阅读说明：本技术 一种具有生物活力的人工合成材料及其农业应用 (Artificially synthesized material with biological activity and agricultural application thereof ) 是由 林敏� 柯秀彬 燕永亮 王劲 战嵛华 周正富 于 2021-09-15 设计创作，主要内容包括：本发明提供了一种应用于农业的具有生物活力的人工合成材料,含有本发明设计合成的人工亲水蛋白、人工血红蛋白以及聚羟基脂肪酸酯(PHA)。所述材料可用于制备微米级活性人工微囊,包裹具有固氮、解磷解钾、抗病虫和促生等特性的农用微生物优良菌株形成新型菌肥,还可用于制备新型农作物种子活性包衣剂等农业领域。所述材料具有保持菌体活力、定向结合根表和施肥精准高效等独特性能,适用于机械化播种、水肥一体化滴灌和无土栽培等现代农业生产方式。(The invention provides an artificial synthetic material with biological activity for agriculture, which contains artificial hydrophilic protein, artificial hemoglobin and Polyhydroxyalkanoate (PHA) designed and synthesized by the invention. The material can be used for preparing micron-sized active artificial microcapsules, wrapping agricultural microbial excellent strains with the characteristics of nitrogen fixation, phosphate and potassium dissolution, disease and insect resistance, growth promotion and the like to form novel bacterial fertilizers, and can also be used for preparing agricultural fields such as novel crop seed active coating agents and the like. The material has the unique properties of keeping the activity of thalli, directionally combining with a root table, applying fertilizer accurately and efficiently and the like, and is suitable for modern agricultural production modes such as mechanized sowing, water and fertilizer integrated drip irrigation, soilless culture and the like.)

1. An artificial synthetic material with biological activity for agricultural use contains the following three components:

artificial hydrophylin with amino acid sequence shown as SEQ ID No.2, artificial hemoglobin with amino acid sequence shown as SEQ ID No.4, and Polyhydroxyalkanoate (PHA).

2. The synthetic material of claim 1, wherein the three components are present in a volume ratio of:

PHA aqueous solution (concentration 50-200g/L), artificial hydrophilic protein solution (concentration 0.1-1g/ml) and artificial hemoglobin solution (concentration 0.1-1g/ml) are respectively mixed in a ratio of 10:1: 1.

3. The artificially synthesized material as set forth in claim 2, wherein three components of PHA aqueous solution (concentration 100g/L), artificial hydrophilic protein solution (concentration 0.5g/ml) and artificial hemoglobin solution (concentration 0.5g/ml) are mixed in a volume ratio of 10:1:1 when used in a seed coating agent.

4. The amino acid sequence of the protein is shown as SEQ ID No.2 or SEQ ID No. 4.

5. DNA with a nucleotide sequence shown as SEQ ID No.1 or SEQ ID No. 3.

6. The protein with the amino acid sequence shown as SEQ ID No.2 and/or the protein with the amino acid sequence shown as SEQ ID No.4 are applied to agriculture.

7. The application of claim 6, which comprises preparing seed coating agent, preparing novel bacterial manure of agricultural microorganism with the characteristics of nitrogen fixation, phosphate and potassium dissolution, disease and insect resistance and growth promotion, and preparing preparation for promoting crop growth.

The technical field is as follows:

the invention relates to an artificial synthetic material with biological activity and application thereof in agriculture.

Background art:

the ideal agricultural microbial product (preparation) should have the excellent characteristics of plasticity, degradability, water retention, root surface affinity, oxygen and carbon supply and the like so as to realize the functions of fixing nitrogen, dissolving phosphorus and potassium, resisting diseases and insects and the like and promote the growth of plants.

At present, the microbial fertilizer product has the problems of short shelf life, single dosage form, difficult long-distance transportation, complex application mode, unstable effect and the like caused by poor survival and low activity of strains. In addition, products such as nitrogen-fixing microbial agents are greatly influenced by stress factors such as rhizosphere ammonia nitrogen and adversity, so that the field nitrogen-fixing efficiency is low, the fertilizer-saving and yield-increasing effects are unstable, artificial sowing can be adopted, and the urgent requirements of the mechanized and industrialized agricultural development of China at present are difficult to meet. The seed coating is used to mix the filming agent, plasticizer, coloring agent, etc. with antiseptic, pesticide, plant growth regulator, etc. to coat the seed, so as to reach the aims of preventing and controlling diseases and pests, promoting growth, raising crop yield, etc. However, the existing seed coating agents lack the function of protecting the activity of microorganisms, the survival rate of the coated agricultural microorganisms is low, and the effects of fixing nitrogen, dissolving phosphorus and potassium, resisting diseases and insects, promoting growth and the like are rapidly lost. Aiming at the defects that the field application effect of the traditional microbial fertilizer is unstable, the requirement of adapting to the agricultural modernization development cannot be met, and the like, a novel synthetic biological material needs to be developed, or a micron-sized active microcapsule is synthesized to wrap active microorganisms to form a novel bacterial fertilizer, or the microbial fertilizer is used as a novel crop seed active coating agent, and the like.

Polyhydroxyalkanoate, referred to as PHA for short, is a biodegradable intracellular polyester synthesized in vivo by many microorganisms under the condition of nutrient imbalance, and is mainly used as a carbon source and an energy reserve of the microorganisms. PHA structure diversification, through changing the strain, feeding and fermentation process can change the PHA composition conveniently, and the diversity of the composition structure brings about the performance diversification, so that the PHA has obvious advantages in application. As the surface of the PHA microsphere is easy to modify and reform, more and more functional proteins are presented on the surface of the PHA microsphere through fusion expression with the protein on the surface of the PHA microsphere, so that the PHA microsphere is a novel technology for high-efficiency protein immobilization and presentation. In addition, because PHA microspheres can be completely degraded and metabolized, the toxicity problem of other matrix materials in large dose can be avoided, and carbon sources required by growth of microorganisms in the environment can be provided. Due to the characteristics of good biodegradability, biocompatibility and the like, PHA plays an important role in the research and development of drug sustained-release systems and novel coating materials.

The hydrophilic protein is a biological macromolecule with polar groups and has strong affinity to water. The surface of the solid material formed by the molecules is easy to be wetted by water, keeps high hydrophilicity and has excellent water retention performance. As an important component of extreme response to stress, hydrophilic proteins have become one of the hot spots in the field of life science research.

Hemoglobin is widely present in animals, plants and microorganisms, has typical molecular structures and physiological functions, and is widely applied to industrial production of foods and medicines. Under normal physiological conditions, heme exists in the form of ferrous iron, called oxyhemoglobin, and is responsible for oxygen binding during physiological respiration. In addition, hemoglobin also has an antibacterial function.

If the biodegradable material Polyhydroxyalkanoate (PHA) can be combined with artificially synthesized protein components with hydrophilic and oxygen-carrying functions to be used as a novel material to wrap nitrogen-fixing microorganisms, the effects of protecting the activity of the microorganisms, fixing nitrogen, promoting the growth of crops and the like can be expected to be realized.

In the prior art, only PHA is reported to be used as seed coating alone, but reports related to the adoption of hydrophilic protein and/or hemoglobin are not found.

Disclosure of Invention

The invention aims to provide a biologically active artificially synthesized material for agricultural application.

The material contains the following three components:

the artificial hydrophilic protein is designed and synthesized by the invention, and the amino acid sequence is shown as SEQ ID No. 2;

the artificial hemoglobin is designed and synthesized by the invention, and the amino acid sequence is shown as SEQ ID No. 4;

polyhydroxyalkanoates (PHA).

Wherein the artificial hydrophilic protein is a protein with high water retention property, contains 8 motifs consisting of 11 amino acid residues, contains a connecting peptide between the two motifs, can be used as a molecular chaperone to protect cells under stress and protect the activity of enzymes (lactate dehydrogenase, malate dehydrogenase, citrate synthase, and the like) or proteins (alpha-casein), and the HD structure can play a role of a physical barrier between biomolecules to reduce the collision between the biomolecules, thereby reducing the aggregation of the biomolecules and preventing the change of the structure. The protein not only can protect other various proteins in cells, but also has good protection effect on other proteins or cells in an in vitro environment.

The artificial hemoglobin has high oxygen carrying capacity. Can provide an anti-dropping water environment for PHA and hemoglobin and provide a proper oxygen environment for hydrophilic protein.

Polyhydroxyalkanoate (PHA) is a biodegradable material and used as a carrier to wrap azotobacter, artificial hydrophilic protein and artificial hemoglobin components to artificially construct micron-sized azotobacter microcapsules (figure 1), and the capsule material used as a novel efficient azotobacter and a coating processing technology (figure 2) for replacing a traditional seed coating agent are further provided by regulating and controlling the supply of carbon sources and oxygen, maintaining water and effectively relieving external adversity stress to realize the function of maintaining the activity of azotobacter.

The material has wide agricultural application range, can be used for preparing the agricultural fields such as preparing novel bacterial fertilizers by wrapping agricultural microorganisms with the micron-sized active microcapsules, which have the characteristics of nitrogen fixation, phosphorus and potassium dissolution, disease and insect resistance, growth promotion and the like, and preparing novel crop seed active coating agents, can embody the unique performances of keeping the activity of thalli, directionally combining root tables, applying fertilizer accurately and efficiently and the like, is suitable for modern agricultural production modes such as mechanized sowing, water-fertilizer integrated drip irrigation, soilless culture and the like, and replaces the traditional agricultural microorganism inoculation method with high cost and low efficiency.

The dosage ratio of the three components is as follows: short chain PHA aqueous solution (concentration range is 50-200g/L), artificial hydrophilic protein solution (concentration range is 0.1-1g/ml) and artificial hemoglobin solution (concentration range is 0.1-1g/ml) are mixed according to the volume ratio of 10:1: 1.

The short-chain PHA (short-chain-length, SCL-PHA) is PHA containing 3 to 5 carbon atoms in total, and such monomers form common homopolymers after polymerization, such as Polyhydroxybutyrate (PHB), Polyhydroxyvalerate (PHV) and the like.

When the biomaterial of the present invention is used in a seed coating agent, the three components are mixed in a ratio of 10:1:1 by volume of an aqueous solution of short-chain PHA (concentration 100g/L), an artificial hydrophilic protein solution (concentration 0.5g/ml) and an artificial hemoglobin solution (concentration 0.5 g/ml).

The preparation method of the seed coating agent comprises the following steps: crop seeds were coated with a slurry consisting of 43.1 wt.% film coating formulation, 43.3 wt.% water and 13.6 wt.% pigment concentrate, the amount of slurry applied being 5.5g/kg of seeds.

The film coating formulation consisted of water (60% wt/wt), rheological additives (0.2% wt/wt), defoamer (0.2% wt/wt), 50% vinyl acetate binder emulsion (15% wt/wt), and biomaterial of the invention (24.6% wt/wt).

In order to distinguish between different varieties of seeds, colorants such as pigment concentrates have to be added as indicators. Pigment Red 112(CAS No.6535-46-2), pigment Red 2(CAS No.6041-94-7), pigment Red 48: 2(CAS No.7023-61-2), pigment blue 15: 3(CAS No.147-14-8) and pigment green 36(CAS No. 14302-13-7).

Rheological additives can modify the rheological properties of the material during the coating process, for reducing friction and improving seed appearance. One or more of montmorillonite clay, hectorite, carbomer, propylene glycol, DEA, and PEG-200 glycerol fatty acid can be used.

The anti-foaming agent can cause the liquid film to drain during the coating process, thereby causing the bubbles to collapse. One or more of polyethylene glycol, glycerol, mineral oil antifoam, silicone antifoam, and non-silicone antifoam (such as polyethers, polyacrylics), dimethylpolysiloxane (silicone oils), arylalkyl modified polysiloxanes, polyether siloxane copolymers (containing fumed silica) may be used.

The invention has the beneficial effects that:

aiming at the key bottleneck problem in the production of agricultural microbial manure, the PHA material with multiple structures, biodegradability and high biocompatibility, the artificial hydrophilic protein with high water retention property and the artificial hemoglobin with high oxygen carrying capacity are selected, and a novel artificial synthetic material with biological activity is created.

The material has outstanding characteristics and complementary functions, the PHA microspheres have the characteristic of binding protein, the artificial hydrophilic protein can provide an anti-drop water environment for PHA and hemoglobin, and the artificial hemoglobin provides a suitable oxygen environment for the hydrophilic protein. In addition, the novel artificial active material and the good agricultural microorganism strain are adopted in the application, products such as artificial microcapsules or novel seed coating agents with the functions of nitrogen fixation, phosphorus and potassium decomposition, disease and insect resistance, growth promotion and the like are developed, the biological activity and the survival capability of the strain can be obviously improved, the unique performances such as maintaining the activity of the strain, directionally combining with a root table and accurate and efficient fertilization are realized, the application range is wide, and the application is suitable for modern agricultural production modes such as mechanized seeding, water and fertilizer integrated drip irrigation and soilless culture.

Drawings

FIG. 1 is a schematic diagram of a construction of a biologically active synthetic material.

FIG. 2 is a nitrogen-fixing microcapsule prepared based on artificially synthesized materials and further processed into an encapsulating material of a novel nitrogen-fixing microbial inoculum and a coating replacing a traditional seed coating agent.

FIG. 3 is a flow chart of the preparation of biologically viable synthetic materials.

FIG. 4 is a photomicrograph of a biologically active synthetic material.

FIG. 5 the survival rate (A) and the nitrogenase activity (B) of active microorganisms in the nitrogen-fixing microcapsules are changed.

FIG. 6 the number of active microorganisms colonizing the root system of rice (A) and maize (B) in nitrogen-fixing microvesicles.

FIG. 7 survival (A) and nitrogenase activity (B) changes of active microorganisms in coated seeds.

FIG. 8 shows the amino acid sequence homology analysis of the artificial hydroprotein MHYD of SEQ ID No. 2;

FIG. 9 shows the amino acid sequence homology analysis of MHGB of the artificial hemoglobin of SEQ ID No. 4.

Sequence information

SEQ ID No. 1: artificial hydrophilic protein gene mhyd nucleotide sequence

SEQ ID No. 2: amino acid sequence of artificial hydrophilic protein MHYD

SEQ ID No. 3: artificial hemoglobin gene mhgb nucleotide sequence

SEQ ID No. 4: amino acid sequence of artificial hemoglobin MHGB

Detailed Description

The relevant protein sources mentioned in the examples below are as follows:

source of artificial hydrophilic protein MHYD of SEQ ID No.2 and artificial synthesis

The structural domain of the deinococcus radiodurans hydrophilic protein DosH is truncated and recombined, the protein protection function is optimized, and the expression efficiency is improved. FIG. 8 shows amino acid sequence homology analysis.

Source of artificial hemoglobin MHGB of SEQ ID No.4 and artificial synthesis

The yak myoglobin is used as a skeleton, and the novel artificial myoglobin with higher oxygen carrying capacity is artificially designed through structural domain recombination. FIG. 9 shows amino acid sequence homology analysis.

Example 1 inducible expression and purification of Artificial hydrophilic proteins in E.coli

The experimental process comprises the following steps:

IPTG induced recombinant protein expression

(1) The successfully constructed recombinant strain BL-DosHM was streaked and activated. Single colonies were picked up in 3mL of fresh LB medium (containing 50mg/mL kanamycin) and cultured overnight at 37 ℃ with shaking at 220 rmp.

(2) The next day, the seed liquid is mixed according to the proportion of 1: 100 were transferred to 20mL LB medium containing Km (50mg/mL) and cultured at 37 ℃ with shaking at 220rmp with an OD600 of about 0.6 to 0.8.

(3) A final concentration of 0.5mM IPTG was added overnight at 16 ℃ (together with a blank control without IPTG), and E.coli was induced to express exogenously.

(4) The induced bacterial liquid is taken out, centrifuged for 5min at 5,000rmp, and the thallus is collected. The collected cells were suspended with NTA-0 buffer (1/20 culture volume) by shaking, mixed well and ice-cooled for 30 min.

(5) Adding Triton X-100 with final concentration of 0.05%, mixing, and placing on ice for 15 min.

(6) Cells were disrupted by ultrasound. The ultrasonic disruption time is 15min, and the disrupted cell frequency is 2s and 3s at intervals. After disruption, the cells were centrifuged at 12000rpm for 10min in a refrigerated centrifuge.

(7) And sucking the supernatant for purification.

(II) purification of His-tag-containing fusion protein

(1) The column was prepared in advance, 2-3mL of NTA resin was added to the column, and elution was performed with NTA-0 Buffer in a volume 10 times that of NTA.

(2) The total cellular protein after disruption was applied to a column at a flow rate of 15mL per hour, and the penetrating fraction was collected. To increase the purification efficiency, the sample was applied 3 times. The eluted fractions were analyzed by SDS-PAGE.

(3) Elution was performed by adding NTA-0 buffer in an amount of 5 resin volumes. The liquid was collected for detection of protein binding to the resin.

(4) Elution was carried out by adding 5 volumes of NTA-200 buffer solution, respectively, at a flow rate of 15mL per hour. And collecting the elution buffer for SDS-PAGE analysis to determine the expression of the target protein.

(5) Adding small amount of 5 Xprotein sample buffer solution, respectively, mixing, boiling in boiling water bath for 10min at 12000rpm for 10 min. 10 μ L of the supernatant was subjected to SDS-PAGE to determine the final elution concentration.

(6) The protein buffer was replaced by ultracentrifugation filter tubes and the protein was dissolved in 50mM Tris-HCl buffer.

Example 2 induced expression and purification of Artificial hemoglobin in E.coli

The experimental process comprises the following steps:

IPTG induced recombinant protein expression

(1) And (3) streaking and activating the successfully constructed recombinant strain BL-SYMB. Single colonies were picked up in 3mL of fresh LB medium (containing 50mg/mL kanamycin) and cultured overnight at 37 ℃ with shaking at 220 rmp.

(2) The next day, the seed liquid is mixed according to the proportion of 1: 100 were transferred to 20mL LB medium containing Km (50mg/mL) and cultured at 37 ℃ with shaking at 220rmp with an OD600 of about 0.6 to 0.8.

(3) Then, IPTG was added to the cells at final concentrations of 0.5mM, respectively, and the cells were cultured at 20 ℃ for 10 hours to induce E.coli to perform exogenous expression.

(4) The induced bacterial liquid is centrifuged for 5min at 5,000rmp, and the thallus is collected. The collected cells were suspended with NTA-0 buffer (1/20 culture volume) by shaking, mixed well and ice-cooled for 30 min.

(5) Adding Triton X-100 with final concentration of 0.05%, mixing, and placing on ice for 15 min.

(6) Cells were disrupted by ultrasound. The ultrasonic disruption time is 15min, and the disrupted cell frequency is 2s and 3s at intervals. After disruption, the cells were centrifuged at 12000rpm for 15min in a refrigerated centrifuge.

(7) And sucking the supernatant for purification.

(II) purification of His-tag-containing fusion protein

(1) The column was prepared in advance, 2-3mL of NTA resin was added to the column, and elution was performed with NTA-0 Buffer in a volume 10 times that of NTA.

(2) The total cellular protein after disruption was applied to a column at a flow rate of 15mL per hour, and the penetrating fraction was collected. To increase the purification efficiency, the sample was applied 3 times. The eluted fractions were analyzed by SDS-PAGE.

(3) Elution was performed by adding NTA-0 buffer in an amount of 5 resin volumes. The liquid was collected for detection of protein binding to the resin.

(4) Elution was carried out by adding 5 volumes of NTA-200 buffer solution, respectively, at a flow rate of 15mL per hour. And collecting the elution buffer for SDS-PAGE analysis to determine the expression of the target protein.

(5) Adding small amount of 5 Xprotein sample buffer solution, respectively, mixing, boiling in boiling water bath for 10min at 12000rpm for 10 min. 10 μ L of the supernatant was subjected to SDS-PAGE to determine the final elution concentration.

(6) The protein buffer was replaced by ultracentrifugation filter tubes and the protein was dissolved in 50mM Tris-HCl buffer.

Example 3 preparation of biologically active synthetic Material

The experimental process comprises the following steps:

the raw materials comprise the following components:

the short-chain PHA is a commercial product and is purchased from Shenzhen Italian Manchu Biotech, Inc.; dissolving the artificial hydrophilic protein in 50mM Tris-HCl buffer solution, wherein the concentration is 0.5 g/ml; the artificial hemoglobin was dissolved in 50mM Tris-HCl buffer at a concentration of 0.5 g/ml.

The second step is that the raw materials are prepared by the following steps (figure 3):

(1) carrying out sound treatment on a proper amount of short-chain PHA aqueous solution for 5min at a frequency of 20kHz-100kHz by using an ultrasonic instrument to form a first solution;

(2) mixing artificial hydrophilic protein and artificial hemoglobin dissolved in 50mM Tris-HCl buffer solution in a ratio of 1:1, and stirring for 15min to form a second solution;

(3) adding the first solution after the sound treatment into the second solution according to the volume ratio of 5:1 to form a third solution, wherein the volume ratio of the PHA aqueous solution, the artificial hydrophilic protein solution and the artificial hemoglobin solution in the third solution is 10:1: 1;

(4) neutralizing the third solution with a suitable neutralizing agent 0.1mM sodium carbonate;

(5) and (3) performing sound treatment on the neutralized solution for 15min at the frequency of 20kHz-100kHz by using an ultrasonic instrument to obtain the neutralized solution, wherein the sound treatment causes the PHA in the neutralized solution and the artificial protein encapsulated in the PHA to generate self-assembly. The neutralization solution was filtered using a disposable 0.45 μm microporous filter without significant effect on particle yield or particle size distribution.

(6) Vacuum drying at 40 deg.c for 48 hr to obtain artificially synthesized material with bioactivity.

The experimental results are as follows:

the structure of the artificially synthesized material of the invention is observed by using an optical microscope under the conventional research conditions, the particle size is about 100-200 μm, the finished product particles are uniform, and the structure is in a porous honeycomb shape (figure 4).

Example 4 preparation of Nitrogen-fixing microcapsules and determination of the viability and Activity of the Strain

The experimental process comprises the following steps:

(1) nitrogen-fixing strains A and B were grown overnight to OD1.0 (approximately 10 in number)⁸-10⁹/mL)。

(2) Mixing the artificially synthesized material with the nitrogen-fixing strains A and B, standing for 24h, centrifuging, removing supernatant, collecting precipitate, and naturally air drying to obtain nitrogen-fixing microcapsule. Meanwhile, PHA materials and nitrogen-fixing strains A and B are mixed for treatment, artificial proteins and the nitrogen-fixing strains A and B are mixed, and the nitrogen-fixing strains A and B are not treated, and the like are used as controls.

(3) Dissolving the nitrogen-fixing microcapsule and different control treatments in physiological saline, and storing at room temperature. Taking out proper amount of samples on d0, d5, d10, d15, d20, d40, d60, d80 and d100 days respectively, measuring the number of viable bacteria by a plate colony counting method after shaking to calculate the survival rate of the strain.

(4) Dissolving the nitrogen-fixing microcapsule and different control treatments in physiological saline, and storing at room temperature. Taking out proper amount of samples respectively at d0, d5, d10, d15, d20, d40, d60, d80 and d100 days, shaking and measuring the nitrogen-fixing enzyme activity of the microcapsule under the nitrogen-fixing condition by using an acetylene reduction method.

(5) And respectively adding 9ml of K nitrogen-free culture medium into the small bottles for measuring the activity of the azotobacter, and then adding 1ml of sample to be measured.

(6) The sterilized rubber plug is clamped by tweezers burned by an alcohol lamp to seal the small bottle, and the bottle is covered and sealed.

(7) The vial was filled with 5 minutes of argon to exhaust the air from the vial, and then with 1mL of oxygen and 10mL of acetylene.

(8) And (3) placing the small bottle at 30 ℃, carrying out shake culture at 200rpm, respectively, after 4 hours, 6 hours and 8 hours, absorbing 2.5mL of gas in the bottle after 10 hours to detect the ethylene peak area, and calculating the azotobacter activity of the azotobacter microcapsule by using the formula of azotobacter activity, namely ethylene peak area x (total gas phase volume/sampling volume of a triangular flask)/(1 nmol ethylene standard peak area x reaction time x total mycoprotein amount).

The experimental results are as follows:

the survival rates of the azotobacter A and the azotobacter B in the azotobacter microcapsule are the highest, after 20 days, the survival rates are 67.5 percent and 64.3 percent respectively, and after 100 days, the survival rates are 5.4 percent and 7.5 percent respectively (fig. 5A). The survival rates of the azotobacter A and the azotobacter B treated by the PHA material after 100 days are respectively 2.9 percent and 1.0 percent. The survival rates of the azotobacter A and the azotobacter B treated by the artificial protein after 100 days are 0.1 percent and 0.08 percent respectively. The survival rate of the untreated strain was the lowest, and the number of strains after 20 days was close to 0.

After 20 days, the azotobacter A and B have highest azotobacter activity, and are respectively maintained at 6235 and 6547nmol ethylene (mg protein h)^-1About 7.6% and 6.8% respectively, compared to the initial culture (FIG. 5B); the azotobacter activity of the microencapsulated azotobacter A and B is maintained at 5382 and 5453nmol ethylene (mg protein h) after 100 days^-1About 20.1% and 22.4% lower than at the beginning of the culture. The enzyme activity of azotobacteria A and B treated by PHA material is decreased by 35% after 20 days and by 89% after 100 days. The enzyme activity of azotobacter A and azotobacter B treated by artificial protein is reduced by about 91% in 20 days, and is reduced by about 97% after 100 days. The enzyme activity of the untreated strain decreased by about 98.2% after 20 days.

The results show that the nitrogen-fixing microcapsule formed by wrapping the nitrogen-fixing bacteria with the artificially synthesized material can effectively improve the survival rate of the nitrogen-fixing bacteria and the activity of the nitrogen-fixing enzyme.

Example 5 determination of efficiency of colonization of root System by Nitrogen-fixing microcapsule

The experimental process comprises the following steps:

(I) efficiency of rice root colonization

(1) The experiment is carried out in intelligent greenhouse, can control temperature and humidity in full time. The number of experimental treatments is 8, including bacterium A + PHA + artificial protein, bacterium B + PHA + artificial protein, bacterium A + PHA, bacterium B + PHA, bacterium A + artificial protein, bacterium B + artificial protein, bacterium A and bacterium B; the experiment was set up in 8 replicates.

(2) Soaking and cleaning rice seeds in sterile water for 30min, transferring to 5% NaClO solution, soaking for 1min, treating in 75% ethanol for 2min, and cleaning with sterile water for 5 times. The rice seeds are cultured in sterile filter paper, the filter paper is kept wet during the culture, and the rice seeds emerge 5 to 7 days after germination. The rice seedlings were placed in plastic pots (internal diameter 20cm, height 20cm) containing 2.5 kg of soil matrix (Klasmann-Deilmann), 2 seedlings per pot, 8 replicates per group. Dissolving each treated microcapsule or microbial inoculum in a quantitative Hoagland solution, and pouring the solution into soil.

(3) The rice was taken out on days d0, d5, d10, d15, d20, d40, d60, d80 and d100, respectively, and the roots of the rice were washed with sterile water. The roots of the rice were cut, blotted dry with sterile filter paper, and weighed. Put into a centrifuge tube to which 10ml of 0.85% physiological saline is added. The tube was sonicated for 100s, at 5s intervals, repeated 5 times, and vortexed for 10 min. The suspension is diluted in a gradient (10)^-3、10^-4、10^-5) And (5) coating the plate. After 24h of incubation at 30 ℃ the colonies were counted and the number of microorganisms colonized on the root surface was calculated.

(II) corn root colonization efficiency

(1) The experiment is carried out in intelligent greenhouse, can control temperature and humidity in full time. The number of experimental treatments is 8, including bacterium A + PHA + artificial protein, bacterium B + PHA + artificial protein, bacterium A + PHA, bacterium B + PHA, bacterium A + artificial protein, bacterium B + artificial protein, bacterium A and bacterium B; the experiment was set up in 8 replicates.

(2) Soaking and cleaning semen Maydis in sterile water for 30min, transferring to 5% NaClO solution, soaking for 1min, treating in 75% ethanol for 2min, and cleaning with sterile water for 5 times. Surface sterilized corn seeds were placed in plastic pots (20 cm id, 20cm height) containing 2.5 kg of soil matrix (Klasmann-Deilmann) with 4 seeds per pot, 8 replicates per group. The experiment was set up in 8 replicates. Thinning the corn seedlings to 2 grains/pot after the corn seedlings emerge. Dissolving each treated microcapsule or microbial inoculum in a quantitative Hoagland solution, and pouring the solution into soil.

(3) Taking out the corns on d0, d5, d10, d15, d20, d40, d60, d80 and d100 days respectively, and washing the roots of the corns with sterile water. The corn roots were cut, blotted dry with sterile filter paper, and weighed. Put into a centrifuge tube to which 10ml of 0.85% physiological saline is added. The tube was sonicated for 100s, at 5s intervals, repeated 5 times, and vortexed for 10 min. The suspension is diluted in a gradient (10)^-3、10^-4、10^-5) And (5) coating the plate. After 24h of incubation at 30 ℃ the colonies were counted and the number of microorganisms colonized on the root surface was calculated.

The experimental results are as follows:

after the rice is inoculated with the nitrogen-fixing bacteria A and the bacteria B which are encapsulated by the microcapsules, the number of the nitrogen-fixing bacteria A and the bacteria B on the root system respectively reaches 1.48 multiplied by 10 after 20 days⁵And 2.46X 10⁵Perg root, the number of the azotobacter A and B in the root system of the rice reaches 4.41 multiplied by 10 respectively after 100 days⁶And 3.21X 10⁶In g root (FIG. 6A). After the azotobacter A and the azotobacter B treated by PHA material are inoculated, the number of the azotobacter A and the azotobacter B in the root system of the rice reaches 1.58 multiplied by 10 after 20 days⁴And 9.87X 10³Root/g, the amount reached 1.25X 10 after 100 days⁵And 7.25X 10⁴In grams root. After inoculating azotobacteria A and B treated by artificial protein, the number of bacteria A and B in rice root system after 20 days reaches 3.27 × 10³And 1.34X 10³Root/g, the amount reached 2.21X 10 after 100 days⁴And 1.24X 10⁴In grams root. When the untreated fungus A and B were inoculated, the number of the fungus A and B in the rice root system after 20 days was 8.54X 10²And 8.76X 10²Root/g, number 8.52X 10 after 100 days²And 8.72X 10²/g root。

After the corn is inoculated with the nitrogen-fixing bacteria A and the bacteria B which are encapsulated by the microcapsules, the number of the nitrogen-fixing bacteria A and the bacteria B of the root system reaches 5.49 multiplied by 10 after 20 days⁵And 7.36X 10⁵Perg root, the number of azotobacter A and B in corn root system reaches 4.31 multiplied by 10 after 100 days⁶And 6.42X 10⁶In g root (FIG. 6B). When connectingAfter the azotobacteria A and the azotobacteria B are treated by the PHA material, the number of the azotobacteria A and the azotobacteria B in the root system of the corn reaches 2.74 multiplied by 10 after 20 days⁴And 8.96X 10³Root/g, the amount reached 9.84X 10 after 100 days⁴And 6.99X 10⁴In grams root. After the azotobacter A and the azotobacter B treated by artificial protein are inoculated, the number of the azotobacter A and the azotobacter B in the root system of the corn reaches 1.83 multiplied by 10 after 20 days³And 2.76X 10³Root/g, the amount reached 7.89X 10 after 100 days³And 1.63X 10⁴In grams root. When the untreated bacteria A and B are inoculated, the number of the bacteria A and B in the root system of the corn is 1.09 multiplied by 10 after 20 days³And 1.26X 10³Root/g, number 1.03X 10 after 100 days³And 1.37X 10³/g root。

The results show that the affinity of the nitrogen-fixing microcapsule formed by wrapping nitrogen-fixing bacteria with the artificially synthesized material and the plant root system is obviously enhanced.

Example 6 evaluation of growth promoting Effect of Nitrogen-fixing microcapsules on plants

The experimental process comprises the following steps:

first, growth promotion test of rice

(1) The experiment is carried out in intelligent greenhouse, can control temperature and humidity in full time. The number of experimental treatments is 8, including bacterium A + PHA + artificial protein, bacterium B + PHA + artificial protein, bacterium A + PHA, bacterium B + PHA, bacterium A + artificial protein, bacterium B + artificial protein, bacterium A and bacterium B; the experiment was set up in 8 replicates.

(2) Soaking and cleaning rice seeds in sterile water for 30min, transferring to 5% NaClO solution, soaking for 1min, treating in 75% ethanol for 2min, and cleaning with sterile water for 5 times. The rice seeds are cultured in sterile filter paper, the filter paper is kept wet during the culture, and the rice seeds emerge 5 to 7 days after germination. Dissolving the treated micro-capsules or microbial inoculum in normal saline, and soaking the rice seedlings in the normal saline for 30min respectively. The soaked rice seedlings were placed in plastic pots (inner diameter 20cm, height 20cm) containing 2.5 kg of soil matrix (Klasmann-Deilmann), 2 seedlings per pot, and 8 seedlings per group were replicated. The experiment was set up in 8 replicates. And (5) performing normal management in the rice growth period. And measuring growth indexes of the rice, such as overground and underground growth amount, thousand seed weight, nitrogen content and the like after planting for 90 days.

(II) corn growth promotion test

(1) The experiment is carried out in intelligent greenhouse, can control temperature and humidity in full time. The number of experimental treatments is 8, including bacterium A + PHA + artificial protein, bacterium B + PHA + artificial protein, bacterium A + PHA, bacterium B + PHA, bacterium A + artificial protein, bacterium B + artificial protein, bacterium A and bacterium B; the experiment was set up in 8 replicates.

(2) Soaking and cleaning semen Maydis in sterile water for 30min, transferring to 5% NaClO solution, soaking for 1min, treating in 75% ethanol for 2min, and cleaning with sterile water for 5 times. Dissolving the treated microcapsules or microbial inoculum in normal saline, and soaking the seeds with surface disinfection in normal saline for 30 min. The soaked corn seeds were placed in plastic pots (20 cm id, 20cm height) containing 2.5 kg of soil matrix (Klasmann-Deilmann) with 4 seeds per pot, 8 replicates per group. The experiment was set up in 8 replicates. Thinning the seedlings of the corns to 2/pot after the seedlings emerge, and managing the corns in the growth period according to the normal condition. And respectively measuring the growth indexes of the overground and underground growth quantities of the corns, the yield of the single-plant grains, the nitrogen content and the like after planting for 100 days.

The experimental results are as follows:

after the rice is inoculated with the nitrogen-fixing bacteria A and the bacteria B which are encapsulated by the microcapsules, the plant height, the dry weight, the root length, the thousand seed weight and the nitrogen content of the seeds are all obviously higher than those of PHA material treatment, artificial protein treatment and untreated control (Table 1). After the micro-encapsulated azotobacter A and B are inoculated, the plant height of the rice is increased by 22-38% compared with other treatments, the root length is increased by 18-66%, the dry weight of the plant is increased by 9-41%, the thousand seed weight is increased by 17-47%, and the nitrogen content is increased by 25-106%.

TABLE 1 influence of Nitrogen-fixing microcapsules on growth, yield and nitrogen content of rice under greenhouse potting conditions

After the maize is inoculated with the nitrogen-fixing bacteria A and the bacteria B which are encapsulated by the microcapsules, the plant height, the dry weight, the root length, the thousand seed weight and the nitrogen content of the seeds are all obviously higher than those of PHA material treatment, artificial protein treatment and untreated control (Table 2). After the micro-encapsulated azotobacter A and bacteria B are inoculated, the plant height of the corn is improved by 7-18% compared with other treatments, the root length is improved by 26-64%, the dry weight of the plant is increased by 0.8-8%, the yield of single grains is increased by 5-45%, and the nitrogen content is increased by 11-60%.

TABLE 2 influence of nitrogen-fixing microcapsules on maize growth, yield and nitrogen content under greenhouse potting conditions

The results show that the growth quantity, yield and nitrogen content of the overground part and the underground part of the plant can be obviously improved after the nitrogen-fixing micro-capsules formed by wrapping nitrogen-fixing bacteria with the artificially synthesized material enter the rhizosphere of the plant.

Example 7 seed coating preparation Using Nitrogen fixation coating and Strain viability and Activity assays

The experimental process comprises the following steps:

(1) film coating formulations were prepared according to table 3. Rice and corn seeds were coated with a slurry consisting of 43.1 wt.% film coating formulation, 43.3 wt.% water and 13.6 wt.% pigment concentrate. The amount of slurry applied was 5.5g/kg of seeds.

TABLE 3 composition of film coating formulation

Composition (I)	Composition ratio (% wt/wt)
		Water (W)	60
Rheological additive	0.2
		Defoaming agent	0.2
Vinyl acetate adhesive emulsion (50%)	15
		Artificial synthetic material with biological activity	24.6

(2) Taking out proper amount of seed samples in d0, d5, d10, d15, d20, d40, d60, d80 and d100 days respectively, crushing and shaking the seed samples by a wall breaking machine, dissolving the crushed and shaken seed samples in physiological saline, and measuring the number of viable bacteria by a plate colony counting method to calculate the survival rate of the strain.

(4) Taking out a proper amount of seed samples in d0, d5, d10, d15, d20, d40, d60, d80 and d100 days respectively, crushing and shaking the seed samples by using a wall breaking machine, dissolving the crushed seed samples in physiological saline, and measuring the nitrogenase activity of the microcapsule under the nitrogen fixation condition by using an acetylene reduction method.

(5) And respectively adding 9ml of K nitrogen-free culture medium into the small bottles for measuring the activity of the azotobacter, and then adding 1ml of sample to be measured.

(6) The sterilized rubber plug is clamped by tweezers burned by an alcohol lamp to seal the small bottle, and the bottle is covered and sealed.

(7) The vial was filled with 5 minutes of argon to exhaust the air from the vial, and then with 1mL of oxygen and 10mL of acetylene.

(8) And (3) placing the small bottle at 30 ℃, carrying out shake culture at 200rpm, respectively, after 4 hours, 6 hours and 8 hours, absorbing 2.5mL of gas in the bottle after 10 hours to detect the ethylene peak area, and calculating the azotobacter activity of the azotobacter microcapsule by using the formula of azotobacter activity, namely ethylene peak area x (total gas phase volume/sampling volume of a triangular flask)/(1 nmol ethylene standard peak area x reaction time x total mycoprotein amount).

The experimental results are as follows:

the survival rates of azotobacter A and azotobacter B in the coated seed coating are highest, after 20 days, the survival rates are 41.3% and 43.6% respectively, and after 100 days, the survival rates are 7.5% and 7.6% respectively (fig. 7A). The survival rate of the strain without seed coating treatment after 20 days is 0.11%, and the survival rate after 100 days is 0.0011%.

The azotobacter A and B have the highest azotobacter activity in the seed coating after coating treatment, and the azotobacter A and B have the azotobacter activity maintained at 3235 and 2997nmol ethylene (mg protein h) after 20 days^-1About 39% and 42% decrease, respectively, compared to the initial culture (FIG. 7B); the azotobacter activity of azotobacter A and B in the coated seeds is maintained at 2382 and 2053nmol ethylene (mg protein h) after 100 days^-1About 55% and 60% decrease from the start of culture. The enzyme activity of the uncoated strain decreased by about 89% after 20 days as 563nmol ethylene (mg protein h)^-1After 100 days, the enzyme activity is only 55nmol of ethylene (mgprotein h)^-1Relative to 1.1% of the initial phase.

The results show that the seed coating formed by the nitrogen fixation coating treatment can obviously improve the survival rate of the nitrogen-fixing bacteria and the activity of the nitrogen-fixing enzyme.

Example 8 evaluation of coating Effect of Nitrogen-fixing coated seeds

The experimental process comprises the following steps:

evaluation of Rice seed coating Effect

(1) The experiment is carried out in intelligent greenhouse, can control temperature and humidity in full time. 3 experimental treatments including bacteria A coated seeds, bacteria B coated seeds and non-coated seeds; the experiment was set up in 8 replicates.

(2) Soaking and cleaning rice seeds in sterile water for 30min, transferring to 5% NaClO solution, soaking for 1min, treating in 75% ethanol for 2min, and cleaning with sterile water for 5 times. And (4) culturing the rice seeds in sterile filter paper, keeping the filter paper wet during the culture, germinating 5-7 days after germination, and counting the germination rate. The rice seedlings were transplanted into plastic pots (inner diameter 20cm, height 20cm) containing 2.5 kg of soil matrix (Klasmann-Deilmann), 2 seedlings per pot, 8 replicates per group. The experiment was set up in 8 replicates. And (5) performing normal management in the rice growth period. And measuring growth indexes of the rice, such as overground and underground growth amount, thousand seed weight, nitrogen content and the like after planting for 90 days.

(II) evaluation of corn seed coating effect

(1) The experiment is carried out in intelligent greenhouse, can control temperature and humidity in full time. 3 experimental treatments including bacteria A coated seeds, bacteria B coated seeds and non-coated seeds; the experiment was set up in 8 replicates.

(2) Soaking and cleaning semen Maydis in sterile water for 30min, transferring to 5% NaClO solution, soaking for 1min, treating in 75% ethanol for 2min, and cleaning with sterile water for 5 times. The surface sterilized corn seeds were placed in plastic pots (20 cm id, 20cm height) containing 2.5 kg of soil matrix (Klasmann-Deilmann) with 4 seeds per pot, 8 replicates per group. The experiment was set up in 8 replicates. And counting the germination rate after the corn seedlings emerge, thinning the seedlings to 2 seedlings/pot, and normally managing the seedlings in the growth period of the corn. And respectively measuring the growth indexes of the overground and underground growth quantities of the corns, the yield of the single-plant grains, the nitrogen content and the like after planting for 100 days.

The experimental results are as follows:

the germination rate of the rice seeds formed by coating the azotobacter A and the azotobacter B is 98.6 percent and is obviously higher than the germination rate of the rice seeds without coating by 96.3 percent. The plant height of the plant in the mature period is about 109cm, which is increased by 24 percent compared with the control of the seed without the coating; the root length is about 32cm, which is improved by 60 percent compared with the uncoated seed; the dry weight of the plant is about 87g, which is increased by 38% compared with the uncoated seed control; the thousand kernel weight is about 28g, which is increased by 56% compared with the uncoated seed control; nitrogen content was about 2.5g/plant, a 92% increase over the non-coated seed control (Table 3).

The germination rate of the corn seeds formed by coating the azotobacter A and the azotobacter B is 99.5 percent and is obviously higher than the germination rate of the corn seeds without coating 97.2 percent. The plant height of the plant in the mature period is about 206cm, which is increased by 102 percent compared with the control of the non-coated seed; the root length is about 76cm, which is increased by 41 percent compared with the uncoated seed; the dry weight of the plant is about 492g, which is increased by 6% compared with the non-coated seed control; the yield of single grains is about 186g, which is 39% higher than that of non-coated seeds; nitrogen content was about 3.2g/plant, 167% higher than the uncoated seed control (Table 4).

The results show that the seed coating formed by nitrogen fixation coating treatment can obviously improve the germination rate of the seeds, and the growth quantity, yield and nitrogen content of the overground part and the underground part of the mature plants are also obviously increased.

TABLE 4 germination percentage, growth amount, yield and nitrogen content of nitrogen-fixing coated seeds under greenhouse potting conditions

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> artificially synthesized material with biological activity and agricultural application thereof

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<170> PatentIn version 3.1

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ATGTTTGAAC GCGATGAACA TCACTTTCCG GTTAAGCGTC TGTTGCTGCT CGGTGCCCTC 60

GTCGGGGCCG GCGCCTACTA CCTGAGCCGC GAGCAAAACC GCAAGGCGCT CGACGCCAAG 120

CTGGCCGAAC TTGGCCTGAA AGACGCCGCG CAGGACGTGG GCAGCAGCGT GACCAAGGGC 180

TGGGAAAAGA CCAAGGACGC CGCTCAGAAC GCCGGAAGTG TCATCGCCGA CAAAGCGCAG 240

GACGTGGCGG GCGAAGTGAA GAGCGCCGTG GCGGGCGCGA CCGCCGAAAT CAAGGACGCG 300

GGCAAGGAAG TGGCCGACAC CGCCAAGGAC GCCGGTCAGA ACGTGGGCCA GAACGTCAAG 360

CGCGAAGCTG CCGACCTCGC TGACCAGGCG AAGGACAAAG CCCAGGACGT GAAGGCTGAT 420

GTCAGCAAGG CTGCCGACCA GGCCAAAGAC AAAGCTCAGG ATGTCGCCCA GAACGTGCAA 480

GCCGGGGCCC AGCAGGCCGC CGCCAACGTC AAGGACAAGG TTCAGGATGT GAAGGCTGAC 540

GCCAGCAAGG ACGCCGAAGC GGGCAAGCAG GGCGGCCAGA CCGGCAGCAC CACGAACAAT 600

GCTGGTACGG CGGGCAACAC CGGCATGACG GGCAACACCA ACACCCGCAA GAACTGA 660

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CTGGAAAAAT TCGATAAATT TAAGCATTTA AAAACGGAAG CAGAAATGAA GGCAAACCCT 180

AAATTAGCGG GCCACGCTGA AAAACTCTTT GGACTTGTGC GTGATAGTGC AATACAATTA 240

CGTGCCAAAA AGAAGGGGCA TCACGAAGCG GAGGACGCTG CTTTGGGAAG TATTCATGCA 300

CAAAATAAGC ATAAAATACC CGTTAAATAC CTCGAATTCA TATCCGACGC AATAATTCAT 360

GTTCTACATG CCAAACATCC CTCAGATTTT GGTGCCGATG CTCAGGCTGC GATGTCGAAA 420

GCTCTTGAAT TATTCAGAAA TGATATGGCT GCCCAGTATA AAGTACTTGG GTTTCATGGT 480

TAA 540

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