Application of AtACX2 gene in regulating plant branching development

Abstract

This invention discloses the application of the AtACX2 gene in regulating plant branching development, belonging to the field of plant genetic engineering technology. This invention provides the application of the AtACX2 gene in any of the following: (1) its application in regulating plant branching development; (2) its application in preparing products that regulate plant branching development; the nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1. This invention is the first to discover the ACX gene AtACX2 associated with branching development in Arabidopsis thaliana. Experimental verification shows that the AtACX2 gene negatively regulates plant branching development, and this gene is of great significance in regulating plant architecture and creating new germplasm.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the AtACX2 gene in regulating plant branching development. Background Technology

[0002] Branching is a crucial factor in determining the morphology of plants. It refers to the process by which axillary meristems (AMs) in the leaf axils initiate and form axillary buds, which then rapidly grow and develop into lateral branches. After axillary bud formation, a critical developmental decision arises: whether to continue growing and developing into branches or to remain dormant in the leaf axils. This process is influenced by factors such as genetic factors, plant hormones, natural environment, and nutritional conditions, with genetic factors having a particularly significant impact on plant branching development. In recent years, researchers have cloned a series of important genes related to branching from mutants of Arabidopsis thaliana, rice, peas, petunias, tomatoes, and maize, deepening our understanding of the regulatory mechanisms of plant branching development.

[0003] Acyl-CoA oxidase (ACX) is a peroxisome flavonoid protein that catalyzes the β-oxidation of fatty acids in peroxisomes and is a key enzyme in the jasmonic acid synthesis pathway. Multiple ACX isoenzymes exist in plants, differing in size and subunit composition, and exhibiting substrate (short-chain, medium-chain, and long-chain fatty acid) specificity. Two ACXs were first identified in maize, and their activity was found to be regulated by growth, development, and metabolism, exhibiting tissue specificity. In rice, OsACX1 and OsACX3 were found to be expressed in various tissues and organs except seeds, while OsACX2 was predominantly expressed in seeds. OsACX1 expression was induced by injury and jasmonic acid (JA), suggesting its potential involvement in JA biosynthesis. In Arabidopsis thaliana, five ACXs were identified, catalyzing the degradation of fatty acids of different carbon chain lengths. AtACX1 and AtACX5 genes are involved in jasmonic acid synthesis, showing high expression in stamens and pollen, and are associated with anther development, pollen fertility, and stress response. No research has been reported to date on the role of ACX in regulating plant branching development. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the AtACX2 gene in regulating plant branching development, so as to solve the problems existing in the prior art. The AtACX2 gene negatively regulates the branching development of plants, and this gene is of great significance in regulating plant architecture and creating new germplasm.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of the AtACX2 gene in any of the following:

[0007] (1) Application in regulating plant branching development;

[0008] (2) Application in the preparation of products that regulate plant branching development;

[0009] The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1.

[0010] This invention also provides the use of the protein encoded by the AtACX2 gene in any of the following:

[0011] (1) Application in regulating plant branching development;

[0012] (2) Application in the preparation of products that regulate plant branching development;

[0013] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0014] This invention also provides the use of recombinant vectors containing the AtACX2 gene in any of the following:

[0015] (1) Application in regulating plant branching development;

[0016] (2) Application in the preparation of products that regulate plant branching development;

[0017] The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1.

[0018] The present invention also provides the use of a host bacterium containing a recombinant vector in any of the following:

[0019] (1) Application in regulating plant branching development;

[0020] (2) Application in the preparation of products that regulate plant branching development;

[0021] The recombinant vector is a vector that integrates the AtACX2 gene into the genome, and the nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1.

[0022] Preferably, the regulation is negative regulation.

[0023] Optionally, the plant includes Arabidopsis thaliana.

[0024] The present invention also provides a method for regulating plant branching development, comprising any one of the following methods:

[0025] (1) Increase the number of branches in the plant by knocking out the AtACX2 gene;

[0026] (2) The number of branches in the plant was reduced by overexpressing the AtACX2 gene in the plant;

[0027] The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1, and the plant includes Arabidopsis thaliana.

[0028] The present invention also provides a method for cultivating plants with a high number of branches, comprising the steps of knocking out the AtACX2 gene in the plant, reducing the expression level of the AtACX2 gene, and obtaining plants with a high number of branches.

[0029] The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1, and the plant includes Arabidopsis thaliana.

[0030] The present invention also provides a method for cultivating plants with low branching numbers, comprising the steps of overexpressing the AtACX2 gene in plants, increasing the expression level of the AtACX2 gene, and obtaining plants with low branching numbers.

[0031] The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1, and the plant includes Arabidopsis thaliana.

[0032] The present invention discloses the following technical effects:

[0033] This invention marks the first discovery of the AtACX2 gene, an ACX gene associated with branching development in Arabidopsis thaliana. Experimental results show that, compared to wild-type Arabidopsis plants, the acx2 mutant exhibits a significantly increased number of branches. Overexpression of the AtACX2 gene into the acx2 mutant significantly reduces the number of branches, indicating that the AtACX2 gene negatively regulates branching development in plants. The AtACX2 gene plays a crucial role in regulating plant architecture and creating new germplasm. Attached Figure Description

[0034] 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.

[0035] Figure 1 Phenotypes of wild-type Arabidopsis thaliana, acx2 mutant, and AtACX2 overexpression mutant. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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 readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] 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.

[0041] Example 1: Gene Cloning of AtACX2

[0042] 1. RNA extraction

[0043] Arabidopsis thaliana stem segments were collected, ground into powder using liquid nitrogen, and RNA was extracted using the RN38EASY spin plus plant RNA rapid extraction kit. First-strand cDNA synthesis was performed according to the instructions of the Goldenstar RT6 cDNA Synthesis Kit.

[0044] Based on the CDS sequence of AtACX2 obtained from Arabidopsis thaliana genome sequencing, primers were designed: Primer F: 5'-ATGGAATCGCGGCGAGAGAAG-3' (SEQ ID NO.3); Primer R: 5'-TTATACAAGAAAACAAACCTT-3' (SEQ ID NO.4). The cDNA sequence of AtACX2 was amplified using MCLAB high-fidelity enzyme.

[0045] Table 1 PCR reaction system

[0046] reagents Dosage <![CDATA[I-5 TM 2×High-Fidelity Master Mix]]> 25μL primer F (10μM) 2μL primer R (10μM) 2μL cDNA template 1μL <![CDATA[ddH2O]]> 20μL Total 50μL

[0047] Reaction program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 5 min. PCR products were agarose gel electrophoresis followed by DNA gel extraction to recover the target fragment.

[0048] Cloning vectors were constructed using the pClone007Blunt Vector Kit, and the reaction system is shown in Table 2.

[0049] Table 2 Reaction System

[0050] reagents Dosage pClone007Blunt Vector Kit 1μL PCR product recovery 40ng <![CDATA[ddH2O]]> to 10μL Total 10μL

[0051] Add the sample at room temperature, mix gently, centrifuge briefly, and let stand at 25°C for 10 minutes.

[0052] Add 5 μL of the ligation product to 50 μL of E. coli DH5α competent cells in an ice-water mixture, gently mix, and incubate on ice for 30 min; heat shock at 42℃ for 60 s; incubate on ice for 2–3 min; add 700 μL of antibiotic-free LB broth; incubate at 37℃ with shaking at 200 rpm for 1 h; centrifuge at 3,000 rpm for 1 min; discard 600 μL of supernatant, resuspend the bacterial cells, and spread an appropriate amount of the bacterial suspension onto a medium containing 50 mg·L⁻¹ of LB broth. -1 On LB solid medium of Amp, the cells were incubated upside down at 37°C overnight until a single colony grew. Positive clone detection and sequencing were performed to obtain the nucleotide sequence of AtACX2 as shown in SEQ ID NO.1 and the amino acid sequence as shown in SEQ ID NO.2.

[0053] SEQ ID NO.1:

[0054]

[0055] SEQ ID NO.2:

[0056] .

[0057] Example 2: Overexpression of the Arabidopsis thaliana AtACX2 gene in the Arabidopsis thaliana mutant acx2

[0058] Both the Arabidopsis thaliana mutant and the Colombian wild-type seeds were purchased from AraShare (http: / / www.arashare.cn). The SALK number for the Arabidopsis thaliana acx2 mutant is SALK_063490C. AtACX2 expression is reduced in the Arabidopsis thaliana acx2 mutant.

[0059] An AtACX2-pCambia1300S overexpression vector was constructed. Agrobacterium tumefaciens culture containing AtACX2-pCambia1300S was added to 50 mL of LB medium and cultured until OD500.600 When the concentration is 1.0, collect the bacterial cells and use a suspension (acetylsyleugenol 150 μmol·L⁻¹). -1 Resuspend the bacterial culture to OD using MgCl2 10mM and MES 10mM. 600 The concentration was 0.6–0.8, with the addition of 0.03% SilwetL-77, used to infect the inflorescences of the Arabidopsis mutant acx2.

[0060] Select robust Arabidopsis thaliana acx2 mutants in full bloom. Before infection, remove the pods and completely immerse the inflorescences in a suspension containing Agrobacterium. After 1 minute of infection, return them to the artificial climate chamber for further cultivation. Generally, 2-3 infections are required, with each infection spaced 2-3 days apart. When the Arabidopsis seeds mature, collect them and dry them in a 37℃ incubator for 3-7 days. Before sowing, place them in a 4℃ refrigerator for about 2 days, and finally, incubate them at 35 mg / L... -1 Positive seedlings were initially screened in hygromycin-resistant medium. Once the seedlings had four true leaves, they were transferred to nutrient soil for further cultivation. DNA and RNA were extracted from the initially screened positive seedlings for verification, and further screening was conducted to select the remaining positive seedlings that met the requirements. After harvesting seeds from the T1 generation of positive seedlings, the screening process continued until the T3 generation, after which the seeds were used for subsequent experiments.

[0061] like Figure 1 As shown, the Arabidopsis mutant acx2 has significantly more branches than the wild type. Overexpression of the Arabidopsis AtACX2 gene (AtACX2OE) into the Arabidopsis mutant acx2 resulted in a significant decrease in the number of branches. This result indicates that the AtACX2 gene negatively regulates branching development in plants.

[0062] This invention discovers that AtACX2 negatively regulates branching development in plants, and can be applied to the regulation of branching development and molecular breeding in Arabidopsis thaliana and other plants.

[0063] 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 AtACX2 gene in any of the following: (1) Application in regulating plant branching development; (2) Application in the preparation of products that regulate plant branching development; The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1; The regulation is negative regulation; the plant is Arabidopsis thaliana.

2. The application of the protein encoded by the AtACX2 gene in any of the following: (1) Application in regulating plant branching development; (2) Application in the preparation of products that regulate plant branching development; The amino acid sequence of the protein is shown in SEQ ID NO.2; The regulation is negative regulation; the plant is Arabidopsis thaliana.

3. Application of recombinant vectors containing the AtACX2 gene in any of the following: (1) Application in regulating plant branching development; (2) Application in the preparation of products that regulate plant branching development; The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1; The regulation is negative regulation; the plant is Arabidopsis thaliana.

4. The use of host bacteria containing recombinant vectors in any of the following: (1) Application in regulating plant branching development; (2) Application in the preparation of products that regulate plant branching development; The recombinant vector is a vector that integrates the AtACX2 gene, and the nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1; The regulation is negative regulation; the plant is Arabidopsis thaliana.

5. A method for regulating plant branching development, characterized in that, Including any of the following methods: (1) Increase the number of branches in the plant by knocking out the AtACX2 gene; (2) The number of branches in the plant was reduced by overexpressing the AtACX2 gene in the plant; The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.

6. A method for cultivating plants with a high number of branches, characterized in that, The method includes the steps of knocking out the AtACX2 gene in plants, reducing the expression level of the AtACX2 gene, and obtaining plants with a high number of branches. The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.

7. A method for cultivating plants with a low number of branches, characterized in that, The method includes the steps of overexpressing the AtACX2 gene in plants to increase the expression level of the AtACX2 gene and obtain plants with low branching number. The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.

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