CN100368432C - Glycoprotein elicitor of magnaporthe grisea in rice leaves and its purification method - Google Patents
Glycoprotein elicitor of magnaporthe grisea in rice leaves and its purification method Download PDFInfo
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- CN100368432C CN100368432C CNB2005100340926A CN200510034092A CN100368432C CN 100368432 C CN100368432 C CN 100368432C CN B2005100340926 A CNB2005100340926 A CN B2005100340926A CN 200510034092 A CN200510034092 A CN 200510034092A CN 100368432 C CN100368432 C CN 100368432C
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Abstract
The present invention takes hyphae of magnaporthe grisea as materials to obtain coarse exciton by culture freeze thawing, homogenate centrifuging and PM-10 film ultrafiltration. After the exciton is in freeze thawing, novel glucoprotein exciton of rice blast bacteria is obtained by series of chromatography and purification, the molecular weight of the exciton is 49.08kDa, and the isoelectric point is 6.01 to achieve silver staining electrophoresis purity. The exciton which is identified by mass spectrometry is hypothetical protein MG05155.4 of open reading frame hypothetical codes of rice blast bacteria MG05155.4. The property of the exciton is stable, the raised enzyme activity of phenylalnine ammonialyase in an induced non-compatible interactive rice line is obviously higher than an induced compatible interactive rice line, and raised lowest effective concentration of enzyme activity of peroxidase of induced rice is 1.5 nmol/L. The research has good application prospects in biological control of rice blast, and can provide new references for developing engineering strains for broad-spectrum disease resistance and harmless insecticide.
Description
Technical field
The present invention relates to plant protection and biological technical field, relate in particular to the purifying that a kind of new Pyricularia oryzae glycoprotein excites son.
Background technology
Rice blast (rice blast disease) is caused by ascomycetes Magnaporthe grisea (Hebert) Barr (its asexual generation is Pyricularia grisea (Cooke) Sacc.), is distributed widely in the countries and regions of rice cropping.It is one of paddy rice three big diseases, seriously limits rice high yield, stable yields, high-quality.Rice blast all has generation in various degree throughout the year, general underproduction 10-20% of popular time, and serious reaches more than 5 0%.Since the nineties, area having taken place all more than 3,800,000 hectares China's rice blast year, loses paddy every year and reach several hundred million kilograms.
At present rice blast mainly being taked breeding resistant variety and chemical prevention is main prophylactico-therapeutic measures.Long-term production facts have proved that the seed selection of the anti-pest kind of paddy rice and utilization are the effective measures of control rice blast.But because the genetic background complexity of Pyricularia oryzae, be easy to variation, and the relative simplification of anti-pest kind, the genetic homogeneity of main breed, make breeding for disease resistance be difficult to catch up with the speed of mutation of pathogenic physiological races of rice blast fungus, usually cause a new rice varieties just seriously susceptible behind establishing in large scale 3-5 with anti-rice blast.Chemical prevention usually causes pesticide residue and environmental pollution in the rice, constantly cause people's attention, therefore, disclose the essence of rice anti-rice blast from profound level, seeking more effective, safe, prophylactico-therapeutic measures economically and reasonably is a vital task that urgency is to be solved.Be subjected to the exciton research of extensive concern in recent years, for control rice blast provides new approach.
Exciton is the signaling molecule that can inducing plant produces defense response.Do mutually in the system plant and pathogen, plant combines with the pathogen exciton by the acceptor molecule in cell surface or the ubcellular surface component, starts the cascade signal system, regulation and control defence genetic expression, and final performance is to the disease resistance of pathogen.The exciton inducing anti-disease has characteristics such as pollution-free, disease-resistant spectrum is wide, longer duration, has broad application prospects in the control of rice blast undoubtedly.
Glycoprotein is the important composition composition of fungal cell wall, and the glycoprotein that obtains in the Pyricularia oryzae hyphal cell wall has the exciton activity.At present relevant Pyricularia oryzae glycoprotein excites the research report of son to have: Kanoh etc. (1993) utilize methods such as Celite535 filtration, DEAE-Toyo PEARL650M ion exchange column, gel permeation chromatography, to obtain a kind of molecular weight be that the glycoprotein of 25kDa excites son to purifying from Pyricularia oryzae, can induced activity oxygen set out; Schaffrath etc. (1995) utilize methods such as ultrafiltration, affinity chromatography purifying from Pyricularia oryzae to obtain molecular weight and excite son for the glycoprotein of 15.6kDa, and avtive spot is a glycosyl group; Li Yunfeng etc. (2004) purifying acquisition molecular weight from Pyricularia oryzae is that the glycoprotein of 101.2kDa excites son.Different with these excitons of having reported, it is 49.08kDa that our purifying from Pyricularia oryzae hyphal cell wall obtains a kind of new molecular weight, and iso-electric point is that 6.01 glycoprotein excites son, and its character and identification of proteins are studied.
Summary of the invention
The research that at present domestic relevant Pyricularia oryzae glycoprotein excites son is not seen other report as yet except that this research department.We are from Pyricularia oryzae ZC
13Through serial chromatography, it is that 49kDa, iso-electric point are that 6.01 new glycoprotein excites son that purifying obtains a kind of molecular weight in the microspecies hyphal cell wall extracting solution, and (Chinese name: Pyricularia oryzae glycoprotein excites sub-MGJ1 to called after MGJ1; The Chinese phonetic alphabet: Dao wen bing jun tang dan baiji fa zi MGJ1; English name: Glycoprotein elicior MGJ1 isolated fromMaganporthe grisea), identify it is the hypothetical protein MG05155.4 of the MG05155.4 open reading frame supposition coding in the Pyricularia oryzae 70-15 karyomit(e) III contig 2954 through mass spectrum.℃ preservation biological activity free of losses half a year of this exciton liquid sample-20, the disease-resistant relevant enzyme that can induce different affinities to make rice leaf is mutually lived and is strengthened.We are intended to provide new foundation for the engineering strain and the public nuisance-free agricultural chemicals of exploitation broad-spectrum disease resistance to the research of this exciton.
Pyricularia oryzae glycoprotein excites MGJ1 and the purification process thereof in the son
(1) extraction of the thick exciton of Pyricularia oryzae
Activatory Pyricularia oryzae mycelium was carried out liquid culture 8-10 days, filtered through gauze, distilled water repetitive scrubbing 3 times, frozen under-20 ℃.With frozen mycelia multigelation 5-7 time, broken hyphal cell.With the mycelia of distilled water suspension freeze thawing, with high-speed tissue mashing machine 12,000r/min homogenate 5-7 time repeatedly.With homogenate magnetic stirrer 10-12 hour, centrifuging and taking supernatant liquor.With PM-10 membrane ultrafiltration supernatant liquor, the concentrated solution that is obtained is thick exciton.Thick exciton further uses protein chromatographic system (can use the full-automatic protein chromatographic of the AKTA Purifier-100 system of Amersham Biosiciences) to carry out following chromatographic step after freeze thawing 1-2 time.
(2) DEAE-Sepharose FF ion-exchange chromatography
With 2 times of column volumes of the initial damping fluid balance of the 20mmol/LTis-HCl of pH7.4 HiPrep 16/10 DEAE FF ion exchange column, flow velocity 3mL/min, with sample on the thick exciton, not in conjunction with 2 times of column volumes of wash-out, with 10 times of column volumes of 0.75mol/LNaCl gradient elution, detect (A280: the special absorption peak of albumen) at the A280 wave band.Collect each peak, the ultra-filtration centrifuge tube concentrating and desalinating carries out 4 leaf inoculations of rice seedling peroxidase and surveys than biopsy, gets active peak, and-20 ℃ of preservations are standby.
(3) ConA-Sepharose 4B affinity column chromatography
Dress The addition of C onA-Sepharose 4B (Pharmacia) filler contains 0.15mmol/LNaCl, 1mmol/LMgCl with pH7.4 in the XK16/20 void column
2, 1mmol/LCaCl
2, 1mmol/LMnCl
2The initial damping fluid balance of 20mmolTris-HCl, will be on the activated protein peak sample that the DEAE ion-exchange chromatography obtains sample, the D-seminose that adds 0.15mmol/L in above-mentioned initial damping fluid carries out gradient elution, collect each peak, the ultra-filtration centrifuge tube concentrating and desalinating, carry out 4 leaf inoculations of rice seedling peroxidase and survey than biopsy, get active peak ,-20 ℃ of preservations are standby.
(4) the S-100 gel filtration chromatography obtains MGJl Pyricularia oryzae glycoprotein and excites son
The initial damping fluid balance of 20mmo/LlTi s-HCl Sephacryl S-100 HighResolution gel column with pH7.4, will be on the activated protein peak sample that ConA-Sepharose 4B affinity column chromatography obtains sample, 1.5 times of column volumes of wash-out, collect each peak, the ultra-filtration centrifuge tube concentrating and desalinating, carry out 4 leaf inoculations of rice seedling peroxidase and survey, get active peak, be Pyricularia oryzae glycoprotein in-20 ℃ of preservations after the freeze-drying and excite sub-MGJ1 than biopsy.
Excite sub-MGJ1 to carry out determining the protein quantity (Bradford method) to this new Pyricularia oryzae glycoprotein that is obtained, sugar assay (anthrone-vitriol oil method), purity is identified (dyeing of SDS-PAGE electrophoresis silver and Periodic acid-Schiff sugar dyeing), mass spectrum identification of proteins and the mensuration of inducing host rice disease resistance (to inducing of peroxidase and phenylalanine ammonia lyase).
Pyricularia oryzae glycoprotein excites the feature of the MGJ1 in the son
Measure with mass spectrograph (Applied Biosystems Voyager System 4307), it is 49.08kDa that Pyricularia oryzae glycoprotein excites the relative molecular weight of sub-MGJ1, is 6.01 through its iso-electric point of thin layer isoelectric focusing cataphoretic determination.Obtain the peptide finger printing through mass spectrum (Finnigan LTQ) test, in the Pyricularia oryzae Protein Data Bank, analyze, identify that it is the hypothetical proteinMG05155.4 of the MG05155.4 open reading frame supposition coding in the Pyricularia oryzae 70-15 karyomit(e) III contig 2954 that this glycoprotein excites sub-MGJ1, total number of atnino acid is 449, exciton MGJ1 and the hypothetical proteinMG05155.4 eclipsed peptide section total mass number that is complementary is 1547.7, accounts for 32.58% of hypothetical proteinMG05155.4 total mass number.This exciton is to C101A51 (non-affine mutual work fully), C101LAC (highly non-affine mutual work), disease-resistant relevant enzyme phenylalanine ammonia lyase (PAL) in C104PKT (the non-affine mutual work of moderate) and 4 near isogenic line rice leafs of CO39 (affine mutual work) all has inducing action, induce and fully non-ly affinely do the increment of living of C101A51 and the highly non-affine PAL enzyme of making the C101LAC strain mutually mutually and be respectively 127.59% and 121.945%, affinely do the increment of living of the non-affine PAL enzyme of making the C104PKT strain mutually of CO39 and moderate mutually and be respectively 105.38% and 91.72% and induce, induce non-affinity to do the rising of PAL in the rice strain mutually apparently higher than affinity is done inducing in the rice strain mutually.
This exciton inducing paddy rice POD enzyme minimal effective concentration that raises alive is 1.5nmol/L.℃ active free of losses of preservation exciton half a year of this exciton liquid sample-20, its stable in properties.
Purifying Pyricularia oryzae glycoprotein of the present invention excites the advantage of sub-MGJ1
1, the present invention is a raw material with the Pyricularia oryzae mycelia, and raw material sources are easily cultivated, price is honest and clean, is fit to Application and Development.
2, the present invention adopts protein purification chromatography platform (AKTA Purifier-100), and steps such as last sample, wash-out, collection are carried out automatically, avoid artificially waiting external influence factor, and the purifying highway route design is reasonable, and circulation ratio is stable.
3, to induce the host rice POD enzyme enhanced minimal effective concentration of living be 1.5nmol/L to the MGJ1 exciton of purifying of the present invention, the induced activity height.Liquid sample-20 ℃ active the free of losses of preservation exciton half a year, stable in properties is beneficial to and preserves and application and development.
Description of drawings
Fig. 1: Pyricularia oryzae glycoprotein excites sub-MGJ1 DEAE-Sepharose FF ion-exchange chromatography figure
Fig. 2: Pyricularia oryzae glycoprotein excites sub-MGJ1 ConA-Sepharose 4B affinity column chromatography figure
Fig. 3: Pyricularia oryzae glycoprotein excites sub-MGJ1 Sephacryl S-100 gel filtration chromatography figure
Fig. 4: Pyricularia oryzae glycoprotein excites the SDS-PAGE electrophoresis silver of sub-MGJ1 to dye collection of illustrative plates
Fig. 5: Pyricularia oryzae glycoprotein excites Periodic acid-Schiff method sugar dyeing collection of illustrative plates of sub-MGJ1
Fig. 6: the relative molecular weight collection of illustrative plates of mass spectroscopy exciton MGJ1
Fig. 7: thin layer isoelectric focusing cataphoretic determination Pyricularia oryzae glycoprotein excites the iso-electric point collection of illustrative plates of sub-MGJ1
Fig. 8: Pyricularia oryzae glycoprotein excites the LTQ mass-spectrogram of sub-MGJ1
Fig. 9: exciton MGJ1 does alive the inducing of rice leaf PAL enzyme mutually to different affinities
Figure 10: the exciton MGJ1 of different concns is to inducing that rice leaf POD enzyme is lived
Embodiment
Activatory Pyricularia oryzae mycelium is carried out liquid culture (liquid nutrient medium: yeast extract 5g; DEXTROSE MONOHYDRATE BP 22g; Supply distilled water to 1000mL.), 25 ℃ constant-temperature shaking culture 8-10 days, double gauze filters, distilled water repetitive scrubbing 3 times is frozen under-20 ℃.Frozen mycelia is thawed for 15 ℃-25 ℃, place again-20 ℃ down freezing, multigelation 5-7 time, abundant broken hyphal cell.With the mycelia (5mL/g) of distilled water suspension freeze thawing, with high-speed tissue mashing machine (DS-1 tissue mashing machine) 12,000r/min homogenate 5-7 time repeatedly.Use magnetic stirring apparatus in 4 ℃ of stirrings 10-12 hour homogenate, 10, the centrifugal 30min of 000g gets supernatant liquor.The filter paper filtering supernatant liquor, gained filtrate is used the PM-10 membrane ultrafiltration, high pure nitrogen pressure 0.45MPa, (MW＞10kDa) be thick exciton preserves standby down for one 20 ℃ the concentrated solution that is obtained.Thick exciton after freeze thawing 1-2 time, with full-automatic protein chromatographic system (the AKTA Purifier-100 of AmershamBiosiciences) to the further chromatography purification of thick exciton:
(1) DEAE-Sepharose FF ion-exchange chromatography obtains the active peak of D4
With 2 times of column volumes of initial damping fluid balance HiPrep 16/10 DEAE FF ion exchange column of the 20mmol/LTis-HCl of pH7.4, flow velocity 3mL/min.With sample 4mL on the thick exciton,,, detect (A280: the special absorption peak of albumen) at the A280 wave band with 10 times of column volumes of 0.75mol/LNaCl gradient elution not in conjunction with 2 times of column volumes of wash-out.Elution curve mainly contains 4 tangible peaks (D1, D2, D3, D4), and wherein D1, D2 peak can not be adsorbed by DEAE-Sepharose FF, and D3, D4 peak all can be adsorbed by DEAE-Sepharose FF.Collect each peak, the ultra-filtration centrifuge tube concentrating and desalinating is surveyed than biopsy through 4 leaves of rice seedling inoculation peroxidase, and the D4 peak has strong exciton activity, collects the elutriant (see figure 1) at point place, D4 peak, preserves down that to be the active peak of D4 sample standby for-20 ℃.
(2) ConA-Sepharose 4B affinity column chromatography obtains the active peak of C2
Dress The addition of C onA-Sepharose 4B (Pharmacia) filler is in the XK16/20 void column, with pH7.4, contain 0.15mmol/LNaCl, 1mmol/LMgCl
2, 1mmol/LCaCl
2, 1mmol/LMnCl
2The initial damping fluid balance of 20mmolTris-HCl, will be on the active peak of the D4 sample that the DEAE ion-exchange chromatography obtains sample 2mL, flow velocity 0.2mL/min, the D-seminose that adds 0.15mmol/L in above-mentioned initial damping fluid carries out gradient elution, elution curve mainly contains 2 tangible peaks (C1, C2), and C1, C2 all can not be adsorbed by ConA-Sepharose 4B.Collect each peak, the ultra-filtration centrifuge tube concentrating and desalinating is surveyed than biopsy through 4 leaves of rice seedling inoculation peroxidase, and the C2 peak has strong exciton activity, collects the elutriant (see figure 2) of C2 peak point, preserves down that to be the active peak of C2 sample standby for-20 ℃.
(3) the active peak of S-100 gel filtration chromatography acquisition S1 is that MGJ1 Pyricularia oryzae glycoprotein excites the initial damping fluid balance Sephacryl S-100HighResolution gel column of son with the 20mmo/LlTis-HCl of pH7.4, will be on the activated protein sample that ConA-Sepharose 4B affinity column chromatography obtains sample 1mL, flow velocity 0.5mL/min, 1.5 times of column volumes of wash-out, elution curve mainly contain 2 tangible peaks (S1, S2).Survey than biopsy through 4 leaf inoculations of rice seedling peroxidase, the S1 peak has strong exciton activity, collects the elutriant (see figure 3) at point place, S1 peak, ultrafiltration and concentration, and lyophilize ,-20 ℃ of following preservations promptly obtain new Pyricularia oryzae glycoprotein and excite sub-MGJ1.
The Pyricularia oryzae mycelium obtains thick exciton through steps such as multigelation, homogenate, centrifugal, PM-10 membrane ultrafiltration.Thick exciton obtains new Pyricularia oryzae glycoprotein through DEAE-Sepharose FF ion-exchange chromatography, ConA-Sepharose FF affinity column chromatography, S-100 gel filtration chromatography and excites sub-MGJ1.Excite sub-MGJ1 to carry out the silver dyeing of SDS-PAGE electrophoresis and Periodic acid-Schiff method sugar dyes and all shows single band (seeing Fig. 4, Fig. 5) to the Pyricularia oryzae glycoprotein that is obtained, show that to reach electrophoresis pure.
Through Applied Biosystems Voyager System 4307 mass spectroscopies, the MGJ1 molecular weight is the 49.07968kDa (see figure 6).Iso-electric point through thin layer isoelectric focusing cataphoretic determination MGJl is 6.01 (see figure 7)s.Through LTQ mass spectrometric measurement, peptide fingerprint map analyzing identification of M GJ1 is the hypothetical proteinMG05155.4 (see figure 8) of the MG05155.4 open reading frame supposition coding in the Pyricularia oryzae 70-15 karyomit(e) III contig 2954, MGJ1 and the hypothetical protein MG05155.4 eclipsed peptide section total number of atnino acid that is complementary is 147, account for 32.74% (seeing Table 1, table 2) of total amino acid residue number.
Pyricularia oryzae glycoprotein excites sub-MGJ1 that 4 near isogenic line rice leaf phenylalanine ammonia lyase PAL enzymes work are all had inducing action.MGJ1 induces that fully non-affine to make C101A51 and the highly non-affine PAL enzyme of the making the C101LAC strain mutually increment increasing degree of living mutually bigger, be respectively 127.59% and 121.945%, and induce the affine non-affine PAL enzyme of making the C104PKT strain mutually of CO39 and the moderate increment increasing degree of living of doing mutually less, be respectively 105.38% and 91.72% (see figure 9).
With different concns (1 μ g/mL, 10 μ g/mL, 50 μ g/mL, 100 μ g/mL) Pyricularia oryzae glycoprotein excites sub-MGJ1 to adopt and weighs the rice leaf that the spot method is inoculated the mutual work of different affinities wounded, induce the POD enzyme as shown in figure 10 than the variation of living, the result shows, the rising that the MGJ1 exciton of 10 μ g/mL and above concentration all can induce the rice leaf POD enzyme of different strains to live, and rising along with exciton concentration, induced activity strengthens, l μ g/mL concentration and following MGJ1 thereof handle then the rising of can not inducing paddy rice blade POD enzyme living, and infer that the effective concentration of MGJ1 exciton induced activity is 1.5nmol.Pyricularia oryzae glycoprotein excites son, and the MGJ1 liquid sample is preserved the active free of losses of exciton half a year down at-20 ℃, and its stable in properties is beneficial to and preserves and application and development.
Table 1: exciton MGJ1 and the hypothetical protein MG05155.4 overlapping peptide piecewise analysis that is complementary
The aminoacid sequence table of table 2:hypothetical protein MG05155.4
Table 1
| 1 | MAAPAHKFKV | ADLSLAAFGR | KEIELAENEM | PGLMQTREKY | AADQDQKGAR |
| 51 101 151 201 251 301 351 401 | IAGCLHMTIQ FAWKGETEEE ADCYGVSEET SLVDGIKRAT NALQAAMAGF NIGHFDIEID GCATGHSSFV YVLKILDEE | TAVLIETLTA YNWCLEQQLL TTGVHHLYKM DVMIAGKIAV QVTTMEKAAS VAWLKANAKS MSCSFTNQVL VAKLHLAHCN | LGAEVTWTSC AFKDGKKLNL LKEGKLLVPA VAGYGDVGKG VGQIFVTTTG VQNIKPQVDR AQIMLYKNND VELSTLTPVQ | NIFSTQDHAA ILDDGGDLTH INVNDSVTKS CAMALHGMGA CRDILVGKHF FLMANGRHII AAFGQKYVEF AEYLSLPAEG | AAIAAAGVPV LVHDKYPEML KFDNLYGCRE RVIVTEIDPI EAMPNDAIVC LLAEGRLVNL AKSGKLEKKV PYKPEHYRY |
Table 2 sequence table
＜110〉Agricultural University Of South China
＜120〉a kind of new Pyricularia oryzae glycoprotein excites son and purification process thereof
<130> MG
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 449
<212> PRT
<213> Magnaporthe grisea
<400> 1
Met Ala Ala Pro Ala His Lys Phe Lys Val Ala Asp Leu Ser Leu Ala
1 5 10 15
Ala Phe Gly Arg Lys Glu Ile Glu Leu Ala Glu Asn Glu Met Pro Gly
20 25 30
Leu Met Gln Thr Arg Glu Lys Tyr Ala Ala Asp Gln Pro Leu Lys Gly
35 40 45
Ala Arg Ile Ala Gly Cys Leu His Met Thr Ile Gln Thr Ala Val Leu
50 55 60
Ile Glu Thr Leu Thr Ala Leu Gly Ala Glu Val Thr Trp Thr Ser Cys
65 70 75 80
Asn Ile Phe Ser Thr Gln Asp His Ala Ala Ala Ala Ile Ala Ala Ala
85 90 95
Gly Val Pro Val Phe Ala Trp Lys Gly Glu Thr Glu Glu Glu Tyr Asn
100 105 110
Trp Cys Leu Glu Gln Gln Leu Leu Ala Phe Lys Asp Gly Lys Lys Leu
115 120 125
Asn Leu Ile Leu Asp Asp Gly Gly Asp Leu Thr His Leu Val His Asp
130 135 140
Lys Tyr Pro Glu Met Leu Ala Asp Cys Tyr Gly Val Ser Glu Glu Thr
145 150 155 160
Thr Thr Gly Val His His Leu Tyr Lys Met Leu Lys Glu Gly Lys Leu
165 170 175
Leu Val Pro Ala Ile Asn Val Asn Asp Ser Val Thr Lys Ser Lys Phe
180 185 190
Asp Asn Leu Tyr Gly Cys Arg Glu Ser Leu Val Asp Gly Ile Lys Arg
195 200 205
Ala Thr Asp Val Met Ile Ala Gly Lys Ile Ala Val Val Ala Gly Tyr
210 215 220
Gly Asp Val Gly Lys Gly Cys Ala Met Ala Leu His Gly Met Gly Ala
225 230 235 240
Arg Val Ile Val Thr Glu Ile Asp Pro Ile Asn Ala Leu Gln Ala Ala
245 250 255
Met Ala Gly Phe Gln Val Thr Thr Met Glu Lys Ala Ala Ser Val Gly
260 265 270
Gln Ile Phe Val Thr Thr Thr Gly Cys Arg Asp Ile Leu Val Gly Lys
275 280 285
His Phe Glu Ala Met Pro Asn Asp Ala Ile Val Cys Asn Ile Gly His
290 295 300
Phe Asp Ile Glu Ile Asp Val Ala Trp Leu Lys Ala Asn Ala Lys Ser
305 310 315 320
Val Gln Asn Ile Lys Pro Gln Val Asp Arg Phe Leu Met Ala Asn Gly
325 330 335
Arg His Ile Ile Leu Leu Ala Glu Gly Arg Leu Val Asn Leu Gly Cys
340 345 350
Ala Thr Gly His Ser Ser Phe Val Met Ser Cys Ser Phe Thr Asn Gln
355 360 365
Val Leu Ala Gln Ile Met Leu Tyr Lys Asn Asn Asp Ala Ala Phe Gly
370 375 380
Gln Lys Tyr Val Glu Phe Ala Lys Ser Gly Lys Leu Glu Lys Lys Val
385 390 395 400
Tyr Val Leu Pro Lys Ile Leu Asp Glu Glu Val Ala Lys Leu His Leu
405 410 415
Ala His Cys Asn Val Glu Leu Ser Thr Leu Thr Pro Val Gln Ala Glu
420 425 430
Tyr Leu Ser Leu Pro Ala Glu Gly Pro Tyr Lys Pro Glu His Tyr Arg
435 440 445
Tyr
Claims (2)
1. Pyricularia oryzae glycoprotein excites sub-MGJ1, it is characterized in that: this exciton relative molecular weight is 49.08kDa, its iso-electric point is 6.01, identify that through mass spectrum this exciton is the hypothetical protein MG05155.4 of the MG05155.4 open reading frame supposition coding in the Pyricularia oryzae 70-15 karyomit(e) III contig 2954, total number of atnino acid is 449, exciton MGJ1 and the hypothetical protein MG05155.4 eclipsed peptide section total mass number that is complementary is 15947.7, account for 32.58% of hypothetical protein MG05155.4 total mass number, exciton MGJ1 and the hypothetical protein MG05155.4 eclipsed peptide section that is complementary is represented with italic:
1 MAAPAHKFKV ADLSLAAFGR KEIELAENEM PGLMQTREKY AADQPLKGAR
51 IAGCLHMTIQ TAVLIETLTA LGAEVTWTSC NIFSTQDHAA AAIAAAGVPV
101 FAWKGETEEE YNWCLEQQLL AFKDGKKLNL ILDDGGDLTH LVHDKYPEML
151 ADCYGVSEET TTGVHHLYKM LKEGKLLVPA INMNDSVTKS KFDNLYGCRE
201 SLVDGIKRAT DVMIAGKIAV VAGYGDVGKG CAMALHGMGA RVIVTEIDPI
251 NALQAAMAGF QVTTMEKAAS VGQIFVTTTG CRDILVGKHF EAMPNDAIVC
301 NIGHFDIEID VAWLKANAKS VQNIKPQVDR FLMANGRHII LLAEGRLVNL
351 GCATGHSSFV MSCSFTNQVL AQIMLYKNND AAFGQKYVEF AKSGKLEKKV
401 YVLPKILDEE VAKLHLAHCN VELSTLTPVQ AEYLSLPAEG PYKPEHYRY
2. the described Pyricularia oryzae glycoprotein of claim 1 excites the purification process of sub-MGJ1, it is characterized in that: activatory Pyricularia oryzae mycelium liquid was cultivated 8-10 days, the distilled water repetitive scrubbing is removed impurity, through 15-25 ℃ with-20 ℃ of multigelations to complete broken hyphal cell, mycelia with distilled water suspension freeze thawing, with high-speed tissue mashing machine 12,000r/min homogenate 5-7 time repeatedly, with homogenate magnetic agitation 10-12 hour, 4 ℃ of centrifuging and taking supernatant liquors, supernatant liquor PM-10 membrane ultrafiltration, the concentrated solution that obtains is thick exciton; After thick exciton freeze thawing 1-2 time, last sample HiPrep 16/10 DEAE FF ion exchange column, with 2 times of column volumes of the initial damping fluid balance of the 20mmol/LTis-HCl of pH7.4, flow velocity 3mL/min, not in conjunction with 2 times of column volumes of wash-out, with 10 times of column volumes of 0.75mol/LNaCl gradient elution, collect the elutriant at the 4th peak-to-peak point place in the elution curve, ultrafiltration and concentration obtains active peak sample; Active peak sample is further gone up sample ConA-Sepharose 4B affinity column, with pH7.4, contain 0.15mmol/LNaCl, 1mmol/LMgCl
2, 1mmol/LCaCl
2, 1mmol/LMnCl
2The initial damping fluid balance of Tris-HCl of 20mmol, the D-seminose that adds 0.15mmol/L in above-mentioned initial damping fluid carries out gradient elution, collects the elutriant at second peak-to-peak point place in the elution curve, ultrafiltration and concentration obtains active peak sample; Active peak sample is further gone up sample SephacrylS-100 High Resolution gel column, initial damping fluid balance with the 20mmo/L1Tis-HCl of pH7.4,1.5 times of column volumes of wash-out, collect the elutriant at first peak-to-peak point place in the elution curve, ultrafiltration and concentration is preserved down in-20 ℃ after the freeze-drying.
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| CN1471356A (en) * | 2000-09-07 | 2004-01-28 | �ձ��̲ݲ�ҵ��ʽ���� | Disease-resistant plants and method of constructing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1471356A (en) * | 2000-09-07 | 2004-01-28 | �ձ��̲ݲ�ҵ��ʽ���� | Disease-resistant plants and method of constructing the same |
Non-Patent Citations (2)
| Title |
|---|
| 稻瘟菌GP66激发子诱导的水稻膜脂过氧化及其保护酶活性变化. 李云锋等.植物病理学报，第35卷第1期. 2005 * |
| 稻瘟菌细胞壁激发子活性物质的提取与性质. 李云锋等.华中农业大学学报，第23卷第3期. 2004 * |
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