CN114507271A - Small molecular protein for improving tobacco leaf quality - Google Patents
Small molecular protein for improving tobacco leaf quality Download PDFInfo
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- CN114507271A CN114507271A CN202210176701.5A CN202210176701A CN114507271A CN 114507271 A CN114507271 A CN 114507271A CN 202210176701 A CN202210176701 A CN 202210176701A CN 114507271 A CN114507271 A CN 114507271A
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
Aiming at the problems of increasing planting cost, reducing fertilizer utilization rate, aggravating environmental pollution, having limited effect of improving potassium content and the like existing in the prior art of improving the potassium content of tobacco leaves by additionally applying potassium fertilizer, the invention provides a small molecular protein for improving the quality of the tobacco leaves, which is any one or the combination of more than two of 25 small molecular proteins (EPUT-1 to EPUT-25) and is prepared into an EPUT aqueous solution containing Silwet series organosilicon surfactants when in use. The invention is proved by experiments that: the EPUT solution sprayed on the leaf surfaces can obviously improve the potassium ion content in the tobacco leaves, the nitrogen content is not increased, and the effect on improving the quality of the tobacco leaves is obvious. Compared with the conventional mode of increasing the potassium fertilizer, the mode of improving the potassium ion content in the tobacco leaves by spraying the EPUT solution on the leaf surfaces is simpler, more environment-friendly and more efficient.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a small molecular protein for improving tobacco leaf quality.
Background
The tobacco is a potassium-loving economic crop, and the application amount of potassium fertilizer in field production is generally 2.5-3 times of that of nitrogen fertilizer. The potassium content of the tobacco leaves is one of the most direct key indexes for evaluating the quality of the tobacco, and the increase of the potassium content of the tobacco leaves can obviously improve the yield and the quality of the tobacco leaves, improve the combustibility of the tobacco leaves, improve the identity color of the tobacco leaves and reduce the tar content of the tobacco leaves. The nitrogen content of the tobacco leaves is in negative correlation with the quality of the tobacco leaves, the higher the nitrogen content of the tobacco leaves is, the worse the quality is, and the nitrogen content in the tobacco leaves can be reduced, so that the quality of the tobacco leaves can be improved. The potassium content of the high-quality tobacco leaves is generally not lower than 3 percent, and the potassium content of the tobacco leaves in high-quality tobacco leaf producing countries such as Brazil, America and the like can be as high as 4 to 6 percent; while the average value of the potassium content of the tobacco leaves in China is less than 2 percent, and the average value of the potassium content of the tobacco leaves in Shandong province is only about 1.5 percent. The low content of potassium ions in the tobacco leaves becomes a key for limiting the quality of the tobacco leaves in Shandong and even China.
The potassium absorption capacity of the tobacco root system and the potassium content in the soil are key factors influencing the potassium content of the tobacco leaves. Reasonable fertilization and effective supply of potassium ions in the root zone of the tobacco are important ways for improving the content of the potassium ions in the tobacco leaves. However, the addition of potash fertilizer increases the planting cost, decreases the fertilizer utilization rate, increases environmental pollution, and has a limited effect on increasing the content of potassium ions in tobacco leaves. Therefore, the research and development of the technology for improving the utilization rate of the tobacco potassium fertilizer has important significance for improving the content of the tobacco potassium and promoting the green and continuous development of tobacco production.
Small molecular substances such as plant hormones, polypeptides and the like play very important roles in plant growth and development and nutrient absorption. By spraying IAA, GA and other hormones on the leaf surfaces, the potassium absorption of plants can be improved, the potassium content of the plants can be improved, and the fertilizer utilization rate can be improved. Recently, some small molecule proteins have been found to have hormone-like functions. For example, tobacco CLE can regulate plant senescence and arabidopsis CLE25 can regulate plant drought resistance. Therefore, screening and developing novel micromolecular protein for regulating and controlling potassium ion absorption, establishing a suitable in-vitro application method, and being a novel strategy for improving the utilization rate of the tobacco potassium fertilizer and improving the content and quality of the tobacco potassium ions.
The applicant has previously developed a small molecule substance (patent application No. 2021110799970) for improving cotton yield and fiber quality, the active ingredient of which is any one or a combination of two or more of small molecule proteins ZLC1 to ZLC20, and the last 6 amino acids of these small molecule proteins are SPGMGH. The cotton leaves are sprayed with the aqueous solution containing the micromolecule substance ZLC in the full-bloom stage, so that the nitrogen, phosphorus and potassium absorption capacity of cotton can be enhanced, the growth of the cotton is promoted, and the cotton yield and the fiber quality (fiber length and fiber strength) are improved. However, tobacco is a solanaceous plant of the order tubular flores, while cotton is a malvaceae plant of the order malvaceae, both belonging to different orders and having a far-ranging relationship. When the micromolecule substance ZLC effective to cotton is applied to tobacco, the effect of improving the potassium content of the tobacco leaves is not obvious, but the nitrogen content of the tobacco leaves can be slightly improved (figure 4), and the effect of improving the quality of the tobacco leaves is not achieved. Different from the strategy of simultaneously enhancing the absorption of nitrogen, phosphorus and potassium by cotton, the method needs to find small molecular substances which only improve the potassium content of the tobacco leaves but cannot improve the nitrogen content in the tobacco. Plants generally absorb nitrogen from the root system as nitrate, while plants generally absorb potassium ions and nitrate ions simultaneously in order to maintain cation-anion balance. Therefore, in order to find the micromolecule protein for improving the potassium content of the tobacco leaves, the thought completely different from cotton is utilized, and finally, a large number of experiments prove that 25 micromolecule proteins for improving the quality of the tobacco leaves are found, and the micromolecule protein is more excellent in the aspect of improving the potassium ion content of the tobacco leaves.
Disclosure of Invention
Aiming at the problems of increasing planting cost, reducing fertilizer utilization rate, aggravating environmental pollution, having limited effect of improving potassium content and the like existing in the prior art of improving the potassium content of tobacco leaves by adding potassium fertilizer, the invention provides a small molecular protein (EPUT) for improving the quality of the tobacco leaves. The invention is proved by experiments that: the EPUT solution sprayed on the leaf surfaces can obviously improve the potassium ion content in the tobacco leaves, the nitrogen content is not increased, and the effect on improving the quality of the tobacco leaves is obvious.
The invention provides a small molecular protein for improving the quality of tobacco leaves, which is characterized in that the small molecular protein is any one of or a combination of more than two of 25 small molecular proteins, and the amino acid sequence of the small molecular protein is as follows:
EPUT-1：SIEVEKSGHSPGAGH；
EPUT-2：SIEVEKSGHSPGEGH；
EPUT-3：SIEVEKSGHSPGIGH；
EPUT-4：SIEVEKSGHSPGMGH；
EPUT-5：SIEVEKSGHSPGRGH；
EPUT-6：SIEVEKSGHSPGVGH；
EPUT-7：SIEVEKSGNSPGAGH；
EPUT-8：SIEVEKSGNSPGEGH；
EPUT-9：SIEVEKSGNSPGIGH；
EPUT-10：SIEVEKSGNSPGMGH；
EPUT-11：SIEVEKSGNSPGRGH；
EPUT-12：SIEVEKSGNSPGVGH；
EPUT-13：SIEVEKSGPSPGAGH；
EPUT-14：SIEVEKSGPSPGEGH；
EPUT-15：SIEVEKSGPSPGIGH；
EPUT-16：SIEVEKSGPSPGMGH；
EPUT-17：SIEVEKSGPSPGRGH；
EPUT-18：SIEVEKSGPSPGVGH；
EPUT-19：SIEVEKSGSSPGAGH；
EPUT-20：SIEVEKSGSSPGEGH；
EPUT-21：SIEVEKSGSSPGIGH；
EPUT-22：SIEVEKSGSSPGMGH；
EPUT-23：SIEVEKSGSSPGRGH；
EPUT-24：SIEVEKSGSSPGVGH；
EPUT-25：SIEVQKSGPSPGEGH。
the second purpose of the invention is to provide the application of the small molecular protein in improving the quality of tobacco leaves. The tobacco leaf quality is improved mainly by increasing the content of potassium ions in the tobacco leaves without increasing the content of nitrogen.
The invention also aims to provide a method for improving the quality of tobacco leaves, which is characterized in that an EPUT aqueous solution is sprayed on the leaf surfaces 5 to 10 days before the bud period of the tobacco leaves to promote the potassium ion absorption of the tobacco leaves, so that the potassium ion content in the tobacco leaves is improved, the nitrogen content is not increased, and the quality of the tobacco leaves and the proportion of first-class tobacco leaves are improved.
Further, the EPUT solution is an aqueous solution of EPUT containing Silwet series organosilicon surfactant. Preferably, Silwet L-77 surfactant is used.
Preferably, the working concentration of the EPUT solution is: the final concentration of the single EPUT or EPUT mixture is 0.05-0.2g/L (preferably 0.1g/L), and the concentration of the silwet surfactant is 0.01-0.02%. The components are prepared according to the proportion, and the sprayer is used for spraying the foliage of the tobacco after the components are uniformly mixed.
Aiming at field use, the method for improving the quality of tobacco leaves comprises the following steps: spraying 5-10 days before the bud period of tobacco, and spraying 15-20L of EPUT solution with concentration of 0.05-0.2g/L per mu; the EPUT solution is uniformly sprayed on the front and back surfaces of the tobacco leaves as much as possible when the EPUT solution is sprayed before 10 am or 4 pm.
The invention has the following beneficial effects:
1. at present, the potassium fertilizer is mainly applied to improve the potassium ion content of tobacco leaves and the quality of tobacco leaves. Along with the application of a large amount of potash fertilizer, the utilization rate of the tobacco potash fertilizer is gradually reduced, the environmental pollution is easily caused, the excessive application of the potash fertilizer also has adverse effect on the growth of tobacco leaves, and the increase range of the potassium content is limited. The method breaks through the limitation of the prior art, improves the potassium ion content of the tobacco leaves and the quality of the tobacco leaves by regulating the potassium ion absorption capacity of the tobacco, and improves the proportion of first-class tobacco leaves, so that the product can improve the potassium ion content of the tobacco leaves and the quality of the tobacco leaves, can also improve the utilization rate of potassium fertilizer, reduces environmental pollution, and has wide market application prospect.
2. Experiments prove that after the EPUT solution is sprayed on the tobacco field leaf surfaces, the content of potassium in the tobacco leaves can be obviously improved, the content of nitrogen cannot be increased, and the effect of improving the quality of the tobacco leaves is obvious. Therefore, the method has the functions of improving the utilization rate of the potash fertilizer, improving the quality of the tobacco leaves and improving the proportion of the first-class tobacco leaves, is simpler, more environment-friendly and more efficient compared with the conventional method for applying the potash fertilizer, and has wide application prospect in the aspects of improving the potassium and increasing the aroma (quality) of the tobacco.
Drawings
FIG. 1 shows the results of the test examples of the effect of EPUT (EPUT-1 to EPUT-25) solutions on the potassium ion content of tobacco leaves on the treatment of tobacco seedlings;
FIG. 2 shows the effect of the EPUT (EPUT2, EPUT10) solution on the potassium ion content of tobacco lamina in field tobacco lamina (1 week before the bud stage) treated by the experimental example; wherein, A is potassium ion content of lower leaf, middle leaf and upper leaf of tobacco sampled at 23 days in 7 months; b, the potassium ion content of the middle leaf and the upper leaf of the tobacco sampled on 17 days in 8 months;
FIG. 3 shows the results of the effect of the three-way EPUT solution (EPUT2, EPUT10) on the proportion of upper grade tobacco when used to treat field tobacco lamina (1 week before the bud stage);
FIG. 4 shows the effect of ZLC mixed solution and EPUT mixed solution on potassium ion content and nitrogen content of tobacco leaves of the fourth test example; wherein, the A picture is the result of potassium ion content, and the B picture is the result of total nitrogen content.
Note: CK is clear water sprayed with silwet L-77 surfactant with the same concentration, EPUT (EPUT2, EPUT10) is water solution containing silwet L-77 surfactant sprayed with EPUT (EPUT2, EPUT 10); the ZLC mixed solution is a water solution which is sprayed with ZLC mixed and contains silwet L-77 surfactant.
Detailed Description
The technical solutions and the technical effects thereof will be further described with reference to the following specific embodiments and drawings, and the following description is only for explaining the present invention, but not limiting the present invention in any way, and any modifications or substitutions based on the present invention shall fall within the protection scope of the present invention. The methods of the present invention are conventional in the art unless otherwise specified. The reagents of the present invention are commercially available unless otherwise specified.
Example 1: determination and synthesis of amino acid sequence of small molecule protein
(1) Determination of amino acid sequence of small molecule protein
The inventor of the application searches the protein sequence in solanaceae plants (tobacco, tomato, eggplant and the like) by using a bioinformatics approach, selects 56 polypeptide sequences from the protein sequences to carry out tobacco leaf spraying experiments by combining the expression abundance and the sequence specificity of the protein gene in tobacco, and finds 7 polypeptides which improve the potassium ion content of the tobacco leaves and are arranged at the first 7 positions, wherein the first 8 amino acids of 6 polypeptides are SIEVQKSG, and the first 8 amino acids of the other polypeptide are SIEVEKSG; on the basis that the amino acid at the first 8 th position is SIEVEKSG, the amino acids at the 9 th position and the 13 th position are respectively replaced, 72 different polypeptide sequences are designed, then the 72 polypeptides are subjected to experimental screening, and finally 24 polypeptides with better potassium ion content improving capability are obtained, and in addition, the other polypeptide with the ammonia at the first 8 th position being SIEVQKSG, small molecular proteins of 25 sequences are discovered together, so that the potassium ion content of the tobacco leaves can be obviously improved.
Therefore, 25 small molecule protein sequences were finally determined. The first 8 amino acids of these small molecule proteins are conserved as: SIEV E (Q) KSG. The 25 small molecule protein sequences are named as: EPUT-1 to EPUT-25. Remarking: EPUT is an English acronym for Enhance potassium uptake in tobacao.
(2) Small molecule protein synthesis and preservation
Synthesizing the small molecular protein by using an Fmoc solid-phase polypeptide synthesis method. At least 100mg of crude product is expected to be synthesized according to 10mg of synthesized target small molecular polypeptide. The synthesis steps are as follows:
taking 0.3g of Fmoc-C terminal first amino acid-Wang resin, adding into a 1.5ml centrifuge tube, swelling with DCM for 20 minutes, and then filtering off DCM.
② adding 20 percent piperidine/DMF solution with 3 times of resin volume, blowing nitrogen for 30 minutes, and pumping to dry; then, the resin of the previous step was washed 5 times with 2 resin volumes of DMF.
③ taking 0.27mmol of Fmoc-C end second amino acid-OH, 0.54mmol of DIPEA and 0.26mmol of HBTU, adding a proper amount of solvent DMF, and reacting for 30 minutes to obtain Fmoc-C end second amino acid-C end first amino acid-Queen resin. Amino acid (b): DIPEA: HBTU: resin 3: 6: 2.85: 1 (molar ratio); then washing the resin of the previous step 3 times with 2 resin volumes of DMF; then adding a 20% Pip/DMF solution with the volume 3 times that of the resin, bubbling nitrogen for 30 minutes, draining, removing the Fmoc group to obtain H2N-second amino acid-first amino acid-queen resin; finally, the resin from the previous step was washed 5 times with 2 resin volumes of DMF.
And fourthly, repeating the step 3, adding 1 amino acid for each repeated step 3, and repeating according to the sequence requirement of the small molecular protein to obtain the final target small molecular polypeptide.
Washing the resin with methanol for 3 times, and draining the resin; then adding 6 times of cutting fluid (volume ratio; trifluoroacetic acid: thioanisole: 1, 2-ethanedithiol: phenol: water: 87.5%: 5%: 2.5%: 2.5%: 2.5%) of resin volume, shaking for 2 hours by a shaker, filtering out the resin, precipitating the filtrate by using ice anhydrous ether, washing the precipitate by using ice anhydrous ether for 3 times, finally putting the precipitate into a vacuum drying kettle, and drying for 24 hours at normal temperature to obtain a crude product of the micromolecule protein.
Purifying by an HPLC instrument to obtain the micromolecule protein with the purity of more than 95 percent:
first, the crude small molecule protein was analyzed by fast gradient using C18 analytical column. The mobile phase is as follows: an aqueous phase A: 0.1% TFA/water; and (3) organic phase B: 0.1% TFA/acetonitrile; gradient: 5-70 percent, 20 minutes, and the main peak of the crude product is about 7 minutes.
Secondly, the small molecule protein is separated by using a C18 preparation column, and a target peak is collected. And (5) measuring a mass spectrum, finding that the molecular weight of the collected target peak is consistent with the molecular weight of the target small molecular protein, and determining that the HPLC purity is more than 95 percent, wherein the HPLC purity is preliminarily qualified.
And then freezing the collected target peak solution by using liquid nitrogen, putting the target peak solution on a freeze dryer for vacuum drying to form powder, taking 1mg of the freeze-dried powder micromolecule protein, dissolving the powder by using water, and carrying out HPLC and MS retest, wherein the HPLC purity is over 95 percent, and the MS molecular weight is not changed to be finally qualified.
The small molecular protein can be stored at-80 deg.C for about 2 years, and at-20 deg.C for about half a year.
Example 2: preparation and application of foliar fertilizer EPUT
Dissolving EPUT (single substance or mixture) 10g with 1L deionized water to obtain 100 × mother liquor, and standing the mother liquor at-20 deg.C for 1-2 weeks, if it is required to be prepared at room temperature.
When the EPUT is applied to fields, 100ml of EPUT mother solution is taken, tap water is used for dilution, the final dilution volume is up to 10L, then 1ml of silwet L-77 surfactant is added, the mixture is uniformly mixed, the final concentration of the mixture (single EPUT or EPUT mixture) reaches 0.10g/L, and the EPUT working solution contains 1/10000 of the silwet L-77 surfactant.
Spraying the foliage 1 week before the bud period of the tobacco, wherein the spraying amount is 15-20L/mu; the spraying is carried out before 10 am or after 4 pm. And (3) uniformly spraying the EPUT solution on the front surface and the back surface of the tobacco leaves as much as possible during spraying.
Test example I, influence of foliar application of EPUT solution on growth and yield of field cotton
The method comprises the steps of sowing full and full seeds of the specially fragrant 301 tobacco in a plastic box (the diameter is 10cm, the height is 10cm), placing the box in an artificial climate chamber at the temperature of 28 ℃, thinning the seedlings after 1 true leaf is grown out after germination, and reserving 1 strong tobacco seedling in each pot. After the 6 th main leaf of the tobacco grows out, selecting tobacco seedlings with consistent growth vigor for treatment, setting 25 treatments of spraying EPUT (EPUT-1 to EPUT-25 are 25 types in total) solutions, taking clear water sprayed with silwet L-77 surfactant with the same concentration as a reference, setting 3 times of treatment for each treatment, and repeating 1 tobacco seedling for each time. After 10 days of treatment, the tobacco leaves of each treatment were collected, dried, weighed, and then the potassium ion content was determined. In the test, the EPUT solutions are 25 kinds of small molecular protein solutions respectively containing EPUT-1 to EPUT-25 (the preparation method is shown in example 2), the final concentration is 0.10g/L, the EPUT solutions can be uniformly sprayed on the front and back surfaces of the tobacco leaves as much as possible during spraying, and the spraying dosage of each plant is 10 ml.
And (3) test results: after 10 days of tobacco treatment, sampling was performed to determine the content of potassium ions in the tobacco leaves, and it was found that the content of potassium ions in the tobacco leaves was significantly increased by applying EPUT compared to the control CK (FIG. 1). The fact that the leaf surface spraying of the EPUT can promote the root system of the tobacco to absorb potassium ions and increase the potassium content of the leaves is proved, wherein the effect of increasing the potassium content of the leaves by spraying of the EPUT-2 solution and the EPUT-10 solution is the best.
Test example II, influence of foliar-sprayed EPUT solution on potassium ion content of field tobacco leaves
And (3) sowing the full 301 tobacco seeds in a seedling tray in 2021 year 2 month 20, growing the seedling tray in a tobacco seedling greenhouse of a tobacco company in Feizian county, and transplanting the tobacco seedlings to a field in 4 months and 25 days. Spraying treatment is carried out one week before budding of tobacco in 30 days in 6 months, and after 4 pm, 15L of EPUT solution of 0.10g/L is sprayed on each mu of leaf surface. Setting 2 kinds of EPUT (EPUT-2 and EPUT-10) spraying treatments, and randomly arraying in groups by using clear water sprayed with silwet L-77 surfactant with the same concentration as a control, and repeating for 3 times. Cell area 60m2The method adopts 4-row area, and plants with equal row spacing, the row spacing is 120cm, and the planting spacing is 50 cm. And performing daily management on the tobacco according to local tobacco planting habits. And respectively taking the lower leaf, the middle leaf and the upper leaf of the tobacco on 23 days in 7 months, and respectively taking the middle leaf and the upper leaf of the tobacco on 17 days in 8 months to perform potassium ion determination. In the test, the preparation methods of the EPUT-2 and EPUT-10 solutions are shown in example 2, and the EPUT solution can be uniformly sprayed on the front and back surfaces of the tobacco leaves as much as possible during spraying.
And (3) test results: compared with the control CK, the treatment of the EPUT-2 and EPUT-10 solutions improves the potassium ion content of the lower leaf, the middle leaf and the upper leaf of the tobacco sampled on 23 days in 7 months (figure 2A), and also improves the potassium ion content of the middle leaf and the upper leaf of the tobacco sampled on 17 days in 8 months (figure 2B), and the EPUT-2 and EPUT-10 have little effect on improving the potassium ion content, and the EPUT-2 has a little good effect on improving the potassium ion content of the middle leaf of the tobacco, but the EPUT-2 has a little difference on improving the potassium ion content of the lower leaf and the upper leaf compared with the EPUT-10.
Test example influence of spaying of EPUT solution on tobacco leaf quality of field tobacco
Sowing the full 301 tobacco seeds in 2021 year 20 month in seedling tray, and growing the seedling tray in tobacco seedling greenhouse of Fei county tobacco companyAnd 4, transplanting the tobacco seedlings to the field in 25 days in 4 months. Spraying treatment is carried out one week before budding of tobacco in 30 days in 6 months, and after 4 pm, 15L of EPUT solution of 0.10g/L is sprayed on each mu of leaf surface. Setting 2 kinds of EPUT (EPUT-2 and EPUT-10) spraying treatments, and randomly arraying in groups by using clear water sprayed with silwet L-77 surfactant with the same concentration as a control, and repeating for 3 times. Cell area 60m2The method adopts 4-row area, and plants with equal row spacing, the row spacing is 120cm, and the planting spacing is 50 cm. And performing daily management on the tobacco according to local tobacco planting habits. And harvesting tobacco leaves according to a local planting mode, baking, and finally counting the equal tobacco leaf proportion. In the test, the preparation methods of the EPUT-2 and EPUT-10 solutions are shown in example 2, and the EPUT solution can be uniformly sprayed on the front and back surfaces of the tobacco leaves as much as possible during spraying.
And (3) test results: both EPUT-2 and EPUT-10 solution treatments increased superior tobacco leaf ratios compared to control CK (FIG. 3).
Remarking: the evaluation criteria of first-class tobacco leaves are as follows: the tobacco leaves are divided into 42 grades according to the growth parts (upper part, middle part and lower part) and the leaf colors (lemon yellow, orange yellow and red brown); the first middle orange C1F, the second middle orange C2F, the third middle orange C3F, the first middle lemon C1L, the second middle lemon C2L, the first upper orange B1F, the second upper orange B2F, the first upper lemon B1L, the first upper red B1R, the first fully cooked H1F and the first lower orange X1F are first-class tobacco leaves.
Test example four, influence of foliar spray of ZLC and EPUT solutions on potassium ion content and nitrogen content of tobacco leaves
Sowing the full and full seeds of the specially fragrant 301 tobacco in a plastic box (the diameter is 10cm, the height is 10cm), placing the box in an artificial climate room at 28 ℃, thinning the seedlings after 1 true leaf is grown in each pot, and reserving 1 strong tobacco seedling. After the 6 th true leaf of the tobacco grows out, selecting tobacco seedlings with consistent growth vigor for treatment, setting the treatment of spraying ZLC (20 types of equal weight mixtures from ZLC-1 to ZLC-20) solution and the treatment of spraying EPUT (25 types of equal weight mixtures from EPUT-1 to EPUT-25) solution, taking spraying clear water containing silwet L-77 surfactant with the same concentration as a contrast, setting 3 times of treatment for each treatment, and repeating 1 tobacco seedling for each time. After 10 days of treatment, the tobacco leaves of each treatment were collected, dried, weighed, and then the potassium ion content and nitrogen content were determined. In the test, the preparation method of the EPUT solution is shown in example 2, the preparation method of the ZLC solution is the same as that of the patent with the patent application number of 2021110799970, the final concentration of the EPUT solution and the ZLC solution is 0.10g/L, liquid medicine can be uniformly sprayed on the front side and the back side of the tobacco leaf as much as possible during spraying, and the spraying dosage of each plant is 10 ml.
And (3) test results: after 10 days of tobacco treatment, sampling and measuring the content of potassium ions in the tobacco leaves, and finding that the content of the potassium ions in the tobacco leaves is remarkably improved by spraying the EPUT solution compared with the control CK, but the potassium increasing effect is not obvious by spraying the ZLC solution (figure 4A); spraying the EPUT solution slightly reduced the nitrogen content of the tobacco leaves, but spraying the ZLC solution slightly increased the nitrogen content of the tobacco leaves (FIG. 4B). The potassium content and the nitrogen content in the tobacco leaves respectively present positive correlation and negative correlation with the quality of the tobacco leaves. The small molecular protein EPUT can obviously improve the content of potassium ions in tobacco leaves and slightly reduce the nitrogen content, so that the effect of improving the quality of the tobacco leaves is obvious. Meanwhile, the mode of improving the quality of the tobacco leaves by spraying the EPUT solution on the tobacco leaf surfaces is simpler, more environment-friendly and more efficient compared with the conventional mode of increasing the potassium fertilizer, so that the method has wide application prospect in the aspect of improving the quality of the tobacco leaves.
Claims (9)
1. The small molecular protein for improving the quality of tobacco leaves is characterized by being any one or a combination of more than two of 25 small molecular proteins, and the amino acid sequence of the small molecular protein is as follows:
EPUT-1：SIEVEKSGHSPGAGH；
EPUT-2：SIEVEKSGHSPGEGH；
EPUT-3：SIEVEKSGHSPGIGH；
EPUT-4：SIEVEKSGHSPGMGH；
EPUT-5：SIEVEKSGHSPGRGH；
EPUT-6：SIEVEKSGHSPGVGH；
EPUT-7：SIEVEKSGNSPGAGH；
EPUT-8：SIEVEKSGNSPGEGH；
EPUT-9：SIEVEKSGNSPGIGH；
EPUT-10：SIEVEKSGNSPGMGH；
EPUT-11：SIEVEKSGNSPGRGH；
EPUT-12：SIEVEKSGNSPGVGH；
EPUT-13：SIEVEKSGPSPGAGH；
EPUT-14：SIEVEKSGPSPGEGH；
EPUT-15：SIEVEKSGPSPGIGH；
EPUT-16：SIEVEKSGPSPGMGH；
EPUT-17：SIEVEKSGPSPGRGH；
EPUT-18：SIEVEKSGPSPGVGH；
EPUT-19：SIEVEKSGSSPGAGH；
EPUT-20：SIEVEKSGSSPGEGH；
EPUT-21：SIEVEKSGSSPGIGH；
EPUT-22：SIEVEKSGSSPGMGH；
EPUT-23：SIEVEKSGSSPGRGH；
EPUT-24：SIEVEKSGSSPGVGH；
EPUT-25：SIEVQKSGPSPGEGH。
2. the use of the small molecule protein of claim 1 to improve tobacco leaf quality.
3. Use according to claim 2, wherein the improvement in tobacco quality is achieved primarily by increasing the potassium ion content of the tobacco without increasing the nitrogen content.
4. A method for improving the quality of tobacco leaves is characterized in that the aqueous solution of the small molecular protein EPUT disclosed in claim 1 is sprayed on the leaf surfaces 5-10 days before the bud period of the tobacco leaves to promote the potassium ion absorption of the tobacco leaves, so that the potassium ion content in the tobacco leaves is improved, the nitrogen content is not increased, and the quality of the tobacco leaves and the proportion of first-class tobacco leaves are improved.
5. The method for improving the quality of tobacco leaves according to claim 4, wherein the aqueous solution of the small molecular protein EPUT is an EPUT aqueous solution containing Silwet series organic silicon surfactants.
6. The method for improving the quality of tobacco leaves as claimed in claim 5, wherein the Silwet series silicone surfactant is Silwet L-77 surfactant.
7. The method according to claim 5, wherein the working concentration of the aqueous solution of the small molecule protein EPUT is: the final concentration of single EPUT or EPUT mixture is 0.05-0.2g/L, and the concentration of Silwet series silicone surfactant is 0.01-0.02%.
8. The method of claim 7, wherein the final concentration of the single EPUT or mixture of EPUTs is 0.1 g/L.
9. The method for improving the quality of the tobacco leaves according to claim 7, which is used in a field, and comprises the following steps: spraying 15-20L of EPUT solution with concentration of 0.05-0.2g/L per mu 5-10 days before the bud stage of tobacco; the EPUT solution is uniformly sprayed on the front and back surfaces of the tobacco leaves as much as possible when the EPUT solution is sprayed before 10 am or 4 pm.
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