CN116444621B - Small peptide for promoting plant root system development and application thereof - Google Patents
Small peptide for promoting plant root system development and application thereof Download PDFInfo
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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
- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Health & Medical Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Dentistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a small peptide for promoting plant root system development and application thereof, wherein the amino acid sequence of the small peptide is Glu-Pro-Asp-Ser-Glu-Glu-Glu-Pro-Lys-Glu-Pro-Glu-Ala-IlE. The small peptide is a novel plant root development growth promoting peptide RDPP from bacillus. The application separates and purifies a plant root system development peptide RDPP with the molecular weight of 1598Da from bacillus pumilus, searches an optimal culture medium for RDPP fermentation, fermentation conditions and a separation method, and identifies the amino acid sequence of the RDPP; the activity of RDPP was verified by a growth-promoting test for the growth and development of the model plant Arabidopsis thaliana. The RDPP has broad-spectrum growth promoting effect on grain crops such as soybean, corn, rice and the like and leaf vegetable crops such as tomatoes and the like, and remarkably improves the yield of the crops.
Description
Technical Field
The invention belongs to the fields of agricultural science and technology and bio-fertilizer science, and particularly relates to a small peptide for promoting plant root system development and application thereof.
Background
The problems of field environmental pollution, food safety hazard and the like caused by excessive application of chemical fertilizers in agricultural production in China cause continuous reduction of the quality of the tillable soil, and influence the future tilling potential of the soil. The biological fertilizer is used as a substitute and supplement of the traditional fertilizer, and plays an indispensable role in the aspects of fertilizer synergism and application reduction, soil fertility optimization, quality improvement and yield increase of crops and mineral resource efficient utilization in China.
With the development and popularization of emerging biofertilizers, novel biofertilizers based on microbial metabolites are increasingly receiving attention at home and abroad. Wherein the microbial metabolites used are largely divided into two classes: firstly, plant hormone substances mainly comprising auxin (IAA), cytokinin (CTK), abscisic acid (ABA), gibberellin (GA) and the like; secondly, protein substances mainly comprising bioactive proteins and bioactive polypeptides. The bioactive polypeptide is used as a new plant growth regulating substance except five plant hormones, is nontoxic and harmless to human and livestock in natural environment, and does not pollute the environment; in the growth and development of plants, the polypeptide optimizes the configuration of plant root systems, improves the yield of crops, improves the quality of crops, enhances the immunity of the crops and improves the utilization rate of fertilizer.
Bacillus pumilus (Bacillus pumilus) is widely distributed in nature as an excellent bio-fertilizer resource, has strong reproductive capacity and strong antagonistic capacity to extreme environmental stress in the field. The research shows that bacillus has broad-spectrum growth promoting activity, fast growth speed and high stability, and can secrete various plant life promoting substances, such as indoleacetic acid (IAA), cytokinin (CTK), volatile Organic Compounds (VOCs) and the like.
Disclosure of Invention
The invention aims to provide a small peptide which is produced by fermenting Bacillus pumilus and is used for promoting plant root system development, and the small peptide is plant root system development and growth promotion peptide (Root Development Promoting Peptide, abbreviated as RDPP).
The invention also aims to provide an extraction and separation method of the plant root development promoting peptide RDPP.
It is a further object of the present invention to provide the use of the plant root development promoting peptide RDPP as described above.
The aim of the invention can be achieved by the following technical scheme:
a small peptide for promoting plant root development has an amino acid sequence of Glu-Pro-Asp-Ser-Glu-Glu-Glu-Pro-Lys-Glu-Pro-Glu-Ala-IlE. The small peptide is a novel plant root development growth promoting peptide RDPP from bacillus.
The small peptide sequence codes provided by the invention are also in the protection scope of the invention.
A method for extracting and separating small peptide for promoting plant root system development includes such steps as fermenting Bacillus pumilus, collecting supernatant, sephadex LH-20 chromatography, freeze drying, dissolving in deionized water, concentrating, separating the concentrated solution by dialysis bag with 1-2 kDa molecular cutoff, removing impurities by HPLC, and separating and purifying. In vitro artificial synthesis verification shows that the plant growth regulator has stronger plant life promotion property.
As a preferable technical scheme, the eluent of the Sephadex LH-20 gel chromatographic column chromatography is water-methanol with the volume ratio of 9:1.
As a preferable technical scheme, the active substance is freeze-dried for 40-55 h after being subjected to Sephadex LH-20 gel chromatographic column chromatography.
In the experimental process of the invention: subjecting the supernatant to Sephadex LH-20 gel chromatographic column chromatography, eluting with water and waterMixing different proportions of methanol (water: methanol=9:1, 8:2,7:3,6:4,5:5, v: v), separating the eluate (water: methanol=9:1, 8:2,7:3,6:4, 5:5) after Sephadex LH-20 gel chromatography, and determining the section (water: methanol=9:1) of the eluate where the living matter is located by an activity tracking method; freeze-drying the active substance-containing segment for 48 hr, and subjecting the freeze-dried powder or extract to ddH 2 O is dissolved and concentrated; segmenting substances with different molecular weights by using dialysis bags with molecular cut-off amounts of 100-500 Da, 1kDa, 2kDa, 5kDa and 10kDa, and adopting ddH as dialysis matrix 2 O. Determining the section (1000-2000 Da) where the living matter is located, removing impurities from active dialysate of the 1000-2000Da interception section by using HPLC to the active matter obtained by segmenting the dialysis bag, and finally obtaining the pure product of the plant root system development promoting peptide RDPP.
As a preferred technical scheme, a fermentation medium for fermenting and culturing Bacillus pumilus (Bacillus pumilus) is an MSgg medium.
As a preferable technical scheme, the fermentation culture method of the Bacillus pumilus (Bacillus pumilus) comprises the following steps: the Bacillus pumilus seed solution was transferred to fresh MSgg medium at 1% inoculum size and fermented at 37℃and 170rpm for 36h. Further preferred is: the fermentation broth was centrifuged in a collection centrifuge bowl at 10000rpm for 20min to collect the supernatant.
The application of the small peptide in promoting plant growth and development or promoting plant root system development.
The application of the small peptide in preparing a preparation for promoting plant growth and development.
The plant is model plant such as Arabidopsis thaliana; grain crops, such as soybean, corn or rice; or leaf vegetables such as tomato.
The small peptide is plant root system development growth promoting peptide (Root Development Promoting Peptide, abbreviated as RDPP), wherein the RDPP is taken as an effective component, the optimal application concentration in plant growth is 0.03 mu M-10 mu M, and according to the strain of plants, different growth stages of plants and different application modes and concentrations, the RDPP can be used for crop root systems or cultivation media, the growth of crops is obviously promoted, and the field yield of crops is increased.
The invention has the beneficial effects that:
the application separates and purifies a plant root system development peptide RDPP with the molecular weight of 1598Da from bacillus pumilus, searches an optimal culture medium for RDPP fermentation, fermentation conditions and a separation method, and identifies the amino acid sequence of the RDPP; the activity of RDPP was verified by a growth-promoting test for the growth and development of the model plant Arabidopsis thaliana. The RDPP has broad-spectrum growth promoting effect on grain crops such as soybean, corn, rice and the like and leaf vegetable crops such as tomatoes and the like, and remarkably improves the yield of the crops.
Drawings
FIG. 1 is a graph showing the growth-promoting effect of RDPP on semi-solid culture of Arabidopsis thaliana.
FIG. 2 is a graph showing the growth promoting effect of RDPP on water-cultured Arabidopsis thaliana.
FIG. 3 is a graph showing the growth promoting effect of RDPP on sterile soil-cultivated Arabidopsis.
FIG. 4 is a graph of the pro-effect of RDPP on semi-solid cultured corn.
FIG. 5 is a graph of the RDPP on the pro-effect of hydroponic corn.
FIG. 6 is a graph of the RDPP's pro-effect on potted corn.
FIG. 7 is a graph of the RDPP's pro-efficacy effect on field-stage corn.
FIG. 8 is a graph of the results of the RDPP on the half-solid cultured rice.
Detailed description of the preferred embodiments
The invention discloses a plant root system development promoting peptide RDPP, which is implemented by adopting other various specific embodiments according to the disclosure of the invention or falls into the protection scope of the invention even if the design structure and thought of the invention are adopted to make simple changes. The present invention will be further described in detail with reference to specific examples for better understanding of the technical solution of the present invention by those skilled in the art.
The plant root system development promoting peptide RDPP can be obtained by a chemical synthesis mode, and can also be obtained by separating and purifying fermentation liquor of bacillus pumilus.
Example 1:
seed solution of Bacillus pumilus Wh4 (a common commercially available strain) was transferred at 1% (v/v) into a conical flask containing 400ml of fresh MSgg medium and fermented at 37℃and 170rpm for 36h. The fermentation broth was centrifuged in a collection centrifuge bowl at 10000rpm for 20min to collect the supernatant. Subjecting the supernatant to Sephadex LH-20 gel chromatographic column chromatography, mixing the eluent with different proportions of water and methanol, uniformly mixing the eluent (water: methanol=9:1, 8:2,7:3,6:4,5:5, v/v) subjected to Sephadex LH-20 gel chromatographic column chromatography in a 1/2MS semi-solid culture medium, transplanting wild type Arabidopsis seedlings (7 days old) growing first on the surface of the solid culture medium, and measuring Arabidopsis root system development indexes after five days of cultivation to determine the section (water: methanol=9:1) of the eluent where the living matter is located; freeze-drying the active substance-containing segment for 48 hr, and subjecting the freeze-dried powder or extract to ddH 2 O is dissolved, resuspended and concentrated; segmenting substances with different molecular weights by using dialysis bags with molecular cut-off amounts of 100-500 Da, 1kDa, 2kDa, 5kDa and 10kDa, and adopting ddH as dialysis matrix 2 O. Concentrating the segmented dialysate according to a uniform proportion, uniformly mixing in a 1/2MS semi-solid culture medium, transplanting wild arabidopsis seedlings (7 days old) with one growth vigor on the surface of a solid culture medium, measuring the root system development index of the arabidopsis after five days of cultivation, determining the section (1000-2000 Da) where the living matter is located, detecting the active dialysate of the 1000-2000Da interception section by using a Nano-HPLC-MS mass spectrum, and separating and purifying to obtain the small peptide containing 14 amino acids and promoting the plant root system development.
The extraction and separation method of the finally screened small peptide for promoting plant root system development comprises the following steps: fermenting and culturing Bacillus pumilus (Bacillus pumilus) and centrifugally collecting supernatant of the fermentation liquor, wherein the supernatant is subjected to Sephadex LH-20 gel chromatographic column chromatography, and water is adopted as eluent: methanol=9:1; freeze-drying the eluent after Sephadex LH-20 gel chromatographic column chromatography for 48 hours, dissolving, re-suspending and concentrating the freeze-dried powder or extract by deionized water, separating the concentrated solution by a dialysis bag with molecular cutoff of 1 kDa-2 kDa, removing impurities from active substances obtained by dialysis of the dialysis bag by HPLC, and finally obtaining the pure product of the plant root system development promoting peptide RDPP.
Example 2:
the plant root system development growth promoting peptide RDPP provided by the invention has the effect of promoting growth of model dicotyledon Arabidopsis thaliana:
1. semi-solid culture assay of arabidopsis:
surface disinfection of arabidopsis seeds: the Arabidopsis seeds are placed in 75% (v/v) absolute ethanol, soaked for 4 to 5 minutes, washed for 4 to 5 times by deionized water, then placed in 2% (v/v) sodium hypochlorite, and soaked for 4 to 5 minutes. And (5) cleaning for 4-5 times by using deionized water, and removing the cleaning liquid for standby.
Sowing Arabidopsis thaliana: the Arabidopsis seeds are sown on the surface of a 1/2MS culture medium, and placed in an illumination incubator after Wen Chunhua days at 4 ℃. Culture conditions: the temperature is 22/18 ℃, the photoperiod (16 h light/8 h dark) and the illumination intensity is 150 mu mol/m 2 And/s, the humidity is 55-70%.
RDPP preparation: RDPP was diluted with deionized water to final concentrations of 1. Mu.M, 3. Mu.M and 10. Mu.M and spread evenly over 1/2MS solid sterile medium.
RDPP treatment Arabidopsis thaliana: arabidopsis seedlings with consistent growth vigor and 7 days old are selected and transplanted on 1/2MS solid culture media coated with RDPP with different concentrations to be a treatment group, and the 1/2MS solid culture media coated with double distilled water is used as a control group. Five days later, the physiological index of each group of arabidopsis thaliana was measured.
The results are shown in fig. 1 and table 1, compared with the control group, RDPP has a remarkable promoting effect on the growth and development of arabidopsis thaliana, when the final concentration of RDPP is 3 μm, the promoting effect is optimal, the root length is increased by 23.9%, the lateral root number is increased by 121.4%, and the lateral root density is increased by 78.9%.
TABLE 1 Effect of RDPP at different concentrations in tissue culture on Arabidopsis root development
2. Arabidopsis hydroponic experiments:
surface disinfection of arabidopsis seeds: according to the method.
Sowing Arabidopsis thaliana: according to the method.
RDPP preparation: RDPP was diluted with liquid 1/2MS (without sucrose) to final concentrations of 1. Mu.M, 3. Mu.M and 10. Mu.M and added evenly to 12 well plates (1 ml per well).
RDPP treatment Arabidopsis thaliana: arabidopsis seedlings with consistent growth vigor and 7 days old are selected and transplanted into 1/2MS liquid culture medium (without sucrose) containing RDPP with different concentrations as a treatment group, and 1/2MS liquid culture medium (without sucrose) containing RDPP as a control group. After six days, the physiological index of each group of Arabidopsis thaliana was measured.
The results are shown in fig. 2 and table 2, compared with the control group, RDPP has a remarkable promoting effect on the growth and development of arabidopsis thaliana, when the final concentration of RDPP is 3 μm, the promoting effect is optimal, the root length is increased by 51.3%, the lateral root number is increased by 248.6%, and the lateral root density is increased by 134.9%.
TABLE 2 influence of RDPP at different concentrations on Arabidopsis root development in hydroponics
3. Sterile soil culture experiments of arabidopsis thaliana:
surface disinfection of arabidopsis seeds: according to the method.
Sowing Arabidopsis thaliana: according to the method.
RDPP preparation: RDPP was diluted to a final concentration of 3. Mu.M with liquid 1/2MS (without sucrose) and added to sterile soil evenly.
RDPP treatment Arabidopsis thaliana: arabidopsis seedlings with consistent growth vigor and 7 days old are selected and transplanted into sterile soil filled with RDPP to be used as a treatment group, and sterile soil without RDPP is used as a control group. After six days, the physiological index of each group of Arabidopsis thaliana was measured.
The results are shown in FIG. 3 and Table 3, and compared with the control group, RDPP has remarkable promotion effect on the growth and development of Arabidopsis thaliana, and when the concentration of bioactive small peptide is 3 μm, the root length is increased by 50.4%, the lateral root number is increased by 197.09%, the lateral root density is increased by 101.8%, and the fresh weight of root is increased by 170.4%
TABLE 3 Effect of RDPP on Arabidopsis root development in sterile soil culture
Example 3:
verification of the plant root growth promoting peptide RDPP provided by the invention has the effect of promoting the effect on dicotyledonous tomatoes:
tomato field culture experiment
Selection of tomato varieties: the variety planted in the field tomatoes is preferably low temperature resistant, excellent in disease resistance and good in commodity property, such as cooperation 903.
Preparation of tomato seedlings: before sowing tomatoes, placing the seeds in sunlight for drying for 2-3 days, immersing the seeds in deionized water for 5-6 hours after completely removing water, wrapping the soaked seeds with wet gauze, placing the seeds in a 30 ℃ incubator, replacing the deionized water every 12 hours, and sowing after more than half of the seeds are exposed to white. The seedling raising substrate is purchased nutrient soil, the nutrient soil is filled into seedling raising hole trays, two seeds are sown in each hole, a layer of preservative film is covered on the seedling raising hole trays after sowing so as to be used for preserving moisture and heat, the treated seedling raising hole trays are uniformly placed in a greenhouse, the temperature is 18 ℃ at night in 28 ℃ in daytime, and the preservative film is removed after the seeds bud.
Transplanting tomato seedlings: the test field is arranged in a test field of the academy of agricultural sciences of Jiangsu province, the selection of cultivated field blocks avoids continuous cropping soil of the solanaceae, deep cultivation is carried out on soil in a field district before tomato seedlings are transplanted, the cultivation depth is about 30cm, the whole field district is divided into two large areas, namely a fertilizing area (local conventional fertilizing amount) and a non-fertilizing area, and three areas are respectively arranged in the fertilizing area and the non-fertilizing area: control cells and two polypeptide concentrations treated cells. After dividing, flattening the field, ridging and ridging, transplanting tomato seedlings in 3-5 leaf periods, wherein each plot is provided with four ridges, the row spacing of the planting is about 40-50 cm, and the planting distance is about 30-40 cm.
Preparation of RDPP: the small peptide is dissolved in deionized water to prepare mother solution with the final concentration of 300 mu M for later use, and the mother solution is diluted to 3 mu M and 0.3 mu M by clear water before transplanting.
RDPP treatment of tomato seedlings: and after the tomato seedlings are transplanted, the diluted polypeptide solution is irrigated to the roots of the tomato seedlings, so that the bioactive small peptide can act on the growth of the seedlings in time. And measuring various physiological indexes of the plants before transplanting, after transplanting and in the flowering period of tomatoes respectively.
The results are shown in Table 4, and RDPP has a remarkable promotion effect on the growth and development of tomatoes, and when the RDPP is applied at a concentration of 3 mu M, the RDPP is applied in a non-fertilizing area: compared with the control group, the plant height is increased by 30.1%, the root length is increased by 23.1%, the stem thickness is increased by 20.4%, the chlorophyll value is increased by 8.9%, the fresh weight of the overground part root is increased by 47.3%, the dry weight of the overground part is increased by 46.7%, the fresh weight of the root is increased by 59.1%, and the dry weight of the root is increased by 56.7%.
TABLE 4 influence of RDPP on tomato growth in field trials
Example 4:
the plant root system development growth promoting peptide RDPP provided by the invention has the effect of promoting the growth of dicotyledonous soybean:
soybean field culture experiment:
selection of soybean varieties: the field soybean planting variety is preferably: excellent germination rate, disease resistance, pilling resistance, cold resistance, waterlogging resistance, high yield and other characteristics such as Zhonghuang 37.
Preparation of soybean sowing: the test field is arranged in a laboratory field of the academy of agricultural sciences of Jiangsu province, weeding and deep ploughing are carried out on the field cells, and the whole field cells are divided into three cells: a control cell and two polypeptide processing cells; the soybeans are crops with high water demand, the irrigation frequency in the growth period is required to be improved, the irrigation quantity in each period is ensured, and the emergence rate of the soybeans in the field is improved.
Planting soybeans: the soybean planting mode adopts direct seeding planting and close planting to ensure the emergence rate and the field yield of the soybean. The row spacing of the planting is about 40-50 cm, the planting spacing is about 40-50 cm, and the supply of soybean nutrition absorption is increased. And (5) after 7 days of planting, the soybeans are subjected to seedling supplementing and impurity removal, so that the growth interval of the soybeans and the growth environment of excellent seedlings are ensured.
Preparation of RDPP: the small peptide is dissolved in deionized water to prepare mother solution with the final concentration of 300 mu M for later use, and the mother solution is diluted to 3 mu M and 0.3 mu M by clear water before transplanting.
Bioactive peptide treatment of soybean seedlings: and (3) pouring diluted small peptide solution into the roots of the soybeans from the period from the emergence of the soybeans to 3-5 leaves of the soybeans, so that the bioactive polypeptide can act on the growth of the seedlings in time. And destructive sampling is carried out in the soybean seedling stage, and various physiological indexes of soybean plants are measured.
The results are shown in Table 5, the RDPP has a remarkable promoting effect on the growth and development of soybean compared with the control group before transplanting, and when the RDPP application concentration is 3 mu M, the plant height is increased by 9.3%, the chlorophyll value is increased by 7.9%, the fresh weight of the overground root is increased by 36.3%, the dry weight of the overground root is increased by 35.8%, the fresh weight of the root is increased by 21.9%, and the dry weight of the root is increased by 2.6% compared with the control group.
TABLE 5 Effect of RDPP on Soybean growth in field trials
Example 5:
verification of the plant root development and growth promoting peptide RDPP provided by the invention has the effect of promoting the growth of monocotyledonous corn:
1. semi-solid culture assay of maize:
selecting corn seeds: and selecting a high-yield disease-resistant fine variety Zhengdan 958.
Surface sterilization of corn seeds: selecting corn seeds with the same size, soaking the corn seeds in 75% absolute ethanol for 2-3 minutes, washing the corn seeds with deionized water for 4-5 times, and then soaking the corn seeds in 1% sodium hypochlorite for 20 minutes. And (5) cleaning for 4-5 times by using deionized water, and removing the cleaning liquid for standby.
Sowing corn: dibbling corn seeds on the surface of a 1/2MS solid culture medium, placing the corn seeds in a 30 ℃ incubator, and observing corn germination after dark culture for 72 hours.
RDPP preparation: RDPP was diluted with deionized water to final concentrations of 0.1. Mu.M, 0.3. Mu.M and 1. Mu.M and spread evenly over 1/2MS solid sterile medium.
RDPP treated corn: corn seedlings with consistent germination are selected and transplanted on 1/2MS solid culture media coated with RDPP with different concentrations to form a treatment group, and the 1/2MS solid culture media coated with double distilled water is used as a control group. After 4 days, corn physiological indexes of each group are measured.
The results are shown in fig. 4 and table 6, compared with the control group, the RDPP has a remarkable promotion effect on the growth and development of corn, and when the final concentration of the RDPP is 0.3 μm, the promotion effect on lateral roots is optimal, and 115.64% is increased; when the final concentration of the bioactive polypeptide is 1 mu M, the seed root has optimal effect on promoting the growth of the seed root, and the increase is 63.03 percent.
TABLE 6 influence of RDPP on maize growth in tissue culture experiments
2. Corn water planting test:
surface sterilization of corn seeds: according to the method.
Sowing corn: according to the method.
Seedling raising of corn: corn seedlings with consistent growth vigor are selected and transplanted into a tissue culture bottle with 100ml of 1/2MS semi-solid culture medium for 48 hours.
RDPP preparation: RDPP was diluted with 1/2MS liquid to final concentrations of 0.003. Mu.M, 0.03. Mu.M and 0.3. Mu.M and dispensed into 50ml conical flasks (20 ml polypeptide dilution per flask) for further use.
RDPP treated corn: corn seedlings with consistent germination are selected, transplanted from a tissue culture bottle into 1/2MS liquid culture media with RDPP with different concentrations, marked as a treatment group, and only 1/2MS liquid culture media are filled as a control group. After 5 days, corn physiological indexes of each group are measured.
The results are shown in fig. 5 and table 7, and compared with the control group, RDPP has a remarkable promoting effect on the growth and development of corn, when the final concentration of RDPP is 0.3 μm, the plant height is increased by 30.7%, the root length is increased by 27.1%, the stem thickness is increased by 38.5%, the fresh weight of the overground part is increased by 70.1%, the dry weight of the overground part is increased by 144.4%, the root fresh weight is increased by 209.8%, and the dry weight of the root is increased by 235.7%.
TABLE 7 influence of RDPP on maize growth in hydroponic experiments
3. Corn pot culture test:
surface sterilization of corn seeds: according to the method.
Sowing corn: according to the method.
Seedling raising of corn: corn seedlings with consistent growth vigor are selected and transplanted into a tissue culture bottle with 100ml of 1/2MS semi-solid culture medium for 48 hours.
Test soil: the soil to be tested is the barren land soil of Nanjing Shang Shan, and the cultivated layer soil of 20-40 cm of the field is selected, naturally air-dried, ground and passed through 5mm; the air-dried soil after sieving is put into a PVC flowerpot, the upper caliber is 16cm, the bottom inner diameter is 12cm, the height is 11.5cm, 5-8 small holes are uniformly distributed on the bottom of the bucket, and a gauze is paved on the bottom of the bucket so as to ensure good water-permeable and air-permeable conditions.
RDPP preparation: RDPP was diluted with deionized water to final concentrations of 0.003. Mu.M, 0.03. Mu.M, and 0.3. Mu.M for use.
RDPP treated corn: selecting corn seedlings with consistent growth vigor, transplanting the corn seedlings into tested soil with sufficient water content, irrigating the roots of the corn seedlings with diluted RDPP solution, marking the corn seedlings as a treatment group, and irrigating deionized water as a control group. And the corn seeds are placed in a sunlight room, sufficient illumination time, illumination intensity and irrigation frequency are ensured during planting, and physiological indexes of each group of corn are measured after 21 days.
The results are shown in FIG. 6 and Table 8, and compared with the control group, RDPP has a remarkable promotion effect on the growth and development of corn, when the final concentration of RDPP is 0.3 mu M, the plant height is increased by 30.7%, the stem thickness is increased by 38.5%, the dry weight of the overground part is increased by 144.4%, and the dry weight of roots is increased by 235.7%.
TABLE 8 Effect of RDPP on maize growth in potting experiments
4. Corn field culture test:
preparation of corn sowing: the test land is arranged in a test field of the academy of agricultural science in Jiangsu province, deep ploughing is carried out on the soil before planting, and the fertility and the air permeability of the soil are increased so as to allow corn seedlings to grow normally; dividing the whole field cell into two large areas, namely a fertilizing area (local conventional fertilizing amount) and a non-fertilizing area, wherein three cells are respectively arranged in the fertilizing area and the non-fertilizing area: control cells and two polypeptide concentrations treated cells.
Sowing corn: corn seeds are directly sown in the divided cells, and the planting row spacing is 80cm and the plant spacing is 25cm.
Preparation of RDPP: the small peptide is dissolved in deionized water to prepare mother solution with the final concentration of 300 mu M for standby, and the mother solution is diluted to 3 mu M and 0.3 mu M by clear water before polypeptide treatment.
RDPP treated corn: after field management, adding polypeptide when the height of the corn plant is about 25cm, adding polypeptide once every 7 days, adding polypeptide three times, performing destructive sampling in seedling stage, jointing stage and bell mouth stage respectively, and measuring physiological indexes such as plant height, stem thickness, dry fresh weight and the like.
The results are shown in fig. 7 and table 9, and RDPP significantly promotes the growth and development of corn compared to the control group. Jointing period: in non-fertilized cells, when the RDPP final concentration is 3. Mu.M, the plant height is increased by 8.2%, the root length is increased by 7.9%, the stem thickness is increased by 14.8%, the chlorophyll content is increased by 14.1%, the fresh weight of the overground part is increased by 33.4%, the dry weight of the overground part is increased by 31.7%, the fresh weight of the root is increased by 61.4%, and the dry weight of the root is increased by 53.2%.
TABLE 9 Effect of RDPP on maize growth at different times in field experiments
Measurement of corn field yield:
when corns are mature, 5 representative plants are selected from each cell, the number of ears in the cell, the number of ears harvested per mu, the average number of ears (grains) and hundred grains are measured, threshing and yield measurement are carried out on all corns in each cell, and the yield per mu is reduced according to 14% of water content.
The results are shown in Table 10, and RDPP significantly improved the yield of corn in the field compared to the control group. In non-fertilizing cells, when the RDPP final concentration is 3 mu M, the number of spikes in the cells is increased by 28.6%, the number of spikes harvested per mu is increased by 39.9%, the average number of spikes (grains) is increased by 2.5%, the hundred grains weight is increased by 4.5%, and the theoretical yield (kg/mu) is increased by 50.1%.
TABLE 10 influence of RDPP on corn yield in field experiments
Example 5:
verification of the plant root growth promoting peptide RDPP provided by the invention has the effect of promoting the growth of monocotyledonous rice:
semi-solid culture assay for rice:
selecting rice seeds: and selecting a high-yield and disease-resistant fine variety Long Ping 206.
Surface sterilization of rice seeds: selecting rice seeds with the same size, soaking the rice seeds in 75% absolute ethanol for 2-3 minutes, washing the rice seeds with deionized water for 4-5 times, and then soaking the rice seeds in 1% sodium hypochlorite for 30 minutes. And (5) cleaning for 4-5 times by using deionized water, and removing the cleaning liquid for standby.
Sowing rice: the rice seeds are sowed on the surface of a 1/2MS solid culture medium, placed in a 30 ℃ incubator, and are subjected to dark culture for 72 hours, and then the germination condition of the rice is observed.
RDPP preparation: RDPP was diluted with deionized water to final concentrations of 0.1. Mu.M, 0.3. Mu.M and 1. Mu.M and spread evenly over 1/2MS solid sterile medium.
RDPP treatment of rice: selecting rice seedlings with consistent germination, transplanting the rice seedlings on 1/2MS solid culture media coated with RDPP with different concentrations to form a treatment group, and taking the 1/2MS solid culture media coated with double distilled water as a control group. After 4 days, the physiological indexes of each group of rice are measured.
The results are shown in fig. 8 and table 11, compared with the control group, the RDPP has a remarkable promotion effect on the growth and development of rice, and when the final concentration of the RDPP is 0.3 mu M, the promotion effect on lateral roots is optimal, and the promotion effect is increased by 64.8%; when the final RDPP concentration is 1 mu M, the seed root has optimal effect on the seed root, and the increase is increased by 26.1%.
TABLE 11 influence of RDPP on growth and development of Rice in tissue culture experiments
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Claims (7)
1. A small peptide for promoting plant root system development, characterized in that: the amino acid sequence of the small peptide is Glu-Pro-Asp-Ser-Glu-Glu-Glu-Pro-Lys-Glu-Pro-Glu-Ala-IlE.
2. The method for extracting and separating small peptide according to claim 1, which is characterized by fermenting and culturing bacillus pumilus and collecting supernatant of fermentation liquor, wherein the supernatant is subjected to Sephadex LH-20 gel chromatographic column chromatography and then freeze-dried, freeze-dried powder or extract is dissolved, resuspended and concentrated by deionized water, a dialysis bag with molecular cutoff of 1 kDa-2 kDa is used for separating concentrated solution, HPLC is used for removing impurities from the dialysis solution, and the small peptide for promoting plant root system development is obtained through separation and purification.
3. The method according to claim 2, wherein the eluent of the Sephadex LH-20 gel chromatographic column chromatography is water-methanol in a volume ratio of 9:1.
4. The method according to claim 2, wherein the active substance is freeze-dried for 40-55 h after Sephadex LH-20 gel chromatography.
5. The method of claim 2, wherein the fermentation medium for fermenting bacillus pumilus is MSgg medium.
6. The use of the small peptide of claim 1 for promoting plant growth or promoting plant root system development, wherein the plant is arabidopsis, tomato, soybean, maize or rice.
7. The use of the small peptide of claim 1 for preparing a formulation for promoting plant growth and development, wherein the plant is arabidopsis thaliana, tomato, soybean, maize or rice.
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