Application of CsESE3 gene or protein coded thereby in regulating plant shoot development

Abstract

The application discloses application of CsESE3 gene or a protein coded by the same in regulation of branch development of plants and belongs to the technical field of plant genetic engineering. Traditional citrus hybrid breeding for cultivating ideal plant type plants is long in period, low in selection efficiency and few in cultivatable traits. Artificial control of branches is high in labor cost, and chemical control of branches has the risk of drug damage. The application proposes a new scheme for regulating citrus branch development based on CsESE3 gene expression, that is, the expression of CsESE3 gene is improved to significantly promote the development of branches, so that CsESE3 can become a target for regulating the development of citrus branches.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the CsESE3 gene or its encoded protein in regulating plant shoot development. Background Technology

[0002] Citrus is the world's highest-yielding fruit. As a perennial woody fruit tree, its tree structure directly affects yield, quality, and cultivation management efficiency. Currently, shoot control mainly relies on manual pruning or chemical treatment, which is not only time-consuming and labor-intensive but also poses potential environmental risks. In recent years, plant genetic engineering technologies (such as gene editing, transgenics, and marker-assisted breeding) have made significant progress in improving citrus stress resistance, fruit quality, and agronomic traits. However, research on shoot growth control remains relatively weak, and the available gene targets are limited. Therefore, it is urgent to conduct research on the mechanisms of citrus shoot development to provide a theoretical basis and genetic resources for breeding new varieties with superior plant types and developing simple and efficient shoot control techniques. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the CsESE3 gene or its encoded protein in regulating plant shoot development, thereby addressing the problems existing in the prior art. This invention provides a new gene target that can be used in hybridization breeding to cultivate ideal plant types or for the development of shoot growth regulation technologies.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] One of the technical solutions of this invention is the application of the CsESE3 gene or its encoded protein in regulating plant shoot development.

[0006] The second technical solution of the present invention is a method for regulating the development of plant branches by overexpressing the CsESE3 gene or upregulating the level of its encoded protein to increase the number of branches of the plant.

[0007] The third technical solution of this invention is the application of the CsESE3 gene in the cultivation of multi-branched citrus plant varieties.

[0008] The fourth technical solution of the present invention is a method for cultivating multi-branched citrus varieties, which involves overexpressing the CsESE3 gene using a recombinant vector containing the CsESE3 gene, an expression cassette, a transgenic cell line or recombinant bacteria.

[0009] The fifth technical solution of this invention is the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsESE3 gene in regulating plant shoot development.

[0010] The sixth technical solution of this invention is the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsESE3 gene in the cultivation of multi-branched citrus plant varieties.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] This invention proposes a new scheme for regulating citrus shoot development based on CsESE3 gene expression: increasing the expression of CsESE3 gene significantly promotes shoot development, therefore CsESE3 can be a target for regulating citrus shoot development. Attached Figure Description

[0013] 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.

[0014] Figure 1 To induce an increase in axillary bud germination in lemon plants that overexpress CsESE3, white arrows indicate new shoots emerging from axillary buds.

[0015] Figure 2 To enhance shoot development in lemon plants that overexpress CsESE3, red arrows indicate growing lateral branches. Detailed Implementation

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0022] This invention provides the application of the CsESE3 gene or its encoded protein in regulating plant shoot development.

[0023] In some specific implementations, the plant is a citrus plant.

[0024] In some specific embodiments, the nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2.

[0025] This invention also provides a method for regulating plant shoot development by overexpressing the CsESE3 gene or upregulating the level of its encoded protein to increase the number of branches in the plant.

[0026] In some specific implementations, the plant is a citrus plant.

[0027] This invention also provides the application of the CsESE3 gene in the breeding of multi-branched citrus varieties.

[0028] This invention also provides a method for cultivating multi-branched citrus varieties, which involves overexpressing the CsESE3 gene using a recombinant vector containing the CsESE3 gene, an expression cassette, a transgenic cell line, or a recombinant bacterium.

[0029] This invention also provides the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsESE3 gene in regulating plant shoot development.

[0030] This invention also provides the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the CsESE3 gene in the cultivation of multibranched citrus plant varieties.

[0031] Traditional citrus hybridization breeding suffers from long cycles, low selection efficiency, and a limited range of selectable traits. Manual shoot control is labor-intensive, while chemical shoot control carries the risk of pesticide damage. This invention addresses these problems by proposing a novel approach based on the expression regulation of the CsESE3 gene to promote citrus shoot development: increasing CsESE3 gene expression significantly promotes shoot development; therefore, CsESE3 can serve as a target for regulating citrus shoot development.

[0032] The Newhall navel oranges used in this embodiment of the invention were obtained from the germplasm resource nursery of our research group at Huazhong Agricultural University in Wuhan, Hubei Province (E 114°21′2″, N 30°28′35″).

[0033] Example 1

[0034] 1. Cloning of the Citrus CsESE3 Gene

[0035] 1.1 Material selection: Axillary buds were collected from mature branches of healthy Newhall navel orange (Citrus sinensis L. Osbeck).

[0036] 1.2 RNA extraction and cDNA synthesis: Total RNA was extracted from axillary buds using the Trizol method, and first-strand cDNA was synthesized using a reverse transcription kit.

[0037] Take fresh, mature shoots of Newhall navel oranges and grind them into powder in liquid nitrogen. Add 100 mg of the axillary shoot powder to 1 mL of TRI pure, vortex to mix, and let stand for 5 min. Centrifuge at high speed (4℃, 12000 rpm) for 10 min. Transfer the supernatant to a new 1.5 mL RNAase-free centrifuge tube and add 200 μL of pre-chilled (-20℃) chloroform. Vortex to mix for 15 s and let stand for 3 min. Centrifuge at high speed for 15 min. Transfer the supernatant to a new 1.5 mL RNAase-free centrifuge tube, add an equal volume of pre-chilled isopropanol, invert to mix, and place in a 4℃ freezer for 10 min. Transfer the solution to the adsorption column, centrifuge at high speed for 30 seconds, and discard the waste liquid; add 500 μL of pre-cooled anhydrous ethanol to the adsorption column, let stand for 1 min, centrifuge at high speed for 30 seconds, and discard the waste liquid. Repeat this step three times; add 500 μL of GT solution to the adsorption column, let stand for 1 min, centrifuge at high speed for 30 seconds, and discard the waste liquid; add 500 μL of NT solution to the adsorption column, let stand for 1 min, centrifuge at high speed for 30 seconds, and discard the waste liquid; centrifuge at high speed for 2 min; open the cap of the adsorption column and let stand for 5 min to dry the liquid; place the adsorption column into a new 1.5 ml RNAase-free centrifuge tube, add 20 μL of PEPC-Treated ddH2O to the center of the adsorption membrane, let stand for 2 min, centrifuge at high speed for 2 min; collect the liquid product (RNA), take 2 μL of the solution for electrophoresis to detect the quality of RNA, and store at -80℃. The TRI pureReagent was purchased from Kolpu Biotechnology Co., Ltd., while the adsorption column and GT / NT solution were products from Sangon Biotech's column-based plant total RNA extraction and purification kit.

[0038] Using Beijing Quanshijin Bio The One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit is used for reverse transcription. The reaction steps are as follows:

[0039] Prepare 8 μL of the mixture in RNase-free centrifuge tubes, as shown in Table 1.

[0040] Table 1

[0041]

[0042] Incubate at 65°C for 5 min in a PCR instrument; place on ice for 2 min; add 10 μL 2×TS Reaction Mix, 1 μL gDNA Remover, and 1 μL... RT / RI Enzyme Mix; incubate at 42°C for 30 min in a PCR instrument, and store the product at -20°C after the reaction is complete.

[0043] 1.3 Gene Cloning: Specific primers were designed based on the coding sequence of the sweet orange CsESE3 gene (accession number: Cs_ont_4g001780) in the CPBD database (http: / / citrus.hzau.edu.cn / ); PCR amplification was performed using cDNA as a template to obtain the target fragment.

[0044] Primers for CsESE3 amplification were designed based on the coding region sequence information of the Cs_ont_4g001780 gene in the sweet orange genome (Citrus sinensis v3.0). PCR amplification was performed using a high-fidelity enzyme from Vazyme, and the PCR reaction system is shown in Table 2.

[0045] Table 2

[0046]

[0047]

[0048] The PCR reaction procedure is shown in Table 3.

[0049] Table 3

[0050]

[0051] PCR products were subjected to electrophoresis. After confirming that the product size was correct, gel extraction was performed. The gel extraction method was based on that of OmegaBio-Tek. Gel & PCR Clean Up Kit Instruction Manual.

[0052] 1.4 Vector construction and transformation: The purified PCR product was ligated into the pTOPO vector; Escherichia coli DH5α competent cells were transformed by heat shock, and single clones were picked and propagated on antibiotic plates. Positive clones were verified by colony PCR.

[0053] Target fragment ligated to pTOPO vector

[0054] Hieff from Yisheng Biotechnology Co., Ltd. The Zero TOPO-Blunt Simple Cloning Kit was used to construct recombinant vectors. The systems shown in Table 4 were prepared in 200u centrifuge tubes.

[0055] Table 4

[0056]

[0057] After low-speed centrifugation, ligate the sample in a PCR instrument at 25-37℃ for 5 minutes.

[0058] 1.5 Sequence Validation: Positive clones are tested, and the similarity between the amplified sequence and the reference sequence in the database is analyzed by sequence alignment.

[0059] Transformation was performed using DH5α Chemically Competent Cell from Weidi Biotechnology. The specific steps were as follows: Competent E. coli were removed from -80℃ and thawed on ice; 5 μL of ligation product was added and gently mixed by tapping the bottom of the EP tube; the tube was incubated on ice for 5 minutes; a heat shock was performed at 42℃ for 1 minute; the tube was immediately returned to ice and incubated for 2 minutes; 500 μL of LB liquid medium was added to the centrifuge tube, mixed, and then incubated at 37℃, 200 rpm for 20 minutes; approximately 100 μL was plated onto AMP (100 μg / L) resistant LB solid medium; the plate was inverted and incubated overnight at 37℃. After incubation, single colonies were picked and added to 500 μL of LB liquid medium containing AMP (100 μg / L) and incubated for 5 hours; 1 μL of the bacterial culture was used to identify positive clones using M13F / R primers. Five positive clones were sent to the company for sequencing to confirm sequence accuracy.

[0060] The nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the corresponding protein amino acid sequence is shown in SEQ ID NO.2.

[0061] SEQ ID NO.1: ATGGCCAGACCACAGCAGCGATACAGGGGCGTTCGACAGAG;

[0062] SEQ ID NO.2: MARPQQRYRGVRQRHWGSWVSEIRHPLLKTRIWLGTFETAED AARAYDEAARLMCGPRARTNFPYNPNASHSSSSKLSATKLHRCYMASLQMTK SSAANEPQKAPASHVSTPSGNATRINEMGKHLLEMRPMQVQGSTEPINRAVKKEQVE IAQQFKPLEDDHIEQMIEELLDYGSLELCSVVSPQAL*.

[0063] The preparation method for LB liquid medium is as follows (1 liter): 10g tryptone, 5g yeast extract, 10g NaCl, sterilize at 121℃ for 20min; LB solid medium is prepared by adding 16-20g agar to LB liquid medium, adding antibiotics after sterilization, and the plate will solidify to form LB solid medium containing the corresponding antibiotics.

[0064] 2. Construction of pK7WG2D-CsESE3 overexpression vector and Agrobacterium-mediated transformation

[0065] 2.1 Amplification of the target fragment: Using the pTOPO-CsESE3 recombinant plasmid that has been verified by sequencing as a template, the CsESE3 fragment with a specific adapter sequence was amplified by PCR using high-fidelity DNA polymerase. After the product was verified by 1% agarose gel electrophoresis, the target fragment was purified and recovered.

[0066] Referencing Omega Bio-Tek The Plasmid DNAMini Kit I method was used to extract the correctly sequenced pTOPO-CsESE3 E. coli liquid plasmid. Using the CsESE3-pTOPO plasmid as a template, and Adapter-CsESE3-F / Adapter-CsESE3-R primers, a first round of PCR amplification was performed. The amplification system and reaction procedure are shown in Table 2-3. A second round of PCR amplification was performed using the first round amplification product as a template and Adapter-attB1 / Adapter-attB2 primers. The reaction system and procedure are shown in Table 2-3. The amplification products were separated by 1% agarose gel electrophoresis, and the target fragment was recovered.

[0067] 2.2 Ligation of the target fragment to the initiation vector: The recovered target fragment was ligated to the initiation vector pDONOR221 via a BP reaction to form the pDONOR221-CsESE3 recombinant plasmid. After transformation with E. coli and sequence confirmation, the plasmid was extracted from the positive bacterial solution for subsequent reactions.

[0068] The BP reaction system was prepared and connected in a PCR instrument at 25°C for 4 hours. The BP reaction system is shown in Table 5.

[0069] Table 5

[0070]

[0071] The ligation product was transformed into competent E. coli, and after M13F / R positive detection, it was sent for sequencing.

[0072] pDONR221 is resistant to kanamycin, and E. coli culture is performed using 100 mg / L resistant medium.

[0073] 2.3 Construction of pK7WG2D-CsESE3 recombinant vector: The CsESE3 gene sequence in the pDONOR221-CsESE3 recombinant plasmid was ligated into the pK7WG2D overexpression vector via LR reaction. After transformation with E. coli and sequence confirmation, the positive bacterial solution plasmid was extracted for subsequent reactions.

[0074] DNA was extracted from the correctly sequenced positive clone pDONOR221-CsESE3 plasmid; the LR reaction system was prepared and ligated in a PCR instrument at 25℃ for 4 hours. The LR reaction system is shown in Table 6.

[0075] Table 6

[0076]

[0077] The ligation product was transformed into competent E. coli, and after positive tests with 35S-F and CsESEE-R, it was sent for sequencing. The pK7WG2D vector was spectinomycin resistant, and the E. coli were cultured using 100 mg / L resistant medium.

[0078] 2.4 Transformation of Agrobacterium tumefaciens with recombinant vector: pK7WG2D-CsESE3 plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells. Transformants were screened under spectinomycin and rifampicin resistance and the positive results were verified by 35S-F / CsESE3-R. The positive bacterial culture was stored in a freezer at -80℃.

[0079] Referencing Omega Bio-Tek The plasmid pK7WG2D-CsESE3 *E. coli* was extracted using the Plasmid DNAMini Kit I method. The plasmid was then transformed into GV3101 competent *Agrobacterium* cells, following the instructions for use with the GV3101 Chemically Competent Cell product from Weidi Biotechnology Co., Ltd. *Agrobacterium* pK7WG2D-CsESE3 was screened in spectinomycin (100 mg / ml) and rifampin (50 mg / ml) resistant media, and PCR detection was performed using 35S-F and CsESE3-R primers. Positive *Agrobacterium* culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80°C for subsequent activation before use in plant transformation.

[0080] 3. Genetic transformation of lemons using OE-CsESE3

[0081] 3.1 Preparation of hypocotyls for lemon seedlings: Fresh lemon seeds were sterilized and then sown in test tube culture medium. The hypocotyls of the seedlings were obtained by dark culture.

[0082] Using lemon seedling epicotyls as genetic transformation material, the specific steps are as follows: Seeds were removed from fresh fruit and soaked in a 1 mol / L NaOH solution with stirring for 15 minutes to remove pectin from the seed surface. After discarding the NaOH solution, the seeds were left to soak in clean water overnight. The outer seed coat of the lemon seeds was removed, and the seeds were sterilized and sown in a clean bench. The seeds were then sterilized by soaking in 75% anhydrous ethanol solution for 60 seconds and 30% sodium hypochlorite solution (containing 5% NaClO) for 15 minutes. After discarding the NaClO solution, the seeds were rinsed 5-7 times with sterile water and dried on sterilized filter paper. They were then sown in sterilized test tube culture medium (MT + 30 g / L sucrose, pH 5.8) and cultured in the dark for approximately 25-30 days, followed by light culture for 7-10 days. The greened epicotyls were used for genetic transformation.

[0083] 3.2 Agrobacterium infection and explant culture: Agrobacterium stored at -80℃ was propagated in LB liquid medium. The CsESE3 gene was transformed into lemon explants using Agrobacterium-mediated genetic transformation. The infected explants were cultured in co-culture, SM and other media to obtain callus tissue. New plants were formed by inducing callus development, thereby obtaining transgenic plants.

[0084] 3.2.1 Propagation of Agrobacterium-positive bacteria

[0085] Take 100 μl of Agrobacterium tumefaciens stored at low temperature, inoculate it into 50 ml of sterilized LB liquid medium, add 50 μl each of spectinomycin (100 mg / ml) and rifampin (50 mg / ml), and 25 μl of acetylsuccinone (AS, 50 mg / ml), and incubate upside down in a constant temperature incubator at 28℃ for 16-20 h.

[0086] 3.3.2 Preparation, Infection, and Culture of Lemon Hypocotyl Explants

[0087] In a clean bench, seedlings that have already undergone light-induced greening treatment are placed on sterile filter paper and cut into approximately 1cm equilateral trapezoidal stem segments using a sterile blade. The stem segments are immediately placed in a 30ml Erlenmeyer flask containing suspension culture medium (MT + 30g / L sucrose + 0.5g maltose extract + 1.5g L-Glutamine) to prevent the cut ends from drying out. The prepared Agrobacterium is poured into a sterile 50ml centrifuge tube in the clean bench and centrifuged at 4000rpm / min for 10min. After discarding the supernatant, the OD of the bacterial suspension is adjusted to 0.6 with suspension culture medium. AS (concentration: 20mg / L) is added, and the bacterial suspension is poured into the Erlenmeyer flask containing the stem segments. The flask is sealed with sealing film and incubated at 28℃, 200rpm / min for 20min. After infection, the stem segments were picked out, spread out on filter paper to dry, and then placed in a co-culture medium (MT + 30 g / L sucrose + 20 mg / L AS, pH 5.8) and incubated in the dark at 21°C for 3 days. Afterward, the stem segments were transferred to SM medium (MT + 30 g / L sucrose + 0.5 mg / L KT + 1 mg / L 6-BA + 0.1 mg / L NAA + 50 mg / L Kana + 100 mg / L Tim, pH 5.8) and incubated in the dark at 28°C for 7 days, before being transferred to light for further culture.

[0088] 4. Screening and phenotypic analysis of lemon seedlings with excessive expression of CsESE3

[0089] Seedlings were initially identified as positive by observing the green fluorescence under UV light. After the positive seedlings developed stem segments, they were grafted onto lemon rootstock. DNA and RNA were extracted from the leaves, and successful OE-CsESE3 transformation was confirmed at the DNA sequence and gene expression levels, respectively. When branching appeared in the leaf axils, the shoot development characteristics of wild-type and OE-CsESE3 positive plants were compared.

[0090] After 1-2 months of selection and culture, lemon stem segments formed callus at the cut surfaces. Under hormone induction, the callus tissue differentiated into seedlings. Positive plants showed green fluorescence under UV light due to the GFP fluorescent protein carried by the pK7WG2D vector, confirming their positive status. Once the positive seedlings had developed stem segments, they were grafted onto lemon rootstocks for further growth. DNA was extracted from the plant leaves and amplified by PCR using 35S-F / CsESE3-R primers, confirming successful gene transformation. Simultaneously, RNA was extracted from the leaves, reverse transcribed to obtain cDNA, and qPCR was used to determine the overexpression fold of CsESE3.

[0091] like Figure 1 and Figure 2As shown, compared to wild-type lemons, lemon plants overexpressing CsESE3 exhibit increased branching. Therefore, the citrus CsESE3 gene cloned in this invention has the function of regulating shoot development and can serve as a gene target for hybridization breeding to cultivate ideal plant types or for the research and development of shoot growth regulation technologies.

[0092] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of the CsESE3 gene or its encoded protein in regulating plant shoot development, characterized in that, The plant in question is a lemon; The nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.

2. Overexpression of the CsESE3 gene or upregulation of its encoded protein levels increases the number of branches in the plant.

2. A method for regulating plant shoot development, characterized in that, Overexpression of the CsESE3 gene or upregulation of its encoded protein level increases the number of branches in the plant; the nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.

2. The plant in question is a lemon.

3. The application of the CsESE3 gene in the breeding of multi-branched lemon varieties, characterized in that... Overexpression of the CsESE3 gene or upregulation of its encoded protein level increases the number of branches in lemons; the nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.

2.

4. A method for cultivating multi-branched lemon varieties, characterized in that, The CsESE3 gene was overexpressed using a recombinant vector, expression cassette, or transgenic cell line containing the CsESE3 gene to increase the number of branches in lemons; the nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.

2.

5. The application of recombinant vectors, expression cassettes, or transgenic cell lines containing the CsESE3 gene in regulating plant shoot development, characterized in that... The CsESE3 gene is overexpressed using a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria containing the CsESE3 gene, thereby increasing the number of branches in the plant. The nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.

2. The plant is lemon.

6. The application of recombinant vectors, expression cassettes, or transgenic cell lines containing the CsESE3 gene in the cultivation of multi-branched lemon varieties, characterized in that... The CsESE3 gene was overexpressed using a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria containing the CsESE3 gene, thereby increasing the number of branches in lemons; the nucleotide sequence of the CsESE3 gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2.

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