CN100352833C - Polypeptide for suppressing weed seed germination and rootage, and separation method and uses - Google Patents
Polypeptide for suppressing weed seed germination and rootage, and separation method and uses Download PDFInfo
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- CN100352833C CN100352833C CNB2005101047862A CN200510104786A CN100352833C CN 100352833 C CN100352833 C CN 100352833C CN B2005101047862 A CNB2005101047862 A CN B2005101047862A CN 200510104786 A CN200510104786 A CN 200510104786A CN 100352833 C CN100352833 C CN 100352833C
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Abstract
The present invention discloses a polypeptide for inhibiting the germination and the rootage of weed seeds, a separation method and the application thereof. The present invention relates to a polypeptide, particularly to a polypeptide for inhibiting the germination and the rootage of weed seeds, a separation method and the application of the polypeptide for inhibiting the germination and the rootage of weed seeds to the preparation of inhibitors or weed killers of the germination and the rootage of weed seeds. An amino acid sequence of the polypeptide is Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala. During separation, zein powder is collected and pulverized, and then undersized powder is used as enzymolysis substrate; alkali protease is added for hydrolysis to obtain hydrolyzate, ion exchange chromatography is carried out on the hydrolyzate to obtain active constituents; gel chromatography is carried out to obtain active constituents of zein powder polypeptide with the high inhibition of the germination and the rootage of weeds. Reversed-phase high-performance liquid chromatography is carried out for two times so as to filter out high inhibition active constituents. The polypeptide has the activity for inhibiting the germination and the rootage of weeds and can be used for preparing inhibitors or weed killers of the germination and the rootage of weed seeds.
Description
Technical field
The present invention relates to a peptide species, especially relate to a kind of inhibition suppressing weed seed germination take root polypeptide and separation method and suppress the suppressing weed seed germination polypeptide of taking root and give birth to inhibitor or herbicide applications at the preparation suppressing weed seed germination.
Background technology
Being extensive use of for the control weeds of chemical herbicide brought into play vital role to the intrusion and the harm of farmland and urban lawn.Publication number is that the application for a patent for invention of CN1181184A discloses a kind of high effective weeding composition for non-cultivating land, be a kind of contain to weed seed, young shoot, young grass have killing action ethyl methyl and to weeds on the ground green portion the composite series of high efficiency herbicidal composition that form such as the Paraquat of killing action, cogongrass are sticking, hexazinone are arranged.Publication number is that the application for a patent for invention of CN1687086A discloses a kind of substituted benzene oxygen acetoxy fragrant heterocyclic radical hydrocarbon phosphinate Salt And Preparation Method with bactericidal and herbicidal activity.Publication number is that the application for a patent for invention of CN1631156 discloses a kind of weedicide, it is by N-(phosphono-methyl) glycine, 1,1-dimethyl-4,4 '-dipyridyl sun is from 5-triazine-2-yl)-N-methyl ammonia phosphinylidyne] aminosulfonyl] methyl benzoate, 3-sec.-propyl-2,1,3-benzothiadiazine ketone-(4)-Li forms.Publication number has optionally herbicidal composition for the application for a patent for invention of CN1679396 provides to cereal crop.Yet a large amount of uses of traditional chemical agricultural chemicals cause the resistance of weeds and cause environmental pollution, increase year by year as the chemical herbicide in soil, underground water and the agricultural-food is residual, have endangered the safety of the mankind and animal.Therefore, develop natural, have no side effect, degradable weedicide has important ecological benefits and application prospects.Early 1990s, the gardening scholar N.E Christians (.Liu of American I woa state university, D.L.-Y.and N.E.Christians.Isolation and identification of root-inhibiting compounds fromcorn gluten hydrolysate.J.Plant Grown Regul., 1994, the formation of root system when 13:227-230) the discovery Zein powder can suppress plant seed germination, and develop Zein powder and sprout preceding weedicide, but Zein powder is a pressed powder, water insoluble, thereby it can not resemble and can spray and be easy to infiltrate soil performance effect the liquid herbicide, N.E.Christans (.Christians afterwards, N.E., J.T.Garbutt and D.Liu.Preemergence weed control using plant proteinhydrolysate.U.S.Patent No.5,290,749,1994a) wait the hydrolyzate of human Hydrolysis of Corn Gluten Meal by Alkaline Protease powder preparation higher weeding activity to be arranged than Zein powder, and and then separation and purification go out the weeding activity peptide monomer, weeding activity experiment in the laboratory culture ware shows that the restraining effect that the weeding activity peptide monomer is taken root to plant seed germination than Zein powder hydrolysis crude extract is more remarkable.They from the hydrolyzate of Zein powder, isolate 5 have weeding activity 2 peptides and 5 peptides that weeding activity is stronger, and two patents of SEPARATE APPLICATION.The sequence of these polypeptide is respectively: Gln-Gln, Ala-Asa, Ala-Gln, Gly-Ala, Ala-Ala and Leu-Ser-Pro-Ala-Gln.The weeding mechanism of active polypeptide is to suppress the formation of seed root system by the merismatic mitotic division of blocking-up seed root system, no matter this restraining effect is dicotyledons weeds or monocotyledon weed all produces effect, to then not influence of the ripe root system after sprouting, and environment do not produced any contamination and can be used as the source of manure of lawn plant.Their research is also inferred, also may exist other to have the polypeptide monomer of weeding activity in the corn hydrolysate.But after this, the progress of this research field is but very slow.
Summary of the invention
The object of the present invention is to provide a kind of inhibition suppressing weed seed germination polypeptide of taking root; Another object of the present invention is to provide a kind of inhibition suppressing weed seed germination polypeptide separation method of taking root; Another object of the present invention is to the inhibition suppressing weed seed germination polypeptide of taking root is used to prepare weed germination take root inhibitor or weedicide.
Inhibition suppressing weed seed germination of the present invention its aminoacid sequence of polypeptide of taking root is:
Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala，
Its molecular weight is 970.6855, and theoretical iso-electric point is 5.52, is white powder, and is soluble in water, can be made into different concns and spray soil and come the management of weeds seed germination.
The take root separation method of polypeptide of inhibition suppressing weed seed germination of the present invention the steps include:
1) the raw material Zein powder is collected undersized pale yellow powder after crushed as the enzymolysis substrate;
2) in Zein powder, add hydrolysis by novo and get hydrolyzed solution, the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: (0.03～0.05AU), pH is 7～9, and hydrolysis temperature is 50～70 ℃, and hydrolysis time is 7～8h;
3) hydrolyzed solution separates obtaining active ingredient through ion exchange chromatography;
4) active ingredient that separation is obtained gets the high inhibition suppressing weed seed germination polypeptide active component of taking root through gel chromatography;
5) height that separation is obtained suppresses suppressing weed seed germination and takes root the polypeptide active component through twice RPLC, filters out to have the high active component that suppresses, and through measuring, its sequence is Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala.
In step 1), the most well after crushed 200 mesh sieves of raw material Zein powder.
In step 2) in, the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.042AU, and pH is 8.0, and hydrolysis temperature is 60 ℃, and hydrolysis time is 6h, Sumizyme MP is selected from Alacalase 2.4L.
The inhibition suppressing weed seed germination of the present invention polypeptide of taking root has high weed germination take root activity or the weeding activity of suppressing, and can be used for preparing weed germination take root inhibitor or weedicide.
Compare with the aminoacid sequence of the zein of having reported and show by the inhibition suppressing weed seed germination that the present invention the is obtained polypeptide of taking root, the polypeptide that the present invention obtains does not all come from zein, but come from other albumen, and this peptide sequence has not yet to see report.
Embodiment
Following examples will the present invention is further illustrated.
Embodiment 1
1) with the zein is raw material, it is bigger brown platy shaped particle, enzyme-to-substrate can fully contact during for the ease of enzyme digestion reaction, raw material carries out pre-treatment as follows: the raw material Zein powder is crossed 200 mesh sieves after pulverizer is pulverized, collect undersized pale yellow powder as the enzymolysis substrate.
2) in Zein powder, add hydrolysis by novo, the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.042 AU, pH are 8.0, and hydrolysis temperature is 60 ℃, hydrolysis time is 6h, and Sumizyme MP is selected from Alacalase 2.4L.
3) hydrolyzed solution is through ion exchange chromatography, ion-exchange packing is QAE-A25, processing step is: filler pre-treatment → dress post → last sample → gradient elution, processing condition are: (1) filler pre-treatment: take by weighing 7g QAE-A25 anionite-exchange resin powder in beaker, the elutriant A that adds certain volume, swelling 2h in boiling water bath, outwell buoyant fine particle on the liquid level after the cooling, clean repeatedly several times with elutriant A, be made into 75% suspension, it is standby to vacuumize the bubble of catching up with in the suspension then in vacuum chamber.(2) dress post: with chromatography column (C16/40, internal diameter 16mm, long 40cm) vertically is put on the iron stand after cleaning up with deionized water, adds a certain amount of elutriant A, open water outlet to drive the bubble of bottom away, when the damping fluid of column bottom has 2cm high approximately, close chromatography column bottom water outlet.With disposable the pouring in the chromatography column of swelling gel suspension good, that stir, connect peristaltic pump then rapidly, open water outlet, with the flow velocity of 2mL/min elutriant A is pumped into and to make the rapid sedimentation of filler in the chromatography column, treat to use the flow velocity of 5mL/min to push filler again after the whole sedimentations of filler fully, treat that the post bed height no longer changes the back termination of pumping, connects Adaptor and suitably compress rapidly, make bed a surperficial no redundant space and an elutriant, this moment, the bed volume height was about 13cm.With using behind 2 bed volumes of elutriant A balance.(3) go up sample: sample on the peristaltic pump, applied sample amount are 300mg, and last sample speed is 0.5mL/min.(4) gradient elution: elutriant A is 30mmol/LpH8.0Tris-HCl, and the ionic strength of elutriant B is 2mol/LNaCl, ionic strength gradient (NaCl) be 0.024mol/L.min, elution flow rate be 0.9mL/min.
4) separate the height inhibition suppressing weed seed germination that obtains by step 3) and take root the polypeptide active component through gel chromatography, filler is Superdex 30p.g..Processing step is: filler pre-treatment → dress post → last sample → wash-out.Processing condition are: (1) filler pre-treatment: swelling is good in advance in 20% ethanol for filler, therefore only needs in use earlier ethanol is removed and can be used.With cleaning filler repeatedly,, put into vacuum drying oven then and vacuumize and to adorn post until ethanol is removed fully through the filterable deionized water of 0.45 μ m filter membrane.(2) dress post: the chromatography column that the water washed with de-ionized water is clean (XK16/70, internal diameter 16mm, long 70cm) vertically is positioned on the iron stand.In post, add a certain amount of elutriant earlier, the bubble of post bottom is discharged, close column outlet then, and connect Revisior on the top of post.The filler of having handled well is transferred to the uniform suspension liquid of the about 475mL of volume with deionized water, pour in the chromatography column along glass stick is disposable then, rapidly peristaltic pump is connected, with elutriant with the speed of 1mL/min flushing pillar 2h, with the sedimentation of quickening filler and make the uniform filling sedimentation.Treat carefully to take off Revisior after filling settlement fully, connect Adaptor,, treat that the post height gets final product termination of pumping when no longer changing again with the speed punching press pillar of 4mL/min.Adaptor is a little firmly compressed downwards, make it not interspace with tight contact the in bed surface.Can use with two bed volumes of damping fluid balance again.With this method dress post good repeatability is arranged.(3) go up sample: sample on the peristaltic pump, applied sample amount are 2% of bed volume, and last sample speed is 0.5mL/min.(4) wash-out: elutriant is pH6.0 50mol/Lm acetate-ammonium acetate buffer solution, and elution flow rate is 0.9mL/min.
5) separated the height inhibition suppressing weed seed germination that obtains by step 4) and taken root the polypeptide active component through twice RPLC, chromatographic column is Everest C18 reversed-phase column (4.6mm * 250mm, 238TP54), adopt gradient elution, mobile phase A is for being 5% acetonitrile+0.1% trifluoroacetic acid, and B is pure acetonitrile+0.1% trifluoroacetic acid mutually.Chromatographic separation condition is for seeing Table 1.Filter out and have the high active component that suppresses, through measuring, its sequence is Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala.
Table 1 chromatographic separation condition
| Time (min) | A(％) | B(％) | Flow velocity (mL/min) | Temperature (℃) |
| 0.00 2.00 35.00 | 75.0 70.0 55.0 | 25.0 30.0 45.0 | 0.800 0.800 0.800 | 30 30 30 |
Embodiment 2
Similar to Example 1, its difference is that the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.05AU, and pH is 7～9, and hydrolysis temperature is 50 ℃, and hydrolysis time is 8h, and Sumizyme MP is selected from Alacalase2.4L.
Embodiment 3
Similar to Example 1, its difference is that the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.03AU, and pH is 7～9, and hydrolysis temperature is 70 ℃, and hydrolysis time is 7h, and Sumizyme MP is selected from Alacalase2.4L.
Embodiment 4
Below provide and suppress the suppressing weed seed germination polypeptide of taking root and suppress that weed germination is taken root or the detection method and the result of weeding activity.
Get weeds mother chrysanthemum (Dendranthema indicum) seed and be placed on the culture dish that places 6cm on the double-deck filter paper with 75% alcohol sterilization and with behind the distilled water flushing 3 times, add the 1ml deionized water earlier, cover loam cake and cultivate after 5 days, seal cultivation with the Parafilm sealing compound after the polypeptide liquid that adds the 1ml respective concentration is respectively handled in set experiment.Culture condition: illumination every day 16h, 25 ℃ of temperature, intensity of illumination 15001x.Incubation time totally 7 days.Suppress the suppressing weed seed germination polypeptide of taking root and respectively establish 4 concentration and handle, be respectively 0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/l is contrast with the deionized water, each is handled and 20 seeds are selected in contrast for use, repeats 3 times.Incubation time finishes the back and measures in each seedlings root the length of the longest root as statistical standard with electronic digital indicator.
Below provide experimental result and application.
Inhibiting rate is as shown in the formula calculating:
Inhibiting rate (%)=long * 100% all quantitative targets of (control group root length-treatment group root is long)/control group root all use x ± s to represent.
Experimental result sees Table 1, and the polypeptide solution of different concns is taken root to the mother chrysanthemum seed germination all the obvious suppression effect, and polypeptide Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala concentration inhibiting rate when 2mg/ml is about 91%.The formation of root system also had the obvious suppression effect when in addition, further experiment also showed this polypeptide also to 4 kinds of monocotyledonss and 5 kinds of dicotyledons suppressing weed seed germinations.Since the polypeptide thing during to weed germination root system be formed with restraining effect, and to the ripe root system of plant without any influence, therefore weedicide was applied in the farmland before it can be used as a kind of natural sprouting, particularly on the control of weeds of organic conversion hysteria vegetables, urban lawn, important use is arranged, can reduce like this and use chemical herbicide, have good economic benefit, ecological benefits and social benefit the pollution that environment produces.
The polypeptide solution of table 1 different concns is to the mother chrysanthemum seed germination restraining effect of taking root
| Concentration (mg/ml) | Inhibiting rate (%) |
| 0.5 | 69.05±5.33 |
| 1.0 | 82.07±3.60 |
| 1.5 | 87.85±3.60 |
| 2.0 | 100 |
Embodiment 5～13
Below provide the inhibition suppressing weed seed germination of the present invention polypeptide of taking root and suppress monocotyledons Bermuda grass (Cynodondactylon (L.) Pers), light squama Shortleaf Kyllinga Herb (Kyllinga brevifolia Rottb.var.leiolepis (Franch.etSavat.) Hara), Herba Eleusines Indicae (Eleucine indica L.Gaertn) and annual bluegrass (Poa annu L.) and the green amaranth of dicotyledons (Amaranthus viridisL.), Sowthistle Tasselflower Herb (Emilia sonchifolia (L.) DC.), Herba Agerati Conyzoidis (Ageratumconyzoides L), the experimental result of Herba Dichodrae (Dichondra repens Fors) and plumage awns chrysanthemum weeds kinds such as (Tridax procumbens L) sees table 2 for details.
Table 2 2mg/ml polypeptide is taken root to different weed seeds and is suppressed effect (%)
| The weeds kind | Affiliated class | Inhibiting rate (%) |
| Bermuda grass (Cynodon dactylon (L.) Pers) | Monocotyledons | 95±6.29 |
| Light squama Shortleaf Kyllinga Herb (Kyllinga brevifolia Rottb.var.leiolepis (Franch.et Savat.) Hara) | Monocotyledons | 100 |
| Herba Eleusines Indicae (Eleuclne indica L.Gaertn) | Monocotyledons | 90±5.26 |
| Annual bluegrass (Poa annu L.) | Monocotyledons | 100 |
| Green amaranth (Amaranthus viridis L.) | Dicotyledons | 100 |
| A bit (Emilia sonchifolia (L.) DC.) | Dicotyledons | 100 |
| Herba Agerati Conyzoidis (Agera tum conyzoides L) | Dicotyledons | 100 |
| Herba Dichodrae (Dichondra repens Fors) | Dicotyledons | 99±4.26 |
| Plumage awns chrysanthemum (Tridax procumbens L) | Dicotyledons | 100 |
This shows, polypeptide does not produce any side effect to ripe root system because the inhibition suppressing weed seed germination is taken root, no matter and be that dicotyledons or monocotyledonous seed germination are taken root restraining effect is arranged to plant, therefore can be developed into nuisanceless, can degrade sprout preceding weedicide.This product is white powder, and is soluble in water, can be made into different concns and spray soil and come the management of weeds seed germination.
Claims (1)
1, aminoacid sequence is: the inhibition suppressing weed seed germination of Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala is taken root polypeptide in preparation weed germination take root inhibitor or herbicide applications.
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| CNB2005101047862A CN100352833C (en) | 2005-12-30 | 2005-12-30 | Polypeptide for suppressing weed seed germination and rootage, and separation method and uses |
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| CN100352833C true CN100352833C (en) | 2007-12-05 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04164094A (en) * | 1990-10-26 | 1992-06-09 | Oji Koonsutaac Kk | New peptide, its production and angiotensin converting enzyme inhibitor containing the same peptide as active ingredient |
| JPH05331072A (en) * | 1992-05-27 | 1993-12-14 | Agency Of Ind Science & Technol | Prolyl endopeptidase inhibitor |
| CN1314945A (en) * | 1998-08-12 | 2001-09-26 | 麦克西根股份有限公司 | DNA shuffling to produce herbicide selective crops |
| CN1359422A (en) * | 1999-04-29 | 2002-07-17 | 辛甄塔有限公司 | Herbicide resistant plants |
-
2005
- 2005-12-30 CN CNB2005101047862A patent/CN100352833C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04164094A (en) * | 1990-10-26 | 1992-06-09 | Oji Koonsutaac Kk | New peptide, its production and angiotensin converting enzyme inhibitor containing the same peptide as active ingredient |
| JPH05331072A (en) * | 1992-05-27 | 1993-12-14 | Agency Of Ind Science & Technol | Prolyl endopeptidase inhibitor |
| CN1314945A (en) * | 1998-08-12 | 2001-09-26 | 麦克西根股份有限公司 | DNA shuffling to produce herbicide selective crops |
| CN1359422A (en) * | 1999-04-29 | 2002-07-17 | 辛甄塔有限公司 | Herbicide resistant plants |
Non-Patent Citations (1)
| Title |
|---|
| 草坪杂草无公害防治的研究进展 于兴娜等.草业科学，第22卷第5期 2005 * |
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