CN115850413B

CN115850413B - Polypeptide CYS3 for influencing seed germination

Info

Publication number: CN115850413B
Application number: CN202211007486.2A
Authority: CN
Inventors: 王国平; 张立猛; 郑昀晔; 牛永志; 索文龙; 许杰
Original assignee: YUXI ZHONGYAN TOBACCO SEED CO Ltd
Current assignee: YUXI ZHONGYAN TOBACCO SEED CO Ltd
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2025-09-16
Anticipated expiration: 2042-08-22
Also published as: CN115850413A

Abstract

The invention relates to a polypeptide CYS3 for influencing seed germination, which is a CYS3 polypeptide molecule for influencing the germination of immature tobacco seeds, wherein the immature tobacco seeds picked in advance 20 days after pollination are taken as test materials, the tobacco seeds are treated after the CYS3 polypeptide is expressed, and the CYS3 protein is found to have the effect of promoting the germination of the immature tobacco seeds, so that a practical technology is provided for promoting the germination and emergence of the tobacco seeds in production.

Description

Polypeptide CYS3 for influencing seed germination

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to the technical field of polypeptide CYS3 for influencing seed germination.

Background

Most flowering plants reproduce their offspring by sexual reproduction and seed production, and seed germination is a complex process in which stationary, dry seeds rapidly resume metabolic activity, complete cellular events of embryo emergence from surrounding structures, and prepare for subsequent seedling growth. The successful germination of seeds into seedlings determines the reproduction and survival of plant populations and also determines the time for the plants to enter the natural and agroecological systems, directly affecting the yield of crops. Therefore, seed germination is of great economic and ecological significance.

Over the past several decades, a large class of novel protein small molecule-polypeptides has been found in plants. Polypeptides are found to be widely involved in plant growth processes such as root development, fertilization of plants, and stress responses. However, little is currently known about polypeptide molecules that play a critical role in seed germination, mainly due to the smaller number of amino acids of polypeptide molecules, which are more difficult to identify.

The following patent applications (in part) were found to be related to polypeptides, seeds, germination. Polypeptide and separation method for inhibiting weed germination and rooting corn gluten meal of publication No. CN1807448A and application thereof, polypeptide and separation method for inhibiting weed seed germination and rooting of publication No. CN1800205A and application thereof, polypeptide and separation method for inhibiting weed seed germination and rooting of publication No. CN1800204A and application thereof, wherein the polypeptide related to the patent application can inhibit weed germination. The patent applications include polypeptide as a part of additives in the formula components, such as an amino acid polypeptide water-soluble fertilizer disclosed in publication No. CN109704819A, a preparation method of a plant growth regulator disclosed in publication No. CN108849992A for seeds, a photinia fraseri seed germination process disclosed in publication No. CN110278736A, and a photinia fraseri early seedling planting process disclosed in publication No. CN 110214623A. A living algae composition with publication No. CN114806589A, a preparation method and application thereof, wherein the amino acid sequence related to the polypeptide disclosed in the patent application is RSS, and the living algae composition with the polypeptide as a component can be used for improving soil and stimulating plant rooting and sprouting.

The polypeptide and the application thereof disclosed above are independent of tobacco, and are further independent of tobacco seeds.

Although the germination rate of the seeds collected in the 27 th to 33 th days after pollination of tobacco seeds can reach more than 90%, the seed vitality and the storage characteristics of the seeds at different collecting times in the period are greatly different. Thus, the study of the germination process of tobacco seeds, the excavation of functional substances or protein molecules that play a critical role in the germination process, will facilitate the understanding of the germination events of tobacco seeds.

Disclosure of Invention

At present, a large class of novel protein small molecules-polypeptides are found in plants to be widely involved in the growth and development process of plants, but research on polypeptides in tobacco has not been started yet, in order to further research polypeptide molecules which play a role in the germination process of tobacco seeds. The invention aims to provide a polypeptide molecule capable of influencing germination of tobacco seeds, and improving uniformity and robustness of seed emergence.

The invention aims to solve the defects and provides a polypeptide affecting the germination of tobacco seeds.

The invention is realized by adopting the following technical scheme.

A nucleotide coding sequence of the polypeptide CYS3 is shown in SEQ ID NO. 1:

ATGAGAGTATTTGGTAACACCACACTGCTATTTGCTTTAATTCTGTCATTAAGTTTTCTGTTCTCTGCGTTTGGGTTAAGCGAACAAGGGAAAACCGAAGGATTCTGCGGGGAAGAGGAAGGGAAAGGAAATAATCTGATTGAGATGACTACTCTTGGTGGGATTCGTGATTCTCATGCTTCGTCCCAGAACAGCGACGAGATCCATAACCTTGCAAAATTTGCCGTCGACGAGCACAACAAGAAGGAGAATGCGATGATTGAGTTGGCCAGAGTTGTGAAGGCGCGAGAGCAAGTTGTTGCTGGTACACTGCACCATCTGACTCTTGAGGTCATAGATGCTGGAAAAAAGAAACTCTATGAGGCTAAGGTTTGGGTCAAACCATGGTTGAATTTCAAGGAACTTCAAGAGTTCAGTCATGTTGAAGATGTTCCTACCTTAACTTCTTCAGATCTAGGTGTTAAGCAAGAAGAGGAAGGCTCTGGATTGAAATCAGTGCCTGTGCATGACCCGGTGGTTCAAGAAGCTGCAGAGCATGCAATTAAGACCATCCAGCAGAGATCCAACTCACTACTTCCATATGAACTCCAAGAGATTGTTCATGCAAATGCTGAGGTCATTGAGGAGGACAATATGAAGCTTCATATGCTCATCAAAACTAGCAGGGGAGGGAAGCAAGAAAAGTTCAAAGTTCAAGTGCACCACAATAAAGAAGGTGCCTTCCAACTGAATCATATGGAGCCTGACCACTCCTAA.

the amino acid sequence of the polypeptide CYS3 is shown in SEQ ID NO. 2:

MRVFGNTTLLFALILSLSFLFSAFGLSEQGKTEGFCGEEEGKGNNLIEMTTLGGIRDSHASSQNSDEIHNLAKFAVDEHNKKENAMIELARVVKAREQVVAGTLHHLTLEVIDAGKKKLYEAKVWVKPWLNFKELQEFSHVEDVPTLTSSDLGVKQEEEGSGLKSVPVHDPVVQEAAEHAIKTIQQRSNSLLPYELQEIVHANAEVIEEDNMKLHMLIKTSRGGKQEKFKVQVHHNKEGAFQLNHMEPDHS.

The application of the polypeptide CYS3 disclosed by the invention is to influence the germination rate and vigour of seeds.

The application of the polypeptide CYS3 provided by the invention is to influence the germination rate and vigour of tobacco seeds.

The tobacco seeds of the invention are immature tobacco seeds, namely tobacco seeds harvested 20 days after pollination.

The action concentration of the polypeptide CYS3 is more than 0 mu M and less than or equal to 10 mu M.

The action concentration of the polypeptide CYS3 is more than 0 mu M and less than or equal to 1 mu M, and the polypeptide CYS3 is applied to promote the germination of immature tobacco seeds.

The polypeptide CYS3 of the invention has the action concentration of 1 mu M or 0.75 mu M or 0.5 mu M or 0.25 mu M.

The action concentration of the polypeptide CYS3 is more than 1 mu M and less than or equal to 10 mu M, and the polypeptide CYS3 is applied to inhibiting the germination of immature tobacco seeds.

The polypeptide CYS3 of the invention has the action concentration of 10 mu M or 7.5 mu M or 5 mu M or 2.5 mu M or 2 mu M.

A biological agent affecting germination of tobacco seeds, the biological agent of the present invention is any one of the following b 1) to b 6);

b1 An expression cassette comprising a nucleic acid molecule as set forth in SEQ ID NO. 1;

b2 A biological agent comprising a protein molecule as set forth in SEQ ID NO. 2;

b3 A recombinant vector comprising the expression cassette of b 1);

b4 Recombinant microorganism comprising the nucleic acid molecule of SEQ ID NO. 1;

b5 Recombinant microorganism comprising the expression cassette of b 3).

The novel polypeptide molecule CYS3 provided by the invention has the beneficial effects that 1) the germination rate and vigor of the immature tobacco seeds can be promoted. 2) The CYS3 polypeptide provided by the invention can promote the germination of immature (20-day early harvest) seeds at low concentration (0-1 mu M), and 3) the CYS3 protein provided by the invention can inhibit the germination of the seeds of the immature (20-day early harvest) seeds at high concentration (1-10 mu M).

The invention is further explained below with reference to the drawings and the detailed description.

Drawings

FIG. 1 shows the result of SDS-PAGE detection of the polypeptide of the present invention, CYS3 protein.

FIG. 2 is a graph showing the results of the first round of screening according to example 2 of the present invention.

FIG. 3 is a graph showing the results of the second round of screening according to example 2 of the present invention.

FIG. 4 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 0. Mu.M).

FIG. 5 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 0.25. Mu.M).

FIG. 6 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 0.5. Mu.M).

FIG. 7 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 0.75. Mu.M).

FIG. 8 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 1. Mu.M).

FIG. 9 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 1.5. Mu.M).

FIG. 10 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 2. Mu.M).

FIG. 11 is a photograph showing an example 2 of the present invention (CYS 3 protein concentration 5. Mu.M).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 expression and purification of Small peptides

The CYS3 small peptide is connected with a pET32a vector for prokaryotic expression.

The method comprises the following specific steps:

(1) The corresponding primers for the small peptides were designed by the Infusion ligation method (the design of the primers is conventional to those skilled in the art), and the target fragment was amplified by PCR using cDNA of the K326 sample as a template. The empty pET32a was cut with BamHI and EcoRI, and the PCR product was ligated to pET32a by the infion method. After heat shock is transformed into host bacteria DH5 alpha, single colony is selected for culture, plasmid DNA is extracted by using a Dongsheng plasmid small-quantity extraction kit (N1011) for PCR detection and double enzyme digestion detection, and the recombinant plasmid detected as positive is sent to the Songger sequencing and comparison sequence of the recombinant plasmid.

(2) The plasmid with correct sequence is transferred into an escherichia coli strain BL21 (DE 3), and single colony is picked and inoculated into 5mL of LB/AMP liquid medium for culturing for 12h at 37 ℃ and 220 r/min. 1mL of bacterial liquid is inoculated into 100mLLB/AMP liquid culture medium, and 1mL of bacterial liquid before induction is taken for subsequent detection when the bacterial liquid is cultured until the OD600 is approximately equal to 0.6-0.8 in a shaking way at 37 ℃. IPTG was added to a final concentration of 0.5mmol/L, followed by shaking culture at 16℃and 110rpm for 12-16h, and 1mL of the post-induction bacterial liquid was taken for SDS-PAGE to detect protein expression.

(3) After confirming that the expression was smooth, the bacterial cell pellet was resuspended by 10mLLysis Buffer per 100mL of bacterial liquid, PMSF was added to 1mM, cells were disrupted by sonication, and the bacterial liquid supernatant was incubated with nickel column for 4-8h (column hanging was possible overnight). Loading the nickel column into a protein purification column, standing for precipitation, and standing for supernatant to flow out. The permeate was collected by washing twice with 10mLLysisBuffer and twice with 10mL Wash Buffer. The incubation was stopped for 10min with 2.5mL Elution Buffer elution per 500 μl collected. Protein purification was detected using SDS-PAGE. The purified protein was diluted to 15mL and concentrated to 500. Mu.L using an Amicon-Ultra-15 ultrafiltration tube (MWCO 10kD, UFC 901096). Again diluted, concentrated, and repeated 5 times.

Results and analysis SDS-PAGE detection results show that CYS3 can be successfully and effectively expressed and purified (figure 1).

EXAMPLE 2CYS3 protein promotes tobacco immature seed germination

Referring to FIG. 2, in the first round of screening, the protein concentrated in example 1 was diluted to a target concentration of 10. Mu.M, 7.5. Mu.M, 5. Mu.M, 2.5. Mu.M, 1. Mu.M, 0. Mu.M, and K326 tobacco seeds 110 were counted, K326 tobacco seeds harvested in advance 20 days after soaking pollination with the corresponding polypeptide solution were used for 24 hours, the seeds were spotted on wet filter paper and the germination rate of the tobacco seeds was counted daily, photographs were taken, and experiments found that CYS3 at low concentrations (0-1. Mu.M) could promote germination of immature (20 days of early harvest) seeds.

Referring to FIG. 3, the second round of screening was performed by further narrowing down the polypeptide concentration range to 5. Mu.M, 2. Mu.M, 1. Mu.M, 0.75. Mu.M, 0.5. Mu.M, 0.25. Mu.M, 0. Mu.M according to the results of the first round of screening, and then treating tobacco seeds in the same manner.

Results and analysis in preliminary screening experiments with CYS3 treated seeds, it was found that CYS3 at low concentrations (0-1. Mu.M) promoted germination of immature (20 days of early harvest) seeds, while at high concentrations (1-10. Mu.M) suppressed germination of seeds of immature (20 days of early harvest) seeds.

At treatment concentrations of 0-1 μm, it was found that with increasing concentration of polypeptide CYS3, the germination rate and eventually the seed was significantly increased.

The novel polypeptide molecule CYS3 provided by the invention can promote the germination rate and vigor of the immature tobacco seeds.

In conclusion, the CYS3 polypeptide has a germination promoting effect on K326 immature seeds picked in advance 20 days after pollination, so that the CYS3 polypeptide has a good value in promoting the utilization of the immature seeds to germinate.

The foregoing description is only a few specific embodiments of the present application (since the formulation of the present application includes numerical ranges, the embodiments are not intended to be exhaustive, and the scope of the present application includes numerical ranges and other technical gist ranges), and the details or common knowledge of the present application is not described in any more detail herein. It should be noted that the above embodiments do not limit the present application in any way, and it is within the scope of the present application for those skilled in the art to obtain the technical solution by equivalent substitution or equivalent transformation. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims

1. A method for influencing the germination rate and germination potential of tobacco seeds using a polypeptide CYS3, wherein the nucleotide coding sequence of the polypeptide CYS3 is as shown in SEQ ID NO. 1;

The amino acid sequence of the polypeptide CYS3 is shown in SEQ ID NO.2;

The tobacco seeds are immature tobacco seeds, i.e. tobacco seeds harvested 20 days after pollination;

The effective concentration of the polypeptide CYS3 is greater than 0.25 μM and less than or equal to 1 μM; and the application thereof is to promote the germination of immature tobacco seeds.

2. The use according to claim 1, characterized in that the effective concentration of the polypeptide CYS3 is 1 μM, 0.75 μM, 0.5 μM or 0.25 μM.