CN113841700B

CN113841700B - SENPP 1-3 mature polypeptide plant senescence promoter, preparation method and application thereof

Info

Publication number: CN113841700B
Application number: CN202111158479.8A
Authority: CN
Inventors: 郭永峰; 田冬冬; 邓智超; 刘涛; 李伟; 张增林
Original assignee: Qingzhou Tobacco Research Institute of China National Tobacco Corp of Institute of Tobacco Research of CAAS
Current assignee: Qingzhou Tobacco Research Institute of China National Tobacco Corp of Institute of Tobacco Research of CAAS
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2022-11-18
Anticipated expiration: 2041-09-30
Also published as: CN113841700A

Abstract

The invention provides a SENPP 1-3 mature polypeptide plant senescence promoter and a preparation method and application thereof, belonging to the field of plant senescence promoters, wherein the SENPP 1-3 mature polypeptide plant senescence promoter takes SENPP 1-3 mature polypeptide as a main functional component, and the amino acid sequence of the SENPP 1-3 mature polypeptide is as follows: H-D-P-D-D-V-F-P-G-L-V-L-K-I. The plant senescence promoter provided by the invention can promote the leaf senescence of plants, accelerates the leaf senescence, has no other additional adverse performance, and has extremely strong field operability.

Description

SENPP 1-3 mature polypeptide plant senescence promoter, preparation method and application thereof

Technical Field

The invention belongs to the field of plant senescence promoters, and particularly relates to a SENPP 1-3 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 tobacco entering a growth later stage, the maturation periods of the upper, middle and lower leaves of the tobacco are different, if the harvesting time is delayed by adopting a strategy of harvesting different parts at different periods, 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 Dexamethasone (DEX) induces the gene to be over-expressed, the senescence of the transgenic arabidopsis leaves 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 on the fact that senescence-associated genes are 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 SENPP 1-3 mature polypeptide plant senescence promoter can promote leaf senescence of plants, enables senescence to be accelerated, and has no other additional adverse performance.

In order to solve the technical problems, the invention provides a SENPPE 1-3 mature polypeptide plant senescence promoter, which takes the SENPP 1-3 mature polypeptide as a main functional component, wherein the SENPP 1-3 mature polypeptide has the following amino acid sequence:

H-D-P-D-D-V-F-P-G-L-V-L-K-I。

preferably, the plant senescence promoter comprises SENPP 1-3 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 SENPP 1-3 mature polypeptide because it is a novel biological buffer that better facilitates the provision of the corresponding function by the polypeptide.

Preferably, the concentration of the SENPP 1-3 mature polypeptide is 0.5-5 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 is understood that the concentration of the mature SENPP 1-3 polypeptide may also be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5. Mu. Mol/L or any of the above ranges, the concentration of the 2- (N-morpholine) ethanesulfonic acid solution may be 2.8, 2.9 or 3mmol/L, and the pH of the 2- (N-morpholine) ethanesulfonic acid solution may be 5.8 or 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 foliar wetting and foliar spreading, and the solution is not particularly limited in the scheme as long as the solution can be spread on the surface of the plant and osmotically wetted 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 mill.

The invention also provides a preparation method of the SENPP 1-3 mature polypeptide plant senescence promoter, which specifically comprises the following steps:

s1: dissolving SENPP 1-3 mature polypeptide powder in water to prepare SENPP 1-3 mature polypeptide mother liquor with the concentration of 1 Ag/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 above steps is purchased from Beijing Sorley company, and when in use, the formula 4.42 Ag powder is dissolved in 1L of water, and the culture medium can fully dissolve 2- (N-morpholine) ethanesulfonic acid, so that 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 SENPP 1-3 mature polypeptide mother liquor into a 2- (N-morpholine) ethanesulfonic acid solution, and uniformly stirring to obtain the SENPP 1-3 mature polypeptide plant senescence promoter. Wherein, the concentration of the SENPP 1-3 mature polypeptide is 0.5-5 mu mol/L, and the concentration of the 2- (N-morpholine) ethanesulfonic acid is 2.8-3mmol/L.

It is understood that the above steps S1 and S2 may be replaced with each other, and S1 or S2 may be prepared first, which is not specifically required here. In addition, the prepared SENPP 1-3 mature polypeptide mother liquor needs to be prepared as it is, and if the 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 thousandths of tween-20 after adding the SENPP 1-3 mature polypeptide mother liquor into a 2- (N-morpholine) ethanesulfonic acid solution.

The invention also provides the application of the SENPPE 1-3 mature polypeptide plant senescence promoter in the aspect of promoting plant senescence, and during application, the SENPPE 1-3 mature polypeptide plant senescence promoter is directly sprayed on the leaf surfaces of the plant leaves in the complete stretching period, or the SENPP 1-3 mature polypeptide plant senescence promoter is used for soaking cotton balls and smearing the cotton balls on the leaf surfaces of the plant leaves in the complete stretching period.

The invention also provides the application of the SENPPE 1-3 mature polypeptide plant senescence promoter in the aspect of promoting plant senescence, wherein when the SENPP 1-3 mature polypeptide plant senescence promoter is applied, the SENPP 1-3 mature polypeptide plant senescence promoter is directly sprayed on the leaf surface of the tobacco leaf at the early stage of maturation, or the SENPP 1-3 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 direct spray on the leaf surface of the plant/crop at the early stage of leaf maturation is 0.5-5 mu mol/L.

The invention has the following positive advantages and beneficial effects:

1. the SENPP 1-3 mature polypeptide in the SENPP 1-3 mature polypeptide plant senescence promoter provided by the invention is a mature polypeptide obtained by artificial synthesis, 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 required for application personnel, 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 isolated leaves of cultivated tobacco K326 with an artificially synthesized SENPPE 1-3 mature polypeptide plant senescence promoter;

FIG. 2 is a schematic diagram showing the results of treating the leaf of tobacco leaves in vivo with the SENPPE 1-3 mature polypeptide plant senescence promoter.

Detailed Description

In order to more clearly and fully describe the SENPPE 1-3 mature polypeptide plant senescence promoter and the method and use thereof provided in the examples of the present invention, the technical solutions in the examples of the present invention will be described below clearly and completely. 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

The SENPP 1-3 mature polypeptide plant senescence promoter comprises 0.5 mu mol/L SENPP 1-3 mature polypeptide and 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 SENPP 1-3 mature polypeptide powder, dissolving in water, and preparing SENPP 1-3 mature polypeptide mother liquor with concentration of 1 Ag/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 31.3 mu L of SENPP 1-3 mature polypeptide mother liquor obtained in the step (1) into 40mL of 2- (N-morpholine) ethanesulfonic acid solution prepared in the step (2), and uniformly stirring by using a glass rod to obtain 40mL of SENPP 1-3 mature polypeptide plant senescence promoter.

Example 2

The SENPP 1-3 mature polypeptide plant senescence promoter comprises 1 mu mol/L SENPP 1-3 mature polypeptide and 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:

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

Example 3

SENPPE 1-3 mature polypeptide plant senescence promoter, comprising 5 mu mol/L SENPP 1-3 mature polypeptide, 3 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 of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8.

The preparation method comprises the following steps:

(2) Weighing a certain amount of 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 (3) adding 313 mu L of SENPP 1-3 mature polypeptide mother liquor obtained in the step (1) into 40mL of 2- (N-morpholine) ethanesulfonic acid solution prepared in the step (2), and uniformly stirring with a glass rod to obtain 40mL of SENPP 1-3 mature polypeptide plant senescence promoter.

Performance test

Laboratory test

Using example 1 as an example, the SENPPE 1-3 mature polypeptide plant senescence promoter prepared in example 1 was treated separately with the isolated leaves of Arabidopsis thaliana and the isolated leaves of tobacco according to the application method.

Treatment 1: treatment of Arabidopsis leaves: growing the arabidopsis thaliana for about 30 days by continuous illumination, obtaining in-vitro leaves (sixth leaf position) of the same leaf position, processing the leaves by using a perforator with the diameter of 0.5cm, horizontally placing the leaves on a culture dish, processing the leaves by using 2.8mM 2- (N-morpholine) ethanesulfonic acid (MES) as a control, simultaneously uniformly and directly spraying a polypeptide reagent on the surfaces of the in-vitro leaves of the arabidopsis thaliana at the speed of 1 mu mol/L, and observing the phenotype after standing for 8 days.

And (3) treatment 2: tobacco flourishes for a long time: treating the tobacco leaves in vitro, treating the leaves in vitro at the same leaf position (the seventh leaf position) by using a perforator with the diameter of 0.5cm, flatly placing the leaves on a culture dish, treating the leaves by using 2.8mM 2- (N-morpholine) ethanesulfonic acid (MES) as a control, uniformly and directly spraying a polypeptide reagent on the surfaces of the tobacco leaves at the same time at the concentration of 1 mu mol/L, and observing the phenotype after the leaves are placed for 8 days.

As shown in FIGS. 1A-D, the tobacco leaves with the same leaf position (sixth rosette leaf) and consistent growth vigor were selected, treated with a punch having a diameter of 0.5cm, and then placed in a petri dish in a regular manner, and the SENPPE 1-3 mature polypeptide plant senescence promoter was treated in a spraying manner while the control was treated in the same manner, and left for 8 days under continuous light.

As a result, it can be seen that the SENPPE 1-3 mature polypeptide plant senescence promoter can promote tobacco leaf senescence. After 8 days, the leaf of the control group has no obvious change compared with 0 day and is in a green state, and the tobacco leaves in vitro sprayed with the SENPPE 1-3 mature polypeptide plant senescence promoter show obvious yellowing phenomenon, even the whole leaf turns yellow. While the green leaf content was determined using an ethanol method, as shown in fig. 1E, the chlorophyll content in the leaves treated with the SENPEP1-3 mature polypeptide plant senescence promoter decreased compared to the control group, which fell to 122.524m and the group to 85.052 m.

Field test

As shown in FIGS. 2A-C, a cultured tobacco K326 variety growing for about 60 days under field conditions is taken as a material, the SENPIP 1-3 mature polypeptide plant senescence promoter is sprayed on the tobacco at 1 mu mol/L, meanwhile, the control is sprayed with a solution not containing the SENPIP 1-3 mature polypeptide, after 14 days, the tobacco sprayed with the SENPIP 1-3 mature polypeptide plant senescence promoter is obviously yellowed on the whole, particularly on the lower leaves (FIG. 2A-B), and a single plant is obviously yellowed, wherein the upper, middle and lower leaves are all yellowed than the control group tobacco (FIG. 2C); meanwhile, the photosynthetic rate of the different leaf positions of the polypeptide-treated and control plants was measured by a photosynthesis analyzer, and the results showed that the photosynthetic rate of the tobacco leaves (upper leaf 0.716, middle leaf 0.716, lower leaf 0.753) treated with the SENPEP1-3 mature polypeptide plant senescence promoter was significantly reduced (fig. 2D) as shown in fig. 2E, compared to the control group (upper leaf 0.798, middle leaf 0.800, lower leaf 0.804). The ethanol method is used for determining the chlorophyll content of the tobacco leaves 14 days after the SENPEP1-3 mature polypeptide phytosenescence promoter is sprayed and the control, and the result shows that the chlorophyll content (274.701) of the tobacco leaves after the SENPEP1-3 mature polypeptide phytosenescence promoter is treated is obviously reduced compared with the control (413.830 ml Ag/Ag) (fig. 2E). The results show that the SENPP 1-3 mature polypeptide plant senescence promoter and the mature polypeptide plant senescence promoter have obvious senescence-promoting effect on the leaves of living plants.

Claims

A SENPPE 1-3 mature polypeptide plant senescence promoter, which comprises SENPP 1-3 mature polypeptide and 2- (N-morpholine) ethanesulfonic acid solution; wherein the amino acid sequence of the SENPP 1-3 mature polypeptide is as follows: H-D-P-D-D-V-F-P-G-L-V-L-K-I; the 2- (N-morpholine) ethanesulfonic acid solution is obtained by dissolving 2- (N-morpholine) ethanesulfonic acid monohydrate in MS liquid culture medium.
2. The plant senescence promoter according to claim 1, wherein the mature SENPP 1-3 polypeptide has a concentration of 0.5 to 5 μmol/L,2- (N-morpholine) ethanesulfonic acid has a concentration of 2.8 to 3mmol/L, and the pH of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8 to 5.9.
3. The plant senescence promoter according to claim 2, wherein the mature SENPP 1-3 polypeptide has a concentration of 1 μmol/L,2- (N-morpholine) ethanesulfonic acid has a concentration of 2.8mmol/L, and the pH of the 2- (N-morpholine) ethanesulfonic acid solution is 5.8.
4. The plant senescence promoter according to claim 1, further comprising an additive, wherein the additive is tween-20, and the additive is added in an amount of 1 to 2 ‰.
5. The SENPP 1-3 mature polypeptide plant senescence promoter of any of claims 1-4, which comprises the following steps:

dissolving SENPP 1-3 mature polypeptide powder in water to prepare SENPP 1-3 mature polypeptide mother solution with the concentration of 1 Ag/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 SENPP 1-3 mature polypeptide mother liquor into a 2- (N-morpholine) ethanesulfonic acid solution, and uniformly stirring to obtain the SENPP 1-3 mature polypeptide plant senescence promoter.
6. The method of claim 5, further comprising the step of adding 1-2% o tween-20 after the obtained SENPP 1-3 mature polypeptide mother liquor is added to the 2- (N-morpholine) ethanesulfonic acid solution.
7. The SENPPE 1-3 mature polypeptide plant senescence promoter of any one of claims 1-4, which is used for promoting plant senescence, wherein the SENPPE 1-3 mature polypeptide plant senescence promoter is directly sprayed on the leaves of the plants in the period of complete leaf extension, or the SENPP 1-3 mature polypeptide plant senescence promoter is used for soaking cotton balls and then is smeared on the leaves of the plants in the period of complete leaf extension.
8. The SENPPE 1-3 mature polypeptide plant senescence promoter for promoting tobacco leaf senescence according to any one of claims 1-4, wherein the SENPPE 1-3 mature polypeptide plant senescence promoter is directly sprayed on the leaf surface of the tobacco leaf at the early stage of maturation or the SENPP 1-3 mature polypeptide plant senescence promoter is soaked in cotton balls and then the cotton balls are smeared on the leaf surface of the tobacco leaf at the early stage of maturation.
9. The use as claimed in claim 7 or 8, wherein the SENPEP1-3 mature polypeptide plant senescence promoter is directly sprayed on the leaf surface of the tobacco leaves at the early stage of maturation, and the spraying concentration of the SENPEP1-3 mature polypeptide plant senescence promoter is as follows: 0.5-5 mu mol/L.