Use of MaCLE16 gene and / or synthetic short peptide MaCLE16p in regulating fruit ripening of plants

Abstract

The application discloses application of MaCLE16 gene and / or synthetic short peptide MaCLE16p in regulation of fruit ripening of plants and belongs to the technical field of genetic engineering. The nucleotide sequence of the MaCLE16 gene is shown as SEQ ID NO. 3; and the amino acid sequence of the synthetic short peptide MaCLE16p is RLVPTGPNPLHN. The experiment proves that the banana fruit ripening time is shortened by 2-3 days, and the soluble solid content and the pulp hardness are respectively increased by 15% and 20% when the MaCLE16 gene is overexpressed. The further synthesized core short peptide MaCLE16p can dose-dependently accelerate the banana fruit yellowing, and the effect is consistent with that of the gene overexpression, and there is no chemical residue risk. The application first discloses the regulation effect of the CLE gene in fruit ripening, provides a new strategy for developing a green and safe ripening agent, and has significant industrial application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the MaCLE16 gene and / or the synthetic short peptide MaCLE16p in regulating the ripening of plant fruits. Background Technology

[0002] Banana (Musa nana Lour.) is a perennial monocotyledonous herbaceous plant belonging to the genus Musa in the family Musaceae. Native to Southeast Asia, it is an important tropical fruit crop. As a typical climacteric fruit, bananas are harvested from the field as green bananas after they have fully matured, transported to the market, and ripened with ethylene before being sold. Currently, the ripening process of bananas is mainly regulated by controlling environmental temperature and ethylene concentration. For example, low-temperature treatment can reduce the respiration rate of bananas and significantly inhibit the ripening process. However, bananas are quite sensitive to low temperatures; excessively low temperatures or prolonged exposure can easily cause chilling injury, reducing the commercial value of the fruit. Ethylene inhibitors such as 1-methylcyclopropene (1-MCP) can also delay the ripening and senescence of bananas, but the concentration used varies greatly among different types and varieties of bananas. Improper treatment concentrations can lead to ripening disorders and quality decline. Therefore, how to effectively regulate the postharvest shelf life of bananas, improve postharvest quality, identify key regulatory factors for ripening and postharvest quality, and develop new green, environmentally friendly, and safe ripening agents has become an important topic in banana postharvest physiological research and high-quality breeding.

[0003] The CLAVATA3 / EMBRYO SURROUNDING REGION (CLE) is one of the most representative short peptide signaling molecules in plants, playing a crucial regulatory role in plant growth and development. The CLE precursor protein is relatively large; after protease hydrolysis, the signal peptide and variable domain disappear, producing the biologically active CLE peptide. Treatment of plants with the synthesized CLE short peptide can induce a phenotype similar to overexpression of the corresponding CLE gene. Currently, there are no reports on the regulation of fruit ripening by the CLE gene. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the MaCLE16 gene and / or the synthetic short peptide MaCLE16p in regulating fruit ripening in plants, thereby addressing the problems existing in the prior art. The MaCLE16 gene disclosed in this invention is a potential fruit ripening promoter, and the MaCLE16 short peptide can also be developed and utilized as a novel, green, safe, and environmentally friendly ripening agent for bananas.

[0005] To address the above problems, the present invention provides the following solution:

[0006] Technical Solution 1: Application of the MaCLE16 gene in regulating plant fruit ripening, wherein the nucleotide sequence of the MaCLE16 gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the MaCLE16 gene is shown in SEQ ID NO.4.

[0007] SEQ ID NO.3:

[0008] ATGGAGAAGAGATGGGCTCGAGTAGCCCTGGTCGCCTGGTTCATCTTGTTCGTCGCCGGCTCGCATGGGTTGGCTCGAAGTGTCAGCCGGAAGCACAGTCACCACCGTCACAAGGCAGATGAGAAGGCATCGGTGGAGGAGTTGGCTGCC GGCGTTGCCACCATCCACCGCCGTGGGTGCAGGTTCCAGAATCTCGATCTAGTTTGCGGCTATCTCACGCAGGCGTGCAAGAACGGGAGCTCGGTCGACGACGACAAGCGACTCGTACCAACCGGCCCCAACCCCTTGCATAACAGATGA;

[0009] SEQ ID NO.4:

[0010] MEKRWARAALVAWFILFVAGSHGLARSVSRKHSHHRHKADEKASVEELAAGVATIHR RGCRSQNLDLVCGYLTQACKNGSSVDDDKRLVPTGPNPLHNR.

[0011] Technical Solution 2: A recombinant expression vector pCAMBIA1300-MaCLE16, containing the MaCLE16 gene.

[0012] Technical Solution 3: A method for promoting the ripening of plant fruits, comprising the step of overexpressing the MaCLE16 gene in plant fruits.

[0013] Further, the step includes introducing the MaCLE16 gene into plants by introducing the recombinant expression vector pCAMBIA1300-MaCLE16 into plants via Agrobacterium-mediated genetic transformation.

[0014] Furthermore, the Agrobacterium is Agrobacterium tumefaciens EHA105.

[0015] Technical Solution 4: A synthetic short peptide MaCLE16p, the amino acid sequence of which is RLVPTGPNPLHN.

[0016] The research on the MaCLE16 gene provided by this invention provides a target for molecular breeding, while the development of the synthetic short peptide MaCLE16p provides a genetically modified, green and safe ripening agent strategy. The two complement each other in basic research and industrial applications.

[0017] Technical Solution 5: A method for promoting the ripening of plant fruits, comprising the step of exogenously spraying the synthetic short peptide MaCLE16p onto plant fruits.

[0018] Technical Solution Six: A ripening agent comprising the MaCLE16 gene or the synthetic short peptide MaCLE16p.

[0019] Technical Solution 7: Application of the MaCLE16 gene in regulating fruit ripening or improving plant quality.

[0020] Furthermore, the plant includes bananas.

[0021] The present invention discloses the following technical effects:

[0022] This invention utilizes omics analysis to identify the MaCLE16 gene's involvement in banana fruit ripening via ethylene signaling. Analysis of the phenotypes and related physiological indicators of bananas transiently overexpressing MaCLE16 and those treated with MaCLE16 short peptides revealed that, compared to control fruits, bananas transiently overexpressing MaCLE16 showed earlier activation of ethylene synthesis-related genes, thus promoting fruit ripening and increasing soluble solids and fruit firmness. Treatment with MaCLE16 short peptides exhibited similar effects, indicating that the MaCLE16 gene is a potential fruit ripening promoter, and the MaCLE16 short peptide could be developed and utilized as a novel, green, safe, and environmentally friendly ripening agent for bananas. Specifically, bananas overexpressing the MaCLE16 gene turned yellow 2-3 days earlier, and soluble solids content and firmness increased by 15% and 20%, respectively. Further synthesis of the core short peptide MaCLE16p (RLVPTGPNPLHN) and exogenous spraying at a concentration of 10 μM can dose-dependently accelerate the ripening of 'Jiali' and 'Brazil' varieties of bananas, with effects consistent with gene overexpression and without the risk of chemical residues. The technology provided by this invention overcomes the limitations of traditional ethylene regulation, offering an innovative solution for post-harvest green ripening and quality improvement of bananas, and also providing a theoretical basis for studying the molecular regulatory mechanisms of banana fruit ripening. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Electrophoresis image of the banana MaCLE16 clone; where M is the marker, P1 is a sample matured for 1 day, P2 is a sample matured for 3 days, and P3 is a sample matured for 5 days.

[0025] Figure 2 Phylogenetic tree of banana CLE protein and Arabidopsis CLE protein;

[0026] Figure 3 Sequence alignment of banana MaCLE16, Arabidopsis thaliana AtCLE19, rice OsCLE19 and radish RsCLE19 (A) and expression analysis of banana MaCLE16 gene during fruit ripening (B);

[0027] Figure 4 The study investigated the effect of MaCLE16 overexpression on banana fruit ripening. In this study, A represents the relative expression level of the MaCLE16 gene in control and overexpressed banana fruits; B represents the ripening status of control and overexpressed MaCLE16 banana fruits under ethylene treatment; C and D represent the color, soluble solids content, and fruit firmness of control and overexpressed MaCLE16 banana fruits under ethylene treatment, respectively; *, **, and *** indicate p < 0.05, 0.01, and 0.001, respectively.

[0028] Figure 5 The expression levels of ethylene synthesis-related genes (MaACS1, MaACS12, MaACO1, MaACO4, MaACO5, and MaACO8 genes) in MaCLE16 overexpression and control fruits (control);

[0029] Figure 6 The ripeness of 'Gale' and 'Brazil' bananas after treatment with MaCLE16p. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Example 1

[0036] 1. Cloning of the MaCLE16 gene and construction of an overexpression vector

[0037] (1) Experimental materials

[0038] After harvesting bananas of the 'Musa acuminata' (AA) variety at 80% maturity (approximately 45-50 days after flowering) from the field, individual bananas were cut (head and tail combs removed). Bananas of similar size, maturity, and free from pests and diseases were selected, washed clean, and dried. They were then placed in sealed polyethylene bags and ripened at 25°C for 5 days. Samples were taken at stages P1 (1 day ripening), P3 (3 days ripening), and P5 (5 days ripening). The pulp from five bananas was chopped, thoroughly mixed, frozen in liquid nitrogen, and stored at -80°C for later use.

[0039] (2) Experimental methods

[0040] RNA extraction: Total RNA was extracted from banana fruits at different stages using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to the instructions.

[0041] cDNA reverse transcription: Following the manufacturer's instructions, use the HiScript III 1st Strand cDNASynthesis Kit (+gDNA wiper) (Nanjing Novizan Biotechnology Co., Ltd.) to reverse transcribe the RNA sample into cDNA.

[0042] (3) Cloning of the MaCLE16 gene

[0043] Using cDNA from 'Jiali' banana fruit as a template, PCR was performed with Takara Taq enzyme using upstream primer MaCLE16-F and downstream primer MaCLE16-R. The reaction system is as follows:

[0044] 10 μL system, including 10×PCR Buffer (containing Mg 2+ Add 1.0 μL of dNTPs (10 μmol / L), 0.2 μL of MaCLE16-F (10 μmol / L) and MaCLE16-R (10 μmol / L), 0.1 μL of Taq enzyme (5 U / μL, TaKaRa), 1.0 μL of template DNA (10 ng / μL), and ddH2O to a final volume of 10 μL. Mix well and centrifuge.

[0045] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 50 s, 56℃ annealing for 50 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 10 min, and detection by 1.2% agarose gel electrophoresis.

[0046] PCR products of the MaCLE16 gene CDS sequence obtained by amplification from different samples were subjected to gel electrophoresis. Figure 1 The PCR product of the CDS sequence of the MaCLE16 gene was ligated into a sticky-terminated T vector (pMD-19T), and the ligation product was transformed into E. coli DH5α by heat shock and sent to Qingke Biotechnology Co., Ltd. for sequencing to obtain the plasmid containing pMD-19T-MaCLE16.

[0047] The specific primers are as follows:

[0048] MaCLE16-F: ATGGAGAAGAGATGGGCTCGAGTAG (SEQ ID NO. 1);

[0049] MaCLE16-R: TCATCTGTTATGCAAGGGGTTGGG (SEQ ID NO. 2).

[0050] The cloned MaCLE16 gene has a full-length base sequence of 300 bp, as shown in SEQ ID NO.3, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.4.

[0051] SEQ ID NO.3:

[0052] ATGGAGAAGAGATGGGCTCGAGTAGCCCTGGTCGCCTGGTTCATCTTGTTCGTCGCCGGCTCGCATGGGTTGGCTCGAAGTGTCAGCCGGAAGCACAGTCACCACCGTCACAAGGCAGATGAGAAGGCATCGGTGGAGGAGTTGGCTGCC GGCGTTGCCACCATCCACCGCCGTGGGTGCAGGTTCCAGAATCTCGATCTAGTTTGCGGCTATCTCACGCAGGCGTGCAAGAACGGGAGCTCGGTCGACGACGACAAGCGACTCGTACCAACCGGCCCCAACCCCTTGCATAACAGATGA;

[0053] SEQ ID NO.4:

[0054] MEKRWARAALVAWFILFVAGSHGLARSVSRKHSHHRHKADEKASVEELAAGVATIHR RGCRSQNLDLVCGYLTQACKNGSSVDDDKRLVPTGPNPLHNR.

[0055] (4) Construction of hyperexpression vectors

[0056] The cloned *E. coli* containing the pMD-19T-MaCLE16 plasmid was added to liquid LB medium and cultured overnight. The plasmid was extracted using the FastPure Plasmid Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.). PCR was performed using MaCLE19-1300-F and MaCLE19-1300-R primers, pMD-19T-MaCLE16 plasmid as a template, and TakaraTaq polymerase. The PCR reaction system was consistent with that used for gene cloning. Specific primers are as follows:

[0057] MACLE19-1300-F:ATACACCAAATCGACTCTAGAATGGAGAAGAGATGGGCTCGAG(SEQ IDNO.5);

[0058] MACLE19-1300-R: GCCCTTGCTCACCATGGTACCTCTGTTATGCAAGGGGTTGGG (SEQ ID NO. 6).

[0059] The amplified fragment was separated using a 1.2% agarose gel, and the target gene of uniform size was recovered using the FastPure Gel DNA Extraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.). The recovered target gene product was ligated into the pCAMBIA1300 vector linearized with Kpn I / Xba I using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.). The ligation reaction mixture was as follows: 10 μL system, including 5.0 μL of 2×ClonExpress Mix, 3 μL of the recovered target gene product, and 2 μL of the linearized vector fragment, mixed thoroughly, and centrifuged. Ligation temperature: 50℃, 5 min.

[0060] The ligation product (10 μL) was added to 100 μL of DH5α *E. coli* competent cells using a heat shock method. The mixture was gently mixed, incubated on ice for 30 min, then at 42°C for 45 s, followed by another 5 min on ice. 1 mL of antibiotic-free liquid LB was added, and the mixture was incubated at 37°C for 1 h on a shaker at 220 rpm. The resulting solution was then plated onto solid LB plates containing kanamycin and incubated at 37°C for 1 day. Single clones that were correctly identified after PCR were sent to Ketone sequencing company for sequencing. The plasmid containing pCAMBIA1300-MaCLE16 was transformed into *Agrobacterium tumefaciens* EHA105 using a freeze-thaw method. Positive single clones were identified by culture PCR.

[0061] The MaCLE16 sequence was amplified from diploid banana 'Gale' at different ripening stages. The full-length gene is 300 bp. Figure 1 The protein, encoding 99 amino acids, has a molecular weight of 10.97 kDa. Phylogenetic analysis of the banana CLE protein and the Arabidopsis CLE protein was performed using MEGA 6.0. Figure 2It is known that the MaCLE16 protein belongs to type A, Class IV CLE protein. Sequence analysis of the MaCLE16 protein revealed that it also possesses the significant characteristics of CLE proteins, namely, an N-terminal signal peptide, a variable domain in the middle, and a conserved CLE motif at the C-terminus. Furthermore, the CLE motif of MaCLE16 is most similar to that of Arabidopsis thaliana AtCLE19 and rice OsCLE19. Figure 3 Expression analysis revealed that MaCLE16 was almost not expressed in plump green fruits, but its expression increased rapidly as the fruit matured, reaching its highest level during the yellow ripening stage. Figure 3 (B).

[0062] 2. Transient genetic transformation of banana fruit

[0063] Single colonies of Agrobacterium tumefaciens pCAMBIA1300-MaCLE16, correctly identified, were selected for banana fruit infection. Banana fruits of the 'Jiali' variety, approximately 80% plump (about 45-50 days after flowering), were chosen as test materials. The fruit infection was performed using an injection method, as detailed below:

[0064] 1) Inoculate a single Agrobacterium-positive clone of pCAMBIA1300-MaCLE16 into 15 mL of LB liquid medium (containing 50 mg / L Kan and 25 mg / L Rif), and shake at 28°C and 220 rpm until OD. 600 =2.0.

[0065] 2) Centrifuge the bacterial culture at 6000 rpm for 5 minutes at room temperature and remove the supernatant;

[0066] 3) Add pH 5.6-5.7 infection buffer (10mM MgCl2, 10mM MES, 200μM acetylsylcholine) and resuspend to OD. 600 =1.0, after standing at room temperature in the dark for 3 hours, use a sterile syringe to draw up the resuspension and inject it into the fruit of 'Jiali' from the pistil end;

[0067] 4) Place the injected fruit in a sealed polyethylene bag and allow it to mature at 25°C for 5 days.

[0068] 3. Analysis of ripening time and determination of corresponding indicators of MaCLE16 transgenic fruit

[0069] To verify whether the MaCLE16 gene is involved in the banana fruit ripening process, the constructed pCAMBIA1300-MaCLE16 recombinant plasmid was introduced into Agrobacterium EHA105. 'Jiali' 'Jiali' ripe fruits (MaCLE16-OE) at 80% plumpness were transiently infected via injection, with the empty vector pCAMBIA1300 serving as a control. Samples were collected after 5 days for relevant testing.

[0070] RT-qPCR results showed that, compared with the control fruit, the transcriptional level of MaCLE16 in MaCLE16-OE fruit was significantly upregulated. Figure 4 (A); Infection results showed that fruits injected without a vector only began to turn yellow on the 3rd day and were completely yellow on the 5th day, while fruits transiently overexpressed with MaCLE16 began to turn yellow on the 1st day and were completely yellow on the 3rd day. Figure 4 (B). Peel color analysis showed that from day 1, the color of MaCLE16-OE fruit was significantly higher than that of the control (B). Figure 4 The results (C) indicate that overexpression of the MaCLE16 gene promotes banana fruit ripening. Fruit quality analysis on day 5 revealed that the soluble solids content and pulp firmness of MaCLE16-OE fruit were significantly higher than the control. Figure 4 D and Figure 4 The results (E) indicate that the MaCLE16 gene not only positively regulates the post-ripening of banana fruit, but also improves the post-harvest quality of the fruit.

[0071] Furthermore, this invention used RT-qPCR to detect the expression trends of some ethylene pathway-related genes during the ripening process of control and MaCLE16-OE fruits. The results showed that, compared with control fruits, the expression peaks of MaACS1, MaACS12, MaACO1, MaACO4, MaACO5, and MaACO8 genes were significantly earlier in fruits overexpressing MaCLE16. Figure 5 This indicates that overexpression of MaCLE16 prematurely activates the expression of ethylene synthesis-related genes, thereby promoting fruit ripening.

[0072] 4. Analysis of fruit ripening time after treatment with MaCLE16 short peptide

[0073] Exogenous treatment with the synthesized CLE peptide resulted in a phenotype similar to overexpression of the corresponding CLE gene. To further investigate the bioactive form of MaCLE16, a 12-amino acid short peptide (RLVPTGPNPLHN (SEQ ID NO. 8), MaCLE16 peptide, MaCLE16p) corresponding to the MaCLE16 motif was synthesized by Nanjing GenScript Biotech Co., Ltd. The synthesized MaCLE16p (purity >99%) was dissolved in ultrapure water and prepared at concentrations of 1, 10, and 100 μM. Water was used as a control, and the solution was sprayed onto unripe 'Jiali' and 'Brazil' (Musa spp. AAA) banana fruits.

[0074] The results showed that 10 μM MaCLE16p treatment promoted the ripening process of 'Gale' and 'Brazil' bananas, while treatments with excessively low (1 μM) or high (100 μM) MaCLE16p treatments did not promote fruit ripening. Figure 6This indicates that the MaCLE16 gene participates in the banana fruit ripening process in the form of MaCLE16p, and that the effect of MaCLE16p on banana fruit ripening is dose-dependent.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the MaCLE16 gene in regulating plant fruit ripening, characterized by, The nucleotide sequence of the MaCLE16 gene is shown in SEQ ID NO.3; The regulation is achieved by overexpressing the MaCLE16 gene to increase the fruit maturity of the plant. The plant in question is a banana.

2. A method for promoting the ripening of plant fruits, characterized in that, The step of overexpressing the MaCLE16 gene as described in claim 1 in plant fruit; The plant in question is a banana.

3. The method according to claim 2, characterized in that, The step includes introducing the MaCLE16 gene into plants via Agrobacterium-mediated genetic transformation using a recombinant expression vector pCAMBIA1300-MaCLE16 containing the MaCLE16 gene as described in claim 1.

4. The method according to claim 3, characterized in that, The Agrobacterium is Agrobacterium tumefaciens EHA105.

5. A method for promoting the ripening of plant fruits, characterized in that, This includes the step of exogenously spraying the synthetic short peptide MaCLE16p onto plant fruits; The amino acid sequence of the synthesized short peptide MaCLE16p is RLVPTGPNPLHN; The concentration of the synthetic short peptide MaCLE16p was 10 μM; The plant in question is a banana.

6. A ripening agent, characterized in that, Contains the MaCLE16 gene or the synthetic short peptide MaCLE16p as described in claim 1; The amino acid sequence of the synthesized short peptide MaCLE16p is RLVPTGPNPLHN; The concentration of the synthetic short peptide MaCLE16p was 10 μM; The ripening agent is a banana ripening agent.

7. The application of the MaCLE16 gene as described in claim 1 in regulating fruit ripening and improving plant quality, characterized in that, The regulation is achieved by overexpressing the MaCLE16 gene to promote fruit ripening in the plant. The plant quality refers to the soluble solids content of the fruit or the firmness of the pulp. The plant in question is a banana.

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