for the pH value of the tobacco suspension cell of process MoHrip1 processing and water treatment (contrast).
Fig. 3 is the isolating protein sequence of gained clonal expression of the present invention, and wherein underscore partly is the peptide section sequence through the mass spectrum acquisition.
Fig. 4 inoculates the photo of rice blast fungus spore after 20 days through the paddy rice seedling of MoHrip1 processing and water treatment (contrast) respectively.
Embodiment:
Further specify characteristics of the present invention through specific examples below.
The cultivation of embodiment 1 rice blast fungus and fermented liquid preparation
Rice blast fungus (Magnaporthe oryzae) bacterial strain line bacterium piece is inoculated in the tomato medium oatmeal, and [preparation method of tomato medium oatmeal is: get rolled oats 30 grams, add 800 milliliters in water and boiled 30 minutes, elimination oat residue; Other gets two in tomato, smashes and squeezes the juice, and gets 150 milliliters of Tomato juice and adds in the oat filtrating; Supply water to 1000 milliliter, add agar 15 grams, dissolve the back packing; 121 ℃ of sterilizations 30 minutes] flat board, cultivated for 2 weeks for 28 ℃, choose the colony edge place and be inoculated in 200mL and fill the YPD liquid nutrient medium [preparation method of YPD liquid nutrient medium is: the 10g yeast extract paste; The 20g peptone, 20g glucose (Beijing Chemical Plant, analytical pure); Add deionized water to 1L] the 500ml triangular flask in, 26 ℃, 130r/min were cultivated 14 days, obtained the fermented liquid of rice blast fungus.
The preparation and the monitoring of embodiment 2 crude protein excitons
Get the fermented liquid of gained among the embodiment 1, centrifugal 1h under 4 ℃, 5000rpm condition (or 13000rpm, 30min), collect supernatant, filter membrane (Whatman product) suction filtration twice with 0.45mm until there not being thalline, obtains fermented supernatant fluid.Add the ammonium sulfate powder, making in the fermented supernatant fluid ammonium sulfate final concentration is 80% to precipitate target protein, and 4 ℃ of dialysis 48h and freeze-drying obtain the exoprotein behind the desalination, i.e. crude protein.At last the exoprotein that obtains is dissolved in the Mes-NaOH damping fluid (20mM, pH 6.0), gets crude protein solution, wherein the exoprotein final concentration is 10 μ mol/L.Protein contnt is measured at wavelength 590nm place through GF-M2000 type ELIASA (Shandong Gaomi Caihong Analytical Instrument Co., Ltd.) with BCATM protein assay kit (Thermo Scientific).
Bioactive monitoring: to grow 2 months; Tobacco with 5-7 sheet true leaf is a material; Mes-NaOH damping fluid and 10 μ mol/L bovine serum albumen solution with 0.2mM, pH 6.0 are contrast respectively; The crude protein strength of solution is 10 μ mol/L, utilizes 1mL not with the syringe of syringe needle, and 50 μ LMes-NaOH damping fluids, 50 μ L BSA solution, 50 μ L crude protein solution are injected into the blade respectively from the leaf back side and go.Observe through 24h whether necrotic plaque formation is arranged.
The result: the blade of injection crude protein solution, water stain spot appears behind the injection site 6h, and injection place blade becomes transparent more thinner behind the 12h, can present typical anaphylaxis behind the 24h, has necrotic plaque to form.Contrast does not have any reaction, and is the same with normal blade.
The separation and purification and the biological activity determination of protein exciton in the embodiment 3 crude protein solution
Use
Explore 10 (GE Healthcare) protein purification appearance is further purified the crude protein of gained, and sample is at first through HP Q HiTrap
TMAnion-exchange column (GE Healthcare); (0%-100% 30min), acquires protein elution peak (see figure 1) to carry out linear elution with NaCl; Each protein elution peak component is carried out bioactive detection; Making protein concn in applied each component is 10 μ mol/L, and the result shows that protein peak a2 (as shown in Figure 1) can cause the anaphylaxis of tobacco by intensive; With the component of this protein peak a2 being further purified through 15%Native-PAGE, rubber tapping purifying and electroelution again; Obtain activated single protein; Need the activity of monitoring protein exciton in the above purge process, monitoring method is undertaken by bioactive monitoring method among the embodiment 2.The single protein of this purifying gained detects through 15% denaturing polyacrylamide gel electrophoresis and is single band, explains that lipidated protein is high.This protein apparent molecular weight is 15kD (pI=4.53), with this protein exciton called after MoHrip1 (Magnaporthe oryzae Hypersensitive response-inducing
P Rotein 1).With MoHrip1 (30 μ Ms, 10 μ Ms, the 5 μ Ms of bioactive monitoring method among the embodiment 2 to different concns; 1 μ M, 500nM, 100nM; 10nM) carry out activity monitoring, the MoHrip1 of 10 μ M can cause typical anaphylaxis, and the protein concentration that low energy causes allergic reaction is 5 μ M.
The result: the MoHrip1 of separation and purification can make tobacco form anaphylactoid necrotic plaque.
Some reactions that the MoHrip1 that separation and purification obtains causes plant:
(1) mensuration of oxidative burst
Oxidative burst refers to the accumulation of reactive oxygen species, and reactive oxygen species comprises H
2O
2This experiment is through monitoring H
2O
2Accumulation the process of oxidative burst is described.Oxidative burst (Reactive Oxygen Species; ROS) in exciton inductive plant disease-resistant defense response, play important effect; They all participate in stomatal movement, promote the cell programmatic dead, directly or indirectly kill the pathogenic bacteria of invasion; Can on transcriptional level, activate and regulate and control various defence Expression of Related Genes and participation system acquired resistance (Systemic acquired resistance, foundation SAR) etc. in the plant materials as the second messenger.
A, to the effect of tobacco suspension cell
Tobacco suspension cell (tobacco BY-2) is given by life science institute of China Agricultural University from Nicotiana tabacum cv.Xanthi.Tobacco suspension cell substratum: MS liquid nutrient medium (MS+100mg/ml, inositol; 0.2%, KH
2PO
40.2mg/ml, 2,4-D; 1mg/ml, VB1; 3% sucrose), 121 ℃ of high pressure steam sterilization 15min, room temperature preservation.Suspension cell is cultivated in 25 ℃, the shaking table of 130rpm, makes cell remain on the Exponential growth phase.
Get above-mentioned gained tobacco suspension cell nutrient solution, centrifugal collection is resuspended in damping fluid, and (buffer formulation is: 175mM N.F,USP MANNITOL, 0.5mM calcium chloride; 0.5mM vitriolate of tartar (Beijing chemical industry, analytical pure), 10mM HEPES (HERES; Sigma) pH 5.75) in; Making tobacco cell concentration is 0.1g/mL (wet cell weight/volume), hatches 1h at 24 ℃, 150rpm.(buffer formulation is: 175mM N.F,USP MANNITOL, 0.5mM calcium chloride, 0.5mM vitriolate of tartar (Beijing chemical industry to get 250 μ L suspension cells adding HERES damping fluid then; Analytical pure), and the 10mM HEPES (HERES, Sigma) pH 5.75; Add 0.3mM luminol (Luminol) simultaneously) in; Adding MoHrip1 simultaneously, to make its final concentration be 10 μ M, begins with chemiluminescence detector (Promega), detects the active oxygen that cell generated.
B, to the effect of tobacco leaf
Method in tobacco leaf treatment process such as the monitoring of embodiment 2 biological activitys, MoHrip1 concentration is 10 μ M, observes through 24h.The blade of getting processing is placed in the 2mL pipe after cleaning with zero(ppm) water, gets an amount of DAB (3,3 '-Diaminbenzidine) (Sigma) dye liquor (1mg.mL
-1, pH 3.8), behind NaOH adjust pH to 5.8, dye liquor is added pretreated blade, room temperature kept in Dark Place 8 hours.Dye liquor in each pipe of subsequent removal adds 95% ethanol, boiling water bath number minute; Remove and respectively manage liquid; Add absolute ethyl alcohol and boiling water bath again till leaf green is sloughed fully, liquid is respectively managed in sucking-off once more, with sloughing green being immersed in 70% glycerine; Drive the iuntercellular bubble out of, use microscopic examination.
C, to the effect of rice leaf
Get 8 all rice leafs, be cut into diameter and be 1 centimetre discoid, in 96 orifice plates that fill 100 microlitre zero(ppm) water; Lucifuge was soaked 6-8 hour; In 96 orifice plates, add respectively subsequently 100 microlitre luminols (luminol, Sigma), (HRP is the MoHrip1 of 10 μ M with 10 microlitre concentration Sigma) to 1 microlitre horseradish peroxidase; Utilize Chemiluminescence Apparatus (Luminometer, Promega) values of chemiluminescence of detection by quantitative reactive oxygen species immediately.
After result: a, MoHrip1 handle about 30min, tobacco suspension cell presence of oxygen outburst phenomenon; B, have tangible brown deposited material to occur, and reddish-brown precipitation also appear in the stomata guard cell on the pore of into treatment sites at the tobacco leaf position that MoHrip1 handles; C, MoHrip1 handled rice leaf 2-3 minute, obvious oxidative burst phenomenon occurred.
(2) alkalization of cell culture medium
Calcium ion flows and generally is considered to one of early response of exciton, and the stream potential of calcium ion must change the pH value variation of cellular environment, and this experiment has detected the alkalization situation of exciton processing back cell culture medium.
Get the tobacco suspension cell that concentration is 0.1g/mL, be transferred in the new tobacco suspension cell substratum (culture medium prescription is seen in the mensuration of oxidative burst the agency part to tobacco suspension cell) 25 ℃ again; 150r/min cultivated after 3 days, added protein exciton MoHrip1 and handled, and making the MoHrip1 final concentration is 10 μ M; Continue to shake training; With the water treatment is contrast, and after this every pH that crosses 10min with pH agent mensuration substratum is up to handling back 180min.
The result: protein exciton MoHrip1 can cause the alkalization of cell culture medium, and the pH of 10min substratum obviously improves (as shown in Figure 2) after processing.
(3) accumulation of the deposition of callose, aldehydes matter and xylogen
The accumulation of the deposition of callose, aldehydes matter and xylogen is considered to the secondary reaction of the host cell that typical exciton causes, the generation of these secondary metabolites or accumulation are relevant with host defense system.The accumulation of callose, aldehydes matter and xylogen can the enhancement of plant cell walls toughness, resist the invasion of pathogenic bacteria.
A, to the tobacco leaf effect
In the monitoring of tobacco leaf treatment process such as embodiment 2 biological activitys method; Blade after the MoHrip1 of 10 μ M handles 24h is put into damping fluid (1% LUTARALDEHYDE, 5mM Hydrocerol A with blade; 90mM Sodium phosphate, dibasic (Beijing chemical industry, analytical pure) pH 7.4) spends the night.Put into absolute ethyl alcohol then and boil 10min, putting into 50% ethanol, then change damping fluid (67mM potassium hydrogenphosphate (Beijing chemical industry, analytical pure), pH 12) over to.At staining fluid (pH 12 for 0.1% aniline blue (Sigma), 67mM potassium hydrogenphosphate (Beijing chemical industry, analytical pure)) dyeing 1h, put into 70% glycerol stationary and observe.
B, to the effect of tobacco suspension cell
Get the 0.5g tobacco suspension cell, it is 10uM that adding MoHrip1 makes its final concentration, behind the processing 108h, handles with the 2.5w/v% Phloroglucinol (Sigma) of 6mol/L, observes immediately.
Result: a, with the tobacco leaf that MoHrip1 handles, the starlike accumulation of callose appears in cell walls; B, the tobacco suspension cell of handling have the accumulation of xylogen.
The acquisition of embodiment april protein exciton MoHrip1 peptide section sequence
The MoHrip1 protein of purifying detects through two-dimensional electrophoresis and is single-point; Cut the protein single-point and modify Trypsin matter enzyme (Sequencing Grade Modified Trypsin through 10ng/ μ l order-checking level respectively; Promega) 30 ℃, pH7.5 enzymolysis 30min～1h, ESI-MS/MS are that the Q-TOF2 quadrature of Britain Micromass company quickens the esi-msn appearance.The spraying source that rises is received in outfit.All mensuration are all carried out under the positive ion mode.Mass spectrograph is proofreaied and correct with the MSMS fragment of Glu-fib, and mass accuracy is less than 0.1Da.Atomization gas is a nitrogen, and collision gas is an argon gas.80 ℃ of source temperature, taper hole voltage 50V.TOF acceleration voltage 9.1kV.The MCP detector voltage is 2150V.Capillary voltage 800V.Obtain this proteinic a plurality of peptide section sequences, peptide section sequence is as follows: (1) NTPTNVNFK; (2) GDSAYSF.
The clone of embodiment 5 protein exciton MoHrip1 encoding soxs
Degeneracy design primer according to mass spectrum sequencing result and codon is MoHrip1-forward 5 '-ATGCGCTTCGCCACCATCACCG-3 ' and the reverse 5 '-CTAAGCGGAGCCGTCAATGGCAATA-3 ' of MoHrip1-; And be template with the extractive mRNA of rice blast bacterial strain, select for use HiFi reverse transcription polysaccharase to carry out PT-PCR (94 ℃ of 3min; 94 ℃ of 30s, 65 ℃ of 30s, 72 ℃ of 30s, 35cycles; 72 ℃ of 10min; 4 ℃ of ∝), amplify the dna fragmentation of 429bp, comprising the complete ORFs of 429bp, with its called after MoHrip1 gene, the order of this gene is shown in SEQ ID NO:1.Express the protein of the available aminoacid sequence of SEQ ID NO:1 shown in SEQ ID NO:2, as shown in Figure 3, SEQ ID NO:2 includes 2 peptide sections that obtain among the embodiment 5.
(1) structure of expression vector
The Auele Specific Primer of design protein exciton gene M oHrip1, introducing has the restriction enzyme site that enzyme is cut the protection base, forward primer: 5 '-
GGATCCATGCGCTTCGCCACCATCACCG-3 ', (underscore is that the enzyme of BamH1 is cut sequence), reverse primer: 5 '-
GTCGACCTAAGCGGAGCCGTCAATGGCAATA-3 ', (underscore is that the enzyme of Sal1 is cut sequence), amplification total length MoHrip1 gene (94 ℃ of 3min; 94 ℃ of 30s, 65 ℃ of 30s, 72 ℃ of 30s, 35cycles; 72 ℃ of 10min; 4 ℃ of ∝).Earlier with MoHrip1 gene purpose fragment cloning to pMD18-T simple cloning vector; After confirming that order-checking is correct, after BamH1 and Sal1 enzyme are cut, with the BamH1/ and the Sal1 site of MoHrip1 fragment cloning to pET-28a (+) expression vector (Novagen); Thermal excitation is transformed into intestinal bacteria Trans 5 α (Transgene); The picking positive colony shakes bacterium and extracts plasmid, and enzyme is cut and sequence verification.
(2) abduction delivering
With in the step (1) the expression vector that obtains be transformed among the intestinal bacteria Transetta (DE3) with thermal excitation.Plasmid extracts and transforms the method for expression host strain with (1), after checking is correct, expresses.With the activation of spending the night of the correct recombinant strains of checking, the bacterial strain of getting the pET-28a empty plasmid simultaneously is as contrast.Get 1mL incubated overnight liquid respectively and join in the 100mL LB liquid nutrient medium that contains 100 μ g/mL kantlex (1% inoculum size), 37 ℃, 200r/min shaking culture 2～3h is cultured to OD
600Be 0.6～0.8.Add inductor IPTG (final concentration is 0.2mmol/L), 16 ℃, 220r/min continues shaking culture 16h, abduction delivering target protein.High speed centrifugation is collected thalline and is added damping fluid.Behind the ultrasonication thalline, 4 ℃ of high speed centrifugations are collected supernatant, obtain recombinant protein liquid.Get 20 μ L supernatants, add the resuspended thalline of 5 μ L, 5 * SDS sample-loading buffers (sex change), heat 10min in the boiling water bath, the centrifugal 10min of 13000r/min gets supernatant and carries out the SDS-PAGE detection, and expression is observed in Coomassie brilliant blue R250 dyeing.
Result: detect through SDS-PAGE, obtain the amalgamation and expression albumen (His-MoHrip1) that comprises Histidine of an about 18-kDa of molecular weight.
(3) purifying of recombinant protein
Utilize
explorer10 protein purification appearance to carry out the purifying of recombinant protein.Select for use Hitrap chelating Column post to carry out affinity chromatography, earlier with cleaning buffer solution balance affinity column; Get the recombinant protein liquid 5mL sample introduction that obtains in the step (2), flow velocity is 1mL/min, and drip washing is to baseline, and elution buffer carries out wash-out; Each elution peak of collecting, 4 ℃ of dialysed overnight in the phosphate buffered saline buffer of a large amount of volumes are carried out the dyeing of SDS-PAGE electrophoresis and Coomassie brilliant blue subsequently and are detected proteic purity.
Biological activity determination: method is measured the activity of recombinant protein exciton to tobacco among the employing embodiment 2.
Result: the amalgamation and expression albumen (His-MoHrip1 recombinant protein) that obtains about 18KD of purifying.
The reaction that embodiment 7 recombinant proteins (His-MoHrip1) cause on tobacco
With 1mL not with the syringe of syringe needle; Will about 50mL; 5 μ M amalgamation and expression albumen (His-MoHrip1) inject tobacco leaf respectively from the leaf back side, simultaneously, and with pET-28a (+) the empty plasmid expressing protein and the Mes-NaOH (20mM of same concentrations; PH 6.0) solution is as contrast, injects different tobacco leafs respectively from the leaf back side.Observe the reaction on the blade behind the 24h.
Like the method for (1) among the embodiment 3, observe recombinant protein and induce H
2O
2In the accumulation of handling on the blade; Like the method for (2) among the embodiment 3, observe the alkalization that recombinant protein causes the tobacco cell substratum.
The result: recombinant protein (His-MoHrip1) can cause tobacco leaf generation anaphylaxis; His-MoHrip1 makes and handles tobacco leaf acquisition H
2O
2Accumulation produces reddish-brown precipitation; His-MoHrip1 makes the tobacco suspension cell substratum alkalization of processing, the basically identical that the time that medium pH is the highest and the MoHrip1 of natural purifying cause.
The reaction that embodiment 8 recombinant proteins (His-MoHrip1) cause on paddy rice
(1) like the method for (1) among the embodiment 3, the detection by quantitative recombinant protein is induced H
2O
2In the accumulation of handling on the rice leaf.
The result: recombinant protein His-MoHrip1 inducing paddy rice blade produces the values of chemiluminescence of reactive oxygen species and the MoHrip1 inductive basically identical of natural purifying.
(2) paddy rice resistance related gene fluorescence quantitative RT-RCR is analyzed
Get required sample liquid nitrogen and pulverize, press 50-100mg/mL and add Trizol, room temperature 5min leaves standstill, and the centrifugal 5min of 12000rpm abandons deposition; By 200 μ L chloroform/mL Trizol, concussion mixing 15min places, and 4 ℃, the centrifugal 15min of 12000rpm draws the upper strata, adds 0.5mL Virahol/mL Trizol, mixing, and-20 ℃ leave standstill 20min; Centrifugal, 75% ethanol rinsing, centrifugal removal ethanol, 5-10min dries.Add diethypyrocarbonate (being abbreviated as DEPC) treating water, survey RNA concentration, the furnishing same concentrations.
The first chain cDNA's is synthetic, adopts the TransScript Two-Step RT-PCR Kit of TransGen company.Getting the total RNA of 500ng, according to the test kit specification sheets, with TransScript RT/RI Enzyme Mix, is primer with Anchored Oligo (dT) 18, hatches 30min for 42 ℃, and 85 ℃ of heating 5min make enzyme deactivation.Get 1 μ L reverse transcription product and be PCR .Osactin is an internal standard gene, is PCR (OsPR-1a gene F:5 '-GTATGCTATGCTACGTGTTTATGC-3 ', R:5 '-GCAAATACGGCTGACAGTACAG-3 ' with the resistance related gene special primer; OsPR-10a gene F:5 '-GGCTTGGTCGACGACATTG-3 ', R:5 '-CAGGGTTAAGCTTCATGGTGTAGA-3 '; OsLOX2 gene F:5 '-AGATGAGGCGCGTGATGAC-3 ', R:5 '-CATGGAAGTCGAGCATGAACA-3 '; OsAOS2 gene F:5 '-TACCAGCCGTGCGCCACCAG-3 ', R:5 '-AGGACGGAGCTGGTTGAGTGG-3 '; OsEDS1 gene F:5 '-CCCCGCATACCACTTACT-3 ', R:5 '-TGTTGATGAAACCACTCCC-3 '; OsPAL1 gene F:5 '-GGTGTTCTGCGAGGTGATGA-3 ', R:5 '-AGGGTGGTGCTTCAGCTTGT-3 '; OsNH1 gene F:5 '-ATCTTGATGATGCGTTTGC-3 ', R:5 '-TCAGCTTGCTCCAGTATTTC-3 '), the expression of observation resistance related gene.
The result: through the visible His-MoHrip1 of fluorescent quantitative PCR result after handling tobacco leaf 2 days, just can the induction of resistance Expression of Related Genes.
(3) induce of the harm of minimizing rice blast fungus to paddy rice
His-MoHrip1 behind 10mL, the purifying (10 μ M) solution evenly is sprayed on the 15 strain rice leafs; Inoculate rice blast fungus after three days; With spray the Mes-NaOH damping fluid and after three days 15 strain rice leafs of inoculation rice blast fungus compare, should test 3 (see figure 4)s of repetition.The inoculation method of rice blast fungus is: get the rice blast fungus spore for preparing in advance with damping fluid, contain 1%Tween20 in this damping fluid, making the rice blast fungus spore concentration is 2 * 10
5Individual spore/ml evenly is sprayed on the rice plant.Statistics plant morbidity number after 7 days.
The inoculation rice blast fungus is after 7 days, and investigation disease plant quantity is calculated sickness rate and morbidity inhibiting rate.
Inhibiting rate (%)=[(control group disease plant number-treatment group disease plant number)/control group disease plant number] * 100
Sickness rate (%)=(disease plant number/plant sum) * 100
Result such as following table.
When * is illustrated in ρ＜0.05 level in the table, compare with control group, treatment group has the significance difference opposite sex.
The above results shows that recombinant protein exciton His-MoHrip1 can induce and reduce the harm of rice blast fungus to paddy rice, after with 10 μ M His-MoHrip1 solution-treated rice leaf 3d, inoculates rice blast fungus, can reach 63.89% to the inhibiting rate of falling ill.
Above embodiment describes preferred implementation of the present invention; Be not that scope of the present invention is limited; Design under the prerequisite of spirit not breaking away from the present invention; Various distortion and improvement that those of ordinary skills make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (9)
1. an isolating protein is characterized in that, its aminoacid sequence is shown in SEQ ID NO:2.
2. the application of the described protein of claim 1 in improving plant resistance to environment stress and inducing plant defensive raction.
3. the application of protein according to claim 2 in improving plant resistance to environment stress and inducing plant defensive raction is characterized in that described plant is tobacco and paddy rice.
4. polynucleotide is characterized in that, its nucleotides sequence one of is classified as what follows:
(1) the proteinic polynucleotide sequence of encoding amino acid sequence such as SED ID NO:2;
(2) with (1) in polynucleotide sequence according to basepairing rule complementary polynucleotide sequence.
5. polynucleotide according to claim 4 is characterized in that, encoding amino acid sequence like the proteinic polynucleotide sequence of SED ID NO:2 shown in SEQ ID NO:1.
6. a recombinant vectors is characterized in that, contains claim 4 or 5 described polynucleotide.
7. recombinant vectors according to claim 6 is characterized in that, said carrier is the recombinant vectors that between the BamH1/ site of pET-28a (+) carrier and Sal1 site, inserts claim 4 or 5 described polynucleotide.
8. the host cell of a genetic engineering is characterized in that, it contains claim 6 or 7 described recombinant vectorss.
9. the host cell of genetic engineering according to claim 8 is characterized in that, said host cell is the intestinal bacteria Transetta (DE3) that contains claim 6 or 7 described recombinant vectorss.
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| CN103175809A (en) * | 2012-12-05 | 2013-06-26 | 云南农业大学 | Method for inducing rice plant system to generate defensive reaction |
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| CN105861540A (en) * | 2016-05-23 | 2016-08-17 | 中国农业科学院植物保护研究所 | Application of magnaporthe grisea protein elicitors in enhancement and improvement of drought-resisting capacity of plants |
| CN108314714A (en) * | 2018-04-18 | 2018-07-24 | 中国农业科学院植物保护研究所 | Verticillium dahliae secreted protein exciton VdPEL1 and its application |
| CN108314714B (en) * | 2018-04-18 | 2020-08-04 | 中国农业科学院植物保护研究所 | Verticillium dahliae secretory protein elicitor VdPE L1 and application thereof |
| CN109180790A (en) * | 2018-09-07 | 2019-01-11 | 中国农业科学院植物保护研究所 | Brevibacillus laterosporus A60 albumen exciton PeBL2 and its encoding gene and application |
| CN109180790B (en) * | 2018-09-07 | 2020-10-02 | 中国农业科学院植物保护研究所 | Brevibacillus laterosporus A60 protein exciton PeBL2 and coding gene and application thereof |
| CN115428684A (en) * | 2022-10-12 | 2022-12-06 | 云南农业大学 | Method for compensating reduction of blast resistance of OsLOX3 knockout rice strain and verification method and application |
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