Apple circular RNA and application in inhibiting ethylene biosynthesis
By overexpressing circCals circular RNA in apple fruit to inhibit ethylene biosynthesis, the problems of rotting and quality decline in apple fruit during storage were solved, achieving precise regulation of fruit ripening and preservation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENYANG AGRI UNIV
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-16
AI Technical Summary
Apples are prone to rotting, softening, and flavor deterioration during storage, resulting in losses of up to 20%. Current technologies lack effective molecular mechanisms to delay fruit ripening.
By overexpressing circular RNA (circCals) in apple fruit, ethylene biosynthesis was inhibited and fruit ripening was delayed. The circCals overexpression vector was introduced using Agrobacterium-mediated infection to promote efficient transcription and circularization of circular RNA in the fruit.
It significantly reduces ethylene production, delays fruit ripening, extends fruit storage period, and improves fruit quality, providing a new method for fruit preservation.
Smart Images
Figure CN122214342A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to an apple circular RNA and its application in inhibiting ethylene biosynthesis and thus delaying fruit ripening. Background Technology
[0002] my country ranks first in the world in both apple cultivation area and yield, playing a pivotal role in the country's agricultural and rural development. However, due to the long storage period from harvest to consumer purchase, fruit suffers from quality degradation such as rotting, softening, and flavor deterioration, resulting in annual apple losses of up to 20% and causing significant economic losses. Ethylene is a key hormone regulating fruit ripening and affecting its storability; therefore, exploring the molecular mechanisms of ethylene biosynthesis is crucial for solving the postharvest storage problem.
[0003] Circular RNAs (RRNAs), widely found in plants and animals and playing a crucial role in regulating their life activities, growth, and development, are also involved in the regulation of fruit development. Sequencing results indicate that RRNAs exhibit expression specificity at different fruit developmental stages, significantly impacting plant hormone signal transduction and fruit coloring and ripening. However, reliable evidence regarding the regulatory mechanism of RRNAs during fruit ripening remains lacking. In-depth exploration of the molecular regulatory mechanism of RRNAs on ethylene biosynthesis in apples could provide new directions for the development of fruit preservatives and antiseptics, not only having significant industrial implications for extending apple shelf life and improving fruit quality but also laying a theoretical foundation for the research and application of plant RRNAs. Summary of the Invention
[0004] One of the objectives of this invention is to provide an apple circular RNA that regulates the ripening of apple fruits.
[0005] The second objective of this invention is to provide the application of the above-mentioned apple circular RNA in inhibiting ethylene biosynthesis.
[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A type of apple circular RNA, named circCals Its nucleotide sequence is shown in SEQ ID NO.1. circCals Its nucleotide sequence consists of 567 bases.
[0007] The primary objective of this invention is to provide the above-mentioned circCals It plays a role in regulating fruit ripening. This mainly involves apple circular RNA (…). circCals Its application in inhibiting ethylene biosynthesis and thus delaying fruit ripening.
[0008] Studies have found that treatment with exogenous or endogenous ethylene can promote… circCals The expression level increased, and the stated circCals Overexpression reduces ethylene synthesis and slows fruit ripening. This is according to the description. circCals It can regulate the synthesis of ethylene in apple fruits. In practical applications, it can be used to overexpress ethylene in apple fruits. circCals This is to delay the ripening of the fruit.
[0009] Additionally, containing the above circCals Gene expression cassettes, recombinant vectors, recombinant microorganisms, and transgenic plants can also inhibit ethylene biosynthesis and delay fruit ripening. Therefore, their functions in inhibiting ethylene biosynthesis and delaying fruit ripening are also within the scope of protection of this invention.
[0010] The above research shows that, circCals It can inhibit the ripening of apples. In practice, this can be achieved through genetic modification. circCals The transfer to other recipient plants is not particularly limited in its suitability for this invention. It includes not only apples, especially 'Golden Delicious' apples, but also other plants with high homology, as long as they are suitable for gene transformation, such as various crops, flowering plants, or forestry plants. The plants mentioned can be, for example (but not limited to): dicotyledons, monocotyledons, woody plants, Rosales plants, Rosaceae plants, Prunus genus, Prunus, Brassicaceae plants, Arabidopsis genus, Arabidopsis, etc.
[0011] This invention measures the rate of fruit ripening by measuring ethylene production, which refers to the amount of ethylene released by an apple fruit in 1 hour, and is detected by gas chromatography.
[0012] The overexpression method used in this invention circCals The method is to build circCals An overexpression vector was used, and then Agrobacterium-mediated infection was employed to directly inject Agrobacterium EHA105 into apple fruits. More specifically, an overexpression vector was constructed by inserting the full-length sequence of apple circular RNA and its upstream and downstream flanking intron regulatory sequences into a eukaryotic expression vector as a backbone. This overexpression vector drove the efficient transcription and circularization maturation of circular RNA in apple cells.
[0013] In addition, the present invention also protects a method for inhibiting ethylene biosynthesis in apples or delaying the ripening of apple fruits, the method being to promote the expression of circular RNA in apple fruits.
[0014] The methods for promoting the expression of circular RNA in the target plant are selected from the following: Method (1) involves introducing circular RNA into the target plant; Method (2) involves introducing strong promoters and / or enhancers; Method (3) includes other common methods in the field, such as small RNA regulation, methylation / demethylation, phosphorylation / dephosphorylation, and promoter binding site regulation.
[0015] Therefore, this invention also protects a method for preserving apple fruit, which involves overexpressing apple circular RNA in the post-harvest stage to inhibit ethylene synthesis in the fruit, delay fruit ripening, and thus extend the storage and preservation period. Since apples begin to release ethylene in large quantities 15 days after harvest, in practice, the overexpression of circular RNA is generally performed immediately after harvesting, and no later than 10 days post-harvest.
[0016] The term "plant" as used in this invention includes the whole plant, its parent and offspring plants, and different parts of the plant, including seeds, fruits, stems, buds, leaves, roots, flowers, tissues, and organs, all of which contain our target gene or nucleic acid. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, and pollen; similarly, each of these objects contains the target gene / nucleic acid.
[0017] This invention includes any plant cell, or any plant obtained or obtainable by the methods described herein, as well as all plant parts and their propagules. This patent also includes transfected cells, tissues, organs, or whole plants obtained by any of the foregoing methods. The only requirement is that the offspring exhibit the same genotype or phenotypic characteristics, and that offspring obtained using the methods of this patent have identical characteristics.
[0018] The invention also extends to the harvestable parts of the plant as described above, but is not limited to seeds, fruits, and peels. Furthermore, it relates to other derivatives of the plant after harvest, such as organic acids, sugars, lycopene, tomatine, volatile substances, vitamins, minerals, and proteins.
[0019] In this invention, there are no particular limitations on the plants or target plants suitable for gene transformation, such as various crops, flowering plants, or forestry plants. The plants may be (but are not limited to): dicotyledons, monocotyledons, or gymnosperms.
[0020] The beneficial effects of this invention are as follows: This invention identified a novel circular RNA associated with apple fruit ripening through analysis, named... circCals This is the first confirmation of Apple's stance. circCals It regulates the function of ethylene biosynthesis during the delay of apple fruit ripening. Compared with control apples, overexpression... circCals The gene reduces ethylene production in apple fruit. This invention demonstrates... circCalsIt is a negative regulator of ethylene synthesis in apple fruit, providing new insights into molecular research on delaying apple fruit ripening. Attached Figure Description
[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0022] Figure 1 for circCals Identification of ring structures. Figure 1 a: circCals and MdCals Agarose gel electrophoresis identification image. The marker is the D2000 molecular weight standard, and the samples are apple fruit cDNA and genomic DNA (gDNA). circCals Specific bands were amplified only in cDNA samples, and no amplified bands were observed in gDNA samples. MdCals Specific bands were amplified in both cDNA and gDNA samples, indicating that... circCals It is a circular RNA. Figure 1 b: RNase R treatment circCals and MdCals Bar chart showing the effect of qPCR on expression levels. Mock represents the untreated control group, and RNase R represents the RNase R enzyme-treated group; there was no significant difference in circCals expression levels between the two groups, while... MdCals The expression level was significantly reduced after RNase R treatment (**P<0.01), demonstrating that... circCals It has RNase R resistance unique to circular RNA (linear RNA is easily degraded by RNase R, while circular RNA is resistant to degradation).
[0023] Figure 2 To study the postharvest storage phenotypes of apple fruits under different treatments and circCals Relative expression level analysis. Figure 2 a: Phenotypic images of apple fruits after 0, 5, 10, 15 and 20 days of postharvest storage under different treatments (CK control group, Ethylene treatment group, 1-MCP treatment group), Scale bar=1 cm; Figure 2 b: Postharvest storage of apples under different treatments circCals Bar chart of qPCR detection of relative expression levels (**P<0.01).
[0024] Figure 3 For overexpression circCals Apple fruit phenotype and circCals The level of expression. Figure 3 a is overexpression circCalsAnd a phenotypic diagram of the apple fruit for comparison, scale bar = 1 cm; Figure 3 b is under the same conditions, overexpression circCals and control apple fruits circCals Analysis of expression levels.
[0025] Figure 4 For overexpression circCals The effect on ethylene biosynthesis in apple fruit. Detailed Implementation
[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. 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.
[0027] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.
[0029] Unless otherwise stated, the present invention will be practiced using conventional botanical techniques, tissue culture, molecular biology, chemistry, biochemistry, and bioinformatics techniques that are readily apparent to those skilled in the art. These techniques are fully explained in the published literature and can be implemented using methods already disclosed in the existing literature.
[0030] An 'expression vector' is a vector that adds expression elements (such as promoters, RBS, terminators, etc.) to the basic skeleton of a cloning vector, enabling the target gene to be expressed.
[0031] Example 1: Apple circular RNA circCals Identification of ring structures A circular RNA responsive to ethylene biosynthesis was screened from delinearized circular RNA sequencing results of 'Golden Delicious' apple fruits at 0 and 15 days post-harvest and named [name missing]. circCals The 'Golden Delicious' apples harvested 15 days post-harvest are those that have undergone the climacteric respiration phase.
[0032] right circCals The specific method for identifying the ring structure is as follows: (1) Based on the molecular characteristics of the circRNA backsplicing site, divergent amplification primers and convergent cloning primers were designed, and PCR amplification was performed using fruit cDNA and gDNA as templates. The amplification products were analyzed by agarose gel electrophoresis. The gel electrophoresis results showed that ( Figure 1 a), circCals The divergent primers amplified a clear target band only in the cDNA template, with no visible amplified band in the gDNA template; while the control polymeric primers amplified the target band of the expected size in both cDNA and gDNA templates. circCals Evidence 1: The ring-shaped structure.
[0033] Diverging primers: circCals-F: 5'-ATGTTCCCATTCATTCGCCG-3'; circCals-R: 5'-TAAGTCCCCCGACCCATTCC-3'.
[0034] Polymer primers: Md Cals-F: 5'-CATTTCGCCGTCTTGGAGAG -3'; Md Cals-F: 5'-CAAACTTGCGAGAGAACGTGG -3'.
[0035] (2) Based on the fact that circular RNA has stronger nuclease stability than linear RNA. circCals and its homologous linear transcripts MdCals Total RNA was extracted from apple fruits by RNase R digestion. An appropriate amount of total RNA was mixed with 2U RNase R (Biovision) and incubated at 37 ℃ for 30 min. After incubation, the reaction products were purified and recovered using an RNA purification kit (DP412; Tiangen Biotech). The results were compared using quantitative real-time PCR. circCals and MdCals Level of expression, results as follows Figure 1 b, after digestion with RNase R MdCals The expression level of decreased significantly, while circCals The expression level did not change significantly, indicating that... circCals Its ability to resist RNase R degradation further confirms that it possesses a cyclic structure. circCals Evidence 2: the ring structure.
[0036] in circCals The sequence is shown in SEQ ID NO.1, and the nucleotide sequence length is 567 bp.
[0037] SEQ ID NO.1: .
[0038] Example 2 Apple circular RNA ( circCals Expression level analysis The phenotypes of apple fruits treated with 1-MCP, ethephon, and the control at 0, 5, 10, 15, and 20 days post-harvest are as follows: Figure 2 As shown in figure a, RNA was extracted from 'Golden Delicious' apple fruits, and cDNA was obtained by reverse transcription. Using cDNA as a template, specific expression primers were designed using Primer 3.0, and the results were detected by qRT-PCR. circCals Expression levels in 1-MCP, ethephon-treated, and control apple fruits. Experimental results are as follows: Figure 2 As shown in b: After 1-MCP treatment circCals Expression was significantly lower than the control after ethephon treatment. circCals The expression was significantly higher than that of the control.
[0039] The 1-MCP treatment involves placing harvested apples in a sealed container and fumigating them with 1-methylcyclopropene (1-MCP) gas at a concentration of 1 μL·L⁻¹ for 12 hours at room temperature (24°C). After treatment, the apples are stored normally.
[0040] The ethephon treatment involves immersing apples in a 0.1% ethephon solution for 15 seconds, then removing them and placing them in a sealed container at room temperature (24°C) for 24 hours to complete the ethylene-induced treatment.
[0041] Example 3 circCals Functional verification (1) circCals Construction of overexpression vectors Synthesis includes parts circCals Upstream and downstream flanking intron sequences and circCals Full-length sequence as overexpression circCals The target gene sequence, the overexpression circCals The target gene sequence is shown in SEQ ID NO.2:
[0042] Two primers, circCals-OE-F and circCals-OE-R, were synthesized and used for PCR amplification with the double-stranded DNA molecule of SEQ ID NO.2 as a template. The PCR amplification products were then recovered.
[0043] circCals-OE-F: 5'-GACCCGGGGGGTACCGGATCCGTAATTTTTATTTTTCACCTCTCCCA-3'; circCals-OE-R: 5'-AGAGTTGTTGATTCAGAATTCCAACATCATAAGTTTATTATGCATTTTTC-3'.
[0044] The pRI101 (TaKaRa) vector was double-digested with restriction endonucleases BamHI and SalI. The digestion products were recovered and ligated with the PCR amplification products from the previous step to obtain the desired results. circCals -OE recombinant vectors were sequenced and identified.
[0045] (2) Obtain circCals Overexpression of apple fruit Take (1) constructed circCals The overexpression vector was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium, which was then inoculated into the infection solution. Apple fruits harvested 10 days before commercial harvest were selected. The peel was gently punctured with a 1 ml sterile syringe, and the infection solution was slowly injected. The empty vector pRI101 was used as a negative control (empty vector control group). Fruits were harvested 10 days after infection and treated in a light incubator for 15 days. The phenotypes are shown below. Figure 3 a, and ethylene production was measured and samples were taken for analysis every 5 days.
[0046] RNA was extracted from the overexpressed and control apple fruits in the previous step, and then quantified after reverse transcription to identify RNA levels. circCals Quantitative detection was performed using specific expression primers circCals-F and circCals-R. circCals The results of the identification of overexpression of apple fruit (circCals-OE) at the RNA level are shown in the figure. Figure 3 b, its expression level was significantly higher than that of the empty control group (pRI101).
[0047] (3) Analysis of overexpression circCals The Influence of Genes on Ethylene Synthesis in Apple Fruit The ethylene production of apple fruits was measured every 5 days during step (2). Figure 4 As shown, overexpression circCals The ethylene production of apple fruits was significantly lower than that of the unloaded control group.
[0048] In summary, the apple described in this invention circCals It can negatively regulate the biosynthesis of ethylene in apples, delay fruit ripening, and extend the shelf life of fruits. It provides a new molecular target and technical path for the research and development of precise regulation of fruit ripening and green preservation technology, and has broad application prospects in plant molecular breeding.
[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. An apple circular RNA, characterized in that, The nucleotide sequence of the apple circular RNA is shown in SEQ ID NO.
1.
2. The application of the apple circular RNA according to claim 1 in inhibiting ethylene biosynthesis in apple fruit.
3. The application according to claim 2, characterized in that, By overexpressing circular RNA in apple fruit, the circular RNA negatively regulates ethylene biosynthesis, inhibiting the production of endogenous ethylene in apple fruit and thus delaying fruit ripening and senescence.
4. The application according to claim 3, characterized in that, The circular RNA overexpression vector is constructed by inserting the full-length sequence of apple circular RNA and its upstream and downstream flanking intron regulatory sequences into a plant eukaryotic expression vector as a backbone. The overexpression vector drives the efficient transcription of circular RNA in apple cells and mediates its circularization to form mature circular RNA.
5. A method for inhibiting the biosynthesis of ethylene in apples, characterized in that, The method involves overexpressing apple circular RNA in apple fruit, the nucleotide sequence of which is shown in SEQ ID NO.
1.
6. A method for delaying the ripening of apple fruits, characterized in that, The method involves overexpressing apple circular RNA in apple fruit, the nucleotide sequence of which is shown in SEQ ID NO.
1.
7. A method for preserving apples, characterized in that, Overexpression of apple circular RNA during the postharvest stage of apple production inhibits ethylene synthesis in the fruit, delays fruit ripening, and extends the fruit's storage and shelf life. The nucleotide sequence of the apple circular RNA is shown in SEQ ID NO.
1.
8. The method for preserving apple fruit according to claim 7, characterized in that, Overexpression of apple circular RNA within 10 days post-harvest.