The application discloses a tobacco NtGLV05 gene, protein and polypeptide coding, and application of the tobacco NtGLV05 gene, protein and polypeptide coding in promoting plant yellowing, and a preparation and a preparation method

Abstract

The application discloses a tobacco NtGLV05 gene, a protein, and polypeptide coding, and application and preparation and a preparation method of the polypeptide in promoting plant yellowing. The tobacco NtGLV05 gene codes the NtGLV05 protein, and the NtGLV05 polypeptide is generated after the NtGLV05 protein is cut and chemically modified. It is found for the first time that the NtGLV05 polypeptide is involved in tobacco yellowing regulation, and no report is found in crops including tobacco at present. The NtGLV05 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 NtGLV01 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 and fall off. The upper, middle, and lower leaves of tobacco plants mature at different times.

[0003] Typically, production employs a strategy of harvesting different parts of the tobacco leaves at different times, which delays the harvest period. Furthermore, environmental factors such as continuous rainy days close to harvest time can cause "secondary greening" of the 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 in tobacco leaves from different parts.

[0004] 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.

[0005] In tobacco, the NtGLV05 gene encodes the NtGLV05 protein, which, after cleavage and chemical modification, produces the NtGLV05 polypeptide. Whether the NtGLV05 polypeptide participates in the regulation of tobacco senescence and yellowing has not yet been reported in crops including tobacco. Summary of the Invention

[0006] This invention comprises six objectives, primarily aimed at improving the senescence and yellowing of leaves in plants, especially tobacco. Specifically, these include:

[0007] First, it provides an application of the tobacco NtGLV05 gene in promoting yellowing in plants;

[0008] The CDS sequence of the NtGLV05 gene described in this invention is shown in SEQ ID NO.1.

[0009] 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.

[0010] Furthermore, the specific object that promotes yellowing is tobacco.

[0011] Secondly, this study provides an application of NtGLV05 protein in promoting yellowing in plants;

[0012] The sequence of the tobacco NtGLV05 protein described in this invention is shown in SEQ ID NO.2.

[0013] The protein sequence also includes protein sequences with similar functions, having a similarity of not less than 95%; the similar functions include promoting yellowing.

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

[0015] Third, a tobacco NtGLV05 polypeptide is provided, the amino acid sequence of which is shown in SEQ ID NO.3. The NtGLV05 polypeptide sequence also includes polypeptide sequences with similar functions and a similarity of not less than 95%; the similar functions include promoting yellowing.

[0016] Furthermore, the specific object that promotes yellowing is tobacco.

[0017] Fourth, provide a preparation to promote the shedding of jaundice.

[0018] The jaundice-promoting preparation of the present invention includes the above-mentioned NtGLV05 polypeptide.

[0019] The concentration of NtGLV05 peptide is 1-10 μmol / L.

[0020] Furthermore, 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. The pH of the 2-(N-morpholine)ethanesulfonic acid solution is 5.6-6.0.

[0021] Furthermore, the 2-(N-morpholine)ethanesulfonic acid solution is 2-(N-morpholine)ethanesulfonic acid dissolved in MS liquid culture medium or water.

[0022] Fifth, provide a method for preparing a preparation that promotes the shedding of jaundice, specifically including:

[0023] The NtGLV05 polypeptide stock solution prepared with ddH2O is added to MS liquid medium or 2-(N-morpholine)ethanesulfonic acid solution prepared with water and mixed well to obtain the final product.

[0024] Further details include the following steps:

[0025] (1) Weigh NtGLV05 peptide powder and dissolve it in ddH2O water to prepare NtGLV05 peptide stock solution with a concentration of 10 mmol / L for later use.

[0026] (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.

[0027] (3) Add the NtGLV05 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 NtGLV05 polypeptide hormone-promoting tobacco aging and yellowing preparation, wherein the concentration of NtGLV05 polypeptide is 10 μmol / L, 2-

[0028] The concentration of (N-morpholine) ethanesulfonic acid was 3 mmol / L, and the pH value was 5.8.

[0029] Sixth, provide applications for agents that promote yellowing of leaves, especially for tobacco.

[0030] Furthermore, a yellowing-promoting agent was sprayed on the plant leaves.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) This invention is the first to discover that NtGLV05 polypeptide participates 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.

[0033] (2) The polypeptides in the NtGLV05 polypeptide hormone-promoting tobacco aging and yellowing preparation of the present invention can be synthesized in large quantities artificially.

[0034] (3) The preparation and use of the NtGLV05 polypeptide hormone-promoting tobacco aging and yellowing preparation of the present invention are simple and easy to operate;

[0035] (4) The NtGLV05 polypeptide hormone preparation of the present invention effectively promotes the aging and yellowing of tobacco and has biological safety. Attached Figure Description

[0036] Figure 1 The expression level of the NtGLV05 gene described in this invention in tobacco plants in the early and late stages of maturity;

[0037] Figure 2This diagram illustrates the results of treating mature tobacco plants with a NtGLV05 polypeptide hormone-induced senescence and yellowing-promoting agent. A represents the control group, and B represents the agent-treated group.

[0038] Figure 3 The image shows the Fv / Fm ratio of tobacco leaves in tobacco plants treated with the NtGLV polypeptide hormone to promote tobacco senescence and yellowing, and in tobacco plants not treated with the formulation described in this invention. Detailed Implementation

[0039] 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.

[0040] This invention provides an NtGLV05 polypeptide hormone-promoting tobacco aging and yellowing-reducing preparation, comprising NtGLV05 polypeptide, the amino acid sequence of which is KVNRGSFTAFSADYHMPRTHP-PKNN. This NtGLV05 polypeptide hormone-promoting tobacco aging and yellowing-reducing preparation comprises: NtGLV05 polypeptide, and 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.

[0041] Understandably, the concentration of NtGLV05 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.

[0042] The embodiments of the present invention provide a method for preparing an NtGLV05 polypeptide hormone preparation for promoting tobacco aging and yellowing, specifically, the method includes the following steps:

[0043] 1. Weigh the synthetically produced NtGLV05 peptide powder and dissolve it in water to prepare a 10 mmol / L NtGLV05 peptide stock solution. The NtGLV05 peptide stock solution should be prepared fresh for each use. If used within 30 days, store the NtGLV05 peptide stock solution at -20°C. If not used within 30 days, store the NtGLV05 peptide stock solution at -80°C.

[0044] 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.

[0045] 3. Add the NtGLV05 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 NtGLV05 polypeptide hormone-promoting tobacco aging and yellowing preparation. The concentration of the NtGLV05 mature polypeptide is 10...

[0046] μmol / L, the concentration of 2-(N-morpholine)ethanesulfonic acid is 3 mmol / L.

[0047] This invention provides an embodiment of the application of an NtGLV05 polypeptide hormone-promoting tobacco senescence and yellowing-promoting preparation in inducing yellowing in tobacco. Specifically, the application method is as follows: spray the tobacco leaves with the NtGLV05 polypeptide hormone-promoting tobacco senescence and yellowing-promoting preparation.

[0048] The NtGLV05 polypeptide preparation described in the above embodiments can promote the aging and yellowing of tobacco, with significant application effects; NtGLV05 polypeptide is easy to synthesize, readily available, and can be synthesized in batches; the preparation and use methods are simple, convenient, and practical.

[0049] To more clearly and in detail illustrate how the NtGLV05 polypeptide formulation provided in this invention promotes tobacco aging and yellowing, the following description will be based on specific embodiments.

[0050] The embodiments of the present invention were performed three biological replicates.

[0051] Example 1

[0052] The NtGLV05 polypeptide hormone-promoting tobacco aging and yellowing preparation comprises: 10 μmol / L NtGLV05 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.

[0053] The preparation method is as follows:

[0054] (1) Weigh an appropriate amount of NtGLV05 polypeptide powder and dissolve it in ddH2O to prepare a NtGLV05 polypeptide hormone stock solution with a concentration of 10 mmol / L.

[0055] (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;

[0056] (3) Take 500 μL of the NtGLV05 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 NtGLV05 polypeptide hormone preparation for promoting tobacco aging and yellowing.

[0057] Example 2

[0058] Transcriptional analysis of leaf tissue from the middle leaves of K326 tobacco plants was performed during the topping stage (early maturity) and one month after topping (late maturity). This revealed a tobacco gene, NtGLV05, whose expression was significantly upregulated in the late maturity stage. The NtGLV05 gene has the accession number Nta12g03610 in the Nicomics multi-omics database (http: / / lifenglab.hzau.edu.cn / Nicomics / ). This gene is annotated to encode a GLV protein, the specific function of which is unknown. Quantitative real-time PCR primers were designed to verify the expression pattern of the NtGLV05 gene in the early and late maturity stages of tobacco leaves. The primer sequences are as follows:

[0059] Front primer F: TGTGGTCGATGAAGCTGGAA

[0060] Terminal primer R: TTCTCAGCTTTCCTTGCCCT

[0061] Using the expression of the NtGLV05 gene in the early stage of tobacco leaf maturity as a control ( Figure 1 The study found that the NtGLV05 gene was significantly upregulated in the late stage of tobacco leaf maturity, 16.5 times that of the control.

[0062] Example 3

[0063] The method for applying the NtGLV05 peptide-based tobacco aging and yellowing-promoting preparation to tobacco was as follows: 10 mL of the NtGLV05 peptide-based tobacco aging and yellowing-promoting preparation prepared in Example 1 was taken and applied to tobacco according to the application method. Simultaneously, the control was a 3.0 mM 2-(N-morpholine) ethanesulfonic acid MS solution (pH = 5.8) without NtGLV05 peptide.

[0064] 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 NtGLV05 peptide-based formulation to promote tobacco senescence and yellowing. Fv / Fm was measured at 0, 5, 10, and 15 days after treatment with the NtGLV05 peptide-based formulation. The results showed that compared with the control... Figure 2 Compared to the left side, the tobacco plants treated with the tobacco senescence and yellowing-promoting agent prepared in Example 1 (on the left side) Figure 2The degree of senescence and yellowing of tobacco plants (on the right side) 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 ).

[0065] 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.

[0066] 1. CDS sequence of the NtGLV05 gene (SEQ ID NO.1)

[0067] >NtGLV05_CDS

[0068] ATGTTGTCCTTAAGTTTTAGTAGCTTTATGCTTGTTCTGCTTTTCATTTTGCTATTCATAAAC

[0069] GATACTATTGCTCATACTGACAAAGTTAAACAAAATTTTGTGGTCGATGAAGCTGGAAATGGT

[0070] ATTGCTAAGACAAGAAGGAGCCACCTTAATGTGGAGAAGGAAGATTTTCATGGTGTAACTCCA

[0071] GCAACTATTCAAGTTGCCAGTGGCAGAATTGGAGGAAGAAAAAGAATGGTGGAGCAAAGGAAT

[0072] ATGAGGCAAAGTACGAAGAAAATTGAAGGTATCATGCCAAATAGAGGGCAAGGAAAGCTGAGA

[0073] ATTCAAAGTAAATTAAATGGCAAAAATAGCCATTCTGATAATGTGAAGGTCAATAGAGGAAGT

[0074] The amino acid sequence of the protein TTCACAGCATTCAGTGCTGATTATCACATGCCAAGAACACACCCTCCAAAGAATAACTGA2.NtGLV05 is SEQ ID NO. 2.

[0075] >NtGLV05_Protein

[0076] MLSLSFSSFMLVLLFILLFINDTIAHTDKVKQNFVVDEAGNGIAKTRRSHLNVEKEDFHGVTP

[0077] ATIQVASGRIGGRKRMVEQRNMRQSTKKIEGIMPNRGQGKLRIQSKLNGKNSHSDNVKVNRGS

[0078] FTAFSADYHMPRTHPPKNN

[0079] 3. The amino acid sequence of the NtGLV05 polypeptide is as shown in SEQ ID NO.3

[0080] >NtGLV05_Pep

[0081] KVNRGSFTAFSADYHMPRTHPPKNN。

Claims

1. An application of the tobacco NtGLV05 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 NtGLV05 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 NtGLV05 polypeptide, characterized in that, The sequence is shown in SEQ ID NO.

3.

8. The tobacco NtGLV05 polypeptide according to claim 7, characterized in that, The NtGLV05 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 NtGLV05 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 the shedding of jaundice, characterized in that, Includes the NtGLV05 polypeptide according to any one of claims 7-9.

11. The preparation for promoting the shedding of jaundice according to claim 10, characterized in that, The concentration of NtGLV05 peptide is 1-10 μmol / L.

12. The preparation for promoting the shedding of jaundice 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 preparation for promoting jaundice reduction according to claim 12, characterized in that, The pH of 2-(N-morpholine)ethanesulfonic acid solution is 5.6-6.

0.

14. The preparation for promoting jaundice reduction 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 anti-jaundice preparation according to any one of claims 10-14, characterized in that, The NtGLV05 polypeptide stock solution prepared with ddH2O is added to MS liquid medium or 2-(N-morpholine)ethanesulfonic acid solution prepared with water and mixed well to obtain the final product.

16. The application of the jaundice-promoting preparation 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 yellowing-promoting agent sprayed on the plant leaves.

Images