Application of MaCLE16 gene and / or synthetic oligopeptide MaCLE16p in regulation and control of plant fruit maturation

Abstract

The invention discloses application of a MaCLE16 gene and / or synthetic oligopeptide MaCLE16p in regulation and control of plant fruit maturation, and belongs to the technical field of gene engineering. The nucleotide sequence of the MaCLE16 gene is as shown in SEQ ID NO. 3; the amino acid sequence of the synthesized oligopeptide MaCLE16p is RLVPTGPNPLHN. The invention further discloses a preparation method of the oligopeptide. Experiments prove that the ripening time of banana fruits over-expressed with the MaCLE16 gene is shortened by 2-3 days, and the soluble solid content and the pulp hardness are improved by 15% and 20% respectively. The further synthesized core oligopeptide MaCLE16p can accelerate the yellowing of banana fruits in a dose-dependent manner, the effect is consistent with that of gene overexpression, and the risk of chemical residues is avoided. The invention discloses the regulation effect of the CLE gene in fruit ripening for the first time, provides a new strategy for developing a green and safe ripening agent, and has remarkable industrial application value.

Description

Technical Field

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

[0002] Banana (Musa nana Lour.) is a perennial monocotyledonous herbaceous plant of the genus Musa in the Musaceae family, native to Southeast Asia and is one of the important tropical fruit crops. As a typical climacteric fruit, the banana fruits are harvested from the field in the form of green bananas when they grow plump, transported to the market and then ripened by ethylene for terminal sales. At present, in production, the postharvest ripening process of banana fruits is mainly regulated by controlling the environmental temperature and ethylene concentration. For example, low-temperature treatment can reduce the respiration rate of banana fruits and significantly inhibit the postharvest ripening process. However, bananas are relatively sensitive to low temperature, and cold damage is likely to occur when the temperature is too low or the time is too long, reducing the commercial value of the fruits. Treatment with ethylene inhibitors such as 1-methylcyclopropene (1-MCP) can also delay the ripening and senescence of banana fruits, but the application concentrations vary greatly for different types and varieties of bananas, and improper treatment concentrations will lead to postharvest ripening disorders and quality degradation of banana fruits. Therefore, how to effectively regulate the postharvest shelf life of banana fruits, improve the postharvest quality of fruits, obtain the key regulatory factors for fruit ripening and postharvest quality, and develop a new type of green, environmentally friendly and safe ripening agent have become important topics in the postharvest physiology research and high-quality breeding of bananas.

[0003] CLAVATA3 / EMBRYO SURROUNDING REGION (CLE) is the most representative short peptide signaling molecule in plants and has an important regulatory function in plant growth and development. The CLE precursor protein is relatively large. After being hydrolyzed by protease, the signal peptide and variable domain disappear, generating a biologically active CLE peptide. Treating plants with synthetic CLE short peptides can exhibit phenotypes similar to those of overexpression of the corresponding CLE genes. At present, there is no relevant report on the regulation of fruit ripening by CLE genes. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of MaCLE16 gene and / or synthetic short peptide MaCLE16p in regulating the ripening of plant fruits to solve the problems existing in the above-mentioned prior art. The MaCLE16 gene disclosed in the present invention is a potential fruit ripening promoting factor, and the MaCLE16 short peptide can also be developed and utilized as a new type of green, safe and environmentally friendly ripening agent for bananas.

[0005] To solve the above problems, the present invention provides the following solutions:

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

[0007] SEQ ID NO.3:

[0008] ATGGAGAAGAGATGGGCTCGAGTAGCCCTGGTCGCCTGGTTCATCTTGTTCGTCGCCGGCTCGCATGGGTTGGCTCGAAGTGTCAGCCGGAAGCACAGTCACCACCGTCACAAGGCAGATGAGAAGGCATCGGTGGAGGAGTTGGCTGCCGGCGTTGCCACCATCCACCGCCGTGGGTGCAGGTTCCAGAATCTCGATCTAGTTTGCGGCTATCTCACGCAGGCGTGCAAGAACGGGAGCTCGGTCGACGACGACAAGCGACTCGTACCAACCGGCCCCAACCCCTTGCATAACAGATGA;

[0009] SEQ ID NO.4:

[0010] MEKRWARAALVAWFILFVAGSHGLARSVSRKHSHHRHKADEKASVEELAAGVATIHR RGCRSQNLDLVCGYLTQACKNGSSVDDDKRLVPTGPNPLHNR.

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

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

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

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

[0015] Technical solution 4: A synthetic short peptide MaCLE16p, whose amino acid sequence is RLVPTGPNPLHN.

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

[0017] Technical solution five: A method for promoting the ripening of plant fruits, including the step of externally spraying the synthetic short peptide MaCLE16p on the plant fruits.

[0018] Technical solution six: A ripening agent, comprising the MaCLE16 gene or the synthetic short peptide MaCLE16p described above.

[0019] Technical solution seven: The application of the MaCLE16 gene in regulating the ripening of plant fruits or improving the quality of plants.

[0020] Furthermore, the plant includes bananas.

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

[0022] The present invention uses omics analysis to identify that the MaCLE16 gene participates in the postharvest ripening process of banana fruits through the ethylene signal. By analyzing the phenotypes and related physiological indexes of banana fruits with transient overexpression of MaCLE16 and banana fruits treated with MaCLE16 short peptides, the results show that: compared with the control fruits, the expression of ethylene synthesis-related genes in banana fruits with transient overexpression of MaCLE16 is activated in advance, thereby promoting fruit ripening and increasing the soluble solids and fruit firmness. The banana fruits treated with MaCLE16 short peptides also have similar effects, indicating that the MaCLE16 gene is a potential fruit ripening promoter, and the MaCLE16 short peptide can also be developed and utilized as a new type of green, safe and environmentally friendly ripening agent for bananas. Specifically, the banana fruits overexpressing the MaCLE16 gene can turn yellow 2-3 days in advance, and the soluble solids content and firmness are increased by 15% and 20% respectively. Further, the core short peptide MaCLE16p (RLVPTGPNPLHN) is synthesized, and external spraying at a concentration of 10 μM can dose-dependently accelerate the ripening of 'Jiali' and 'Brazil' fruits, and the effect is consistent with gene overexpression, and there is no risk of chemical residue. The technology provided by the present invention breaks through the limitations of traditional ethylene regulation, provides an innovative solution for the green ripening and quality improvement of bananas after harvest, and also provides a theoretical basis for studying the molecular regulation mechanism of banana fruit ripening. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 Electrophoretogram for the cloning of banana MaCLE16; among them, M is Marker, P1 is the sample at 1 day of maturity, P2 is the sample at 3 days of maturity, and P3 is the sample at 5 days of maturity.

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

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

[0027] Figure 4 Effect of overexpressing MaCLE16 on banana fruit ripening; among them, A is the relative expression level of MaCLE16 gene in control and overexpressing banana fruits; B is the ripening situation of control and MaCLE16-overexpressing banana fruits under ethylene treatment; C - E are the color, soluble solid content, and fruit firmness of control and MaCLE16-overexpressing banana fruits under ethylene treatment in sequence; *, **, *** respectively indicate p < 0.05, 0.01, 0.001.

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

[0029] Figure 6 Ripening degrees of 'Jiali' and 'Brazil' banana fruits after MaCLE16p treatment Detailed implementation manners

[0030] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

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

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0034] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0035] Example 1

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

[0037] (1) Experimental materials

[0038] After harvesting the fruits of the banana cultivar 'Jiali' (Musa acuminata, AA) with 80% plumpness (about 45 - 50 days after flowering) in the field, cut them into individual banana fruits (removing the head and tail combs), select banana fruits with basically the same fruit size, maturity and no pests and diseases, wash them clean with water, dry them, and put them into an airtight polyethylene bag and ripen at 25°C for 5 days. Sampling was carried out at stages P1 (1 day of ripening), P3 (3 days of ripening) and P5 (5 days of ripening). The pulp from 5 banana fruits was chopped and thoroughly mixed, then frozen in liquid nitrogen and stored at -80°C for later use.

[0039] (2) Experimental methods

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

[0041] cDNA reverse transcription: According to the manufacturer's instructions, the RNA samples were reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novoprotein Biotechnology Co., Ltd.).

[0042] (3) Cloning of the MaCLE16 gene

[0043] Using the cDNA of 'Jiali' banana fruits as a template, PCR reactions were performed with the upstream primer MaCLE16-F and the downstream primer MaCLE16-R and Takara Taq enzyme. The reaction system was as follows:

[0044] 10 μL system, including 10×PCR Buffer (containing Mg 2+ ) 1.0 μL, dNTPs (10 μmol / L) 0.2 μL, MaCLE16-F (10 μmol / L) and MaCLE16-R (10 μmol / L) 0.5 μL each, Taq enzyme (5 U / μL, TaKaRa) 0.1 μL, template DNA (10 ng / μL) 1.0 μL, and ddH2O was added to make up to 10 μL. Mix well and centrifuge.

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

[0046] The PCR products of the MaCLE16 gene CDS sequence obtained by amplification of different samples were subjected to gel electrophoresis ( Figure 1 )). The PCR products of the MaCLE16 gene CDS sequence were ligated to the sticky-end T vector (pMD-19T), and the ligation products were transformed into Escherichia coli DH5α by heat shock method 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. Its base sequence is 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] ATGGAGAAGAGATGGGCTCGAGTAGCCCTGGTCGCCTGGTTCATCTTGTTCGTCGCCGGCTCGCATGGGTTGGCTCGAAGTGTCAGCCGGAAGCACAGTCACCACCGTCACAAGGCAGATGAGAAGGCATCGGTGGAGGAGTTGGCTGCCGGCGTTGCCACCATCCACCGCCGTGGGTGCAGGTTCCAGAATCTCGATCTAGTTTGCGGCTATCTCACGCAGGCGTGCAAGAACGGGAGCTCGGTCGACGACGACAAGCGACTCGTACCAACCGGCCCCAACCCCTTGCATAACAGATGA;

[0053] SEQ ID NO.4:

[0054] MEKRWARAALVAWFILFVAGSHGLARSVSRKHSHHRHKADEKASVEELAAGVATIHR RGCRSQNLDLVCGYLTQACKNGSSVDDDKRLVPTGPNPLHNR.

[0055] (4) Construction of overexpression vector

[0056] The Escherichia coli containing the pMD-19T-MaCLE16 plasmid obtained by cloning was added to a liquid LB medium for overnight culture, and the plasmid was extracted using the FastPure Plasmid Mini Kit (Nanjing Novoprotein Scientific Co., Ltd.). Using MaCLE19-1300-F and MaCLE19-1300-R as amplification primers, the pMD-19T-MaCLE16 plasmid as a template, and Takara Taq enzyme for PCR reaction, the PCR reaction system was the same as that for gene cloning. The specific primers are as follows:

[0057] MACLE19 - 1300 - F: ATACACCAAATCGACTCTAGAATGGAGAAGAGATGGGCTCGAG (SEQ ID NO.5);

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

[0059] The amplified fragments were separated using a 1.2% agarose gel, and the target gene with consistent size was recovered using the FastPure Gel DNA Extraction Mini Kit (Nanjing Novoprotein Scientific Inc.). The recovered target gene product was ligated with the pCAMBIA1300 vector linearized by Kpn I / Xba I using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novoprotein Scientific Inc.). The ligation reaction system 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. Mix well and centrifuge. Ligation system: 50 °C, 5 min.

[0060] The above 10 μL ligation product was added to 100 μL of DH5α Escherichia coli competent cells by heat shock method, gently mixed, ice-bathed for 30 min, incubated at 42 °C for 45 s, and then ice-bathed for 5 min. 1 mL of liquid LB without antibiotics was added, and the cells were resuscitated at 37 °C on a shaker at 220 rpm for 1 h, and then spread on a solid LB plate containing kanamycin antibiotic and cultured in a 37 °C incubator for 1 day. The correct monoclonal strains after PCR reaction were selected and sent to Tsingke Sequencing Company for sequencing. The plasmid with pCAMBIA1300 - MaCLE16 with correct sequencing was transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. Positive monoclonal strains were identified by colony PCR.

[0061] The MaCLE16 sequence was amplified from fruits of diploid banana 'Jiali' at different ripening stages. The full length of this gene is 300 bp ( Figure 1 ), encoding 99 amino acids, and the molecular weight of the protein is 10.97 kDa. The CLE proteins of banana and Arabidopsis thaliana were analyzed by phylogenetic tree using MEGA 6.0. By Figure 2It can be seen that the MaCLE16 protein belongs to the Class IV CLE protein of type A. Sequence analysis of the MaCLE16 protein reveals that the MaCLE16 protein also has the significant characteristics of CLE proteins, namely, there is a signal peptide at the N-terminus, a variable domain in the middle, and a conserved CLE motif at the C-terminus, and the CLE motif of MaCLE16 is most similar to that of Arabidopsis AtCLE19 and rice OsCLE19( Figure 3 A). Expression analysis shows that MaCLE16 is hardly expressed in full green fruits, and its expression increases rapidly as the fruits mature, reaching the highest level at the yellow-ripe stage( Figure 3 B).

[0062] 2. Transient genetic transformation of banana fruits

[0063] Select the correctly identified single colony of Agrobacterium tumefaciens pCAMBIA1300-MaCLE16 for banana fruit infection. Select fruits of the banana cultivar 'Jiali' with 80% fullness (about 45 - 50 days after flowering) as the test materials, and use the injection method for fruit infection. The specific method is as follows:

[0064] 1) Inoculate the positive monoclonal of Agrobacterium tumefaciens 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 liquid at 6000 rpm for 5 min at room temperature, and remove the supernatant;

[0066] 3) Add infection buffer (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone) with pH 5.6 - 5.7 to resuspend to OD 600 = 1.0. After standing in the dark at room temperature for 3 h, use a sterile syringe to extract the resuspended liquid and inject it into the fruits of 'Jiali' from the style end;

[0067] 4) Put the injected fruits into a sealed polyethylene bag and ripen them at 25 °C for 5 days.

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

[0069] To verify whether the MaCLE16 gene is involved in the banana fruit ripening process, the constructed recombinant plasmid pCAMBIA1300-MaCLE16 was introduced into Agrobacterium tumefaciens EHA105. The mature fruits of 'Jiali' with 80% fullness were transiently infected by the injection method (MaCLE16-OE), and the empty vector pCAMBIA1300 was used as a control (Control). Samples were taken after 5 days and relevant detections were carried out.

[0070] RT-qPCR results showed that, compared with control fruits, the transcriptional level of MaCLE16 in MaCLE16-OE fruits was significantly up-regulated ( Figure 4 A); the infection results showed that fruits injected with empty vector began to turn yellow on the 3rd day and all turned yellow on the 5th day, while MaCLE16 transient overexpression fruits began to turn yellow on the 1st day and all turned yellow on the 3rd day ( Figure 4 B). The pericarp color analysis showed that from the 1st day, the color of MaCLE16-OE fruits was significantly higher than that of the control ( Figure 4 C), indicating that overexpression of the MaCLE16 gene promoted banana fruit ripening. The fruit quality analysis on the 5th day found that the soluble solids and pulp firmness of MaCLE16-OE fruits were significantly higher than those of the control ( Figure 4 D and Figure 4 E), indicating that the MaCLE16 gene not only positively regulated banana fruit ripening, but also improved the postharvest quality of fruits.

[0071] In addition, the present invention detected the expression trends of some ethylene pathway-related genes during the ripening process of control and MaCLE16-OE fruits by RT-qPCR. The results showed that, compared with control fruits, the expression peaks of MaACS1, MaACS12, MaACO1, MaACO4, MaACO5 and MaACO8 genes were significantly advanced in MaCLE16 overexpressed fruits ( Figure 5 ), indicating that overexpression of MaCLE16 prematurely activated the expression of ethylene synthesis-related genes, thereby promoting fruit ripening.

[0072] 4. Analysis of the ripening time of fruits treated with MaCLE16 short peptide

[0073] Treatment with synthetic CLE peptides exogenously can exhibit phenotypes similar to those of overexpression of the corresponding CLE genes. To further study the bioactive form of MaCLE16, a 12-amino acid short peptide corresponding to the MaCLE16 motif (RLVPTGPNPLHN (SEQ ID NO.8), MaCLE16 peptide, MaCLE16p) was sent to Nanjing Genscript Biotech Co., Ltd. for synthesis. The synthesized MaCLE16p (purity > 99%) was dissolved in ultrapure water and prepared into concentrations of 1, 10, and 100 μM, and the green fruits of 'Jiali' and 'Brazil' (Musa spp. AAA) bananas were sprayed with water as a control.

[0074] The results showed that treatment with 10 μM MaCLE16p could promote the ripening process of 'Jiali' and 'Brazil' banana fruits, while treatment with too low (1 μM) or too high (100 μM) MaCLE16p could not promote banana fruit ripening ( Figure 6) It shows that the MaCLE16 gene participates in the banana fruit ripening process in the form of MaCLE16p, and the effect of MaCLE16p on banana fruit ripening is dose-dependent.

[0075] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of the MaCLE16 gene in regulating the maturity of plant fruits, characterized in that, The nucleotide sequence of the MaCLE16 gene is shown as SEQ ID NO.3, and the amino acid sequence of the protein encoded by the MaCLE16 gene is shown as SEQ ID NO.

4.

2. A recombinant expression vector pCAMBIA1300-MaCLE16, characterized in that, Comprising the MaCLE16 gene described in claim 1.

3. A method for promoting the ripening of plant fruits, characterized in that, Including the step of overexpressing the MaCLE16 gene described in claim 1 in plant fruits.

4. The method according to claim 3, characterized in that, The step includes introducing the MaCLE16 gene into a plant by Agrobacterium-mediated genetic transformation using the recombinant expression vector pCAMBIA1300-MaCLE16 described in claim 2.

5. The method according to claim 4, wherein The Agrobacterium is Agrobacterium tumefaciens EHA105.

6. A synthetic short peptide MaCLE16p, characterized in that, Its amino acid sequence is RLVPTGPNPLHN.

7. A method for promoting the ripening of plant fruits, characterized in that, Including the step of externally spraying the synthetic short peptide MaCLE16p described in claim 6 on plant fruits.

8. A ripening agent, characterized in that, Comprising the MaCLE16 gene described in claim 1 or the synthetic short peptide MaCLE16p described in claim 6.

9. Use of the MaCLE16 gene described in claim 1 in regulating plant fruit ripening and improving plant quality.

10. The application according to claim 1, the method according to claim 3, the method according to claim 7, or the application according to claim 9, characterized in that, The plant includes bananas.

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