阅读说明：本技术 Idl7成熟多肽植物衰老促进剂、其制备方法和应用 (IDL7 mature polypeptide plant aging promoter, and preparation method and application thereof ) 是由 郭永峰 郭存 王奇 李伟 张增林 文利超 邓智超 刘涛 于 2020-11-27 设计创作，主要内容包括：本发明提出一种IDL7成熟多肽植物衰老促进剂、其制备方法和应用,属于植物衰老促进剂领域,该IDL7成熟多肽植物衰老促进剂以IDL7成熟多肽为主要功能成份,所述IDL7成熟多肽具有如下氨基酸序列：F-G-S-L-V-L-N-A-L-P-K-G-S-R-P-G-S-G-P-S-K-K-T-N。本发明提供的植物衰老促进剂可以促进植物的叶片衰老,使其衰老加速,且无其它额外不良表现,具有极强的田间可操作性。(The invention provides an IDL7 mature polypeptide plant senescence promoter, a preparation method and application thereof, belonging to the field of plant senescence promoters, wherein the IDL7 mature polypeptide plant senescence promoter takes IDL7 mature polypeptide as a main functional component, and the IDL7 mature polypeptide has the following amino acid sequence: F-G-S-L-V-L-N-A-L-P-K-G-S-R-P-G-S-G-P-S-K-K-T-N. The plant senescence promoter provided by the invention can promote leaf senescence of plants, so that the senescence of the plants is accelerated, no other additional adverse performance exists, and the plant senescence promoter has extremely strong field operability.)

The IDL7 mature polypeptide plant senescence promoter, which is characterized in that the plant senescence promoter takes IDL7 mature polypeptide as a main functional component, and the IDL7 mature polypeptide has the following amino acid sequence: F-G-S-L-V-L-N-A-L-P-K-G-S-R-P-G-S-G-P-S-K-K-T-N.

2. The plant senescence promoter according to claim 1, wherein the plant senescence promoter comprises IDL7 mature polypeptide and 2- (N-morpholine) ethanesulfonic acid solution; wherein the 2- (N-morpholine) ethanesulfonic acid solution is a solution obtained by dissolving 2- (N-morpholine) ethanesulfonic acid monohydrate in MS liquid culture medium.

3. The plant senescence promoter according to claim 2, wherein the concentration of the IDL7 mature polypeptide is 10 to 13 μmol/L, the concentration of 2- (N-morpholine) ethanesulfonic acid is 2.8 to 3mmol/L, and the pH of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8 to 5.9.

4. The plant senescence promoter according to claim 3, wherein the concentration of IDL7 mature polypeptide is 10 μmol/L, the concentration of 2- (N-morpholine) ethanesulfonic acid is 2.8mmol/L, and the pH of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8.

5. The plant aging promoter according to claim 2, further comprising an auxiliary agent, wherein the auxiliary agent is tween-20, and the addition amount of the auxiliary agent is 1 to 2% o.

6. The method for preparing IDL7 mature polypeptide plant senescence promoter according to any one of claims 1-5, comprising the following steps:

dissolving IDL7 mature polypeptide powder in water to prepare IDL7 mature polypeptide mother liquor with the concentration of 10-13mmol/L for later use;

adding a certain amount of 2- (N-morpholine) ethanesulfonic acid monohydrate into a prepared MS liquid culture medium to prepare 2.8-3 mmol/L2- (N-morpholine) ethanesulfonic acid solution, adjusting the pH value of the solution to 5.8-5.9, fully mixing, and dissolving uniformly to obtain 2- (N-morpholine) ethanesulfonic acid solution;

adding the obtained IDL7 mature polypeptide mother liquor into 2- (N-morpholine) ethanesulfonic acid solution, and stirring uniformly to obtain IDL7 mature polypeptide plant aging promoter.

7. The method according to claim 6, wherein the method further comprises the step of adding 1-2% o tween-20 after the obtained IDL7 mature polypeptide mother liquor is added into the 2- (N-morpholine) ethanesulfonic acid solution.

8. The use of the IDL7 mature polypeptide plant senescence promoter for promoting plant senescence according to any one of claims 1 to 5, wherein when in use, the IDL7 mature polypeptide plant senescence promoter is directly sprayed on the leaf surface of the plant leaf in the complete expansion period, or the IDL7 mature polypeptide plant senescence promoter is soaked in cotton balls and is smeared on the leaf surface of the plant leaf in the complete expansion period.

9. The use of the IDL7 mature polypeptide plant senescence promoter of any one of claims 1-5, wherein the IDL7 mature polypeptide plant senescence promoter is directly sprayed on the leaf surface of the tobacco leaf at the early stage of maturation or the IDL7 mature polypeptide plant senescence promoter is soaked in cotton balls and then is smeared on the leaf surface of the tobacco leaf at the early stage of maturation.

10. The use according to claim 8 or 9, wherein the concentration directly sprayed on the leaf surface of the tobacco leaf at the early stage of maturity is: 10-12 mu mol/L.

Technical Field

The invention belongs to the field of plant senescence promoters, and particularly relates to an IDL7 mature polypeptide plant senescence promoter, and a preparation method and application thereof.

Background

Leaf senescence is an important factor affecting agronomic traits such as crop yield and quality. Taking tobacco as an example, the leaves are harvesting organs of the tobacco, the leaves enter an aging stage along with the fact that the tobacco enters a growth later stage, the maturity periods of the upper leaves, the middle leaves and the lower leaves of the tobacco are different, if a strategy that different parts are harvested at different periods is adopted, harvesting time can be delayed, and the problems of inconsistent maturity and green turning of the tobacco leaves at different parts can be effectively solved by using the aging accelerator.

At present, the application of the plant senescence control is mainly focused on the regulation of plant hormones, for example, auxin, cytokinin and the like can inhibit leaf senescence, ethylene and abscisic acid can promote leaf senescence, and the law can be mastered to artificially slow down the leaf senescence process. The work of function analysis relating to senescence has been widely and deeply carried out, and a large number of genes promoting leaf senescence have been functionally analyzed. The leaf senescence delay phenotype is found by the deletion of the dioxygenase gene dependent on 2-oxoglutarate in arabidopsis thaliana, such as ligustrum quihoui and the like; after the Dexamethasone (DEX) induces the gene to be over-expressed, the leaf senescence of the transgenic arabidopsis can be advanced; guo et al found that overexpression of the NAP gene, a member of the Arabidopsis AtNAC family, can cause early senescence of leaves, while deletion mutants of the NAP gene delay senescence, and similarly, in rice, the gene can affect leaf senescence. However, to date, there have been few reports that the reported senescence-associated genes are actually directly applied to crop production.

The polypeptide is a small molecular substance with regulation and control functions in animals and plants, and the mature polypeptide is a functional molecule which is formed by cutting and modifying a precursor protein after translation and finally forms short amino acid, and is used as a ligand to identify with a receptor positioned on a cell membrane and start signal transduction. Mature polypeptides can be obtained by artificial synthesis, and direct exo-application can have an effect on plant growth, such as root elongation. However, the application of mature polypeptide in plants, especially crops, is rarely studied at present, and the application of mature polypeptide in the aspect of promoting plant senescence is not studied.

Disclosure of Invention

The IDL7 mature polypeptide plant senescence promoter can promote leaf senescence of plants, enables senescence of the plants to be accelerated, and has no other additional adverse performance.

In order to solve the technical problems, the invention provides an IDL7 mature polypeptide plant senescence promoter, which takes IDL7 mature polypeptide as a main functional component, and the IDL7 mature polypeptide has the following amino acid sequence:

F-G-S-L-V-L-N-A-L-P-K-G-S-R-P-G-S-G-P-S-K-K-T-N。

preferably, the plant senescence promoter comprises IDL7 mature polypeptide and 2- (N-morpholine) ethanesulfonic acid solution; wherein the 2- (N-morpholine) ethanesulfonic acid solution is a solution obtained by dissolving 2- (N-morpholine) ethanesulfonic acid monohydrate in MS liquid culture medium. It is understood that 2- (N-morpholine) ethanesulfonic acid is used as a solvent for dissolving IDL7 mature polypeptide, because it is a novel biological buffer that can better promote the polypeptide to provide the corresponding function.

Preferably, the concentration of the IDL7 mature polypeptide is 10-13 mu mol/L, the concentration of 2- (N-morpholine) ethanesulfonic acid is 2.8-3mmol/L, and the pH of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8-5.9. It will be appreciated that the concentration of the mature polypeptide of IDL7 may be 10, 11, 12, 13. mu. mol/L, the concentration of the 2- (N-morpholine) ethanesulfonic acid solution may be 2.8, 2.9, 3mmol/L, and the pH of the 2- (N-morpholine) ethanesulfonic acid solution may be 5.8, 5.9.

Preferably, the plant senescence promoter further comprises an auxiliary agent, and the auxiliary agent can be a conventional auxiliary agent used in the field for leaf surface wetting and leaf surface spreading, and the scheme is not particularly limited as long as the solution can be spread and osmotically wetted on the surface of the plant as quickly as possible. It is understood that the auxiliary agent can be tween-20, and the addition amount of the auxiliary agent is 1 to 2 per thousand.

The invention also provides a preparation method of the IDL7 mature polypeptide plant senescence promoter, which specifically comprises the following steps:

s1: dissolving IDL7 mature polypeptide powder in water to prepare IDL7 mature polypeptide mother liquor with the concentration of 10-13mmol/L for later use;

s2: adding a certain amount of 2- (N-morpholine) ethanesulfonic acid monohydrate into a prepared MS liquid culture medium to prepare 2.8-3 mmol/L2- (N-morpholine) ethanesulfonic acid solution, adjusting the pH value of the solution to 5.8-5.9, fully mixing, and dissolving uniformly to obtain 2- (N-morpholine) ethanesulfonic acid solution;

the MS culture medium powder (not containing agar and sucrose) used in the steps is purchased from Beijing Soilebao company, when the MS culture medium powder is used, 4.42g of the powder in the formula is dissolved in 1L of water, the culture medium can fully dissolve 2- (N-morpholine) ethanesulfonic acid, a certain penetration type can be formed on the surface of a plant, a foundation is provided for the function exertion of polypeptide, and meanwhile, the liquid culture medium can also be used as a solvent to provide corresponding large and medium element nutrition and guarantee the action effect.

S3: adding the obtained IDL7 mature polypeptide mother liquor into 2- (N-morpholine) ethanesulfonic acid solution, and stirring uniformly to obtain IDL7 mature polypeptide plant aging promoter. Wherein the concentration of IDL7 mature polypeptide is 10-13 μmol/L, and the concentration of 2- (N-morpholine) ethanesulfonic acid is 2.8-3 mmol/L.

It is understood that the steps S1 and S2 may be replaced with each other, and may be prepared in advance at S1 or at S2, which is not specifically required herein. In addition, the prepared IDL7 mature polypeptide mother liquor needs to be prepared as before use, and if the IDL7 mature polypeptide mother liquor is not used for more than 40 days, the mother liquor is stored at-70 ℃; if the mother liquor is used within 40 days, the mother liquor is stored at-20 ℃ and stored at 4 ℃ within one week.

Preferably, the method also comprises the step of adding 1-2 per thousand of tween-20 after adding the obtained IDL7 mature polypeptide mother liquor into a 2- (N-morpholine) ethanesulfonic acid solution.

The invention also provides the application of the IDL7 mature polypeptide plant senescence promoter in the aspect of promoting plant senescence, wherein in the application, the IDL7 mature polypeptide plant senescence promoter is directly sprayed on the leaf surface of a plant leaf in the complete expansion period, or the IDL7 mature polypeptide plant senescence promoter is used for soaking cotton balls and coating the cotton balls on the leaf surface of the plant leaf in the complete expansion period.

The invention also provides the application of the IDL7 mature polypeptide plant senescence promoter in the aspect of promoting plant senescence, wherein in the application process, the IDL7 mature polypeptide plant senescence promoter is directly sprayed on the leaf surface of the tobacco leaf at the early stage of maturation, or the IDL7 mature polypeptide plant senescence promoter is used for soaking cotton balls and smearing the cotton balls on the leaf surface of the tobacco leaf at the early stage of maturation.

It can be understood that whether the plant leaf senescence promoter is directly sprayed on the early leaf surface of the plant/crop leaf or the cotton ball soaked with the plant senescence promoter is coated on the early leaf surface of the plant/crop leaf, the plant leaf senescence promoter can promote the leaf senescence of the plant under the premise that the growth state of the plant is not influenced, and the plant leaf senescence promoter is particularly beneficial to the consistent harvesting of the crops with the asynchronous leaf senescence, such as tobacco. It can be understood that the concentration of the pesticide directly sprayed on the leaf surface of the plant/crop at the early stage of leaf maturation is 10-12 mu mol/L.

The invention has the following positive advantages and beneficial effects:

1. the IDL7 mature polypeptide in the IDL7 mature polypeptide plant senescence promoter provided by the invention is artificially synthesized mature polypeptide, is convenient to obtain and can be synthesized in large batch;

2. the preparation and use method of the plant aging promoter provided by the invention is simple, the field operability is strong, special training is not needed for operators, and the operation is convenient and practical;

3. the plant senescence promoter provided by the invention has moderate influence on leaf senescence (has no obvious difference compared with normal senescent leaves), does not cause adverse reaction, and has beneficial effect.

Drawings

FIG. 1 is a schematic diagram showing the results of treating Arabidopsis thaliana detached leaves with an artificially synthesized IDL7 mature polypeptide plant senescence promoter;

FIG. 2 is a schematic diagram showing the results of IDL7 mature polypeptide plant senescence promoter treatment of living tobacco leaves.

Detailed Description

In order to more clearly and specifically describe the IDL7 mature polypeptide plant senescence promoter and its preparation method and use provided in the examples of the present invention in detail, the technical solutions in the examples of the present invention will be described clearly and completely below, and it is obvious that the described examples are only a part of the examples of the present invention, not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

IDL7 mature polypeptide plant aging promoter comprises 10 μmol/L IDL7 mature polypeptide, 2.8 mmol/L2- (N-morpholine) ethanesulfonic acid solution; the 2- (N-morpholine) ethanesulfonic acid solution is 2- (N-morpholine) ethanesulfonic acid solution monohydrate dissolved in MS liquid culture medium, and the pH value of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8.

The preparation method comprises the following steps:

(1) weighing IDL7 mature polypeptide powder, dissolving in water, and preparing into IDL7 mature polypeptide mother liquor with concentration of 10mmol/L for later use;

(2) weighing a certain amount of 2- (N-morpholine) ethanesulfonic acid monohydrate, adding into the MS liquid culture medium to prepare a solution of 2.8mmol/L, adjusting the pH value of the solution to 5.8, and fully mixing and dissolving uniformly to obtain a 2- (N-morpholine) ethanesulfonic acid solution;

(3) and (3) adding 40 mu L of the IDL7 mature polypeptide mother liquor obtained in the step (1) into 40mL of the 2- (N-morpholine) ethanesulfonic acid solution prepared in the step (2), and uniformly stirring by using a glass rod to obtain 40mL of IDL7 mature polypeptide plant senescence promoter.

Example 2

IDL7 mature polypeptide plant aging promoter comprises 12 μmol/L IDL7 mature polypeptide, 2.9 mmol/L2- (N-morpholine) ethanesulfonic acid solution; the 2- (N-morpholine) ethanesulfonic acid solution is 2- (N-morpholine) ethanesulfonic acid solution monohydrate dissolved in MS liquid culture medium, and the pH value of the 2- (N-morpholine) ethanesulfonic acid solution is 5.9.

The preparation method comprises the following steps:

(1) weighing IDL7 mature polypeptide powder, dissolving in water, and preparing into IDL7 mature polypeptide mother liquor with concentration of 10mmol/L for later use;

(2) weighing a certain amount of 2- (N-morpholine) ethanesulfonic acid monohydrate, adding into the MS liquid culture medium to prepare a solution of 2.5mmol/L, adjusting the pH value of the solution to 5.9, and fully mixing and dissolving uniformly to obtain a 2- (N-morpholine) ethanesulfonic acid solution;

(3) and (2) adding 120 mu L of IDL7 mature polypeptide mother liquor obtained in the step (1) into 100mL of 2- (N-morpholine) ethanesulfonic acid solution prepared in the step (2), and uniformly stirring by using a glass rod to obtain 100mL of IDL7 mature polypeptide plant senescence promoter.

Example 3

IDL7 mature polypeptide plant aging promoter comprises 13 μmol/L IDL7 mature polypeptide, 3mmol/L2- (N-morpholine) ethanesulfonic acid solution; the 2- (N-morpholine) ethanesulfonic acid solution is 2- (N-morpholine) ethanesulfonic acid solution monohydrate dissolved in MS liquid culture medium, and the pH value of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8.

The preparation method comprises the following steps:

(1) weighing IDL7 mature polypeptide powder, dissolving in water, and preparing into IDL7 mature polypeptide mother liquor with concentration of 10mmol/L for later use;

(2) weighing quantitative 2- (N-morpholine) ethanesulfonic acid monohydrate, adding into the MS liquid culture medium to prepare a solution of 3mmol/L, adjusting the pH value of the solution to 5.8, and fully mixing and dissolving uniformly to obtain a 2- (N-morpholine) ethanesulfonic acid solution;

(3) and (2) adding 78 mu L of IDL7 mature polypeptide mother liquor obtained in the step (1) into 60mL of 2- (N-morpholine) ethanesulfonic acid solution prepared in the step (2), and uniformly stirring by using a glass rod to obtain 60mL of IDL7 mature polypeptide plant senescence promoter.

Performance testing

Laboratory test

Using example 1 as an example, the IDL7 mature polypeptide plant senescence promoter prepared in example 1 was treated with the isolated leaf of Arabidopsis thaliana and the living leaf of tobacco, respectively, according to the application method.

Treatment 1: treatment of Arabidopsis leaves: growing the arabidopsis thaliana for about 30 days by continuous illumination, obtaining the in-vitro leaf (the sixth leaf) at the same leaf position, processing the in-vitro leaf by using a puncher with the diameter of 0.5cm, horizontally placing the in-vitro leaf on a culture dish, processing the in-vitro leaf by using 2.8mM 2- (N-morpholine) ethanesulfonic acid (MES) as a control, simultaneously uniformly and directly spraying a polypeptide reagent on the surface of the in-vitro leaf of the arabidopsis thaliana at 10 mu mol/L, and observing the phenotype after standing for 4 days.

And (3) treatment 2: tobacco flourishing for a long time: tobacco leaf live bodies were treated by treating with 2.8mM 2- (N-morpholine) ethanesulfonic acid (MES) as a control, and the polypeptide reagent was sprayed uniformly and directly onto the tobacco leaf surface at 10. mu. mol/L, and the phenotype was observed after leaving for 4 days.

As shown in FIG. 1A, leaves of Arabidopsis thaliana (sixth rosette leaf) having the same leaf position and uniform growth were picked with tweezers, treated with a 0.5cm diameter punch, and then placed in a petri dish in a regular manner, and the IDL7 mature polypeptide plant senescence promoter was treated in the same manner with the control, and left for 4 days under continuous light.

As a result, the IDL7 mature polypeptide plant senescence promoter can promote the senescence of Arabidopsis thaliana leaves. After 4 days, the arabidopsis isolated leaves sprayed with the IDL7 mature polypeptide plant senescence promoter have obvious premature senility phenotype. Meanwhile, the green leaf content was determined by ethanol method, as shown in FIG. 1B, the chlorophyll content in the leaves treated with IDL7 mature polypeptide plant senescence promoter was decreased to 0.517. mu.g/mg in the control group and 0.311. mu.g/mg in the treated group, compared to the control group. .

Field test

As shown in FIG. 2A, the premature senescence phenotype of the tobacco sprayed with the IDL7 mature polypeptide plant senescence-accelerating agent was clearly observed after 14 days when the IDL7 mature polypeptide plant senescence-accelerating agent was sprayed at 10. mu. mol/L to the tobacco as a material of the cultivated tobacco K326 variety growing under field conditions for about 60 days, and the control was sprayed with a solution containing the IDL7 mature polypeptide; meanwhile, the content of chlorophyll in tobacco leaves 14 days after spraying the IDL7 mature polypeptide plant senescence promoter and the control is measured by an ethanol method, and the result shows that the content of chlorophyll in tobacco leaves (0.225 mug/mg) after being treated by the IDL7 mature polypeptide plant senescence promoter is obviously reduced compared with the control (0.598 mug/mg) as shown in figure 2B. The result shows that the IDL7 mature polypeptide plant senescence promoter has obvious senescence-promoting effect on the leaves of living plants.