CN115850413A

CN115850413A - Polypeptide CYS3 influencing seed germination

Info

Publication number: CN115850413A
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: 2023-03-28

Abstract

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

Description

Polypeptide CYS3 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 influencing seed germination.

Background

Most flowering plants reproduce progeny by sexual reproduction and generation of seeds, and seed germination is a complex process in which quiescent dry seeds rapidly regain metabolic activity, complete the cellular events of the embryo extending out of the surrounding structures, and prepare for subsequent seedling growth. Successful seed germination into seedlings determines the propagation and survival of the plant population, and also determines the time for the plant to enter the natural and agricultural ecosystems, directly affecting the yield of the crop. Therefore, seed germination has important economic and ecological significance.

In the past decades, a large class of novel small protein molecules-polypeptides-has been found in plants. The polypeptide is found to be widely involved in the growth and development processes of plants, such as the development of root systems, the fertilization of plants and adverse stress response. However, the polypeptide molecules which play a key role in the seed germination process are not known at present, and the number of amino acids mainly derived from the polypeptide molecules is small, so that the polypeptide molecules are difficult to identify.

It was found by search that the following patent applications (in part) relate to polypeptides, seeds, germination. The corn protein powder polypeptide for inhibiting weed germination and rooting with the publication number CN1807448A, the separation method and the application thereof, the polypeptide with the inhibition effect on weed seed germination and rooting with the publication number CN1800205A, the separation method and the application thereof, and the polypeptide for inhibiting weed seed germination and rooting with the publication number CN1800204A, the separation method and the application thereof; the polypeptides referred to in the above patent application inhibit weed germination. An amino acid polypeptide water-soluble fertilizer disclosed in publication No. CN109704819A and a preparation method thereof, a preparation method of a plant growth regulator for seeds disclosed in publication No. CN108849992A, a photinia seed germination process disclosed in publication No. CN110278736A, and an early seedling cultivation and planting process disclosed in publication No. CN 110214623A; the above patent applications all use polypeptides as part of the additive of the formulation components. The patent application discloses that the related amino acid sequence of the polypeptide is RSS, and the living algae composition using 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 not related to tobacco, and are not related to tobacco seeds.

Although the germination rate of the seeds harvested 27-33 days after pollination of the tobacco seeds can reach more than 90%, the seeds harvested at different harvesting times have great difference in seed activity and storage characteristics in the period. Therefore, the research on the germination process of the tobacco seeds and the excavation of functional substances or protein molecules playing critical roles in the germination process can promote the understanding of the germination events of the tobacco seeds.

Disclosure of Invention

A large novel protein small molecule-polypeptide is found to be widely involved in the growth and development process of plants in plants, but the research on the polypeptide in tobacco is not started, so as to further research the polypeptide molecules which play a role in the germination process of tobacco seeds. The invention aims to provide a polypeptide molecule capable of influencing the germination of tobacco seeds and improving the uniformity and robustness of seed emergence.

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

The invention is realized by adopting the following technical scheme.

A polypeptide CYS3, the 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 polypeptide CYS3 of the invention is applied to influence the germination rate and germination vigor of seeds.

The polypeptide CYS3 of the invention is applied to influence the germination rate and germination vigor of tobacco seeds.

The tobacco seeds 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; the application is to promote the germination of immature tobacco seeds.

The action concentration of the polypeptide CYS3 is 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; the application of the composition is to inhibit the germination of immature tobacco seeds.

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

A biological agent for influencing the germination of tobacco seeds, which is any one of the following b 1) to b 6);

b1 ): an expression cassette comprising a nucleic acid molecule of SEQ id No. 1;

b2 ): biological agent containing protein molecule described by SEQ ID NO. 2;

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

b4 ): a recombinant microorganism comprising a nucleic acid molecule of SEQ id No. 1;

b5 ): a recombinant microorganism comprising the expression cassette of b 3).

The polypeptide has the beneficial effects that 1) the novel polypeptide molecule CYS3 provided by the invention can promote the germination rate and germination vigor of immature tobacco seeds. 2) The CYS3 polypeptide provided by the invention can promote the germination of immature (20 days of 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 immature (20 days of 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 a polypeptide of the present invention: induction of CYS3 protein SDS-PAGE detection results.

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

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

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

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

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

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

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

FIG. 9 is a photograph showing the concentration of CYS3 protein at 1.5. Mu.M in example 2 of the present invention.

FIG. 10 is a photograph showing the concentration of CYS3 protein at 2. Mu.M in example 2 of the present invention.

FIG. 11 is a photograph showing the results of 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 clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 expression and purification of Small peptides

Connecting CYS3 small peptide with pET32a vector for prokaryotic expression.

The method comprises the following specific steps:

(1) The corresponding primer of the small peptide is designed by adopting an Infusion ligation method (the design of the primer is the conventional means of a person skilled in the art), and the target fragment is amplified by PCR by taking the cDNA of the K326 sample as a template. The pET32a empty strand was cleaved with BamHI and EcoRI, and the PCR product was ligated to pET32a by the Infusion method. Thermally transformed into host bacteria DH5 alpha, selecting single colony for culture, extracting plasmid DNA by using Dongsheng plasmid miniprep kit (N1011) for PCR detection and double enzyme digestion detection, sending the recombinant plasmid detected as positive to the Scophyton biotechnology limited company for Sangge sequencing and comparing the sequences.

(2) The correctly sequenced plasmid is transferred into an Escherichia coli strain BL21 (DE 3), a single colony is picked and inoculated into 5mL LB/AMP liquid medium, and the single colony is cultured for 12h at 37 ℃ and 220 r/min. Inoculating 1mL of bacterial liquid into a 100mLLB/AMP liquid culture medium, and taking 1mL of bacterial liquid before induction for subsequent detection when the bacterial liquid is subjected to shaking culture at 37 ℃ until OD600 is approximately equal to 0.6-0.8. Adding IPTG to the final concentration of 0.5mmol/L, then carrying out shaking culture at 16 ℃ and 110rpm for 12-16h, and taking 1mL of induced bacterial liquid for detecting the protein expression by SDS-PAGE.

(3) After confirming that the expression is smooth, resuspending the bacterial cell sediment of each 100mL bacterial solution by using 10mLLysis Buffer, adding PMSF to 1mM, breaking cells by using an ultrasonic method, and incubating the bacterial solution supernatant and a nickel column for 4-8h (the column can be hung overnight). Loading the nickel column into a protein purification column, standing for precipitation, and standing until the supernatant flows out. The transudate solution was collected by washing twice with 10mL lysiss Buffer and twice with 10mL Wash Buffer. Elution was performed using 2.5mL Elution Buffer, and incubation was stopped at 500. Mu.L per harvest for 10min. Protein purification was checked using SDS-PAGE. The purified protein was diluted to 15mL and concentrated to 500. Mu.L using Amicon-Ultra-15 ultrafiltration tube (MWCO 10kD, UFC 901096). Re-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 germination of immature seeds of tobacco

See fig. 2, first round screening: the concentrated protein of example 1 was diluted to a target concentration of 10 μ M,7.5 μ M,5 μ M,2.5 μ M,1 μ M,0 μ M, 110K 326 tobacco seeds were counted, K326 tobacco seeds harvested 20 days in advance after pollination were soaked with the corresponding polypeptide solution for 24h, the seeds were spotted on wet filter paper and the germination rate of tobacco seeds was counted daily, and photographed, and experiments found that CYS3 at low concentration (0-1 μ M) could promote germination of immature (20 days in advance) seeds.

See fig. 3, second round screening: according to the results of the first round of screening, the polypeptide concentration range was further narrowed down 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, and then the tobacco seeds were treated in the same manner.

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

At treatment concentrations of 0-1. Mu.M, it was found that the germination rate and eventually the germination rate of the seeds increased significantly with increasing concentration of the polypeptide CYS 3.

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

In conclusion: the invention discloses that CYS3 polypeptide has the function of promoting germination of K326 immature seeds picked in advance 20 days after pollination, so that the polypeptide has a good value in the aspect of promoting the utilization of the immature seeds.

The above description is only a part of specific embodiments of the present invention (since the formula of the present invention includes numerical ranges, the embodiments are not exhaustive, and the protection scope of the present invention includes the numerical ranges and other technical essential ranges), and the detailed contents or common knowledge known in the schemes are not described herein too much. It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

1. A polypeptide CYS3, wherein the nucleotide coding sequence of the polypeptide CYS3 is as set forth in SEQ ID No. 1:

2. polypeptide CYS3 according to claim 1, wherein the amino acid sequence of polypeptide CYS3 is as set forth in SEQ ID No. 2:

3. use of a polypeptide CYS3 as claimed in claim 2 for influencing the germination rate and vigour of seeds.

4. The use of the polypeptide CYS3 as claimed in claim 2 for influencing the germination rate and vigour of tobacco seeds.

5. The use of claim 4, wherein the tobacco seed is immature tobacco seed, i.e. tobacco seed harvested 20 days after pollination.

6. The use as claimed in claim 5 wherein the polypeptide CYS3 is present at a concentration of greater than 0 μ M and equal to or less than 10 μ M.

7. The use according to claim 6, wherein the polypeptide CYS3 is present in a concentration of greater than 0 μ M and equal to or less than 1 μ M; the application of the method is to promote the germination of immature tobacco seeds.

8. Use according to claim 7, wherein polypeptide CYS3 is present at a concentration of 1 μ M or 0.75 μ M or 0.5 μ M or 0.25 μ M.

9. The use according to claim 6, wherein the polypeptide CYS3 is present in a concentration of greater than 1 μ M and equal to or less than 10 μ M; the application of the composition is to inhibit the germination of immature tobacco seeds.

10. Use according to claim 9, wherein polypeptide CYS3 is present at a concentration of 10 μ M or 7.5 μ M or 5 μ M or 2.5 μ M or 2 μ M.