CN114478705B

CN114478705B - Small molecular protein for improving photosynthesis of wheat and application thereof

Info

Publication number: CN114478705B
Application number: CN202210175053.1A
Authority: CN
Inventors: 孔祥强; 罗振; 唐薇; 尹静; 卢合全
Original assignee: Shandong Academy of Agricultural Sciences
Current assignee: Shandong Academy of Agricultural Sciences
Priority date: 2022-02-24
Filing date: 2022-02-24
Publication date: 2023-05-05
Anticipated expiration: 2042-02-24
Also published as: CN114478705A

Abstract

The invention discloses a small molecular protein for improving photosynthesis of wheat and application thereof. The invention utilizes bioinformatics analysis and research, and replaces partial amino acid, and finally finds 19 small molecular proteins EPW-1-EPW-19 which can obviously improve photosynthesis of wheat flag leaves through repeated screening. In a proper growth period, the content of EPW in wheat can be increased by spraying EPW solution on leaf surfaces, so that the photosynthesis capacity of wheat leaves is improved, and the wheat yield is improved.

Description

Small molecular protein for improving photosynthesis of wheat and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a small molecular protein (EPW, enhance photosynthesis of wheat) for improving photosynthesis of wheat and application thereof.

Background

Wheat is an important grain crop in China, and the improvement of the wheat yield is significant for guaranteeing the grain safety in China. The scientific and reasonable fertilization and irrigation are key to realizing high and stable yield of crops, and in field production, the reasonable fertilization and irrigation can promote the growth of wheat and improve the yield of wheat, but with the massive application of fertilizer, the utilization rate of fertilizer can be obviously reduced, so that massive application of fertilizer remains in soil and causes fertilizer loss, thereby causing environmental pollution. Improving the nutrient absorption efficiency of crops is a key measure for increasing the crop yield and reducing fertilizer pollution. Leaf photosynthesis plays an important role in wheat growth, wherein flag leaves play an important role in yield, and improving flag leaf photosynthesis can remarkably improve wheat yield. Wheat leaves are the main source of kernel carbohydrates, and flag leaf photosynthetic rate and flag leaf area have a significant positive correlation with ear weight and yield. Therefore, improving flag leaf photosynthetic rate or extending flag leaf photosynthesis time has an important role in improving wheat yield.

The plant hormone plays an important role in regulating and controlling plant growth and development, stress tolerance and nutrient absorption. Recently, many small molecule substances have been found to also function like hormones. At present, a large number of small molecular proteins (polypeptides) have been found in animals and plants, and play a very important role in regulating disease resistance, stress tolerance and growth of animals and plants. Therefore, on the basis of fully researching the functions of the small molecular proteins, novel small molecular proteins for regulating and controlling the in-vivo ion transport of wheat are developed, a proper in-vitro application method is established, the nutrient content in wheat flag leaves is improved, the photosynthesis of the wheat is enhanced, and technical support can be provided for improving the yield of the wheat and guaranteeing the grain safety of China.

Disclosure of Invention

The invention utilizes bioinformatics analysis and research, and replaces partial amino acid, and finally finds 19 small molecular proteins EPW-1-EPW-19 which can obviously improve photosynthesis of wheat flag leaves through repeated screening. In a proper growth period, the content of EPW in wheat can be increased by spraying EPW solution on leaf surfaces, so that the photosynthesis capacity of wheat leaves is improved, and the wheat yield is improved.

The technical scheme of the invention is as follows: a small molecule protein for improving photosynthesis of wheat, which is any one or a mixture of more than two of EPW-1 to EPW-19, wherein the amino acid sequence of EPW-1 to EPW-19 is as follows:

EPW-1：DVRPTAPGHSPSIGH

EPW-2：DVRPTAPGHSPSVGH

EPW-3：DVRPTAPGHSPSAGH

EPW-4：DVRPTAPGHSPSMGH

EPW-5：DVRPTAPGHSPGIGH

EPW-6：DVRPTAPGHSPSIGN

EPW-7：DVRPTAPGHSPSVGN

EPW-8：DVRPTAPGHSPSAGN

EPW-9：DVRPTAPGHSPSMGN

EPW-10：DFRPTAPGHSPGIGN

EPW-11：DFRPTAPGHSPSIGN

EPW-12：DFRPTAPGHSPSVGN

EPW-13：DFRPTAPGHSPSAGN

EPW-14：DFRPTAPGHSPSMGN

EPW-15：DFRPTAPGHSPGIGH

EPW-16：DFRPTAPGHSPSIGH

EPW-17：DFRPTAPGHSPSVGH

EPW-18：DFRPTAPGHSPSAGH

EPW-19：DFRPTAPGHSPSMGH

the small molecular proteins EPW-1 to EPW-19 can improve photosynthesis of wheat (especially photosynthesis of flag leaves), thereby improving wheat yield.

The method for improving the photosynthesis of the wheat by adopting the EPW-1 to EPW-19 comprises the following steps: spraying EPW solution on the leaf surfaces of wheat in the flowering period; the EPW solution is EPW aqueous solution containing Silwet series organic silicon surfactant, and is sprayed by 10-15L per mu when used in a field. The addition of Silwet series organic silicon surfactant in EPW solution can promote small molecular protein to enter wheat leaves, promote EPW to exert effect, and better improve yield.

The working concentration of the EPW solution is as follows: the final concentration of any one or more than two of EPW-1 to EPW-19 is 0.1+ -0.05 g/L (preferably 0.1 g/L), and the concentration of Silwet series silicone surfactant is 0.005-0.01%.

The invention has the technical effects that:

1. the prior art mainly improves the wheat yield by a breeding method or improves the wheat yield by a cultivation method such as increasing fertilization amount or fertilizing for a plurality of times. However, as the amount of fertilizer applied increases, the fertilizer utilization rate of wheat decreases, and the increase in the number of fertilizer applications increases labor costs. The method of the invention overcomes the problems existing in the prior art, adopts the leaves to spray the small molecular protein in the key period of the reproductive growth of the wheat, improves the photosynthesis of the wheat flag leaves and improves the wheat yield. Therefore, the product can improve the wheat yield without increasing the fertilizer consumption, not only can prevent environmental pollution, but also can improve the wheat yield, and has wide application prospect.

2. A field experiment shows that after the EPW solution is sprayed on the wheat leaves, the photosynthesis of the wheat leaves can be improved, the thousand seed weight of the wheat can be improved, and the wheat yield can be improved. Therefore, the method not only has the function of improving photosynthesis of wheat flag leaves, but also has the function of improving yield, and provides a realistic and effective product and method for sustainable and efficient development of wheat production.

Drawings

FIG. 1 is a graph showing the effect of treatment of wheat with EPW-1 to EPW-19 solutions, respectively, on net photosynthetic rate of wheat flag leaves; as can be seen from the figures: EPW-1 to EPW-19 solutions can obviously improve the net photosynthetic rate of wheat flag leaves;

FIG. 2 is a graph showing the effect of treating wheat with solutions EPW-1 to EPW-19, respectively, on thousand kernel weight of wheat in test examples; as can be seen from the figures: EPW-1 to EPW-19 can obviously improve thousand grain weight of wheat;

FIG. 3 is a graph showing the effect of treatment of wheat with solutions EPW-1 to EPW-19, respectively, on wheat yield in test example III; as can be seen from the figures: EPW-1 to EPW-19 solutions can obviously improve the wheat yield;

FIG. 4 is a graph showing the effect of treatment of wheat with EPW and ZLC solutions, respectively, on net photosynthetic rate of wheat flag leaves in test example four; as can be seen from the figures: compared with ZLC solution, EPW solution can obviously improve the net photosynthetic rate of wheat flag leaves;

wherein EPW solution: an aqueous solution of EPW containing silwet L-77 surfactant; ZLC solution: an aqueous solution of ZLC containing silwet L-77 surfactant; CK: clear water control containing the same concentration of silwet L-77 surfactant.

Detailed Description

The technical scheme of the present invention 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 invention, but not limiting the present invention in any way, and any changes or substitutions based on the present invention are all within the scope of the present invention. The method of the invention is a conventional method in the art unless specifically stated otherwise. The reagents of the invention 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 protein sequence in crops such as gramineae wheat, barley, oat and the like, the inventor combines the expression abundance of the protein gene in the wheat, uses bioinformatics to analyze and research specificity and the like, changes partial amino acids, preliminarily selects 78 small molecular proteins from 300 small molecular proteins, determines that 78 small molecular proteins are sprayed on wheat flag She Shese and photosynthesis, and finally discovers that 19 small molecular proteins can obviously improve the photosynthesis of wheat flag leaves. The amino acids 3 to 11 of the 19 small molecule proteins are conserved, as follows: RPTAPGHSP. The 19 small molecule protein sequences were respectively named: EPW-1 to EPW-19.

(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) Taking 0.27mmol of Fmoc-C terminal second amino acid-OH, 0.54mmol DIPEA,0.26mmol HBTU, adding into the centrifuge tube, and adding a proper amount of solvent DMF for reaction for 30 minutes to obtain 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

Respectively taking 10g of equal mass mixture of EPW-1 to EPW-19 and 19 small molecular proteins, dissolving with 1L of deionized water to prepare 100x mother liquor, and standing the mother liquor below-20 ℃ for 1-2 weeks, wherein the mother liquor can be prepared at room temperature.

In the field application, 100ml of EPW mother liquor is taken, diluted with tap water, finally diluted to a constant volume of 10L, then 1ml of silwet L-77 surfactant is added, and the mixture is uniformly mixed to make the final concentration of the mixture reach 0.1g/L, EPW working solution contains 1/10000 silwet L-77 surfactant, and the solution is used in the following test example.

Spraying the leaf surface of the wheat in the flowering period, wherein the spraying amount is 10-15L/mu; spraying is carried out before 10 am or after 4 pm and in 1 day without rain. When spraying, EPW solution can be uniformly sprayed on the front and back sides of the wheat leaves as much as possible.

Test example one, influence of foliar spray EPW solution on photosynthesis of wheat

The full Jimai No. 22 wheat seeds are selected, sowed in the field on the verruca mongolica in 10 months and 16 days in 2020, the wheat sowing amount is 25 jin of seeds per mu, the row spacing is 20cm. Treatment with 19 spray EPW (EPW-1 to EPW-19 total 19 types) solutions was set up to spray clean water with the same concentration of silwet L-77 surfactant as a control, randomly arranged in groups, and repeated 3 times. Cell area 36m ² A 6 row area is used. And in the flowering period of the wheat, 10L of EPW solution with the concentration of 0.1g/L is sprayed on each mu of leaves after the rainless weather is selected to be 4 pm. The photosynthetic rate of wheat flag leaf was measured 20 days after spraying. The EPW solution in this experiment was a solution of 19 small molecule proteins containing EPW-1 to EPW-19, respectively (preparation method is shown in example 2).

Test results: the wheat flag leaf photosynthetic rate was measured and it was found that spraying EPW significantly increased the wheat flag leaf photosynthetic rate compared to the control (fig. 1). It is proved that the foliage spraying EPW solution can obviously improve the photosynthetic rate of the wheat flag leaves, and the EPW-1 to EPW-19 solutions have little effect on improving the photosynthetic rate of the wheat flag leaves.

Test example II, influence of foliar spray EPW solution on thousand grain weight of wheat

The full Jimai No. 22 wheat seeds are selected, sowed in the field on the verruca mongolica in 10 months and 16 days in 2020, the wheat sowing amount is 25 jin of seeds per mu, the row spacing is 20cm. Treatment with 19 spray EPW (EPW-1 to EPW-19 total 19 types) solutions was set up to spray clean water with the same concentration of silwet L-77 surfactant as a control, randomly arranged in groups, and repeated 3 times. Cell area 36m ² A 6 row area is used. And in the flowering period of the wheat, 10L of EPW solution with the concentration of 0.1g/L is sprayed on each mu of leaves after the rainless weather is selected to be 4 pm. After wheat maturing, each cell was harvested separately and the thousand kernel weight of wheat was determined after harvest. In the test, the EPW solution is a solution containing 19 small molecular proteins EPW-1 to EPW-19 respectively (the preparation method is as shown in example 2), and EPW solution can be uniformly sprayed on the front and back sides of wheat leaves as much as possible during spraying.

Test results: after wheat harvest, the thousand kernel weight of the wheat was measured, and it was found that spraying EPW solution significantly increased the thousand kernel weight of the wheat (fig. 2) compared to the control, and EPW-1 to EPW-19 solutions were not significantly different in effect in increasing the thousand kernel weight of the wheat.

Test example Effect of three-leaf spraying EPW solution on wheat yield

The full Jimai No. 22 wheat seeds are selected, sowed in the field on the verruca mongolica in 10 months and 16 days in 2020, the wheat sowing amount is 25 jin of seeds per mu, the row spacing is 20cm. Treatment with 19 spray EPW (EPW-1 to EPW-19 total 19 types) solutions was set up to spray clean water with the same concentration of silwet L-77 surfactant as a control, randomly arranged in groups, and repeated 3 times. Cell area 36m ² A 6 row area is used. And in the flowering period of the wheat, 10L of EPW solution with the concentration of 0.1g/L is sprayed on each mu of leaves after the rainless weather is selected to be 4 pm. After the wheat is mature, harvesting is performed and the wheat yield is determined. In the test, the EPW solution is a solution containing 19 small molecular proteins EPW-1 to EPW-19 respectively (the preparation method is as shown in example 2), and EPW solution can be uniformly sprayed on the front and back sides of wheat leaves as much as possible during spraying.

Test results: after wheat harvest, wheat yield was measured and it was found that spraying EPW solution significantly improved wheat yield compared to control (fig. 3), and EPW-1 to EPW-19 solutions were not significantly effective in increasing thousand kernel weight of wheat.

Test example four, influence of foliar spray EPW and ZLC solution on wheat photosynthesis

The applicant previously filed 1 patent with application number 2021110799970, and the effective components of the patent application are any one or more than two of ZLC1 to ZLC 20. The ZLC is adopted to spray cotton leaves, so that the cotton yield and the fiber quality can be improved. In order to compare the functional differences between ZLC and the invention active ingredient EPW, the following experiments were performed:

the full Jimai No. 22 wheat seeds are selected, sowed in the field on the verruca mongolica in 10 months and 16 days in 2020, the wheat sowing amount is 25 jin of seeds per mu, the row spacing is 20cm. The mixture of EPW is sprayed with a solution (EPW mixture is EPW-1 to EPW-19 solution of equal weight mixture of 19 types of small molecule proteins, and the preparation method is as followsAs shown in example 2), ZLC mixture solution treatment (ZLC mixture solution ZLC-1 to ZLC-20 of an equal weight mixture solution of 20 types of small molecule polypeptides, the preparation method is the same as that of the patent with application number 2021110799970) was sprayed, and the spray of clear water containing the same concentration of silwet L-77 surfactant was used as a control, and the spray was repeated 3 times in random block arrangement. Cell area 36m ² A 6 row area is used. And in the flowering period of the wheat, after the rainless weather is selected to be 4 pm, each mu of leaf surfaces is sprayed with 0.1g/L of EPW mixture and 10L of ZLC mixture solution. The photosynthetic rate of wheat flag leaf was measured 20 days after spraying.

Test results: measuring the photosynthetic rate of the wheat flag leaf, and finding that compared with a control, the spraying EPW remarkably improves the photosynthetic rate of the wheat flag leaf, and the EPW treated wheat photosynthetic rate is improved by 19.1 percent compared with the control; the photosynthetic rate of wheat flag leaves can be slightly improved by 7.2% compared with a control by spraying ZLC; however, the photosynthetic rate of EPW treated wheat flag leaves was significantly higher than that of ZLC treatment, 11.9% higher than ZLC treatment (fig. 4). The effect of EPW on improving the photosynthetic rate of wheat flag leaves is proved to be obviously superior to ZLC. Cotton is a dicotyledonous malvaceae plant, while wheat is a monocotyledonous gramineous plant, cotton and wheat are far related, and ZLC developed for cotton cannot efficiently improve wheat photosynthesis.

Claims

1. A small molecule protein for improving photosynthesis of wheat, which is any one or a mixture of more than two of EPW-1 to EPW-19, wherein the amino acid sequence of EPW-1 to EPW-19 is as follows:

EPW-1：DVRPTAPGHSPSIGH

EPW-2：DVRPTAPGHSPSVGH

EPW-3：DVRPTAPGHSPSAGH

EPW-4：DVRPTAPGHSPSMGH

EPW-5：DVRPTAPGHSPGIGH

EPW-6：DVRPTAPGHSPSIGN

EPW-7：DVRPTAPGHSPSVGN

EPW-8：DVRPTAPGHSPSAGN

EPW-9：DVRPTAPGHSPSMGN

EPW-10：DFRPTAPGHSPGIGN

EPW-11：DFRPTAPGHSPSIGN

EPW-12：DFRPTAPGHSPSVGN

EPW-13：DFRPTAPGHSPSAGN

EPW-14：DFRPTAPGHSPSMGN

EPW-15：DFRPTAPGHSPGIGH

EPW-16：DFRPTAPGHSPSIGH

EPW-17：DFRPTAPGHSPSVGH

EPW-18：DFRPTAPGHSPSAGH

EPW-19：DFRPTAPGHSPSMGH。

2. use of the small molecule protein of claim 1 for increasing photosynthesis in wheat.

3. The use according to claim 2, wherein the wheat photosynthesis is wheat flag leaf photosynthesis.

4. Use of the small molecule protein of claim 1 for increasing thousand kernel weight of wheat.

5. Use of the small molecule protein of claim 1 for increasing wheat yield.

6. A method for improving photosynthesis of wheat by adopting the small molecular protein of claim 1, which is characterized in that EPW solution is sprayed on leaves of the wheat in the flowering period; the EPW solution is an aqueous solution of EPW containing Silwet series silicone surfactant; the EPW is any one or a mixture of more than two of EPW-1 to EPW-19.

7. The method of claim 6, wherein the working concentration of the EPW solution is: the final concentration of any one or more than two of EPW-1 to EPW-19 is 0.1+ -0.05 g/L, and the concentration of Silwet series silicone surfactant is 0.005-0.01%.

8. The method of claim 7, wherein the solution of EPW is sprayed in an amount of 10-15L per mu when applied in a field.