A peptide CYS3 that affects seed germination

Abstract

The present invention relates to a polypeptide CYS3 that affects seed germination. The CYS3 polypeptide molecule affects the germination of immature tobacco seeds. Immature tobacco seeds picked 20 days in advance after pollination are used as test materials. The CYS3 polypeptide is expressed and then treated in the tobacco seeds. It is found that the CYS3 protein has a germination-promoting effect on the immature tobacco seeds, providing a practical technology for promoting tobacco seed germination and seedling emergence in production.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the technical field of polypeptide CYS3 that affects seed germination. Background Art

[0002] Most flowering plants reproduce sexually and produce seeds. Seed germination is a complex process during which a dormant, dry seed rapidly regains metabolic activity, completing the cellular events that allow the embryo to extend beyond surrounding structures and prepare for subsequent seedling growth. Successful seed germination and seedling formation determine the reproduction and survival of plant populations, the timing of plant incorporation into natural and agricultural ecosystems, and directly impacts crop yields. Therefore, seed germination has important economic and ecological implications.

[0003] Over the past few decades, a large class of novel small protein molecules—peptides—has been discovered in plants. Peptides are widely involved in plant growth and development, such as root development, fertilization, and responses to environmental stress. However, little is known about the peptides that play a key role in seed germination, primarily due to their limited amino acid count, making their identification difficult.

[0004] After searching, it was found that the following patent applications (partially) involve polypeptides, seeds, and germination. Publication No. CN1807448A: Corn protein powder polypeptide and separation method for inhibiting weed germination and rooting, and its application; Publication No. CN1800205A: Polypeptide and separation method for inhibiting weed seed germination and rooting, and its application; Publication No. CN1800204A: Polypeptide and separation method for inhibiting weed seed germination and rooting; The polypeptides involved in the above patent applications can inhibit weed germination. Publication No. CN109704819A: An amino acid polypeptide water-soluble fertilizer and its preparation method; Publication No. CN108849992A: A preparation method for a plant growth regulator for seeds; Publication No. CN110278736A: A heather seed germination process; Publication No. CN110214623A: A heather early seedling planting process; The above patent applications all use polypeptides as part of the additives in the formula components. Publication No. CN114806589A discloses a living algae composition, a preparation method thereof, and an application thereof. The amino acid sequence of the polypeptide disclosed in the patent application is RSS, and the living algae composition containing the polypeptide as a component can be used to improve soil and stimulate plant rooting and germination.

[0005] The above-mentioned polypeptides and their applications have nothing to do with tobacco, let alone tobacco seeds.

[0006] Although tobacco seeds harvested 27 to 33 days after pollination have a germination rate exceeding 90%, seeds harvested at different times during this period exhibit significant differences in seed viability and storage properties. Therefore, studying the germination process of tobacco seeds and identifying the functional substances or protein molecules that play a key role in germination will advance our understanding of tobacco seed germination events. Summary of the Invention

[0007] A large class of novel small protein molecules, peptides, have been discovered in plants and are widely involved in plant growth and development. However, research on these peptides in tobacco has not yet begun. To further investigate the peptides that play a role in tobacco seed germination, the present invention aims to provide a peptide that can influence tobacco seed germination, thereby improving the uniformity and robustness of seed emergence.

[0008] The present invention is intended to solve the above problems and defects and provides a polypeptide that affects tobacco seed germination.

[0009] The present invention is implemented by adopting the following technical solutions.

[0010] A polypeptide CYS3, the nucleotide coding sequence of the polypeptide CYS3 of the present invention is shown in SEQ ID NO.1:

[0011] ATGAGAGTATTTGGTAACACCACACTGCTATTTGCTTTAATTCTGTCATTAAGTTTTCTGTTCTCTGCGTTTGGGTTAAGCGAACAAGGGAAAACCGAAGGATTCTGCGGGGAAGAGGAAGGGAAAGGAAATAATCTGATTGAGATGACTACTCTTGGTGGGATTCGTGATTCTCATGCTTCGTCCCAGAACAGCGACGAGATCCATAACCTTGCAAAATTTGCCGTCGACGAGCACAACAAGAAGGAGAATGCGATGATTGAGTTGGCCAGAGTTGTGAAGGCGCGAGAGCAAGTTGTTGCTGGTACACTGCACCATCTGACTCTTGAGGTCATAGATGCTGGAAAAAAGAAACTCTATGAGGCTAAGGTTTGGGTCAAACCATGGTTGAATTTCAAGGAACTTCAAGAGTTCAGTCATGTTGAAGATGTTCCTACCTTAACTTCTTCAGATCTAGGTGTTAAGCAAGAAGAGGAAGGCTCTGGATTGAAATCAGTGCCTGTGCATGACCCGGTGGTTCAAGAAGCTGCAGAGCATGCAATTAAGACCATCCAGCAGAGATCCAACTCACTACTTCCATATGAACTCCAAGAGATTGTTCATGCAAATGCTGAGGTCATTGAGGAGGACAATATGAAGCTTCATATGCTCATCAAAACTAGCAGGGGAGGGAAGCAAGAAAAGTTCAAAGTTCAAGTGCACCACAATAAAGAAGGTGCCTTCCAACTGAATCATATGGAGCCTGACCACTCCTAA。

[0012] The amino acid sequence of polypeptide CYS3 according to the present invention is shown as SEQ ID NO.2:

[0013] MRVFGNTTLLFALILSLSFLFSAFGLSEQGKTEGFCGEEEGKGNNLIEMTTLGGIRDSHASSQNSDEIHNLAKFAVDEHNKKENAMIELARVVKAREQVVAGTLHHLTLEVIDAGKKKLYEAKVWV KPWLNFKELQEFSHVEDVPTLTSSDLGVKQEEEGSGLKSVPVHDPVVQEAAEHAIKTIQQRSNSLLPYELQEIVHANAEVIEEDNMKLHMLIKTSRGGKQEKFKVQVHHNKEGAFQLNHMEPDHS.

[0014] The application of the polypeptide CYS3 of the present invention is to influence the germination rate and germination potential of seeds.

[0015] The application of the polypeptide CYS3 of the present invention is to influence the germination rate and germination potential of tobacco seeds.

[0016] The tobacco seeds described in the present invention are immature tobacco seeds, that is, tobacco seeds harvested 20 days after pollination.

[0017] The effective concentration of the polypeptide CYS3 of the present invention is greater than 0 μM and less than or equal to 10 μM.

[0018] The polypeptide CYS3 of the present invention has an effective concentration greater than 0 μM and less than or equal to 1 μM; and is used for promoting the germination of immature tobacco seeds.

[0019] The effective concentration of the polypeptide CYS3 of the present invention is 1 μM, 0.75 μM, 0.5 μM or 0.25 μM.

[0020] The polypeptide CYS3 of the present invention has an effective concentration greater than 1 μM and less than or equal to 10 μM; and is used for inhibiting the germination of immature tobacco seeds.

[0021] The effective concentration of the polypeptide CYS3 of the present invention is 10 μM, 7.5 μM, 5 μM, 2.5 μM or 2 μM.

[0022] A biological agent that affects tobacco seed germination, wherein the biological agent of the present invention is any one of the following b1) to b6);

[0023] b1): an expression cassette containing the nucleic acid molecule described in SEQ ID NO.1;

[0024] b2): A biological preparation containing the protein molecule described in SEQ ID NO. 2;

[0025] b3): a recombinant vector containing the expression cassette described in b1);

[0026] b4): a recombinant microorganism containing the nucleic acid molecule described in SEQ ID NO.1;

[0027] b5): A recombinant microorganism containing the expression cassette described in b3).

[0028] The present invention has the following beneficial effects: 1) the novel polypeptide molecule CYS3 provided by the present invention can promote the germination rate and germination potential of immature tobacco seeds; 2) the CYS3 polypeptide provided by the present invention can promote the germination of immature seeds (harvested 20 days in advance) at low concentrations (0-1 μM); and 3) the CYS3 protein provided by the present invention can inhibit the germination of immature seeds (harvested 20 days in advance) at high concentrations (1-10 μM).

[0029] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of SDS-PAGE detection of the polypeptide of the present invention: CYS3 protein.

[0031] Figure 2 This is a diagram of the first round of screening results of Example 2 of the present invention.

[0032] Figure 3 This is a diagram of the second round of screening results of Example 2 of the present invention.

[0033] Figure 4 This is a photograph of Example 2 of the present invention (CYS3 protein concentration 0 μM).

[0034] Figure 5 This is a photograph of Example 2 of the present invention (CYS3 protein concentration: 0.25 μM).

[0035] Figure 6 This is a photograph of Example 2 of the present invention (CYS3 protein concentration 0.5 μM).

[0036] Figure 7 This is a photograph of Example 2 of the present invention (CYS3 protein concentration: 0.75 μM).

[0037] Figure 8 This is a photograph of Example 2 of the present invention (CYS3 protein concentration 1 μM).

[0038] Figure 9 This is a photograph of Example 2 of the present invention (CYS3 protein concentration: 1.5 μM).

[0039] Figure 10 This is a photograph of Example 2 of the present invention (CYS3 protein concentration 2 μM).

[0040] Figure 11 This is a photograph of Example 2 of the present invention (CYS3 protein concentration 5 μM). DETAILED DESCRIPTION

[0041] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0042] Example 1 Expression and purification of small peptides

[0043] The CYS3 peptide was connected to the pET32a vector for prokaryotic expression.

[0044] The specific steps are as follows:

[0045] (1) The corresponding primers of the small peptide were designed by the infusion connection method (the design of the primers is a routine method for those skilled in the art), and the cDNA of the K326 sample was used as a template to PCR amplify the target fragment. The pET32a empty vector was cut with BamHI and EcoRI, and the PCR product was connected to pET32a by the infusion method. Heat shock transformation was carried out into the host bacteria DH5α. After a single colony was picked and cultured, the plasmid DNA was extracted using the Dongsheng Plasmid Miniprep Kit (N1011) for PCR detection and double enzyme digestion detection. The recombinant plasmids that tested positive were sent to Qingke Biotechnology Co., Ltd. for Sanger sequencing and sequence comparison.

[0046] (2) The above sequenced plasmids were transformed into Escherichia coli strain BL21 (DE3), and a single colony was picked and inoculated into 5 mL LB / AMP liquid medium. The culture was cultured at 37 ° C and 220 r / min for 12 h. 1 mL of bacterial liquid was inoculated into 100 mL LB / AMP liquid medium. The culture was shaken at 37 ° C until OD600 ≈ 0.6-0.8. 1 mL of pre-induction bacterial liquid was taken for subsequent detection. IPTG was added to a final concentration of 0.5 mmol / L, and then the culture was shaken at 16 ° C and 110 rpm for 12-16 h. 1 mL of the post-induction bacterial liquid was taken for SDS-PAGE to detect protein expression.

[0047] (3) After confirming that the expression is successful, resuspend the bacterial pellet with 10 mL Lysis Buffer per 100 mL of bacterial liquid, add PMSF to 1 mM, use ultrasonication to disrupt the cells, and incubate the bacterial supernatant with the nickel column for 4-8 hours (can be hung on the column overnight). Load the nickel column into the protein purification column, let it settle, and wait for the supernatant to flow out. Wash twice with 10 mL Lysis Buffer and twice with 10 mL Wash Buffer, and collect the flowthrough. Elute with 2.5 mL Elution Buffer, and stop collecting and incubate for 10 minutes after collecting 500 μL. Use SDS-PAGE to detect protein purification. Dilute the purified protein to 15 mL and concentrate 500 μL using Amicon-Ultra-15 ultrafiltration tube (MWCO10kD, UFC901096). Dilute and concentrate again, and repeat 5 times.

[0048] Results and analysis: SDS-PAGE results showed that CYS3 could be expressed and purified successfully ( Figure 1 ).

[0049] Example 2 CYS3 protein promotes germination of immature tobacco seeds

[0050] See Figure 2 In the first round of screening, the concentrated protein from Example 1 was diluted to target concentrations of 10 μM, 7.5 μM, 5 μM, 2.5 μM, 1 μM, and 0 μM. 110 K326 tobacco seeds were counted and soaked in the corresponding polypeptide solution for 24 hours. The seeds were spotted on moist filter paper and the germination rate of the tobacco seeds was counted every day. The seeds were photographed. The experiment found that CYS3 at low concentrations (0-1 μM) can promote the germination of immature seeds (harvested 20 days in advance).

[0051] See Figure 3 , Second round of screening: Based on the results of the first round of screening, the peptide concentration range was further narrowed and diluted to 5 μM, 2 μM, 1 μM, 0.75 μM, 0.5 μM, 0.25 μM, and 0 μM, and then the tobacco seeds were treated in the same way.

[0052] Results and Analysis: In the initial screening experiment using CYS3 to treat seeds, it was found that CYS3 at low concentrations (0-1μM) could promote the germination of immature (harvested 20 days in advance) seeds, while at high concentrations (1-10μM) it would inhibit the germination of immature (harvested 20 days in advance) seeds.

[0053] When the treatment concentration was 0-1 μM, it was found that with the increase of the concentration of polypeptide CYS3, the germination rate and final seedlings were significantly improved.

[0054] The novel polypeptide molecule CYS3 provided by the present invention can promote the germination rate and germination potential of immature tobacco seeds.

[0055] In summary, the present invention discloses that the CYS3 polypeptide has the effect of promoting the germination of immature K326 seeds picked 20 days in advance after pollination, so it has good value in promoting the germination of immature seeds.

[0056] The above is only a partial specific embodiment of the present invention (because the formula of the present invention includes a numerical range, the embodiment cannot be exhaustive, and the protection scope recorded in the present invention includes the numerical range of the present invention and other technical key points), and the specific content or common sense known in the scheme are not described in detail here. It should be pointed out that the above embodiment does not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by using equivalent replacement or equivalent conversion methods fall within the protection scope of the present invention. The protection scope required by this application should be based on the content of its claims, and the specific implementation methods in the specification and other records can be used to explain the content of the claims.

Claims

1. An application of a polypeptide CYS3 in affecting the germination rate and germination potential of tobacco seeds, characterized in that: The nucleotide coding sequence of the polypeptide CYS3 is 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.

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