CN116640187A - Small molecular protein for improving nitrogen fixation and yield of peanuts and application thereof - Google Patents
Small molecular protein for improving nitrogen fixation and yield of peanuts and application thereof Download PDFInfo
- Publication number
- CN116640187A CN116640187A CN202310662159.9A CN202310662159A CN116640187A CN 116640187 A CN116640187 A CN 116640187A CN 202310662159 A CN202310662159 A CN 202310662159A CN 116640187 A CN116640187 A CN 116640187A
- Authority
- CN
- China
- Prior art keywords
- enfp
- peanut
- yield
- solution
- nitrogen fixation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000020232 peanut Nutrition 0.000 title claims abstract description 105
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 98
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 49
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 36
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 35
- 241001553178 Arachis glabrata Species 0.000 title claims abstract 16
- 235000017060 Arachis glabrata Nutrition 0.000 claims abstract description 75
- 235000010777 Arachis hypogaea Nutrition 0.000 claims abstract description 75
- 235000018262 Arachis monticola Nutrition 0.000 claims abstract description 75
- 239000000243 solution Substances 0.000 claims abstract description 45
- 239000004094 surface-active agent Substances 0.000 claims abstract description 13
- 230000001965 increasing effect Effects 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 4
- 150000003384 small molecules Chemical class 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 229920001296 polysiloxane Polymers 0.000 claims description 3
- 239000003337 fertilizer Substances 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 4
- 244000105624 Arachis hypogaea Species 0.000 description 90
- 238000005507 spraying Methods 0.000 description 17
- 239000011347 resin Substances 0.000 description 11
- 229920005989 resin Polymers 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 239000007921 spray Substances 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- 238000009331 sowing Methods 0.000 description 5
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 230000024121 nodulation Effects 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012460 protein solution Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000000618 nitrogen fertilizer Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002173 cutting fluid Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- NQRYJNQNLNOLGT-UHFFFAOYSA-N tetrahydropyridine hydrochloride Natural products C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/40—Fabaceae, e.g. beans or peas
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Botany (AREA)
- Wood Science & Technology (AREA)
- Pest Control & Pesticides (AREA)
- General Health & Medical Sciences (AREA)
- Forests & Forestry (AREA)
- Ecology (AREA)
- Organic Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Zoology (AREA)
- Dentistry (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Aiming at the problems of the prior art that the nitrogen fixation capacity of peanut root system is improved, the nitrogen content of root system is gradually increased along with the increase of fertilizing amount, the nitrogen fixation capacity of peanut root system is gradually reduced, and the fertilizer utilization rate is reduced and the environment is polluted, the application provides a small molecular protein for improving the nitrogen fixation and yield of peanut and application thereof according to the biological characteristics that peanut can generate root nodules for nitrogen fixation, and the small molecular protein is any one or more than two of 10 small molecular proteins (ENFP-1 to ENFP-10), and is prepared into an ENFP aqueous solution containing Silwet series organosilicon surfactant during use. The application is proved by experiments: the ENFP solution sprayed on the leaf surface can obviously improve the root nodule quantity of the peanut, the nitrogen fixation capacity of the peanut, the nitrogen content of the peanut leaf, the peanut yield and the yield of the peanut, and has an obvious yield increasing effect.
Description
Technical Field
The application belongs to the technical field of agricultural biology, and particularly relates to a small molecular protein for improving nitrogen fixation and yield of peanuts and application thereof.
Background
Peanut is an important oil crop in China, and the improvement of yield has great significance for guaranteeing the oil supply in China. Most leguminous plants can establish symbiotic relation with nitrogen-fixing rhizobia to form high-efficiency nitrogen-fixing factories, namely rhizobia. Peanut is leguminous plant, root system can form root nodule, fix nitrogen, promote peanut growth. The root nodule formation of peanut starts from 5-6 leaves and is mainly concentrated on main roots and lateral roots formed by epicotyls, and the root nodule formation is influenced by internal factors such as different variety characteristics and external environments such as soil nutrients, soil pH value and soil air permeability. Therefore, the nitrogen in the air is fully utilized, the nitrogen fixation capacity of peanut roots is improved, the nitrogen content of the overground parts of the peanuts is improved, and the method is an important measure for reducing the application amount of nitrogen fertilizer of the peanuts, reducing environmental pollution and improving the yield of the peanuts.
At present, the nitrogen fixation capacity of peanut roots is improved mainly by selecting different peanut varieties or adjusting microenvironments such as peanut root system nutrients, moisture and the like. However, as the fertilizing amount increases, the nitrogen content of the root system gradually increases, and the nitrogen fixation capacity of the peanut root system gradually decreases, so that the fertilizer utilization rate decreases and the environment is polluted.
The small molecular polypeptide plays an important regulatory role in plant growth and development, stress tolerance and nutrient absorption. Many small molecule polypeptides have recently been found to function like hormones. At present, small molecule polypeptides are found to have important roles in regulating root nodule formation in plants. Therefore, on the basis of fully researching the functions of the small molecular polypeptide, the small molecular protein (ENFP, enhance nitrogen fixation of peanut) capable of efficiently regulating and controlling the formation of peanut nodules and the nitrogen fixation capacity is developed, a proper in-vitro application method is established, the nitrogen fixation capacity of peanut roots is improved, the nitrogen content of leaves is enhanced, the application amount of peanut nitrogen fertilizer is reduced, the peanut yield is increased, and technical support can be provided for guaranteeing the oil safety of China.
Disclosure of Invention
The application utilizes bioinformatics means to deeply analyze peanut genome information, fully compares and experimentally screens small molecular polypeptides in peanuts and other plants, and finally finds 10 small molecular polypeptides ENFP-1-ENFP-10 capable of remarkably improving nitrogen fixation capacity and yield of peanuts. The ENFP solution is sprayed in the full bloom stage of the peanut, so that the ENFP content in the peanut body can be increased, the nitrogen fixation capacity of the peanut root system and the nitrogen content of the overground part can be further improved, and the peanut yield can be further improved.
The technical scheme of the application is as follows: the small molecular protein for improving nitrogen fixation and yield of peanuts is characterized by being any one or a mixture of more than two of ENFP-1-ENFP-10, wherein the amino acid sequences of the ENFP-1-ENFP-10 are as follows:
ENFP-1：AFRPTAPSHSPGSMR
ENFP-2：DFRPTAPSHSPGSMR
ENFP-3：NFRPTAPSHSPGSMR
ENFP-4：AFRPTAPGHSPGSMR
ENFP-5：DFRPTAPGHSPGSMR
ENFP-6：NFRPTAPGHSPGSMR
ENFP-7：AFRPTAPGNSPGSMR
ENFP-8：DFRPTAPGNSPGSMR
ENFP-9：NFRPTAPGNSPGSMR
ENFP-10：DFRPTAPGGSPGSMR。
the small molecular proteins ENFP-1 to ENFP-10 can improve the nitrogen fixation capacity of peanut systems, improve the nitrogen content of overground parts and further improve the peanut yield.
The method for improving the nitrogen fixation capacity of the peanut root system by adopting the ENFP-1 to ENFP-10 comprises the following specific steps: spraying ENFP solution on the leaf surfaces of the peanuts in the full-bloom stage; the ENFP solution is an aqueous solution containing Silwet series organic silicon surfactant and ENFP, and is sprayed by 20-30L per mu when the ENFP solution is used in a field. The addition of Silwet series organic silicon surfactant in ENFP solution can promote small molecular proteins to enter peanut leaves, promote ENFP to induce peanuts to produce active substances, and improve the nitrogen fixation capacity of peanut roots.
The working concentration of the ENFP solution is as follows: the final concentration of any one or a mixture of more than two of ENFP-1 to ENFP-10 is 0.02+ -0.005 g/L (preferably 0.02 g/L), and the concentration of Silwet series silicone surfactant is 0.01-0.02%.
The application has the technical effects that:
1. the application overcomes the problems existing in the prior art, adopts the leaves to spray the small molecular protein ENFP solution in the full-bloom stage of the peanuts, improves the nitrogen fixation capacity of the root system, and can improve the peanut yield. Therefore, the product can improve the peanut yield on the premise of reducing the fertilizer consumption, not only can prevent environmental pollution caused by a large amount of fertilizer, but also can improve the peanut yield, and has wide application prospect.
2. A field experiment shows that after the ENFP solution is sprayed on the peanut leaf surfaces, the root nodule number of the peanut root system can be increased, the nitrogen fixation capacity of the peanut and the nitrogen content of the peanut leaf surfaces can be improved, and the peanut yield can be improved. Therefore, the method of the application not only has the function of improving the nitrogen fixation of peanut systems, but also has the function of improving the yield, and provides a realistic and effective product and method for the efficient and sustainable development of peanut production.
Drawings
FIG. 1 is a graph showing the effect of treating greenhouse-grown peanuts with solutions of ENFP-1 to ENFP-10, respectively, on nitrogen content of peanut leaves; as can be seen from the figures: the nitrogen content of peanut leaves can be obviously improved by the ENFP-1 to ENFP-10 solutions;
FIG. 2 is a graph showing the effect of treating field-grown peanuts with solutions of ENFP-1 and ENFP-10, respectively, on root nodules of the peanuts in a test example; as can be seen from the figures: the ENFP-1 and ENFP-10 solutions can obviously improve the quantity of rhizobia of peanut root systems;
FIG. 3 is a graph showing the effect of treating field-grown peanuts with solutions of ENFP-1 and ENFP-10, respectively, on peanut yield for test example III; as can be seen from the figures: both ENFP-1 and ENFP-10 solutions can significantly increase peanut yield;
wherein, ENFP solution: an aqueous solution comprising silwet L-77 surfactant and ENFP; CK: clear water control containing the same concentration of silwet L-77 surfactant.
Detailed Description
The technical scheme of the present application and the technical effects thereof are further described below with reference to specific embodiments and drawings, the following description is only for explaining the present application, but not limiting the present application in any way, and any changes or substitutions based on the present application are all within the scope of the present application. The method of the application is a conventional method in the art unless specifically stated otherwise. The reagents of the application are commercially available unless otherwise specified.
Example 1: determination of amino acid sequence of small molecule protein
(1) Determination of amino acid sequence of small molecule protein
According to the related protein sequences in plants such as leguminous plants peanut, alfalfa and the like and the expression abundance of the protein gene in the peanut, the inventor replaces individual key amino acids through bioinformatics analysis, establishes a small molecule protein library with 150 small molecule protein sequences, respectively sprays the peanut leaves with the 150 small molecule proteins, and finally discovers 10 small molecule proteins capable of obviously improving the root nodule number of the peanut. The 6 amino acids at the C-terminus of the 10 small molecule proteins are conserved: SPGSMR. The above 10 small molecule protein sequences were respectively named: ENFP-1 to ENFP-10.
(2) Synthesis and preservation of small molecule proteins
And synthesizing small molecular protein by Fmoc solid-phase polypeptide synthesis method. The synthesis steps are as follows:
(1) taking 0.3g of Fmoc-C terminal first amino acid-king resin, placing into a centrifuge tube, swelling for 20 minutes by using DCM, and filtering out the DCM; adding 20% piperidine/DMF solution with the volume of 3 times of the resin to dissolve, and blowing nitrogen for 30 minutes by using a nitrogen blowing instrument to dry; 2 volumes of DMF were added, washed 5 times and centrifuged to pour off the liquid.
(2) 0.27mmol Fmoc-C end second was takenAdding the amino acid at the position-OH, 0.54mmol DIPEA,0.26mmol HBTU into the centrifuge tube, adding a proper amount of solvent DMF, and reacting for 30 minutes to obtain the Fmoc-C terminal second amino acid-C terminal first amino acid-king resin. Amino acid: DIPEA: HBTU: resin=3: 6:2.85:1 (molar ratio); the resin of the previous step was then washed 3 times with 2 volumes of DMF; then adding 20% pip/DMF solution with 3 times of resin volume, blowing nitrogen for 30 minutes, pumping out, removing Fmoc group to obtain H 2 N-second amino acid-first amino acid-king resin; finally, the resin of the previous step was washed 5 times with 2 volumes of DMF.
(3) And (3) repeating the step (2), wherein 1 amino acid can be added for each repetition of the step (2), and repeating the steps according to the sequence requirement of the small molecule protein to obtain the final target small molecule polypeptide.
(4) The resin was washed 3 times with methanol, then 6 times of the resin volume of a cutting fluid (volume ratio; trifluoroacetic acid: anisole: 1, 2-ethanedithiol: phenol: water=87.5%: 5%:2.5%: 2.5%), shaking the mixture for 2 hours with a shaker, filtering the resin, precipitating the filtrate with ice anhydrous diethyl ether, and washing the precipitate 3 times with ice anhydrous diethyl ether, finally placing the precipitate in a vacuum drying kettle, and drying at normal temperature for 24 hours to obtain 100mg of a crude small molecular protein product.
(5) Purifying by HPLC instrument to obtain small molecule protein with purity of more than 95%:
first, a C18 analytical column is used for rapid gradient analysis of a small molecular protein crude product. The mobile phase is: aqueous phase a:0.1% tfa/water; organic phase B:0.1% tfa/acetonitrile; gradient: 5% -70%,20 min, and the main peak of the crude product is about 7 min.
Next, small molecule protein separation was performed using a C18 preparative column, and the target peak was collected. And (3) measuring mass spectrum, wherein the molecular weight of the collected target peak is consistent with the molecular weight of the target small molecular protein, and the purity is up to 95% or more and is primarily qualified. Then, the collected target peak solution is quickly frozen by liquid nitrogen, then is placed on a freeze dryer for vacuum drying to be in powder form, 1mg of the freeze-dried powder small molecular protein is taken and dissolved by water, HPLC and MS retest are carried out, the purity is over 95 percent, and the molecular weight is not changed to be finally qualified.
The small molecular proteins can be stored at-80deg.C for about 2 years and at-20deg.C for about half a year.
Example 2: preparation and application of foliar fertilizer EPW
2g of the equal mass mixture of ENFP-1 to ENFP-10 and the 10 small molecular proteins are respectively taken and dissolved by 1L of deionized water to prepare 100x mother liquor, and the mother liquor can be placed below-20 ℃ for 1-2 weeks, and can be prepared at room temperature.
When the ENFP mother liquid is applied in the field, 100ml of the ENFP mother liquid is taken, diluted by tap water, finally diluted to a constant volume of 10L, then 2ml of silwet L-77 surfactant is added, the mixture is uniformly mixed, the final concentration of the mixture reaches 0.02g/L, and the ENFP working liquid contains 2/10000 of silwet L-77 surfactant and is used in the following test examples.
Spraying the leaf surfaces of the peanuts in the full-bloom period, wherein the spraying amount is 20-30L/mu; spraying is carried out before 10 am or after 4 pm and in 1 day without rain. When spraying, the ENFP solution can be uniformly sprayed on the front and back surfaces of the peanut leaves as much as possible.
Test example one, influence of foliar spray of ENFP solution on peanut leaf nitrogen content
Selecting full Luhua No. 23 peanut seeds, sowing the seeds in a small box (10 multiplied by 10 cm) containing seedling culture matrixes in a laboratory, placing the seeds in a greenhouse for germination and growth, and illuminating at the room temperature of 28 ℃ for 12 hours and darkening for 12 hours. After peanut sprouting, the peanut is continuously grown in a greenhouse for 20 days, then the leaf is sprayed with ENFP solution, clean water containing the same concentration of silwet L-77 surfactant is sprayed as a control, and the experiment is repeated for 3 times. And measuring the nitrogen content of the peanut leaf after 10 days of spraying. In this experiment, the ENFP solution was 10 kinds of small molecule protein solutions containing ENFP-1 to ENFP-10, respectively (the preparation method is shown in example 2).
Test results: peanut leaf nitrogen content was measured and it was found that spraying ENFP significantly increased peanut leaf nitrogen content compared to the control (fig. 1). The foliar spray of the ENFP solution can obviously improve the nitrogen content of peanut leaves, and the ENFP-1 to ENFP-10 solutions have little effect on improving the nitrogen content of peanut leaves.
Effect of test example two, foliar spray EPW solution on root nodule of peanut
Full Luhua No. 23 peanut seeds are selected, the seeds are sowed in a field in the Shandong Jinxiang of 2021 in 5 months and 25 days, single-grain precision sowing is carried out on the peanuts, the sowing density of the peanuts is 9000 plants/mu, the row spacing is 40cm, and the row spacing is equal. The treatment of spraying ENFP-1 and ENFP-10 solutions was set, and the treatment was repeated 3 times in a random block arrangement with the control of spraying clear water containing the silwet L-77 surfactant at the same concentration. Cell area 32m 2 8 rows of zones are used. And in the full-bloom period of the peanuts, after a rainless day is selected to be 4 pm, spraying 30L of ENFP solution with the concentration of 0.02g/L per mu of leaf surface. Peanuts are harvested on day 9 and 17, and the number of root nodules is counted during harvesting. In the test, the ENFP solution is a small molecular protein solution containing ENFP-1 and ENFP-10 respectively (the preparation method is shown in example 2), and the ENFP solution can be uniformly sprayed on the front and back surfaces of peanut leaves as much as possible during spraying.
Test results: when peanuts are harvested, the root systems of the peanuts are excavated, the number of the root nodules of the individual plants is counted, and the number of the root nodules of the root systems of the peanuts can be obviously improved by spraying the ENFP-1 solution and the ENFP-10 solution compared with a control (figure 2).
Test example Effect of three-sided spray application of ENFP solution on peanut yield
Full Luhua No. 23 peanut seeds are selected, the seeds are sowed in a field in the Shandong Jinxiang of 2021 in 5 months and 25 days, single-grain precision sowing is carried out on the peanuts, the sowing density of the peanuts is 9000 plants/mu, the row spacing is 40cm, and the row spacing is equal. The treatment of spraying ENFP-1 and ENFP-10 solutions was set, and the treatment was repeated 3 times in a random block arrangement with the control of spraying clear water containing the silwet L-77 surfactant at the same concentration. Cell area 32m 2 8 rows of zones are used. And in the full-bloom period of the peanuts, after a rainless day is selected to be 4 pm, spraying 30L of ENFP solution with the concentration of 0.02g/L per mu of leaf surface. Peanut is harvested on day 9 and 17, and peanut yield is counted. In the test, the ENFP solution is a small molecular protein solution containing ENFP-1 and ENFP-10 respectively (the preparation method is shown in example 2), and the ENFP solution can be uniformly sprayed on the front and back surfaces of peanut leaves as much as possible during spraying.
Test results: after peanut harvesting, peanut yield was measured and it was found that spraying both ENFP-1 and ENFP-10 solutions significantly improved peanut yield compared to the control (FIG. 3).
Claims (8)
1. The small molecular protein for improving nitrogen fixation and yield of peanuts is characterized by being any one or a mixture of more than two of ENFP-1-ENFP-10, wherein the amino acid sequences of the ENFP-1-ENFP-10 are as follows:
ENFP-1：AFRPTAPSHSPGSMR
ENFP-2：DFRPTAPSHSPGSMR
ENFP-3：NFRPTAPSHSPGSMR
ENFP-4：AFRPTAPGHSPGSMR
ENFP-5：DFRPTAPGHSPGSMR
ENFP-6：NFRPTAPGHSPGSMR
ENFP-7：AFRPTAPGNSPGSMR
ENFP-8：DFRPTAPGNSPGSMR
ENFP-9：NFRPTAPGNSPGSMR
ENFP-10：DFRPTAPGGSPGSMR。
2. an ENFP solution comprising the small molecule protein of claim 1, wherein the ENFP solution is an aqueous solution comprising a Silwet-series silicone surfactant and a mixture of any one or two or more of ENFP-1 to ENFP-10.
3. The ENFP solution of claim 2, wherein the final concentration of any one or a mixture of two or more of ENFP-1 to ENFP-10 is 0.02 ± 0.005g/L.
4. The ENFP solution of claim 3, wherein the concentration of the Silwet-series silicone surfactant is from 0.01% to 0.02%.
5. Use of the small molecule protein of claim 1 or the ENFP solution of any one of claims 2-4 for simultaneously increasing the number of root nodules and leaf nitrogen content of a peanut root system.
6. Use of the small molecule protein of claim 1 or the ENFP solution of any of claims 2-4 for increasing peanut yield.
7. A method for improving nitrogen fixation and yield of peanuts, which is characterized in that the ENFP solution of claim 4 is sprayed on leaves of the peanut in the full-bloom stage.
8. The method for improving nitrogen fixation and yield of peanuts as claimed in claim 7, wherein 20-30L per mu is sprayed when used in a field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310662159.9A CN116640187A (en) | 2023-06-06 | 2023-06-06 | Small molecular protein for improving nitrogen fixation and yield of peanuts and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310662159.9A CN116640187A (en) | 2023-06-06 | 2023-06-06 | Small molecular protein for improving nitrogen fixation and yield of peanuts and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116640187A true CN116640187A (en) | 2023-08-25 |
Family
ID=87639743
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310662159.9A Pending CN116640187A (en) | 2023-06-06 | 2023-06-06 | Small molecular protein for improving nitrogen fixation and yield of peanuts and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116640187A (en) |
-
2023
- 2023-06-06 CN CN202310662159.9A patent/CN116640187A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Crews | Phosphorus regulation of nitrogen fixation in a traditional Mexican agroecosystem | |
| Shimshi et al. | Stomatal behavior of wheat cultivars in relation to their transpiration, photosynthesis, and yield 1 | |
| CN108934851B (en) | Saline-alkali-tolerant cold-area stress-resistant rice seedling raising method | |
| CN106171888B (en) | Dendrobium officinale culture medium and culture method | |
| Finke et al. | Efficiency of nitrogen assimilation by N2-fixing and nitrate-grown soybean plants (Glycine max [L.] Merr.) | |
| CN106929522A (en) | Amino acid transport gene OsAAP1 promotes the application of paddy growth under low nitrogen | |
| CN113754732B (en) | Small molecular substance for improving cotton yield and fiber quality | |
| CN116640187A (en) | Small molecular protein for improving nitrogen fixation and yield of peanuts and application thereof | |
| CN110063332B (en) | Promoter for improving nitrate nitrogen absorption of plant root system and application | |
| CN108070601A (en) | Application of the OsNPF8.6b genes in rice yield is improved | |
| CN114478705B (en) | Small molecular protein for improving photosynthesis of wheat and application thereof | |
| CN114711112B (en) | Method for increasing yield of selenium-enriched rice by increasing tillering | |
| Johnson-Flanagan et al. | Frost, abscisic acid, and desiccation hasten embryo development in Brassica napus | |
| CN101597632B (en) | Hyphostroma albumen powder, making method thereof and rice cold resistance albumen fertilizer containing same | |
| CN114287300B (en) | Method for improving mid-term growth vigor of field crops before growth | |
| CN110229213B (en) | Artificially synthesized small molecular peptide, growth regulator containing artificially synthesized small molecular peptide and application method of artificially synthesized small molecular peptide | |
| CN114507271B (en) | Small molecular protein for improving tobacco leaf quality | |
| CN108689778B (en) | Trichoderma full-element bio-organic fertilizer special for pressed-sand watermelon and preparation method and application thereof | |
| CN107628853B (en) | Special biological fertilizer for corn in growing period and preparation method thereof | |
| CN114271080B (en) | Fertilizing method for reducing arginine content of mulberry branches | |
| CN109824757A (en) | A kind of ring (different bright-different bright) dipeptides, preparation method and its application | |
| CN107937433B (en) | Application of OsNPF8.13 gene in promotion of rice growth under high nitrogen | |
| CN107409719B (en) | Method for cultivating strong cowpea seedlings | |
| CN106755068A (en) | Applications of the amino acid transport gene OsANT1 in paddy rice seed selection | |
| CN118725038A (en) | Small molecular substance for improving saline-alkali soil by promoting crop growth in saline-alkali soil |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |