CN1721433A - 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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- CN1721433A CN1721433A CN 200510034092 CN200510034092A CN1721433A CN 1721433 A CN1721433 A CN 1721433A CN 200510034092 CN200510034092 CN 200510034092 CN 200510034092 A CN200510034092 A CN 200510034092A CN 1721433 A CN1721433 A CN 1721433A
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Abstract
The present invention first obtains coarse exciton with rice blast germ mycelium as material and through culturing, freezing-thawing, homogenating, centrifuging and PM-10 membrance ultrafiltering, and then obtain the new rice blast germ glucoprotein exciton through further freezing-thawing and serial chromatographic purifications. The rice blast germ glucoprotein exciton has molecular weight 49.08 kDa, isoelectric point 6.01 and silver staining electrophoresis purity. Mass spectroscopic identification shows that the exciton is rice blast germ MG05155.4 open reading frame coded hypothetical protein MG05155.4. The exciton has stable property, relatively high induction on Phe deaminase activity in non-compatible interactive rice line and lowest effective concentration of 1.5 nmol/L for inducing rice peroxidase. The present invention has excellent application foreground in the biological prevention and control of rice blast.
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 50%.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 elicitor 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 MGJ1 Pyricularia oryzae glycoprotein and excites son
With the initial damping fluid balance of the 20mmo/LlTis-HCl of pH7.4 Sephacryl S-100 HighResolution gel column, 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 15947.7, 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.
Specific implementation method
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-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 son
Initial damping fluid balance Sephacryl S-100 HighResolution gel column 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 MGJ1 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 disconnected peptide section total number of atnino acid of eclipsed 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, 1 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
Figure of description
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 X-coordinate Mass (m/z) of mass spectroscopy exciton MGJ1 is a mass-to-charge ratio, and ordinate zou Intensity is an intensity.
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: it is the time that Pyricularia oryzae glycoprotein excites the LTQ mass-spectrogram X-coordinate Time of sub-MGJ1, and ordinate zou Relative is a relative abundance.
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
Table 1
1 MAAPAHKFKV ADLSLAAFGR KEIELAENEM PGLMQTREKY AADQPLKGAR
51 IAGCLHMTIQ TAVLIETLTA LGAEVTWTSC NIFSTQDHAA AAIAAAGVPV
101 FAWKGETEEE YNWCLEQQLL AFKDGKKLNL ILDDGGDLTH LVHDKYPEML
151 ADCYGVSEET TTGVHHLYKM LKEGKLLVPA INVNDSVTKS 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
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, a kind of new Pyricularia oryzae glycoprotein excites the purification process of son, 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 16/10DEAE 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 Sephacryl S-100High 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, promptly obtain new Pyricularia oryzae glycoprotein in-20 ℃ of following preservations after the freeze-drying and excite son, called after MGJ1.
2, Pyricularia oryzae glycoprotein excites sub-MGJ1 to be characterised in that: respectively through SDS-PAGE silver dye and Periodic acid-dyeing of Schiff sugar all shows single band, it is pure that the Pyricularia oryzae glycoprotein that shows acquisition excites sub-MGJ1 to reach electrophoresis; This exciton relative molecular weight is 49.08kDa, its iso-electric point is 6.01, identify that through mass spectrum this exciton is the hypotheticalprotein 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?INVNDSVTKS?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
℃ preservation biological activity free of losses half a year down of this exciton liquid sample-20, stable in properties; It is 1.5nmol/L that this exciton is induced the disease-resistant relevant enzyme peroxidase of the host rice enzyme minimal effective concentration that raises alive; This exciton is lived to the disease-resistant relevant enzyme phenylalanine ammonia lyase PAL enzyme of 4 near isogenic line rice leafs all 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, promptly Pyricularia oryzae glycoprotein excites sub-MGJ1 to induce non-affinity to do the PAL enzyme is lived in the rice strain rising mutually apparently higher than affinity is done inducing in the rice strain mutually.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558320A (en) * | 2010-12-17 | 2012-07-11 | 中国农业科学院植物保护研究所 | Protein polynucleotide with separated verticillium dahliae and application thereof |
| CN107540733A (en) * | 2017-09-05 | 2018-01-05 | 华南农业大学 | A kind of Pyricularia oryzae exciton Mo65 and its purification process |
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| JP2002325519A (en) * | 2000-09-07 | 2002-11-12 | Japan Tobacco Inc | Disease-resistant plant and method for creating the same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558320A (en) * | 2010-12-17 | 2012-07-11 | 中国农业科学院植物保护研究所 | Protein polynucleotide with separated verticillium dahliae and application thereof |
| CN102558320B (en) * | 2010-12-17 | 2014-03-26 | 中国农业科学院植物保护研究所 | Protein polynucleotide with separated verticillium dahliae and application thereof |
| CN107540733A (en) * | 2017-09-05 | 2018-01-05 | 华南农业大学 | A kind of Pyricularia oryzae exciton Mo65 and its purification process |
| CN107540733B (en) * | 2017-09-05 | 2020-10-23 | 华南农业大学 | Rice blast bacterium exciton Mo65 and purification method thereof |
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