A grape VvCLE11 short peptide and its application method as a leaf plant growth regulator

Abstract

The present invention relates to the field of biotechnology, specifically to a grape VvCLE11 short peptide and its use in promoting fruit color change and as a foliar plant growth regulator. The grape VvCLE11 short peptide can be applied via foliar spraying to indirectly improve fruit quality, shorten the ripening period, and reduce chemical residues on the fruit surface. This approach offers the potential to safely and effectively promote fruit ripening, particularly enhance food safety, and regulate the ripening stage of non-climacteric fruits, potentially replacing the traditional hormone abscisic acid in grape fruit.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a grape VvCLE11 short peptide and its application in promoting fruit color change and as a leaf plant growth regulator. Background Art

[0002] In the field of grape cultivation technology, methods currently exist for using short peptides as plant growth regulators. Existing methods, such as the grape VvCLE1 short peptide disclosed in patent application publication number CN119409786A and its use in promoting fruit color change and as a plant growth regulator, meet the needs of grape cultivation to a certain extent. However, these methods present the following challenges:

[0003] First, the existing method is to exogenously apply grape VvCLE1 short peptides through fruit dipping to promote fruit color change. This method tends to prioritize color change in the treated area, rather than the entire bunch, resulting in treatment blind spots.

[0004] Second: The existing method is that the working liquid directly contacts the surface of the fruit. Improper operation may cause uneven application of the pesticide and lead to problems such as uneven fruit color or deformed fruit.

[0005] Therefore, it is necessary to develop a new application method of foliar plant growth regulator to solve the above problems existing in the existing method. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a grape VvCLE11 short peptide and a method for using the same as a foliar plant growth regulator, so as to solve the problems of grape VvCLE1 short peptide as a plant growth regulator, such as the inability to achieve color change throughout the entire bunch and the possible occurrence of uneven fruit color or deformed fruit.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0008] A grape VvCLE11 short peptide, characterized in that the full-length nucleotide sequence of the grape VvCLE11 short peptide and its corresponding amino acid sequence are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0009] The grape VvCLE11 short peptide is obtained by translating the 205th to 249th positions of its coding gene, namely the CLE motif, and modifying it with hydroxyproline.

[0010] The grape VvCLE11 short peptide can be used as a leaf plant growth regulator to promote the color change of grape fruits.

[0011] The grape fruit is Cabernet Sauvignon grape fruit.

[0012] The grape VvCLE11 short peptide is used as a foliar plant growth regulator in the following manner: before the grape fruit changes color, the grape VvCLE11 short peptide working solution is exogenously applied by foliar spraying to promote the fruit color change;

[0013] The preparation method of the grape VvCLE11 short peptide working solution is as follows:

[0014] According to the ratio of 0.5:1000 (n:V), the corresponding mass of the synthesized grape VvCLE11 short peptide solid powder was calculated and weighed, dissolved in the corresponding volume of deionized water, and then 0.5:1000 (V:V) Tween-20 was added to obtain a grape VvCLE11 short peptide working solution with a concentration of 500 μM.

[0015] The beneficial effects of the present invention are:

[0016] 1. Promote fruit color change: Improve fruit color consistency. Foliar spraying of grape VvCLE11 peptide can promote the color change process of grape fruit. This shows that VvCLE11 peptide plays an important role in regulating endogenous hormones, anthocyanin synthesis and fruit development.

[0017] 2. Shorten the fruit ripening period and improve food safety: Grape VvCLE11 peptide can be applied to the foliage to indirectly improve fruit quality, shorten the ripening period, and reduce chemical residues on the fruit surface. This can safely and effectively promote fruit ripening, especially enhance food safety and regulate the ripening stage of non-climacteric fruits, providing a potential alternative to the traditional hormone abscisic acid in grapes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The nucleotide sequence encoding the full-length grape VvCLE11 short peptide and its corresponding amino acid sequence. In the figure, ATG (▼) is the start codon; TAA (*) is the stop codon; the boxed area is the VvCLE11 functional domain (CLE motif);

[0019] Figure 2 Schematic diagram of the structure of the grape VvCLE11 molecule and the amino acid sequence of the mature VvCLE11 peptide formed after amino acids 5 and 9 are hydroxylated by proline residues. Note: The prolines marked with numbers 5 and 9 in the figure are hydroxyprolines.

[0020] Figure 3 This is a schematic diagram of the foliar spraying status in the field experiment;

[0021] Figure 4The effect of foliar spraying of the peptide VvCLE11 on the color change of Cabernet Sauvignon grapes grown in the field. In the figure, CK is a blank solvent control. From left to right, representative images of Cabernet Sauvignon grapes are shown 2, 5, 8, 12, 17, and 20 days after treatment with VvCLE11 or CK.

[0022] Figure 5 Figure 12 shows the brightness, red-green color index, yellow-blue color index, and red grape color index (CIRG) of grape fruit 12 days after foliar spraying of VvCLE11 short peptide and CK. In the figure, asterisks (*) indicate significant differences, calculated using the t-student test (defined as *P < 0.05, **P < 0.01). Each point represents a single measurement sample.

[0023] Figure 6 Flowchart of the present invention;

[0024] Figure 7 Figure 1 shows the red grape color index (CIRG) of grape fruit 12 days after foliar spraying of grape VvCLE11 short peptide, CK, ABA, and grape VvCLE1 short peptide. In the figure, the bars represent the mean ± SD, and asterisks (*) indicate significant differences calculated using a two-way ANOVA test (defined as ****P < 0.0001). Each point represents a single measurement sample.

[0025] Figure 8 This is a comparative photo of the test of the present invention. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0027] 1. Materials and Reagents

[0028] Specific experimental methods not specified in this example can be carried out according to conventional methods. Unless otherwise specified, the materials and reagents used can be obtained through commercial channels.

[0029] 2. Methods

[0030] The experimental site was selected at the Caoxinzhuang teaching and practice base of Northwest Agriculture and Forestry University in Yangling District, Xianyang City, Shaanxi Province. The "Cabernet Sauvignon" grapes used in the experiment were all cultivated and managed according to the same field model. They grew well and were free of diseases and pests.

[0031] 2.1 Identification of grape VvCLE11 short peptide

[0032] A hidden Markov model was constructed using previously reported CLE short peptide sequences. The obtained gene sequence was then aligned with the Cabernet Sauvignon genome using the bioinformatics tool HMMER3.0. The full-length sequence of the grape VvCLE11 short peptide was identified. The coding region contains 249 nucleotide bases encoding 82 amino acids, with a typical CLE family domain (DYTPPRKKPPIHN) at 205-246 bp.

[0033] The full-length nucleotide sequence of the grape VvCLE11 short peptide and its corresponding amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0034] SEQ ID NO.1

[0035] ATGGCGCCATGCAAATGTCTCATTCTTCTGGCCCTCCTCCTCTGCTTCTTCTTCTTCTTCACCACCGCCAACGCCACTGCGCGGAGTTTTGGAGTGATGGAAAGTAACCAGTATGAATCCGCAG AGAAGGGCCACGACAGTAAGTTTAAGCCAAGTGAAGATGGAGTGCTGCCGGAGCCGGAGGAGATCGGGGACTTGGCGGAGATGGACTACACTCCGCCGAGGAAGAAGCCTCCGATCCATAACTGA

[0036] SEQ ID NO.2

[0037] MAPCKCLILLALLLCFFFFFTTANATARSFGVMESNQYESAEKGHDSKFKPSEDGVLPEPEEIGDLAEMDYTPPRKKPPIHN*

[0038] 2.2. Acquisition of grape VvCLE11 short peptide

[0039] To further investigate the function of the CLE domain, the 12 amino acids comprising the CLE motif in the VvCLE11 peptide were chemically synthesized by GenScript Biotech Co., Ltd. (www.genscript.com.cn). The fifth and ninth proline (Pro) residues in the synthesized peptide were hydroxylated to obtain the VvCLE11 peptide DYTPPRKKPPIHN. Note: The fifth and ninth prolines are hydroxyprolines.

[0040] 2.3. Preparation of grape VvCLE11 short peptide working solution

[0041] Accurately weigh 787.4 mg of the synthesized VvCLE11 solid powder, first add a small amount of deionized water to dissolve it, then add 500 μL / L Tween-20, and finally dilute to 1 L with deionized water to obtain a grape VvCLE11 short peptide working solution with a concentration of 500 μM.

[0042] 2.4. Exogenous application of VvCLE11 to Cabernet Sauvignon grapes grown in the field

[0043] In order to verify the feasibility of this application: the applicant conducted the following experiments:

[0044] The plant growth regulator used in the experimental group (i.e., VvCLE11) is the (grape VvCLE11 short peptide working solution) of this application:

[0045] For the control group (CK), deionized water with corresponding concentration of Tween-20 added was used as the blank solvent.

[0046] The test process is as follows:

[0047] Select Cabernet Sauvignon grape plants that are growing well in the field, free of pests and diseases, and are before the color change period. Bag the target bunches in advance, then select 6 mature leaves from the same plant and branch just above the bagged bunches. Use a spray bottle to evenly spray the grape VvCLE11 short peptide working solution on the front and back of the leaves until the leaves are evenly covered without runoff or slight dripping (the treatment state is as follows: Figure 3 After 48 hours, the fruit bags were removed and cultivation and management were carried out according to the normal mode. The fruit color change was then recorded by taking photos.

[0048] The results of spraying VvCLE11 short peptide on leaves showed that VvCLE11 treatment significantly affected the development period of Cabernet Sauvignon grape berries, such as Figure 4 As shown, the color change of grape fruit is significantly accelerated, the colored area is wider, the color change is obvious, the fruit color is darker, and there is a clear difference from CK.

[0049] 2.5. Measurement of coloration indices of Cabernet Sauvignon grapes after treatment with VvCLE11 short peptide

[0050] On the 12th day after treatment, 3 fruits were randomly selected from the treated and untreated areas of each bunch of fruit using a CR-400 handheld colorimeter, with a total of 5 biological replicates for each treatment. Parameters such as brightness, red-green color index, yellow-blue color index, and red grape color index (CIRG) were obtained 12 days after short peptide treatment. Data analysis was performed using Excel, Graphpad Prism, and other software. Figure 5 As shown in the figure, the CIRG index 12 days after treatment with VvCLE11 short peptide was significantly higher than that of CK, which indicates that VvCLE11 short peptide significantly accelerated and improved the coloring of grape fruit in a short period of time, and accelerated the process of fruit ripening.

[0051] 3. Conclusion

[0052] 3.1. Spraying the VvCLE11 peptide on Cabernet Sauvignon grape leaves before veraison showed a phenotype that promoted fruit color change. Furthermore, color indices were measured using a handheld colorimeter. The results indicate that the VvCLE11 peptide plays a significant role in regulating endogenous hormones, anthocyanin synthesis, and promoting fruit color change and ripening.

[0053] 3.2 The grape VvCLE11 short peptide of the present invention can be indirectly used to improve fruit quality by foliar spraying, thereby shortening the fruit ripening period. This provides new ideas and methods for safely and effectively promoting fruit ripening, reducing chemical residues during the production process, improving food safety, and enhancing the quality of non-climacteric fruits. Future in-depth research targeting different regions and varieties, and the gradual application of this method in production, will provide a theoretical foundation and technical support for the development of the grape industry.

[0054] In order to verify the advanced nature of this application, the applicant conducted a comparative test on grape VvCLE11 short peptide working solution, grape VvCLE1 short peptide working solution, CK, and abscisic acid (ABA); the comparative test process and results are as follows:

[0055] The plant growth regulator used in the experimental group (i.e., VvCLE11) is the (grape VvCLE11 short peptide working solution) of this application:

[0056] Control group 1 (i.e., CK) used deionized water with corresponding concentration of Tween-20 added as the negative control group;

[0057] Control group 2 (i.e., ABA): 500 μM abscisic acid (ABA) solution was used as the positive control group;

[0058] Comparative Group 3 (i.e., VvCLE1 short peptide); the grape VvCLE1 short peptide working solution from the invention patent publication number CN119409786A for plant growth regulators was used:

[0059] The test process is as follows:

[0060] The specific implementation process of the experiment is the same as that of Sections 2.3, 2.4, and 2.5 above. The preparation method of the grape VvCLE1 short peptide working solution refers to the invention patent application publication number CN119409786A. Accurately weigh 711.29 mg of the synthesized VvCLE1 solid powder, first add a small amount of deionized water to aid solubilization, then add 500 μL / L Tween-20, and finally dilute to 1 L with deionized water to obtain a VvCLE1 short peptide working solution with a concentration of 500 μM.

[0061] The test results obtained (see the appendix of the manual) Figure 7 ), Figure 8 These are experimental photos of each group in this experiment after 2 days, 5 days, 8 days, 12 days and 17 days.

[0062] The above experimental results clearly demonstrate that, in the foliar spray test, the grape peptide VvCLE11 working solution and the positive control (abscisic acid) significantly accelerated the color change process of Cabernet Sauvignon grapes, demonstrating significant differences from the negative control (CK) and the grape VvCLE1 working solution. As a foliar plant growth regulator, VvCLE11 plays an important role in promoting grape ripening and offers a potential alternative to the traditional hormone abscisic acid.

[0063] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A grape VvCLE11 short peptide, characterized by: The grape VvCLE11 short peptide is obtained by translating the 205th to 249th positions of its full-length coding gene, namely the CLE motif, and hydroxylating the fifth and ninth proline residues therein. The short peptide sequence is DYTPPRKKPPIHN, and the fifth and ninth prolines are hydroxyprolines.

2. The grape VvCLE11 short peptide according to claim 1, characterized in that: The grape VvCLE11 short peptide can be used as a leaf plant growth regulator to promote the color change of grape fruits.

3. The grape VvCLE11 short peptide according to claim 2, characterized in that: The grape fruit is Cabernet Sauvignon grape fruit.

4. The grape VvCLE11 short peptide according to claim 1, characterized in that: The grape VvCLE11 short peptide is used as a foliar plant growth regulator in the following manner: before the grape fruit changes color, the grape VvCLE11 short peptide working solution is exogenously applied by foliar spraying to promote the fruit color change; The preparation method of the grape VvCLE11 short peptide working solution is as follows: According to the n:V ratio of grape VvCLE11 short peptide solid powder and deionized water (mmol:mL) of 0.5:1000, the corresponding mass of the synthesized grape VvCLE11 short peptide solid powder was calculated and weighed, dissolved in the corresponding volume of deionized water, and then Tween-20 was added thereto at a V:V ratio of 0.5:1000 to obtain a grape VvCLE11 short peptide working solution with a concentration of 500 μM.

Images