Application of AtACX2 gene in regulation and control of plant branching development

Abstract

The invention discloses application of an AtACX2 gene in regulation and control of plant branching development, and belongs to the technical field of plant genetic engineering. The invention provides application of the AtACX2 gene in any one of the following items: (1) application in regulating and controlling plant branching development; (2) application in preparation of products for regulating and controlling plant branching development; the nucleotide sequence of the AtACX2 gene is as shown in SEQ ID NO. 1. The ACX gene AtACX2 related to branch development of arabidopsis thaliana is found for the first time, experiments prove that the AtACX2 gene negatively regulates branch development of plants, and the gene has important significance in regulation of plant types and creation of new germplasm.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of AtACX2 gene in regulating plant branch development. Background Art

[0002] The branching of plants is an important factor in determining their morphological construction. It refers to the process in which the axillary meristem (AM) in the axils of plants initiates and forms axillary buds, which then grow and develop rapidly to form lateral branches. After the axillary buds are formed, they will face a critical developmental decision: whether to continue to grow and develop into branches, or to maintain a silent state in the axils of leaves. This process is affected by factors such as genetic factors, plant hormones, natural environment and nutritional conditions, among which the influence of genetic factors on plant branching development is particularly important. In recent years, people have cloned a series of important genes related to branching from Arabidopsis, rice, pea, petunia, tomato, and corn mutants, and their understanding of the regulatory mechanism of plant branching development has continued to deepen.

[0003] Acyl-CoA oxidase (ACX) is a peroxisomal flavoprotein that catalyzes β-oxidation of fatty acids in peroxisomes and is also a key enzyme in the jasmonic acid biosynthesis pathway. There are multiple ACX isozymes in plants, which differ in size and subunit composition and have substrate (short-chain, medium-chain and long-chain fatty acids) specificity. Two ACXs were first identified in maize, and their activities were found to be regulated by growth and development and metabolism and to have tissue specificity. In rice, OsACX1 and OsACX3 were found to be expressed in multiple tissues and organs except seeds, while OsACX2 was dominantly expressed in seeds. OsACX1 was expressed in response to injury and JA induction, and may be involved in JA biosynthesis. Five ACXs were found in Arabidopsis, which catalyze the degradation of fatty acids of different carbon chain lengths. Among them, AtACX1 and AtACX5 genes are involved in jasmonic acid synthesis, highly expressed in stamens and pollen, and are related to anther development, pollen fertility and stress response. There are no reports on the role of ACX in regulating plant branching development. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the AtACX2 gene in regulating plant branch development to solve the problems existing in the above-mentioned prior art. The AtACX2 gene negatively regulates plant branch development and is of great significance in regulating plant type and creating new germplasm.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of the AtACX2 gene in any of the following:

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

[0008] (2) Application in the preparation of products for regulating plant branch development;

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

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

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

[0012] (2) Application in the preparation of products for regulating plant branch development;

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

[0014] The present invention also provides the use of a recombinant vector containing the AtACX2 gene in any of the following:

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

[0016] (2) Application in the preparation of products for regulating plant branch 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 branch development;

[0020] (2) Application in the preparation of products for regulating plant branch development;

[0021] The recombinant vector is a vector into which the AtACX2 gene is integrated 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 comprises Arabidopsis thaliana.

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

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

[0026] (2) reducing the number of branches of the plant 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 the 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 branch number, comprising the steps of overexpressing the AtACX2 gene in the plant, increasing the expression level of the AtACX2 gene, and obtaining the plants with low branch number;

[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] The present invention first discovered the ACX gene AtACX2 related to the branch development of Arabidopsis thaliana. Experimental verification showed that compared with wild-type Arabidopsis thaliana plants, the number of branches of Arabidopsis thaliana acx2 mutants increased significantly. After overexpressing the Arabidopsis thaliana AtACX2 gene in the Arabidopsis thaliana acx2 mutant, the number of branches decreased significantly, indicating that the AtACX2 gene negatively regulates the branch development of plants. The AtACX2 gene is of great significance in regulating plant type and creating new germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The plant phenotypes of wild-type Arabidopsis, acx2 mutant and AtACX2 overexpression mutant. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1 Gene cloning of AtACX2

[0042] 1. RNA extraction

[0043] Arabidopsis stem tissue was taken, ground into powder with liquid nitrogen, and RNA was extracted using the RN38EASY spin plus plant RNA rapid extraction kit. The first-chain cDNA was synthesized according to the instructions of the Goldenstar RT6cDNA Synthesis Kit.

[0044] According to the CDS sequence of AtACX2 obtained by sequencing the Arabidopsis genome, 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 (10 μM) 2μL Primer R (10 μM) 2μL cDNA template 1μL <![CDATA[ddH2O]]> 20μL Total 50μL

[0047] Reaction procedure: 98℃ pre-denaturation for 2min; 98℃ denaturation for 10s, 60℃ annealing for 15s, 72℃ extension for 30s, 35 cycles in total; 72℃ extension for 5min. The PCR product was run on agarose gel and the target fragment was recovered using a DNA gel recovery kit.

[0048] The pClone007Blunt Vector Kit was used to construct the cloning vector, 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 min.

[0052] Take 5 μL of the ligation product and add it to 50 μL of E. coli DH5α competent cells in an ice-water mixture. Flick gently to mix and place on ice for 30 minutes. Heat shock at 42°C for 60 seconds. Let stand on ice for 2-3 minutes. Add 700 μL of LB liquid medium without antibiotics. Shake and culture at 200 rpm and 37°C for 1 hour. Centrifuge at 3,000 rpm for 1 minute. Discard 600 μL of supernatant, suspend the cells and apply an appropriate amount of the culture solution on a plate containing 50 mg·L -1 Amp was grown on LB solid medium at 37°C overnight until a single colony was grown, and 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 AtACX2 gene in the Arabidopsis mutant acx2

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

[0059] Construct the AtACX2-pCambia1300S overexpression vector, add the Agrobacterium culture containing AtACX2-pCambia1300S into 50 mL LB medium, and culture until OD600 When the value was 1.0, the cells were collected and suspended with a suspension (150 μmol·L -1 , MgCl2 10mM, MES10mM) to resuspend the bacterial solution to OD 600 The concentration of SilwetL-77 was 0.6-0.8, and 0.03% of SilwetL-77 was added to infect the inflorescence of the Arabidopsis mutant acx2.

[0060] Select Arabidopsis acx2 mutants that are growing robustly and in full bloom. Remove the fruit pods before infection and completely immerse the inflorescence in the heavy suspension containing Agrobacterium. After infection for 1 minute, continue to culture in the artificial climate chamber. Generally, infection is required 2-3 times, with an interval of 2-3 days each time. When Arabidopsis seeds mature, collect the seeds and place them in a 37℃ incubator to dry for 3-7 days. Place them in a 4℃ refrigerator for about 2 days before sowing, and finally place them in 35mg·L -1 Positive seedlings were initially screened in a hygromycin resistance medium, and when the seedlings grew 4 true leaves, they were moved to nutrient soil for further cultivation. DNA and RNA of the initially screened positive seedlings were extracted for verification, and positive seedlings that met the requirements were further screened. After harvesting the seeds of the T1 generation positive seedlings, the above method was continued to be screened until the T3 generation for subsequent experiments.

[0061] like Figure 1 As shown, the number of branches of the Arabidopsis mutant acx2 was significantly greater than that of the wild type. When the Arabidopsis AtACX2 gene was overexpressed (AtACX2OE) into the Arabidopsis mutant acx2, it was found that the number of branches of the mutant was significantly reduced. This result indicates that the AtACX2 gene negatively regulates the branching development of plants.

[0062] The invention finds that AtACX2 negatively regulates the branching development of plants and can be applied to the regulation and molecular breeding of the branching development of Arabidopsis and other plants.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of AtACX2 gene in any of the following: (1) Application in regulating plant branch development; (2) Application in the preparation of products for regulating plant branch development; The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.

1.

2. Use of the protein encoded by the AtACX2 gene in any of the following: (1) Application in regulating plant branch development; (2) Application in the preparation of products for regulating plant branch development; The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Use of a recombinant vector containing the AtACX2 gene in any of the following: (1) Application in regulating plant branch development; (2) Application in the preparation of products for regulating plant branch development; The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.

1.

4. Use of host bacteria containing recombinant vectors in any of the following: (1) Application in regulating plant branch development; (2) Application in the preparation of products for regulating plant branch development; The recombinant vector is a vector into which the AtACX2 gene is integrated into the genome, and the nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.

1.

5. The use according to any one of claims 1 to 4, characterized in that: The regulation is negative regulation.

6. The use according to any one of claims 1 to 4, characterized in that: The plants include Arabidopsis thaliana.

7. A method for regulating plant branch development, characterized in that: This includes any of the following methods: (1) Increasing the number of branches of a plant by knocking out the AtACX2 gene in the plant; (2) reducing the number of branches of the plant 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 includes Arabidopsis thaliana.

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

9. A method for cultivating plants with low branch number, characterized in that: The method comprises the steps of overexpressing the AtACX2 gene in the plant, increasing the expression level of the AtACX2 gene, and obtaining a plant with a low number of branches; The nucleotide sequence of the AtACX2 gene is shown in SEQ ID NO.1, and the plant includes Arabidopsis thaliana.

Images