The application discloses a tobacco NtPep03 gene, protein and polypeptide coding, and application of the tobacco NtPep03 gene, protein and polypeptide coding in promoting plant yellowing, and a preparation and a preparation method

Abstract

The application discloses a tobacco NtPep03 gene, a protein, and polypeptide coding, and application of the polypeptide in promoting plant yellowing, and a preparation and a preparation method. The tobacco NtPep03 gene codes the NtPep03 protein, and the NtPep03 polypeptide is generated after the NtPep03 protein is cut and chemically modified. It is found for the first time that the NtPep03 polypeptide is involved in tobacco yellowing regulation, and no report is found in crops including tobacco at present. The NtPep03 polypeptide hormone of the application promotes tobacco senescence and yellowing, and has simple preparation and use methods, strong operability, effectively promotes tobacco senescence and yellowing, and has biological safety.

Description

Technical Field

[0001] This invention relates to the field of formulations that promote the yellowing of tobacco leaves, and more particularly to the application, formulation, and preparation method of the tobacco NtPep03 gene, protein, and encoded polypeptide in promoting the yellowing of plants. Background Technology

[0002] Tobacco is an important economic crop, and its leaves are the harvested organs. As tobacco enters its later growth stage, the leaves begin to turn yellow. The upper, middle, and lower leaves of tobacco plants mature at different times. Typically, production employs a strategy of harvesting different parts at different times, which delays the harvest. Furthermore, environmental factors such as continuous rainy days close to harvest time can cause "secondary greening" of tobacco leaves, affecting the harvest. Therefore, genetic engineering technology can be used to regulate the timing of tobacco leaf yellowing, effectively solving the problems of inconsistent maturity and secondary greening of tobacco leaves in different parts.

[0003] Polypeptides are small, trace molecules composed of amino acids, synthesized by plants themselves, and play important regulatory roles. Due to their small quantity and high efficiency, they are also known as polypeptide hormones. In plants, polypeptides are functional peptides composed of only a few amino acids, resulting from the post-translational cleavage and modification of protein precursors. Acting as ligand and receptor receptors and initiating signal transduction, plant polypeptide hormones have been widely reported to participate in processes such as growth and development, and abiotic stress, including shoot apical meristem development, root apical meristem development, and drought stress. Polypeptide hormones can be synthesized artificially. In plants, exogenous application of polypeptide hormones can produce phenotypes similar to overexpression of the polypeptide hormone-encoding genes. Currently, research on the application of polypeptide hormones in plants, especially crops, is limited.

[0004] In tobacco, the NtPep03 gene encodes the NtPep03 protein, which, after cleavage and chemical modification, produces the NtPep03 polypeptide. Whether the NtPep03 polypeptide participates in the regulation of tobacco senescence and yellowing has not yet been reported in crops including tobacco. Summary of the Invention

[0005] This invention comprises six objectives, primarily aimed at promoting the senescence and yellowing of plant leaves, especially tobacco leaves. Specifically, these include:

[0006] First, it provides an application of the tobacco NtPep03 gene in promoting yellowing in plants;

[0007] The tobacco NtPep03 gene of this invention has a CDS sequence as shown in SEQ ID NO.1. The gene sequence also includes gene sequences with similar functions, possessing a similarity of not less than 95%. These similar functions include the expression of proteins that promote yellowing.

[0008] Furthermore, the specific object that promotes yellowing is tobacco.

[0009] Secondly, this invention provides an application of tobacco NtPep03 protein in promoting yellowing in plants;

[0010] The tobacco NtPep03 protein of this invention has the amino acid sequence shown in SEQ ID NO.2. The protein sequence also includes protein sequences with similar functions, exhibiting a similarity of not less than 95%. These similar functions include promoting yellowing.

[0011] Furthermore, the specific object that promotes yellowing is tobacco.

[0012] Third, a tobacco NtPep03 polypeptide is provided;

[0013] The tobacco NtPep03 polypeptide of this invention has the amino acid sequence shown in SEQ ID NO.3. The NtPep03 polypeptide sequence also includes polypeptide sequences with similar functions, having a similarity of not less than 95%; these similar functions include promoting yellowing.

[0014] Furthermore, the specific object that promotes yellowing is tobacco.

[0015] Fourth, provide a preparation to promote the shedding of yellow leaves in plants;

[0016] The plant yellowing-promoting preparation of the present invention includes the aforementioned NtPep03 polypeptide. The concentration of the NtPep03 polypeptide is 1-10 μmol / L.

[0017] Furthermore, it also contains a 2-(N-morpholine)ethanesulfonic acid solution. The concentration of the 2-(N-morpholine)ethanesulfonic acid solution is 2.8-3.2 mmol / L. The pH of the 2-(N-morpholine)ethanesulfonic acid solution is 5.6-6.0.

[0018] Furthermore, the 2-(N-morpholine)ethanesulfonic acid solution is 2-(N-morpholine)ethanesulfonic acid dissolved in MS liquid culture medium or water.

[0019] Fifth, provide a method for preparing a preparation that promotes the shedding of yellow leaves in plants;

[0020] The preparation method of the plant yellowing-promoting agent of the present invention involves adding the NtPep03 polypeptide stock solution prepared with ddH2O to the MS liquid culture medium solution of 2-(N-morpholine)ethanesulfonic acid and mixing well to obtain the agent.

[0021] Specifically, the following steps are included:

[0022] (1) Weigh NtPep03 polypeptide powder and dissolve it in ddH2O water to prepare NtPep03 polypeptide stock solution with a concentration of 10mmol / L for later use.

[0023] (2) Prepare MS liquid culture medium. Weigh a certain amount of 2-(N-morpholine)ethanesulfonic acid monohydrate and add it to the MS liquid culture medium to prepare a 3 mmol / L solution. Adjust the pH of the solution to 5.8 and mix thoroughly to obtain a 2-(N-morpholine)ethanesulfonic acid solution.

[0024] (3) Add the NtPep03 polypeptide mother liquor obtained in step (1) to the 2-(N-morpholine) ethanesulfonic acid solution prepared in step (2), and stir thoroughly to obtain the NtPep03 polypeptide hormone-promoting tobacco aging and yellowing preparation, wherein the concentration of NtPep03 polypeptide is 10 μmol / L, the concentration of 2-(N-morpholine) ethanesulfonic acid is 3 mmol / L, and the pH value is 5.8.

[0025] Sixth, provide the application of agents that promote the shedding of yellow leaves in plants.

[0026] The application of the plant yellowing-promoting agent described in this invention is for promoting yellowing of plants, especially for promoting yellowing of tobacco.

[0027] Furthermore, spray the plant leaves with a preparation that promotes yellowing of the leaves.

[0028] The beneficial effects of this invention are as follows:

[0029] (1) This invention is the first to discover that the NtPep03 gene is involved in the regulation of tobacco senescence and yellowing. This has not been reported in crops including tobacco, and provides a new solution and approach for promoting yellowing in plants.

[0030] (2) The polypeptides in the NtPep03 polypeptide hormone preparation for promoting tobacco aging and yellowing of the present invention can be synthesized in large quantities artificially.

[0031] (3) The preparation and use of the NtPep03 polypeptide hormone preparation for promoting tobacco aging and yellowing of the present invention are simple and easy to operate;

[0032] (4) The NtPep03 polypeptide hormone preparation of the present invention effectively promotes the aging and yellowing of tobacco and has biological safety. Attached Figure Description

[0033] Figure 1 The expression level of the NtPep03 gene described in this invention in tobacco plants in the early and late stages of maturity.

[0034] Figure 2 This diagram illustrates the results of treating mature tobacco plants with a NtPep03 polypeptide hormone-induced senescence and yellowing-promoting agent. A represents the control group, and B represents the agent-treated group.

[0035] Figure 3The figures show the Fv / Fm ratio of tobacco leaves in tobacco plants treated with the NtPep03 polypeptide hormone to promote tobacco senescence and yellowing, and in tobacco plants without the treatment described in this invention. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] This invention provides an NtPep03 polypeptide hormone-promoting tobacco aging and yellowing-reducing preparation, comprising NtPep03 polypeptide, the amino acid sequence of which is as follows:

[0038] YISYGALSRNRVPCSRRGASY-YNCRPGAQANPYRRGCSAITR-CR, this NtPep03 polypeptide hormone-promoting tobacco aging and yellowing preparation comprises: NtPep03 polypeptide, 2-(N-morpholine)ethanesulfonic acid solution; wherein the 2-(N-morpholine)ethanesulfonic acid solution is 2-(N-morpholine)ethanesulfonic acid dissolved in MS liquid culture medium.

[0039] Understandably, the concentration of NtPep03 peptide can be 1-10 μmol / L, the concentration of 2-(N-morpholine)ethanesulfonic acid solution can be 2.8-3.2 mmol / L, and the pH of 2-(N-morpholine)ethanesulfonic acid solution can be 5.6-6.0.

[0040] The embodiments of the present invention provide a method for preparing an NtPep03 polypeptide hormone preparation for promoting tobacco aging and yellowing, specifically, the method includes the following steps:

[0041] 1. Weigh the artificially synthesized NtPep03 peptide powder and dissolve it in water to prepare a 10 mmol / L NtPep03 peptide stock solution for later use. The NtPep03 peptide stock solution should be prepared fresh for each use. If used within 30 days, store the NtPep03 peptide stock solution at -20℃. If not used within 30 days, store the NtPep03 peptide stock solution at -80℃.

[0042] 2. To prepare MS liquid culture medium, weigh a certain amount of 2-(N-morpholine)ethanesulfonic acid monohydrate and add it to MS liquid culture medium to prepare a 3 mmol / L solution. Adjust the pH of the solution to 5.8 and mix thoroughly to obtain a 2-(N-morpholine)ethanesulfonic acid solution.

[0043] 3. Add the NtPep03 mature polypeptide mother liquor obtained in step (1) to the 2-(N-morpholine)ethanesulfonic acid solution prepared in step (2), and stir evenly with a glass rod to obtain the NtPep03 polypeptide hormone-promoting tobacco aging and yellowing preparation. The concentration of the NtPep03 mature polypeptide is 10 μmol / L, and the concentration of the 2-(N-morpholine)ethanesulfonic acid is 3 mmol / L.

[0044] This invention provides an embodiment of the application of an NtPep03 polypeptide hormone-promoting tobacco senescence and yellowing-promoting preparation in inducing tobacco yellowing. Specifically, the application method is as follows: spray the tobacco leaves with the NtPep03 polypeptide hormone-promoting tobacco senescence and yellowing-promoting preparation.

[0045] The NtPep03 polypeptide preparations described above can promote tobacco aging and yellowing, with significant application effects; NtPep03 polypeptides are easy to synthesize, readily available, and can be synthesized in batches; the preparation and usage methods are simple, convenient, and practical.

[0046] To more clearly and in detail illustrate how the NtPep03 polypeptide preparation provided in this invention promotes tobacco aging and yellowing, the following description will be based on specific embodiments.

[0047] Example 1

[0048] The NtPep03 polypeptide hormone-promoting tobacco aging and yellowing preparation comprises: 10 μmol / L NtPep03 polypeptide and 3 mmol / L 2-(N-morpholine)ethanesulfonic acid solution; wherein the 2-(N-morpholine)ethanesulfonic acid solution is 2-(N-morpholine)ethanesulfonic acid dissolved in MS liquid medium, and the pH of the 2-(N-morpholine)ethanesulfonic acid solution is 5.8.

[0049] The preparation method is as follows:

[0050] (1) Weigh an appropriate amount of NtPep03 polypeptide powder and dissolve it in ddH2O to prepare a NtPep03 polypeptide hormone stock solution with a concentration of 10 mmol / L.

[0051] (2) Prepare 500 mL MS liquid culture medium, weigh a certain amount of 2-(N-morpholine)ethanesulfonic acid monohydrate, add it to the above MS liquid culture medium to prepare a 3 mmol / L solution, adjust the pH of the solution to 5.8, mix thoroughly and dissolve evenly to obtain 2-(N-morpholine)ethanesulfonic acid solution;

[0052] (3) Take 500 μL of the NtPep03 polypeptide hormone mother liquor obtained in step (1) and add it to 500 mL of the 2-(N-morpholine) ethanesulfonic acid solution prepared in step (2). Mix thoroughly to obtain 500 mL of NtPep03 polypeptide hormone preparation for promoting tobacco aging and yellowing.

[0053] Example 2

[0054] Transcriptional analysis of leaf tissue from the middle leaves of K326 tobacco plants was performed during the topping stage (early maturity) and 21 days after topping (late maturity). This revealed a tobacco gene, NtPep03, whose expression was significantly upregulated in the late maturity stage. The NtPep03 gene has the accession number Nta01g25840.1 in the Tobacco Genome Database (http: / / lifenglab.hzau.edu.cn / Nicomics / ). This gene is annotated to encode a polypeptide whose specific function is unknown. Quantitative real-time PCR primers were designed to verify the expression pattern of the NtPep03 gene in the early and late maturity stages of tobacco leaves. The primer sequences are as follows:

[0055] Front primer F:ATCGTTGCGGCAGCGGCGGAAGCT

[0056] Terminal primer R: TTCGGCCAACAACTCACCGATCGAA

[0057] Using the expression of the NtPep03 gene in the early stage of tobacco leaf maturity as a control ( Figure 1 The study found that the NtPep03 gene was significantly upregulated in the late stage of tobacco leaf maturity, reaching 27.9 times that of the control.

[0058] Example 3

[0059] The method for applying the NtPep03 peptide-based tobacco aging and yellowing-promoting preparation to tobacco was as follows: 50 mL of the NtPep03 peptide-based preparation prepared in Example 1 was taken and applied to tobacco plants according to the specified method. Simultaneously, a control was prepared using a 3.0 mM 2-(N-morpholine) ethanesulfonic acid MS solution (pH = 5.8) without NtPep03 peptide.

[0060] Fv / Fm represents the maximum photochemical efficiency of Photosystem II (PSII), and it gradually decreases as leaves age and turn yellow. The yellowing of tobacco leaves was investigated 15 days after treatment with an NtPep03 peptide-based formulation to promote tobacco senescence and yellowing. Fv / Fm was measured at 0, 5, 10, and 15 days after treatment with the NtPep03 peptide-based formulation. The results showed that compared with the control... Figure 2 Compared to Example 1, tobacco plants treated with the agent that promotes tobacco senescence and yellowing (A) Figure 2 The degree of senescence and yellowing in tobacco plants (B) was significantly increased. Furthermore, the Fv / Fm ratio of tobacco plants treated with the senescence-promoting yellowing agent prepared in Example 1 was significantly reduced. Figure 3 ).

[0061] In summary, the preparation for promoting tobacco aging and yellowing prepared in Example 1 has a significant promoting effect on the aging and yellowing of tobacco.

[0062] 1. CDS sequence of the NtPep03 gene (SEQ ID NO.1)

[0063] >NtPep03_CDS

[0064] ATGGCGATGGTGAAAGTGAACTCTTTCACCATTTTGTTGATCTCTTCGATTTTCCTCATCGTT

[0065] GCGGCAGCGGCGGAAGCTACTGGGGCCCACGAGCTCGGCTTTCATCCAATGACGCTGTCCATG

[0066] ACGTCATCGGCCTCATCGATCTATGAAGGTTCGATCGGTGAGTTGTTGGCCGAAAATGGAAGC

[0067] GACGAGGGTTTTCGATGGAGTCTGAGAGCACACGGCGTATTTTAGCGTACCGAAGGAGATAC

[0068] ATTAGCTACGGTGCGCTGAGCAGGAACAGAGTTCCGGTTCGAGAAGAGGTGCTTCATACTAC

[0069] AATTGCCGTCCCGGAGCCCAGGCGAATCCCTACCGACGTGGATGCAGTGCCATCACGCGCTGC

[0070] CGCCGTTAA

[0071] 2. The amino acid sequence of the NtPep03 protein, SEQ ID NO. 2

[0072] >NtPep03_Protein

[0073] MAMVKVNSFTILLISSIFLIVAAAAEATGAHELGFHPMTLSMTSSASSIYEGSIGELLAENGS

[0074] DEEFSMESESTRRILAYRRRYISYGALSRNRVPCSRRGASYYNCRPGAQANPYRRGCSAITRC

[0075] RR

[0076] 3. The amino acid sequence of the NtPep03 polypeptide is shown in SEQ ID NO. 3.

[0077] >NtPep03_Pep

[0078] YISYGALSRNRVPCSRRGASYYNCRPGAQANPYRRGCSAITRCR

Claims

1. An application of the tobacco NtPep03 gene in promoting yellowing in plants, characterized in that, The gene CDS sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The gene sequence also includes gene sequences with similar functions, having a similarity of not less than 95%; the similar functions include the expression of proteins that promote yellowing.

3. The application according to claim 2, characterized in that, The specific object mentioned in the description of promoting yellowing is tobacco.

4. The application of an NtPep03 protein in promoting yellowing in plants, characterized in that, The protein sequence is shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, The protein sequence also includes protein sequences with similar functions, having a similarity of not less than 95%; the similar functions include promoting yellowing.

6. The application according to claim 5, characterized in that, The specific object mentioned in the description of promoting yellowing is tobacco.

7. A tobacco NtPep03 polypeptide, characterized in that, The sequence is shown in SEQ ID NO.

3.

8. The tobacco NtPepO3 polypeptide according to claim 7, characterized in that, The NtPep03 polypeptide sequence also includes polypeptide sequences with similar functions, having a similarity of not less than 95%; the similar functions include promoting yellowing.

9. The tobacco NtPepO3 polypeptide according to claim 8, characterized in that, The specific object mentioned in the description of promoting yellowing is tobacco.

10. A preparation for promoting yellowing and shedding of plants, characterized in that, Includes the NtPep03 polypeptide according to any one of claims 7-9.

11. The plant yellowing-promoting preparation according to claim 10, characterized in that, The concentration of NtPep03 peptide is 1-10 μmol / L.

12. The plant yellowing-promoting preparation according to claim 11, characterized in that, It also contains a 2-(N-morpholine)ethanesulfonic acid solution; preferably, the concentration of the 2-(N-morpholine)ethanesulfonic acid solution is 2.8-3.2 mmol / L.

13. The plant yellowing-promoting preparation according to claim 12, characterized in that, The pH of 2-(N-morpholine)ethanesulfonic acid solution is 5.6-6.

0.

14. The plant yellowing-promoting preparation according to claim 12, characterized in that, The 2-(N-morpholine)ethanesulfonic acid solution is 2-(N-morpholine)ethanesulfonic acid dissolved in MS liquid culture medium or water.

15. The method for preparing the plant yellowing-promoting agent according to any one of claims 10-14, characterized in that, The NtPep03 polypeptide stock solution prepared with ddH2O is added to MS liquid culture medium or 2-(N-morpholine)ethanesulfonic acid solution prepared with water and mixed well to obtain the final product.

16. The application of the plant yellowing-promoting agent according to any one of claims 10-14, characterized in that, Applications used to promote yellowing of plant leaves.

17. The application according to claim 16, characterized in that, For promoting yellowing of tobacco leaves, it is preferable to use a plant yellowing-promoting agent sprayed on the plant leaves.

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