Grape VvCLE1 oligopeptide and application of grape VvCLE1 oligopeptide in promoting fruit color change and serving as plant growth regulator

Abstract

The invention relates to the technical field of biology, in particular to grape VvCLE1 oligopeptide and application of the grape VvCLE1 oligopeptide in promoting fruit coloring and serving as a plant growth regulator. Wherein the full-length nucleotide sequence for coding the grape VvCLE1 short peptide and the corresponding amino acid sequence of the grape VvCLE1 short peptide are shown as SEQ ID NO. 1 and SEQ ID NO. 2. The VvCLE1 oligopeptide is identified and obtained, and experiments of exogenous VvCLE1 application treatment are carried out on cabernet sauvignon cultivated in the field, and it is found that the VvCLE1 oligopeptide can promote color changing of grape fruits. According to the invention, the possibility is provided for accurately controlling the grape ripening time node and replacing the application of the traditional hormone abscisic acid.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to grape VvCLE1 short peptide and its application in promoting fruit color change and as a plant growth regulator. Background Art

[0002] Since short peptides can exert their effects through exogenous application, they are also called plant short peptide hormones. Among them, the CLE (CLAVATA3 / EMBRYO SURROUNDING REGION) short peptide family is one of the earliest and most in-depth small molecule peptides studied. From the perspective of protein structure, the typical structure of the CLE short peptide family is: there is a signal peptide region (Signalpeptide) at the N-terminus for receptor recognition and intracellular localization; the middle part is a non-conserved variable amino acid sequence (Variabledomain); the part near the C-terminus contains a conserved sequence, namely the CLE functional domain (CLE motif), which consists of 12-14 amino acids (KRXVPXGPNPLHNR). This sequence determines the function of the CLE short peptide. Researchers often explore its role in plants by exogenously applying artificially synthesized CLE functional domain fragments, but most of the current research focuses on the function of the CLE short peptide family in plant response to adverse stress. There are relatively few related studies on fruit development, especially in grapes.

[0003] Grape (Vitis Vinifera L.), as one of the most important fruit trees in my country and the world, has extremely high economic and ornamental value. However, the ripening process of grapes is easily affected by the natural environment. How to accurately control the ripening nodes of grapes and maintain high-quality production is one of the challenges facing the current grape cultivation industry. Abscisic acid (ABA) is a key hormone that triggers the ripening of grape fruits. In production, its content in the fruit is often changed by increasing or inhibiting it, which can effectively promote or delay the ripening process of the fruit. It is one of the key factors regulating the quality and ripening period of grapes. Previously, studies have found that Arabidopsis AtCLE25 responds to water stress signals in the roots and is transported to the leaves through the vascular system, inducing the expression of NCED3 in the leaves and promoting the biosynthesis of ABA in the leaves. In addition to CLE25 being proven to induce the synthesis of ABA in leaves over a long distance, the CLE9 / CLE10 short peptides expressed in stomata have also been proven to regulate stomatal closure through the ABA signaling pathway. The conservation of CLE short peptides among species provides the possibility of regulating the ripening nodes and quality of grape fruits by applying CLE. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a grape VvCLE1 short peptide and its use in promoting fruit color change and as a plant growth regulator. This makes it possible to accurately control the time node of grape ripening in production and replace the hormone abscisic acid commonly used in traditional cultivation.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] In a first aspect, a grape VvCLE1 short peptide is provided. The full-length nucleotide sequence encoding the grape VvCLE1 short peptide and the corresponding amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0007] Furthermore, the grape short peptide VvCLE1 is obtained by translating the 256th to 291th position of its coding gene, namely the CLE motif, and modifying it with hydroxyproline.

[0008] In a second aspect, the invention provides the application of grape VvCLE1 short peptide in promoting fruit color change.

[0009] Furthermore, the grape fruit is a "Cabernet Sauvignon" grape fruit.

[0010] Furthermore, before the Cabernet Sauvignon grape berries change color, grape VvCLE1 short peptide is exogenously applied by soaking the berries to promote the color change of the berries.

[0011] Furthermore, the concentration of the VvCLE1 short peptide working solution was 500 μM.

[0012] Furthermore, 500 μL / L Tween-20 was added to the VvCLE1 short peptide working solution to enhance the absorption capacity of the working solution by grape berries.

[0013] In a third aspect, the invention provides the use of grape VvCLE1 short peptide as a plant growth regulator.

[0014] Furthermore, VvCLE1 short peptide can be directly used for exogenous treatment of fruits to shorten the ripening period of fruits.

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

[0016] 1. Promote fruit color change: Exogenous application of grape VvCLE1 short peptide can promote the color change process of grape fruit. This shows that VvCLE1 short peptide plays an important role in regulating endogenous hormones, anthocyanin synthesis and fruit development.

[0017] 2. Shorten the fruit ripening period: Grape VvCLE1 short peptide can be directly used for exogenous treatment of fruits to shorten the fruit ripening period. This provides new ideas and methods for safely and effectively promoting fruit ripening, especially controlling the ripening nodes of non-respiratory climacteric fruits. It also provides a possibility to replace the traditional hormone abscisic acid in the application of grape fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The nucleotide sequence encoding the full-length grape VvCLE1 short peptide and its corresponding amino acid sequence; in the figure, ATG (▼) is the start codon; TAA (*) is the stop codon; the red box area is the VvCLE1 functional domain (CLE motif);

[0019] Figure 2 The short peptide structure of grape VvCLE1 and its amino acid sequence of CLE motif region;

[0020] Figure 3 This is a schematic diagram of the fruit soaking treatment in the field experiment; the yellow box part in the figure is the soaked fruit area (treatment area, Treatment), and the blue box part is the unsoaked fruit area (untreated area, CK), which are distinguished by red ropes during the experiment;

[0021] Figure 4 The effect of grape VvCLE1 short peptide treatment on the color change of Cabernet Sauvignon grape berries in the field; in the figure, NTC is a blank solvent control, and from left to right are representative images of Cabernet Sauvignon grape berries 2, 5, 8, and 12 days after VvCLE1 or NTC treatment;

[0022] Figure 5 The brightness (Brightness), red / green color index (Red-green color index), yellow / blue color index (Yellow-blue color index) and red grape color index (CIRG) of grape fruit after 12 days of treatment with VvCLE1 short peptide; in the figure, the bar graph represents the mean ± SD, and the asterisk (*) indicates a significant difference, which was calculated using the Two-way ANOVA Tukey test (defined as *P<0.05, **P<0.01). DETAILED DESCRIPTION

[0023] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0024] Example

[0025] 1. Materials and reagents

[0026] The experimental methods not specified in this example can be carried out according to conventional methods, and the materials, reagents, etc. used can be obtained through commercial channels unless otherwise specified.

[0027] 2. Methods

[0028] The experimental site was selected at the teaching 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.

[0029] 2.1 Identification of VvCLE1 short peptide

[0030] A hidden Markov model was established with the previously reported CLE short peptide sequence, and the obtained gene sequence was homologously compared with the Cabernet Sauvignon genome using the bioinformatics tool HMMER3.0, and the full-length sequence of the grape VvCLE1 short peptide was screened and identified. Its coding region contains 324 bp of nucleotides, encoding 108 amino acids, and has a typical structural domain unique to the CLE family (RRVPSCPDPLHN) at 256 bp-291 bp.

[0031] 2.2. Obtaining VvCLE1 short peptide

[0032] To further study the function of the CLE domain, the 12 amino acids contained in the CLE motif of the VvCLE1 short peptide were commissioned to GenScript Biotech Co., Ltd. (www.genscript.com.cn) for chemical synthesis. At the same time, the two proline (Pro) residues in the synthetic short peptide were hydroxylated to obtain the VvCLE1 short peptide (RRV{Hyp}SC{Hyp}DPLHN).

[0033] 2.3. Preparation of VvCLE1 short peptide working solution

[0034] Accurately weigh 711.29 mg of the synthesized VvCLE1 solid powder, first add a small amount of deionized water to aid dissolution, then add 500 μL / L of Tween-20, and finally dilute to 1 L with deionized water to obtain a VvCLE1 short peptide working solution with a concentration of 500 mM.

[0035] 2.4. Exogenous application of VvCLE1 to Cabernet Sauvignon grapes grown in the field

[0036] Select Cabernet Sauvignon grapes that are growing well in the field, free of pests and diseases, and before the color change period. Immerse part of each bunch of grapes in the VvCLE1 working solution for about 30 to 40 seconds. Immediately after the immersion treatment, tie a red rope at the boundary to distinguish the immersed area (treated area) from the unimmersed area (untreated area). Figure 3 After treatment, the fruit was bagged, and after 2 days, the bag was removed and managed in the normal mode. Then, the fruit was photographed to observe the color change.

[0037] The results of exogenous application of VvCLE1 short peptide showed that exogenous VvCLE1 treatment significantly affected the development period of Cabernet Sauvignon grape berries, such as Figure 4 As shown, the pigment accumulation of grape fruits in the treated area was significantly accelerated, and the color changed significantly, which was significantly different from the NTC treated and untreated areas.

[0038] 2.5. Measurement of coloration indexes of Cabernet Sauvignon grapes after treatment with VvCLE1 short peptide

[0039] On the 12th day after treatment, 3 fruits were randomly selected from the treated area and the untreated area of ​​each bunch of fruits, with a total of 5 biological replicates for each treatment, and the measurements were performed using a CR-400 handheld colorimeter. The brightness, red-green color index, yellow-blue color index, and red grape color index (CIRG) of the fruits were obtained 12 days after the short peptide treatment. The data were analyzed using software such as Excel and Graphpad prism. Figure 5 As shown, 12 days after VvCLE1 short peptide treatment, the CIRG index of the treated area was significantly higher than that of the untreated area and the NTC treatment, which indicates that VvCLE1 short peptide significantly deepened the color of grape fruit in a short period of time, effectively promoted fruit color change, and accelerated the fruit ripening process.

[0040] 3. Conclusion

[0041] 3.1. Exogenous application of VvCLE1 short peptide to Cabernet Sauvignon grapes before the color change period showed a phenotype of promoting color change. On this basis, the color index was measured by a handheld colorimeter, and the results showed that VvCLE1 short peptide played an important role in regulating endogenous hormones, anthocyanin synthesis, and promoting fruit color change and ripening.

[0042] 3.2. The grape VvCLE1 short peptide of the present invention can be directly used for exogenous treatment of fruits to shorten the fruit ripening period. This provides a new idea and method for safely and effectively promoting fruit ripening, especially improving the quality of non-climacteric fruits. In the future, in-depth research will be conducted on different regions and different varieties, and it will be gradually applied to production, which will provide a theoretical basis and technical support for the development of the grape industry.

[0043] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0044] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Grape VvCLE1 short peptide, characterized in that: The full-length nucleotide sequence encoding the grape VvCLE1 short peptide and its corresponding amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The grape VvCLE1 short peptide according to claim 1, characterized in that The grape short peptide VvCLE1 is obtained by translating the 256th to 291th position of its coding gene, namely the CLE motif, and modifying it with hydroxyproline.

3. Use of the grape VvCLE1 short peptide according to any one of claims 1-2 in promoting fruit color change, characterized in that: It can promote the accumulation of pigments in grape fruits.

4. The use according to claim 3, characterized in that: The grape berries are Cabernet Sauvignon grape berries.

5. The use according to claim 4, characterized in that: Before the Cabernet Sauvignon grapes change color, grape VvCLE1 short peptide is exogenously applied by immersing the fruits to promote the color change of the fruits.

6. The use according to claim 5, characterized in that: The concentration of VvCLE1 short peptide working solution is 500 μM.

7. The use according to claim 6, characterized in that: 500 μL / L Tween-20 was added to the VvCLE1 short peptide working solution to enhance the absorption capacity of the working solution by grape berries.

8. Use of the grape VvCLE1 short peptide according to any one of claims 1-2 as a plant growth regulator.

9. The use according to claim 8, characterized in that: VvCLE1 short peptide can be directly used for exogenous treatment of fruits to shorten the ripening period of fruits.

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