1-aminocyclopropane-1-carboxylic acid oxidase acetylation site mutant for regulating ethylene production and preparation method and application thereof

Abstract

The application discloses a 1-amino cyclopropane-1-carboxylic acid oxidase acetylation site mutant, a preparation method and application thereof, and belongs to the technical field of plant genetic engineering and protein engineering. Three lysine acetylation modification sites on a 1-amino cyclopane-1-carboxylic acid oxidase protein are identified through acetylation omics technology, and site-directed mutants of the corresponding sites are constructed. Through in-vitro enzyme activity detection, the mutant can significantly change ethylene production capacity, wherein the relative enzyme activity of the K293Q mutant is reduced to about 5% of that of the wild type, and the mutant can be applied to fruit preservation and post-harvest life extension as an ethylene synthesis inhibition tool. The application further provides a preparation method of the mutant and application of the mutant in regulating plant ethylene metabolism.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and protein engineering technology, specifically relating to a mutant of 1-aminocyclopropane-1-carboxylic acid oxidase acetylation site for regulating ethylene production, its preparation method and application. Background Technology

[0002] Ethylene is an important gaseous hormone in plants, participating in several key processes of plant growth and development, including seed germination, fruit ripening, leaf senescence and abscission, floral organ development, and adaptive responses to biotic and abiotic stresses. The biosynthesis of ethylene in plants mainly involves two consecutive enzymatic reactions: First, S-adenosylmethionine (SAM) is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) under the catalysis of ACC synthase (ACS); second, ACC is oxidized to ethylene in the presence of molecular oxygen and ascorbic acid under the catalysis of ACC oxidase (ACO), releasing hydrogen cyanide, carbon dioxide, and water. The ACO-catalyzed reaction is the final step in the ethylene synthesis pathway and one of the key rate-limiting steps determining the final yield of ethylene.

[0003] In the field of plant genetic engineering, existing research has focused on reducing the expression level of endogenous ACO genes through methods such as gene silencing, RNAi interference, or gene editing, thereby inhibiting ethylene biosynthesis and applying these techniques to fruit preservation and extending postharvest life. For example, patent WO2014 / 143996 discloses a method for reducing endogenous ACO gene expression using a transgenic composition, which can improve agronomic traits of crops such as maize. Other patents involve exploring the biological role of ACO genes in crop salt tolerance through cloning and functional verification, providing genetic resources for plant stress-resistance breeding.

[0004] Furthermore, the regulatory mechanisms of post-translational modifications (PTMs) on enzyme activity are receiving increasing attention. Lysine acetylation, as a highly dynamic and reversible PTM, has been shown to be widely involved in many important cell biological processes. In the regulation of plant metabolic enzymes, studies have found that acetylated lysine residues located at the active site of Rubisco enzymes can directly lead to the loss of Rubisco enzyme function, revealing that acetylation plays an important role in coordinating the light reaction and carbon assimilation. Other studies have shown that the activity of ACS enzymes is closely related to the acetylation status of key lysine sites, providing a new perspective for understanding the regulatory mechanisms of upstream enzymes in the ethylene synthesis pathway.

[0005] However, existing technologies still have the following shortcomings: (I) Existing technologies focus on the regulation of expression levels, lacking research on the post-translational modification mechanism of ACO protein. Whether through gene silencing to reduce ACO expression or through molecular breeding to screen for salt- and drought-tolerant genes, current strategies remain at the level of "expression regulation" at the transcriptional and translational levels, failing to achieve precise and dynamic regulation of ethylene synthesis. Post-translational modification of proteins is a rapid, reversible, and finely regulated mechanism that can respond to environmental signals in real time at the cellular level; however, there is a lack of systematic reports, both domestically and internationally, on how the acetylation modification of lysine residues within ACO directly regulates the enzyme's activity.

[0006] (ii) Lack of systematic identification and functional verification of acetylation sites in ACO proteins. To date, there are no reports on the comprehensive identification of lysine acetylation modification sites in plant-derived ACO proteins using highly sensitive acetylomics technology. Which lysine residues can be acetylated, how the acetylation state of these sites affects the enzyme's catalytic activity, and whether different sites exhibit functional differentiation and synergistic effects are all questions that remain unanswered by the technology.

[0007] (III) Lack of acetylation site mutants capable of precisely regulating ethylene biosynthesis. Although existing methods exist to inhibit ACO expression using antisense RNA, RNAi, and other technologies, the effectiveness of these methods depends on the stability of gene expression and is difficult to finely regulate the degree of enzyme activity, exhibiting limitations such as operational complexity and uncontrollable effects. Furthermore, existing ACO mutations are mostly focused on the functional studies of conserved active site residues or substrate-binding regions; no studies have yet designed and constructed site-directed mutants that regulate enzyme activity based on acetylation modification regulatory mechanisms.

[0008] (iv) Existing technologies for regulating ACO enzymes are mainly oriented towards “reducing ethylene production”, primarily serving applications such as fruit preservation and extending postharvest life. However, the regulation methods are relatively crude, lacking engineered enzyme mutant tools that can efficiently and precisely inhibit ethylene synthesis. Summary of the Invention

[0009] Therefore, the present invention aims to provide a set of 1-aminocyclopropane-1-carboxylic acid oxidase acetylation site mutants capable of precisely controlling ethylene production, their preparation methods, and applications.

[0010] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a site mutant of acetylation of 1-aminocyclopropane-1-carboxylic acid oxidase, wherein the mutant is formed by mutating one or more lysine residues at positions 167, 268, or 293 in the amino acid sequence of wild-type 1-aminocyclopropane-1-carboxylic acid oxidase to glutamine.

[0011] Preferably, the nucleotide sequence of the primer pair at position 167 used for site-directed mutagenesis is shown in SEQ ID NO:1 and SEQ ID NO:2; The nucleotide sequences of the primer pair at position 268 used for site-directed mutagenesis are shown in SEQ ID NO:3 and SEQ ID NO:4. The nucleotide sequences of the primer pair at position 293 used for site-directed mutagenesis are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0012] The present invention also provides a method for preparing the above-mentioned mutant, comprising the following steps: (1) Identify lysine acetylation modification sites on the 1-aminocyclopropane-1-carboxylic acid oxidase protein using acetylogomics technology; (2) Using overlapping extension polymerase chain reaction or site-directed mutagenesis based on whole plasmid polymerase chain reaction, construct mutant coding sequences in which the lysine codon at the target site is replaced by the glutamine codon; (3) The mutant coding sequence was cloned into a prokaryotic expression vector and transformed into prokaryotic host cells for induced expression; (4) Purify to obtain recombinant mutant protein.

[0013] Preferably, the prokaryotic expression vector in step (3) is pGEX-6p-1, and the prokaryotic host cell is Escherichia coli BL21.

[0014] This invention also provides the application of the above-mentioned site mutants in the preparation of ethylene generation inhibitors.

[0015] This invention also provides the application of the above-mentioned site mutants in the regulation of plant fruit ripening.

[0016] It contains at least the following beneficial technical effects: This invention is the first to systematically identify three key lysine acetylation modification sites (K167, K268, and K293) on the ACO protein using optimized acetylation omics sequencing technology, filling a technical gap in the acetylation modification site mapping of this protein.

[0017] This invention constructs a specific mutant at the above-mentioned site using site-directed mutagenesis technology. In vitro enzyme activity assays confirm that the ethylene production capacity of the K293Q mutant is significantly reduced, with relative enzyme activity only about 5% of that of the wild type, indicating that acetylation modification at this site plays a decisive activating role in ACO enzyme activity.

[0018] The K293Q mutant provided by this invention can be used as a highly efficient tool for inhibiting ethylene synthesis, applied to fruit preservation and extending postharvest life. Compared with traditional gene silencing technology, it has the advantages of being faster and more controllable.

[0019] This invention establishes a complete technical route from "acetylation site identification → site-directed mutagenesis → prokaryotic expression and purification → in vitro functional verification", and has the capability to transform laboratory discovery into industrial application. Attached Figure Description

[0020] Figure 1 This provides an overview of proteomics and acetylation analysis during the ripening process of tomato fruit.

[0021] Figure 2 This is a schematic diagram of the three-dimensional structure of the SlACO1 protein, with the spatial locations of the three acetylation modification sites, K167, K268, and K293, marked.

[0022] Figure 3 The results of SDS-PAGE electrophoresis identification of the purified protein of the mutant at the key acetylation site of ACO protein.

[0023] Figure 4 The amount of ethylene released in vitro by reacting with ACC after any one or more of the three acetylation modification sites (K167, K268, and K293) are mutated to glutamine.

[0024] Figure 5 The results show the in vitro enzyme activity assays for ACO wild-type and various mutants. Detailed Implementation

[0025] 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.

[0026] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0027] 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.

[0028] 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 application specification and embodiments are merely exemplary.

[0029] 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.

[0030] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0031] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0032] Example 1: Identification of ACO protein acetylation modification sites High-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS / MS) combined with an optimized acetylated peptide enrichment strategy was used to analyze the acetylation modification spectra of purified wild-type ACO protein (derived from tomato, UniProt ID P05116). Lysine acetylation modification sites on the ACO protein were precisely identified through mass spectrometry database searches and comparisons, and confidence screening of modification sites.

[0033] This embodiment identified three lysine acetylation modification sites, corresponding to lysine positions 167 (K167), 268 (K268), and 293 (K293) in the wild-type ACO amino acid sequence shown in the P05116 protein. These three sites are distributed in different functional regions within the tertiary structure of the ACO protein, suggesting that they may participate in the regulation of enzyme activity through different mechanisms. Figure 1 , Figure 2 ).

[0034] 2. Construction of site-directed mutants The coding sequences of single-site mutants K167Q, K268Q, and K293Q, as well as the three-site combination mutant K167Q / K268Q / K293Q, were constructed using overlap extension polymerase chain reaction (ELISA) or site-directed mutagenesis based on whole-plasmid PCR. The mutagenesis scheme involved replacing the lysine codon (AAA or AAG) at the target site with the glutamine codon (GAA). Glutamine is electroneutrally neutral and has a spatial structure similar to acetyl groups, which can approximate the physicochemical properties of acetylated lysine, making the observed functional changes primarily attributable to the alteration of the acetylation modification state.

[0035] The primer sequences designed for site-directed mutagenesis are as follows: SlACO1-K167Q-F: 5'-CCATGTCCTGAACCCGATTTG-3' (SEQ ID NO: 1) SlACO1- K167Q-R: 5'-CAAATCGGGTTCAGGACATGG-3' (SEQ ID NO: 2) SlACO1-K268Q-F: 5'-TTGGTTGAAGAAGAGGCAGAG-3' (SEQ ID NO: 3) SlACO1- K268Q-R: 5'-CTCTGCCTCTTCTTCAACCAA-3' (SEQ ID NO:4) SlACO1-K293Q-F: 5'-GCTGGACTCGAATTTCAAGCC-3' (SEQ ID NO:5) SlACO1- K293Q-R: 5'-GGCTTGAAATTCGAGTCCAGC-3' (SEQ ID NO: 6) The PCR amplification conditions are as follows: The purified PCR product and the prokaryotic expression vector pGEX-6p-1 were used separately. XmaI Restriction endonuclease digestion, ligation and transformation into DH5α competent cells, screening for positive clones and sequencing to verify the correct mutation site.

[0036] 3. Prokaryotic expression and purification of recombinant proteins a. Transform the correctly sequenced recombinant expression vector into *E. coli* BL21(DE3) competent cells. Pick single colonies and inoculate them into LB broth containing ampicillin (50 μg / mL), incubating overnight at 37°C with shaking. The next day, transfer the colonies 1:100 to fresh LB broth and incubate at 37°C with shaking until the bacterial culture reaches OD. 600When the concentration was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and expression was induced by shaking at 16℃ for 8 h. b. Collect bacterial cells by centrifugation, resuspend in pre-cooled lysis buffer (1 × PBS, pH 6.8), and sonicate (200W, 3 seconds sonication, 5-second interval, total time 10-15 minutes). Centrifuge the product at 4°C and 12000 rpm for 20 minutes, collecting the supernatant and precipitate separately. After confirming the presence of a large amount of soluble recombinant protein in the supernatant by SDS-PAGE gel electrophoresis, proceed with purification. c. The mutant protein carrying the GT tag was purified using a GST affinity chromatography column. The target protein was then eluted from the column with elution buffer. The purity and molecular weight of the target protein were determined by SDS-PAGE electrophoresis. Figure 3 Protein concentration was determined using the BCA method, and the samples were aliquoted and stored at -80℃ for later use.

[0037] 4. Detection of in vitro enzyme activity The yield of ethylene from ACC substrate catalyzed by the ACO mutant was determined using gas chromatography (GC). The specific steps are as follows: a. The enzyme activation reaction system (total volume 3 mL) includes: 1 × PBS buffer (pH 6.8), 1 mM ACC substrate, 100 μM FeSO4, 30 mM ascorbic acid, 100 μM NaHCO3, and an equal concentration of purified ACO protein sample. Add the reaction mixture (excluding the protein) to the headspace vial, immediately seal with a rubber stopper, then inject the protein sample using a syringe. Immediately seal the needle hole with plastic wrap to prevent leakage of generated ethylene. b. Incubate the sealed reaction system at 28°C with constant temperature shaking for 2 h; c. Using a gas-tight syringe, accurately draw 3 mL of headspace gas from the headspace vial and inject it into the gas chromatograph. The gas chromatographic detection conditions are as follows: use an Rt-5 capillary column or other equivalent hydrophobic capillary column, maintain the column temperature at 60°C, set the injection port temperature to 160°C, and use a flame ionization detector (FID) with a temperature set to 230°C. The retention time of the ethylene standard is confirmed using standards under the same conditions. d. Prepare standard curves using ethylene standards of different concentrations (1 ppm, 5 ppm, 10 ppm, 20 ppm, 50 ppm), and calculate the concentration of ethylene produced in the reaction based on the integrated area of ​​the sample peaks.

[0038] Test results ( Figure 4The results showed that the relative enzyme activity of the K167Q mutant was approximately 60% of that of the wild type; the relative enzyme activity of the K268Q mutant was approximately 50% of that of the wild type; the relative enzyme activity of the K293Q mutant was only about 5% of that of the wild type; and the relative enzyme activity of the three-site combination mutant was 5% lower than that of the wild type. These results indicate that acetylation modification at the K293 site plays a decisive activating role in ACO enzyme activity, and inhibition of acetylation at this site leads to almost complete loss of enzyme activity.

[0039] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of the acetylation site of 1-aminocyclopropane-1-carboxylic acid oxidase, characterized in that, The mutant is formed by mutating one or more lysine residues at positions 167, 268, or 293 in the amino acid sequence of wild-type 1-aminocyclopropane-1-carboxylic acid oxidase to glutamine.

2. The site mutant according to claim 3, characterized in that, The nucleotide sequences of the primer pair at position 167 used for site-directed mutagenesis are shown in SEQ ID NO:1 and SEQ ID NO:

2. The nucleotide sequences of the primer pair at position 268 used for site-directed mutagenesis are shown in SEQ ID NO:3 and SEQ ID NO:

4. The nucleotide sequences of the primer pair at position 293 used for site-directed mutagenesis are shown in SEQ ID NO:5 and SEQ ID NO:

6.

3. The method for preparing the mutant according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Identify lysine acetylation modification sites on the 1-aminocyclopropane-1-carboxylic acid oxidase protein using acetylogomics technology; (2) Using overlapping extension polymerase chain reaction or site-directed mutagenesis based on whole plasmid polymerase chain reaction, construct mutant coding sequences in which the lysine codon at the target site is replaced by the glutamine codon; (3) The mutant coding sequence was cloned into a prokaryotic expression vector and transformed into prokaryotic host cells for induced expression; (4) Purify to obtain recombinant mutant protein.

4. The preparation method according to claim 3, characterized in that, The prokaryotic expression vector in step (3) is pGEX-6p-1, and the prokaryotic host cell is Escherichia coli BL21.

5. The use of the site mutant according to any one of claims 1-2 in the preparation of ethylene production inhibitors.

6. The application of the site mutant according to any one of claims 1-2 in the regulation of plant fruit ripening.

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