Gossypium hirsutum height control gene ghga2ox3 and application thereof

Abstract

The application discloses a land cotton plant height regulation gene GhGA2ox3 and application thereof, and relates to the technical field of genetic engineering; a nucleotide sequence of the regulation gene GhGA2ox3 is shown as SEQ ID NO. 1. The application screens a gene GhGA2ox3 that can regulate plant height from a GhGA2ox family, and experiment verifies that the GhGA2ox3 can respond to GA3 and MeJA; subcellular localization analysis is carried out on the GhGA2ox3 in tobacco, and the result shows that the GhGA2ox3 is located in the cell nucleus and the cytoplasm. The application also proves that the GhGA2ox3 can reduce the active gibberellin content in a plant body, thereby reducing the plant height, and lays a foundation for cultivating dwarf high-yield varieties and in-depth research on a related mechanism of the GhGA2ox3 regulating plant height.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a highly regulatory gene in upland cotton plants, GhGA2ox3, and its applications. Background Technology

[0002] Plant height is an important agronomic trait in cotton, affecting not only yield but also fiber quality. Suitable plant height can increase planting density, optimize field management, and improve yield; furthermore, short stalks are crucial for mechanical harvesting. Plant height is influenced by the number of internodes and the distance between each internode. Plant height is related to cotton genetics and breeding, and increasingly, scholars have conducted in-depth research on traits such as plant height, number of internodes, and internode distance. To study the impact of plant type on cotton yield, Liu Jiguo et al. conducted experiments using Liaomian 10 and 15 and insect-resistant cotton as research materials, finding that the number of fruiting branches and the number of fruiting branch nodes can positively influence cotton production and yield through boll formation rate and boll yield per acre. Cao Xinchuan et al. conducted experiments using 23 upland cotton materials with different genetic backgrounds from domestic and international sources. The results showed that the position of the first fruiting branch node is closely related to fiber quality factors such as lint percentage, single boll weight, fiber length, evenness, micronaire value, and specific strength, and can indirectly serve as a basis for evaluating fiber quality. Qi Zijie et al. conducted principal component and multiple quadratic regression analyses on 17 quantitative traits of 16 spring cotton varieties. The results showed that relatively low initial nodes, numerous branches, and relatively tall plant height could increase lint yield. Tang Feiyu et al. studied the effect of plant type on yield using an additive-dominant genetic model of the interaction between environment and genetics. They selected 9 cotton varieties (or lines) and 20 combinations from the F1 generation for their study. The results showed that the number of fruiting nodes, a plant height-related trait, had the highest dominant genetic contribution rate to lint yield, at 57.8%; while the main stem internode distance had a genetic contribution rate of 17.7%. The contribution rate of cotton variety to lint yield ranged from 0.1% to 11.1%. Ren Yi et al. used the variety "Yanmian 48" to conduct statistical analysis on plant height traits such as plant height, main stem distance, and fruiting branch length, concluding that appropriately reducing plant height is beneficial for dense planting, thereby increasing cotton yield. Cheng Beijiu et al. conducted a correlation analysis of cotton traits using 20 popular varieties and found that the number of fruiting branches, internodes, and plant height can affect cotton yield. Reported literature indicates that suitable plant height can increase cotton's resistance to lodging and planting density, and is also more suitable for mechanized management, thereby increasing yield, reducing planting costs, and increasing cotton farmers' income. Therefore, breeding semi-dwarf varieties is particularly urgent. Summary of the Invention

[0003] The purpose of this invention is to provide a gene GhGA2ox3 that regulates the height of upland cotton and its application, in order to solve the problems existing in the prior art. This invention screens out the gene GhGA2ox3 that may regulate cotton plant height through bioinformatics and tissue expression analysis of the GhGA2ox family, and verifies its plant height regulation function through genetic transformation experiments on Arabidopsis thaliana, rice and cotton VIGS, etc., laying the foundation for breeding dwarf, high-yielding varieties and further studying the relevant mechanisms of GhGA2ox3 regulating plant height.

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

[0005] This invention provides a gene GhGA2ox3 that regulates the height of upland cotton plants, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides a recombinant expression vector comprising the above-mentioned regulatory gene GhGA2ox3.

[0007] The present invention also provides a recombinant microbial strain, comprising the above-described recombinant expression vector.

[0008] This invention also provides the application of the above-mentioned regulatory gene GhGA2ox3, recombinant expression vector, or recombinant microbial strain in reducing the high blood pressure of upland cotton plants.

[0009] The present invention also provides a method for reducing the height of upland cotton plants, including the step of upregulating the expression level of the above-mentioned regulatory gene GhGA2ox3 in upland cotton.

[0010] Furthermore, by genetically transforming the regulatory gene GhGA2ox3 into upland cotton plants to obtain transgenic plants, the expression level of the regulatory gene GhGA2ox3 in upland cotton is upregulated.

[0011] This invention also provides the application of the above-mentioned regulatory gene GhGA2ox3, recombinant expression vector, or recombinant microbial strain in reducing gibberellin activity in upland cotton.

[0012] Furthermore, the gibberellins are GA1 and GA4.

[0013] The present invention discloses the following technical effects:

[0014] This invention identified 25 members of the cotton GhGA2ox family based on reported GA2ox family characteristics. Using public transcriptome data and the relative expression levels of the gene in CRI50 and TM-1, the gene GhGA2ox3, which may regulate plant height, was screened from the GhGA2ox family. Based on hormone-related cis-acting elements located 2000 bp upstream of the GhGA2ox3 promoter, it was verified that GhGA2ox3 responds to GA3 and MeJA. Subcellular localization analysis of GhGA2ox3 in tobacco showed that it is located in the nucleus and cytoplasm.

[0015] Heterologous transformation of Arabidopsis thaliana with GhGA2ox3 reduces plant height, root length, germination rate, and active gibberellin content, but does not affect the thousand-grain weight. Heterologous transformation of rice with GhGA2ox3 reduces seedling height and root length, but does not affect grain length, grain width, or 100-grain weight. Silencing GhGA2ox3 with VIGS in the "CRI50" assay increases plant height and active gibberellin content. This invention demonstrates that GhGA2ox3 can reduce the content of active gibberellin in plants, thereby reducing plant height, laying the foundation for breeding dwarf, high-yielding varieties and further research on the mechanisms by which GhGA2ox3 regulates plant height. Attached Figure Description

[0016] 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.

[0017] Figure 1 This is a schematic diagram of a subcellular localization vector; where A: the gene is downstream of EGFP; B: the gene is upstream of EGFP.

[0018] Figure 2 For the analysis of candidate gene tissue expression patterns; where A: GhGA2ox3; B: GhGA2ox10; C: GhGA2ox14; D: GhGA2ox15; E: GhGA2ox19; different lowercase letters indicate significant differences (P<0.05);

[0019] Figure 3 The height of the six cotton varieties is represented by different lowercase letters; different lowercase letters indicate significant differences (P<0.05).

[0020] Figure 4The figures show the phenotypic values ​​of TM-1 and CRI50, and the expression of GhGA2ox3. A: Phenotypic charts of CRI and TM-1 plant height at the four-leaf stage; B: Phenotypic charts of CRI and TM-1 plant height at the five-leaf stage; C: Phenotypic statistics of CRI and TM-1 plant height at the four-leaf stage; D: Phenotypic statistics of CRI and TM-1 plant height at the five-leaf stage. "*" indicates P < 0.5; "***" indicates P < 0.001; the scale bars in A and B are both 2 cm.

[0021] Figure 5 The changes in GhGA2ox3 expression levels within 24 hours after foliar application of hormones are shown; where A: GA3 application; B: MeJA application; C: ABA application; "*" indicates P<0.5; "***" indicates P<0.001;

[0022] Figure 6 The purpose of this study was to amplify the GhGA2ox3 sequence, detect single-clone positive bacterial suspensions, and validate the recombinant vector. A: PCR amplification sequence, M is Marker III, 1-5 are leaves, 6 is roots, and 7 is stems; B: Single-clone positive bacterial suspension detection, M is Marker III, 1-8 are bacterial suspension PCR; C: Recombinant vector validation, M is Marker III, 1-5 are recombinant vectors.

[0023] Figure 7 Transient expression observation in tobacco epidermal cells; the first column is green fluorescence, the second column is red fluorescence, the third column is normal bright field, and the fourth column is the overlay image; the arrows in the image point to the cytoplasm; all scale bars in the image are 20 μm;

[0024] Figure 8 This is a schematic diagram of the expression vectors; where A: Arabidopsis thaliana overexpression vector; B: rice overexpression vector;

[0025] Figure 9 PCR validation of recombinant vectors; where A: Arabidopsis and rice recombinant vectors, M is Marker III, 1-5 are Arabidopsis overexpression recombinant vectors, 6-10 are rice overexpression recombinant vectors; B: VIGS recombinant vectors, M is Marker III, 1-4 are recombinant vectors.

[0026] Figure 10For the identification of mutants and transgenic Arabidopsis thaliana; where A: mutant detection, M is Marker III, 1 and 2 are detected using water as a template, 3 and 4 are detected using wild-type Arabidopsis thaliana as a template, with atga2ox8LP and atga2ox8RP and atga2ox8LB and atga2ox8RP as primers respectively, and 5 and 6 are detected using Arabidopsis thaliana atga2ox8 as a template, with atga2ox8LP and atga2ox8RP and atga2ox8LB and atga2ox8RP as primers respectively; B: detection of transgenic Arabidopsis thaliana positive strains, M is Marker III, 1 is detected using water as a template, 2 is detected using cotton leaf cDNA as a template, 3 is detected using wild-type Arabidopsis thaliana as a template, 4-6 are detected using overexpressing Arabidopsis thaliana as a template, 7 is detected using Arabidopsis thaliana atga2ox8 as a template, and 8-10 are detected using overexpression in the atga2ox8 background as a template.

[0027] Figure 11 Phenotypes of transgenic Arabidopsis thaliana; where A: bolting stage phenotype; B: flowering stage phenotype; C: 7-week-old phenotype; D: relative expression levels of different lines; E: gibberellin content; lowercase letters indicate significant differences (P<0.05); the scale bar in A and C is 2cm;

[0028] Figure 12 Phenotypic analysis of transgenic Arabidopsis thaliana; where A, B: bolting time; C, D: number of rosette leaves at bolting; E, F: flowering time; G, H: total number of leaves at flowering; I, J: height at flowering; K, L: height at 7 weeks of age; A, C, E, G, I, K: no gibberellin spraying; B, D, F, H, J, L: sprayed with 100 μM gibberellin; different lowercase letters indicate significant differences (P<0.05);

[0029] Figure 13 Root length, germination, and thousand-seed weight of transgenic Arabidopsis thaliana; where A: root length phenotype; B: root length statistics; C: germination phenotype; D: germination statistics; E: thousand-seed weight statistics; different lowercase letters indicate significant differences (P<0.05); the scale bar in A and C is 1 cm;

[0030] Figure 14 To detect the expression levels of GA-related genes in transgenic Arabidopsis thaliana; different lowercase letters in the same group indicate significant differences (P<0.05);

[0031] Figure 15 For the identification of transgenic rice; where A: plant height phenotype; B: positive plant detection results, M is Marker III, 1 is water as template, 2 is cotton leaf cDNA as template, 3 is wild-type rice as template, 4-6 are overexpressing rice as template; C: relative expression level of different lines; "***" indicates P<0.001;

[0032] Figure 16 Phenotypic analysis of positive rice lines; where A: underground part length; B: aboveground part length; C: plant height; D: grain length; E: grain width; F: 100-grain weight; "*" indicates P<0.5; "**" indicates P<0.01; "***" indicates P<0.001;

[0033] Figure 17 The phenotypes of VIGS cotton are shown below; where AC represents the phenotype at the two-leaf to four-leaf stage; D represents the relative expression level at the two-leaf to four-leaf stage; "***" indicates P < 0.001; the scale bar in AC is 4 cm.

[0034] Figure 18 For the determination of gibberellin content in VIGS cotton; where A: GA1 content; B: GA4 content; C: total content of GA1 and GA4; "*" indicates P<0.05; "**" indicates P<0.01; "***" indicates P<0.001. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The nucleotide sequence of the GhGA2ox3 gene in the following examples is shown in SEQ ID NO.1.

[0041] Table 1. GA2ox gene family information in Arabidopsis thaliana, rice, and upland cotton.

[0042]

[0043]

[0044] This invention identifies all members of the GhGA2ox family from the whole genome of upland cotton through bioinformatics analysis (see Table 1 above), and selects GhGA2ox3 as the research object. Preliminary functional analysis is performed using biochemical and molecular biological methods, detailed below:

[0045] Example 1

[0046] 1. Materials and Methods

[0047] 1.1 Plant materials

[0048] The cotton materials selected were "CRI10", "CRI50", "CRI58", "TM-1", "Yu21", and "NM1". Six varieties were planted in the Zhejiang A&F University farmyard, and their boll-opening height was measured. For tissue expression analysis, "CRI50" and "TM-1" were planted in the Zhejiang A&F University farmyard and the Pingshan Greenhouse. "CRI50" was used for exogenous hormone spraying and was planted in a light incubator under the following conditions: 16 / 8h, 28 / 25℃, light intensity of 15000 Lux, and relative humidity of 50%. Subcellular localization was performed using *Nicotiana abenthamiana*, planted in a walk-in plant culture room under the following conditions: 16 / 8h, 28 / 25℃, and light intensity of 300 μmol / m². 2 ·s -1 The relative humidity is 50%.

[0049] 1.2 Vectors and Strains

[0050] *Escherichia coli* DH5α (DL1001), *Agrobacterium* GV3101 (AC1001), and P19 (AC1003L) were purchased from Shanghai Weidi Biotechnology Co., Ltd.; the cloning vector TA / Blunt-Zero Cloning Kit (C601) was purchased from Novizan; the subcellular localization vector pCAMBIA1305::GFP (8600612) was purchased from the BioVector Plasmid Vector Culture Collection Center; and the nuclear colocalization vector p2300-35S-h2b-Mcherry was shared by New England Biolabs. A brief visualization of the subcellular localization vector pCAMBIA1305::GFP-GhGA2ox3 is shown below. Figure 1 .

[0051] 1.3 Experimental Methods

[0052] The short-stemmed material “CRI50” and the tall-stemmed material “TM-1” were selected. “CRI50” and “TM-1” were planted in Pingshan Greenhouse, and their height from the cotyledon node to the terminal bud was measured using a ruler at the four-leaf and five-leaf stages.

[0053] 1.3.1 External hormone spraying

[0054] Prepare 1000x dilution of 100μM GA3 (gibberellin) and ABA (abscisic acid): Weigh 0.3849g GA3 and 0.2935g ABA, dissolve them separately in 200μL anhydrous ethanol, and then bring the volume to 10mL with ddH2O. Prepare 1000x dilution of 100μM MeJA (methyl jasmonate): Pipette 229.22μL and bring the volume to 10mL. Filter the solutions using a 0.22μm aqueous filter membrane. Dilute the hormones 1000x before use. When “CRI50” plants reach the three-leaf stage in the light incubator, spray the hormones until solution drips from the leaves. Take leaf samples at 0h, 6h, 12h, and 24h for real-time quantitative PCR analysis. Perform 10 replicates for each biological sample. Immediately place the samples in liquid nitrogen after sampling for RNA extraction or store at -80℃ for later use.

[0055] 1.3.2 Total RNA extraction from cotton

[0056] Roots, stems, leaves, and terminal buds of "CRI50" at the three-leaf stage were collected; petals, stamens, ovules and fibers collected 3 days before flowering (-3 days postanthesis, -3DPA), 5DPA, 15DPA, and 20DPA were also collected. Stems of "CRI50" and TM-1 at the four-leaf and five-leaf stages were collected from the Pingshan greenhouse. Three biological replicates were taken for each sample, and 10 plants were taken from each biological replicate. After sampling, the samples were immediately placed in liquid nitrogen for RNA extraction or stored at -80℃ for later use.

[0057] For specific RNA extraction methods, refer to the instructions for polysaccharide and polyphenol RNA extraction in the Novizan Plant Total RNA Isolation Kit (RC401). Store the obtained RNA at -20°C for later use or proceed directly to reverse transcription.

[0058] 1.3.3 Reverse transcription to synthesize cDNA

[0059] For specific instructions on reverse transcription of RNA, refer to the Polymerex M5 Super plus qPCR RT kit with gDNA remover (MF166-plus) manual. The obtained cDNA can be used for gene cloning, real-time quantitative PCR, or stored at -20℃ for later use.

[0060] 1.3.4 Real-time quantitative PCR

[0061] qRT-PCR primers were designed on NCBI based on the sequences of GhGA2ox3, GhGA2ox10, GhGA2ox14, GhGA2ox15, and GhGA2ox19. The internal reference gene was GhActin4. The qRT-PCR primers are shown in Table 2.

[0062] Table 2

[0063]

[0064] Note: Lowercase letters indicate recombination sites.

[0065] The cDNA concentration was determined using a Nanodrop 2000 instrument, and the cDNA was diluted to 100 ng / μL with ddH2O. qRT-PCR experiments were performed using Taq Pro Universal SYBR qPCR Master Mix (Novizan, Q712). The experiments were conducted in the dark. The specific procedure was as follows: 2 μL of cDNA, 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix, 0.5 μL of Primer 1 (10 μM upstream primer), 0.5 μL of Primer 2 (10 μM downstream primer), and 7 μL of Nuclease-free ddH2O were added to each well of a 96-well quantitative plate. Set the reaction on the real-time quantitative PCR instrument as follows: 95℃ for 30s, 95℃ for 10s, 60℃ for 30s, 95℃ for 15s, 60℃ for 60s, 95℃ for 15s, and 40℃ for 30s. Among them, the 95℃ for 10s and 60℃ for 30s quantitative PCR were run for 40 cycles, and the melting curve of 95℃ for 15s, 60℃ for 60s, and 95℃ for 15s was run for 1 cycle.

[0066] 1.3.5 Cloning of the GhGA2ox3 gene

[0067] Preparation of a 50-fold stock solution for TAE electrophoresis: Weigh 33.621 g of disodium ethylenediaminetetraacetate dihydrate and 242 g of Tris into a 1 L beaker, add 600 mL of deionized water, stir with a clean glass rod until fully dissolved, then add 57.1 mL of acetic acid, continue stirring, and then bring the volume to 1 L with deionized water. Store at room temperature. Add 1 g of agarose to 100 mL of TAE, heat in a microwave oven for 2 min to prepare the electrophoresis gel, cool to 60 °C, add 10 μL of LGelRed (Solepro, G5560), gently shake to mix, prepare the gel, and cool to room temperature before use. LB medium formula: Add 1 g of peptone, 0.5 g of yeast, and 1 g of NaCl per 100 mL (add 1.5 g of agar powder if solid), autoclave at 121 °C for 15 min before use. Dissolve kanamycin and gentamicin in ddH2O to prepare a 50 mg / mL stock solution. The working solution concentration was 50 μg / mL; rifampicin antibiotic was dissolved in ddH2O to prepare a stock solution with a concentration of 50 mg / mL. The working solution concentration was 25 μg / mL. After preparation, the solution was filtered through a 0.22 μm aqueous filter membrane and stored at -20℃ for later use.

[0068] Cloning primers were designed on NCBI based on the sequence of GhGA2ox3, and the cloning primers are shown in Table 2.

[0069] Using cDNA from stems, leaves, and terminal buds of CRI50 as PCR templates, PCR amplification was performed using KOD FX (TOYOBO, KFX-101). In a 200 μL PCR tube, the following were added: 200 ng cDNA, 25 μL 2×PCR buffer for KOD FX, 5 μL 2 mM dNTPs, 2.5 μL GhGA2ox3-F, 2.5 μL GhGA2ox3-R, 1 μL KOD FX, and Nuclease-free ddH2O to a final volume of 50 μL. The PCR reaction conditions were: 94℃ for 2 min, 98℃ for 10 s, 60℃ for 30 s, 68℃ for 1 min 10 s, and incubation at 16℃. The 98℃ 10 s, 60℃ 30 s, and 68℃ 1 min 10 s cycles were set for 30 cycles, with the 60℃ temperature decreasing by 0.2℃ per cycle.

[0070] After the PCR reaction, agarose gel electrophoresis was performed to observe the bands. After the gel had completely cooled, it was placed in an electrophoresis tank containing TAE working solution. 1 μL of Loading Buffer (Takara, 9157) and 1 μL of PCR amplification product were mixed and spotted into the wells. The time and voltage were set according to the gel size (generally 100-140V, 20 minutes). After completion, the gel was placed in a gel imaging system to view the target bands.

[0071] After electrophoresis confirms the clone fragment size and location are correct, the PCR product is purified using the MiniBEST DNA Fragment Purification Kit Ver. 4.0 (Takara, 9761). Refer to the instruction manual for specific procedures. The obtained product can be used for subsequent experiments or stored at -20°C for later use.

[0072] After purification, the cloning vector is ligated and then transformed into E. coli DH5α. Refer to the instruction manual for specific operating procedures.

[0073] After transformation, single clones grown at 37℃ were subjected to bacterial culture PCR. In a biosafety cabinet, a sterile 1.5mL centrifuge tube was taken, and 1mL of liquid LB medium containing kanamycin was added. Plump single clones were then selected and placed into the centrifuge tube. The tube was sealed with sealing film and incubated at 37℃ on a shaker at 200rpm until the culture became turbid. PCR was then performed to detect the size of the target fragment. PCR detection was performed using 2×Specific Taq Master Mix (Nearshore Protein Technology Co., Ltd., E010). The specific procedure was as follows: 2μL bacterial culture, 10μL 2×Specific™ Taq Master Mix, 0.8μL GhGA2ox3-F, 0.8μL GhGA2ox3-R, and 6.4μL Nuclease-free ddH2O were added to a 200μL PCR tube. The PCR reaction conditions were as follows: 94℃ for 1 min 30 s, 94℃ for 20 s, 60℃ for 20 s, 72℃ for 1 min 10 s, and incubation at 16℃. The 94℃ 20 s, 60℃ 20 s, and 72℃ 1 min 10 s cycles were set for 30 cycles, with the 60℃ temperature decreasing by 0.2℃ in each cycle. Electrophoresis was then used to determine the size of the target fragment, following the same method. Bacterial cultures with correctly determined fragment sizes were sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the target gene sequence. Cultures with successful alignment were used for subsequent experiments or stored in 20% glycerol at -80℃.

[0074] 1.3.6 Construction of Subcellular Localization Vectors

[0075] Add the correctly sequenced cloning vector bacterial culture to liquid LB medium containing kanamycin, and incubate at 37°C with a shaker until turbidity is reached. Then, extract the cloning vector plasmid using the TaKaRa Rapid Plasmid Mini-Prep Kit (DP105). After obtaining the plasmid, measure the plasmid concentration and store it at -20°C for later use or storage.

[0076] The restriction enzyme sites of the subcellular localization vector were examined, and the pCAMBIA1305-35S-GFP vector was digested with the single restriction enzyme site Bgl II and the double restriction enzyme sites Spe I and XbaI. The recombinant sites were introduced into the vector using PCR amplification. Primers were designed as shown in Table 2.

[0077] Using the cloning vector as a template, PCR amplification was performed using the primers in Table 2 and KOD FX. Then, agarose gel electrophoresis was performed to detect the fragment size. After confirming the correct size, the fragment was purified using the same method as in 1.3.5.

[0078] The vector pCAMBIA1305-35S-GFP was digested with enzymes, following the procedure described in TaKaRa QuickCut. TM Bgl II (1606), QuickCut TM Spe I (1631) and QuickCut TM Xba I(1634) instructions. After digestion, purify using the same method as in 1.3.5. Measure the concentration after purification for subsequent experiments or store at -20°C for later use.

[0079] The purified PCR product and enzyme-digested plasmid were recombinantly ligated using the Novizan ClonExpress II One Step Cloning Kit (C112).

[0080] Transform DH5α with 10 μL of the ligation product and sequence it, following the same method as in 1.3.5. Extract the plasmid from the bacterial culture that has been correctly sequenced, following the same method as above. Transform the plasmid into Agrobacterium GV3101; refer to the instruction manual for specific transformation procedures.

[0081] PCR was used to identify positive bacteria, following the same method as in 1.3.5. Bacterial solutions with correct band sizes were stored at -80°C using 20% ​​glycerol.

[0082] 1.3.7 Subcellular localization of tobacco

[0083] Preparation of osmotic resuspension: 2.033 g MgCl2·6H2O and 1.952 g 2-(N-morpholine)ethanesulfonic acid were dissolved in 100 mL ddH2O to prepare a 10-fold stock solution, sterilized at 121 °C for 15 min, and stored at 4 °C for later use; 19.62 mg acetylsyl syringone was dissolved in 1 mL dimethyl sulfoxide to prepare a 5-fold stock solution, which was stored at -20 °C for later use.

[0084] Subcellular localization method: First, Agrobacterium strains 35S::GhGA2ox3-GFP, P19, p2300-35S-h2b-Mcherry, and empty vectors stored at -80℃ were streaked onto solid LB agar plates containing gentamicin, kanamycin, and rifampin for resuscitation. Then, single colonies were placed in 1 mL of LB liquid medium containing kanamycin and rifampin and cultured overnight at 28℃ and 200 rpm. After overnight culture, the bacterial culture was transferred to 25 mL of LB liquid medium containing kanamycin and rifampin and cultured overnight at 28℃ and 200 rpm until OD (digestive activity) was reached. 600Centrifuge at 4000 rpm for 10 min, discard LB medium, resuspend Agrobacterium in osmotic resuspending solution, and adjust OD after resuspending. 600 The culture was allowed to stand at room temperature for 3 hours after reaching a concentration of 0.6. Then, 35S::GhGA2ox3-GFP and P19, and p2300-35S-h2b-Mcherry and P19 were mixed in a 1:1 volume ratio and injected. Using a disposable syringe, 1 mL of the mixed bacterial solution was drawn, and the syringe was gently pumped into the leaf tissue by holding the lower part of the tobacco (1-month-old) leaf with a finger. The injected tobacco was then incubated in the dark at 25°C for 24 hours, followed by normal incubation for 48 hours. The tobacco leaves were then observed under a confocal microscope.

[0085] 1.3.9 Data Measurement and Statistics

[0086] The initial node position and the height from cotyledon to terminal bud were measured using a ruler, with three biological replicates of 10 plants per replicate. A t-test was used to test the significance of the two samples, and a graphical method was used for one-way significance testing. -ΔΔCT Gene expression levels were calculated. GraphPad Prism 5 was used for data visualization and significance analysis. ImageJ was used to annotate the scale bars in the graphs.

[0087] 2 Results and Analysis

[0088] 2.1 Organizational Expression Analysis

[0089] Five genes, GhGA2ox3, GhGA2ox10, GhGA2ox14, GhGA2ox15, and GhGA2ox19, were selected for tissue expression pattern analysis. Transcriptome analysis of GhGA2ox3, GhGA2ox10, GhGA2ox15, and GhGA2ox19 showed high expression levels in stems, suggesting they are potential candidate genes regulating plant height. GhGA2ox14 is a paralog of GhGA2ox3. qRT-PCR analysis was performed on these five genes in roots, stems, leaves, petals, stamens, terminal buds, -3DPA, 5DPA, 15DPA, and 20DPA ovules, and fibers at CRI50. The results showed that GhGA2ox10 was highly expressed in petals, 5-DPA, and 15-DPA; GhGA2ox14 was most highly expressed in leaves; GhGA2ox15 was most highly expressed in petals; GhGA2ox19 showed no specific expression in any tissue; only GhGA2ox3 was highly expressed in stems, terminal buds, and leaves. Figure 2 This suggests that GhGA2ox3 may be involved in the regulation of plant height.

[0090] 2.2 Screening of cotton materials

[0091] This invention selected three shorter varieties, "CRI10", "CRI50", and "CRI58", and three taller varieties, "TM-1", "Yu21", and "NM1". These were planted in an orchard, and the height of the first node of the fruiting branches at the boll-opening stage was measured. Figure 3 The study found that the "TM-1" fruit branch had the highest initial node position, which was significantly different from other materials; the "CRI50" fruit branch had the lowest initial node position, which was significantly different from both "TM-1" and "NM1".

[0092] "CRI50" and "TM-1" were grown in a greenhouse until the five-leaf stage. The height from the cotyledon node to the terminal bud was measured at the four-leaf and five-leaf stages, and stems were taken for qRT-PCR analysis. At the four-leaf and five-leaf stages, "TM-1" was significantly taller than "CRI50". Figure 4 (AC). The expression level of GhGA2ox3 in "TM-1" was significantly lower than that in "CRI50" ( Figure 4 (D). Therefore, “CRI50” and “TM-1” were selected as cotton varieties for studying GhGA2ox3.

[0093] 2.3 Response of GhGA2ox3 to exogenous hormones

[0094] Analysis of the cis-acting elements of the GhGA2ox3 promoter revealed three hormone-related elements: GA3, MeJA, and ABA. Therefore, this invention investigated the response of GhGA2ox3 to hormones by spraying exogenous hormones at the three-leaf stage (CRI50). The relative expression levels of GhGA2ox3 changed after spraying with 100 μM GA3, MeJA, and ABA, respectively. Figure 5 After GA3 treatment ( Figure 5 In the control group (A), the relative expression level of GhGA2ox3 reached its peak at 6 hours, approximately three times that of the control group. The relative expression level decreased at 12 hours but was still significantly higher than the control group; after 24 hours, it returned to a level not significantly different from the control group. After MeJA treatment (A), the relative expression level of GhGA2ox3 reached its peak at 6 hours, approximately three times that of the control group. Figure 5 In the ABA-treated "CRI50" dataset, the relative expression levels of GhGA2ox3 at 6h and 12h were not significantly different from those in the control group; however, the relative expression level at 24h was significantly lower than that in the control group. In the ABA-treated "CRI50" dataset, the relative expression level of GhGA2ox3 was not significantly different from that in the control group within 24h. Figure 5 (C). The above results indicate that GhGA2ox3 can respond to two exogenous hormones, GA3 and MeJA. GA3 can promote the expression of GhGA2ox3 in upland cotton, while MeJA has an inhibitory effect on the expression of GhGA2ox3.

[0095] 2.4 Cloning and Validation of GhGA2ox3 Recombinant Vectors

[0096] Using cDNA from the roots, stems, and leaves of upland cotton “CRI50” as templates, PCR amplification was performed using cloning primers. The PCR product amplified a fragment of approximately 1083 bp, consistent with the CDS size (6A) of the GhGA2ox3 gene. The obtained product was purified and ligated into the TA / Blunt-Zero cloning vector. The resulting recombinant plasmid was transformed into *E. coli* DH5α, and the selected positive clones were confirmed by bacterial PCR. Figure 6 (B) The bacterial culture was sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. Plasmids were extracted from the correctly sequenced bacterial cultures and transformed into GV3101. The resulting single clones were then confirmed to be correct by colony PCR. Figure 6 (C) Store at -80°C for later use or with 20% glycerin.

[0097] Subcellular localization of 2.5GhGA2ox3 in tobacco

[0098] Simultaneous transient expression of 35S::GhGA2ox3-GFP and p2300-35S-h2b-Mcherry in tobacco, followed by confocal microscopy, revealed that 35S::GhGA2ox3-GFP was localized in both the nucleus and cytoplasm; p2300-35S-h2b-Mcherry was localized in the nucleus. Intact cells were clearly visible under bright field microscopy, and the localization results of 35S::GhGA2ox3-GFP and p2300-35S-h2b-Mcherry could be combined and displayed with yellow light. Figure 7 This indicates that GhGA2ox3 is located in the nucleus and cytoplasm, where it interacts with other proteins to regulate plant growth and development.

[0099] Example 2

[0100] atga2ox8 is a homolog of GhGA2ox3 in Arabidopsis thaliana.

[0101] 1. Materials and Methods

[0102] 1.1 Plant materials

[0103] Arabidopsis thaliana were grown using the Columbia wild-type (Clo-0) and atga2ox8 (CS850082) mutants. The mutants were purchased from TAIR-ABRC (https: / / abrc.osu.edu / stocks / number / CS850082). The Arabidopsis were grown in a walk-in plant culture room under the following conditions: 16 / 8h, 23 / 22℃, and a light intensity of 300 μmol / m². 2 ·s -1The relative humidity was 50%. The material used for rice genetic transformation was "Zhonghua 11" ("ZH11"). Rice seedlings were planted in a walk-in plant culture room under the following conditions: 16 / 8h, 28℃, and light intensity of 300 μmol / m². 2 ·s -1 The relative humidity was 50%; later, the plants were grown in a greenhouse in Pingshan. Virus-induced gene silencing (VIGS) was achieved using "CRI50" material. VIGS plants were grown in a walk-in plant culture room under the following conditions: 16 / 8h, 25℃, and a light intensity of 300 μmol / m². 2 ·s -1 The relative humidity is 50%.

[0104] 1.2 Vectors and Strains

[0105] The *E. coli* DH5α and *Agrobacterium* GV3101 used were the same as in Example 1. Schematic diagrams of the *Arabidopsis* overexpression vector pBI121 and the rice overexpression vector pCAMBIA1300s are shown below. Figure 8 In sections A and B; the pTRV vector used in VIGS was a gift from Professor Liu Qibao and is a vector in the Nimble Cloning system.

[0106] 1.3 Experimental Methods

[0107] 1.3.1 Detection of Arabidopsis mutants

[0108] The triple primer sequences used for the mutants were designed using T-DNA Primer Design (http: / / signal.salk.edu / tdnaprimers.2.html) with default parameters. The sequence information is shown in Table 2.

[0109] The product size of atga2ox8LP+atga2ox8RP was 1088 bp, and the product size of atga2ox8LB+atga2ox8RP ranged from 544 to 844 bp. After Arabidopsis thaliana had grown for 18 days, crude DNA was extracted from rosette leaves for PCR amplification and gel electrophoresis. The crude DNA was detected using the M5 Ultra-Fast Mix (MF848). The specific procedure was as follows: 40 μL of the optimal amplification partner was added to a 1.5 mL centrifuge tube, followed by 2 mm of... 2For the left and right samples, grind the solid samples as much as possible and boil for 10 minutes; then centrifuge at 12000 rpm for 2 minutes, and take 2 μL as the template for subsequent PCR; PCR amplification reaction system: add 2 μL of crude DNA extracted in the above steps, 10 μL of 2×M5 Hiper ultra-fast mix (with blue dye), 0.5 μL of GhGA2ox3-F, 0.5 μL of GhGA2ox3-R, and 7 μL of Nuclease-free ddH2O to a 200 μL PCR tube, and then perform PCR. The PCR program is as follows: 95℃ for 3 min, 94℃ for 25 s, 60℃ for 25 s, 72℃ for 1 min 10 s, 72℃ for 5 min, and incubate at 16℃. Among them, 94℃ for 25 s, 60℃ for 25 s and 72℃ for 1 min 10 s are set for 30 cycles, and 60℃ is decreased by 0.2℃ in each cycle; finally, gel electrophoresis is performed, and the method is the same as 1.3.5 of Example 1. The line with the correct band is a homozygous atga2ox8 mutant.

[0110] 1.3.2 Construction of overexpression vectors and VIGS vectors

[0111] The Arabidopsis overexpression vector 35S:GhGA2ox3 selected double restriction sites XbaⅠ and SacⅠ (TaRaKa, 1627); the rice overexpression vector 35S:GhGA2ox3 selected single restriction site XbaⅠ.

[0112] The primers used to construct the overexpression vector and VIGS vector are shown in Table 3.

[0113] Table 3

[0114]

[0115]

[0116] Note: Lowercase letters indicate recombination sites.

[0117] The method for constructing the overexpression vector is the same as 1.3.6 in Example 1.

[0118] The VIGS vector was constructed using the Nimble Cloning kit (Hainan Nixing Biotechnology Co., Ltd., NC001). The specific procedure was as follows: First, the NC adapter was introduced by PCR. Using the cloning vector as a template and pTRV-GhGA2ox3-F and pTRV-GhGA2ox3-R as primers, PCR amplification was performed using KOD FX. Then, agarose gel electrophoresis was performed to check the fragment size. After confirming the correct size, purification was carried out using the same method as in Example 1, section 1.3.5, with a PCR cycle count of 27. The pTRV2 vector plasmid was extracted using the same method as in Example 1, section 1.3.6. Then, 50 ng of the purified PCR product obtained in the above steps and 100 ng of the pTRV2 vector obtained in the above steps were added to a 200 μL PCR tube. 5 μL of Nimble Mix was then added, and the mixture was pipetted 20 times to ensure thorough mixing. The mixture was incubated at 50°C for 30 min and then kept at 4°C. Finally, the product from the above steps was transformed into E. coli, sequenced, plasmid extracted, and transformed into GV3101. PCR detection was performed, following the same method as in 1.3.6 of Example 1. Correctly detected GV3101 samples were placed in 20% glycerol and incubated at -80°C for later use.

[0119] 1.3.3 Genetic transformation in Arabidopsis thaliana

[0120] Prepare 100 mL of Arabidopsis thaliana 1 / 2 MS osmotic buffer as follows: 0.22 g of 1 / 2 MS medium (sucrose and agar-free), 5 g of sucrose, dissolved in 100 mL of ddH2O, and sterilized at 121 °C for 15 min. Add 20 μL of Silwet L-77 before inoculation.

[0121] Arabidopsis inflorescence infection method: One day before infection, cut off the Arabidopsis inflorescences and pods, and water them appropriately to keep the substrate moist; then add the bacterial solution of correctly identified positive colonies to 200 mL of LB liquid medium containing kanamycin and rifampin, and incubate at 28°C and 200 rpm on a constant temperature shaker until OD. 600 1.2; Next, pour the bacterial culture into 50 mL centrifuge tubes, place them symmetrically in a large centrifuge, centrifuge at 4000 rpm for 10 min, discard the supernatant, and retain the bacterial cells; then suspend the bacterial cells in 1 / 2 MS osmotic buffer, vortex until the bacterial cells are completely dissolved in the osmotic buffer, and then adjust the OD to 1 / 2 MS using osmotic buffer. 600 The concentration was between 0.6 and 0.6. Finally, the Arabidopsis flower buds were immersed in the permeate for 45 seconds, cultured in the dark for 24 hours, and then cultured normally. The immersion was repeated once a week for a total of 2 immersions.

[0122] 1.3.4 Genetic transformation of rice

[0123] The methods for rice genetic transformation are referenced in "Lin Yongjun. Research on rice transgenic mediated by Agrobacterium [D]. Huazhong Agricultural University, 2001".

[0124] 1.3.5 Detection of positive strains

[0125] Harvested Arabidopsis (or rice) seeds were cultured on 1 / 2 MS solid medium containing kanamycin (or Hn) resistance for preliminary screening, following the procedure described in 1.1 of this embodiment. Surviving seedlings were transplanted, and once they reached a suitable size, DNA was extracted from young leaves. This DNA was then subjected to PCR amplification and gel electrophoresis, following the same procedure as described in 1.3.1 of this embodiment. Lines with correctly sized bands were considered positive.

[0126] 1.3.6 Cotton VIGS Experiment

[0127] The VIGS resuspension was the same as in Example 1, section 1.3.7. Five days prior to the procedure, TRV1, TRV2 empty vectors, TRV:GhCLA1, and TRV:GhGA2ox3 Agrobacterium, stored at -80°C, were plated onto LB medium containing kanamycin, rifampin, and gentamicin, and incubated at 28°C for 2 days. Single colonies were then picked and incubated overnight at 28°C with kanamycin and rifampin. The activated bacterial culture was then transferred to 25 mL of LB medium containing kanamycin and rifampin and incubated overnight at 28°C and 200 rpm. The culture was then incubated until OD500. 600 1.2; then pour the shaken bacterial culture into a 50mL centrifuge tube, centrifuge at 4000rpm for 10min, discard the supernatant, add an equal volume of resuspension to the centrifuge tube, vortex until the bacteria are completely dissolved in the resuspension, and then adjust the OD with the resuspension. 600 The concentration was increased to 0.6. After standing in the dark at room temperature for 2 hours, cotton with flattened cotyledons was injected. Before injection, TRV1 was mixed with equal volumes of TRV:GhCLA1, TRV2 empty vector, and TRV:GhGA2ox3 Agrobacterium. During injection, the lower epidermis of the cotyledon was scratched with the syringe needle, but the cotyledon should not be punctured. The syringe was used to draw up the mixed resuspension and inject it into the cotyledon to cover more than 90% of the total cotyledon area. After injection, the mixture was incubated in the dark at 25°C overnight, and finally cultured normally at 25°C.

[0128] 1.3.7 Total RNA extraction from Arabidopsis thaliana, rice, and VIGS cotton

[0129] RNA was extracted from Arabidopsis thaliana and rice leaves. Specific procedures for RNA extraction from Arabidopsis thaliana and rice were described in the instructions for the Novizan Plant Total RNA Isolation Kit (for general plant extraction). RNA extraction from cotton stems using VIGS followed the same method as described in section 1.3.2 of Example 1.

[0130] 1.3.8 Reverse transcription synthesis of cDNA in Arabidopsis thaliana, rice, and VIGS cotton

[0131] The method for reverse transcription to synthesize cDNA is the same as 1.3.3 in Example 1.

[0132] 1.3.9 qRT-PCR analysis of Arabidopsis thaliana, rice, and VIGS cotton

[0133] AtUBQ10 was used as an internal control for Arabidopsis thaliana qRT-PCR analysis. OsActin1 was used as an internal control for rice qRT-PCR analysis. Primers used for Arabidopsis thaliana and rice qRT-PCR are shown in Table 3. Primers used for cotton qRT-PCR are the same as those in Table 2.

[0134] The qRT-PCR analysis methods for Arabidopsis thaliana, rice, and VIGS cotton are the same as those in 1.3.4 of Example 1.

[0135] 1.3.10 Determination of gibberellin content in Arabidopsis thaliana and VIGS cotton

[0136] For Arabidopsis thaliana gibberellin content determination, the aboveground parts at flowering stage were selected, while for VIGS plants, the stems at the four-leaf stage were selected. After sampling, the samples were pre-cooled in liquid nitrogen and then transported to Jiangsu Enzyme Immunoassay Co., Ltd. using dry ice. The company then performed gibberellin content determination using the Plant Gibberellin 1 (GA1) ELISA Kit (MM-1189O1), Plant Gibberellin 4 (GA4) ELISA Kit (MM-1532O1), and BCA Protein Quantitative Kit (MM-9227A).

[0137] 1.3.11 Trait Determination and Data Statistics

[0138] For Arabidopsis thaliana, the following data were collected: bolting time; number of rosette leaves before bolting; flowering time; total number of leaves at flowering; above-ground height at flowering; above-ground height at 7 weeks of age; germination rate at 7 days; root length at 10 days; and thousand-grain weight. For rice, the following data were collected: above-ground and underground part length and plant height at tillering; grain length; grain width; and hundred-grain weight. For cotton VIGS plants, the following data were collected: height from cotyledon node to terminal bud at the two-leaf, three-leaf, and four-leaf stages. The method for testing the significance of data differences was the same as in Example 1, section 1.3.7.

[0139] 2 Results and Analysis

[0140] 2.1 PCR Validation of Recombinant Vector

[0141] The recombinant vector of GV3101 was transformed and plated. Single colonies that grew were subjected to culture PCR to confirm successful transformation of the selected colonies. The PCR products of the Arabidopsis and rice overexpression recombinant vectors were approximately 1113 bp in size. Figure 9 The size of the VIGS recombinant vector PCR product is approximately 390 bp (A). Figure 9 (B) Propagate bacterial suspension with the correct fragment size using shaking, for subsequent infection.

[0142] 2.2 Functional verification of GhGA2ox3 in Arabidopsis thaliana

[0143] 2.2.1 Detection of mutants and positive strains

[0144] After surviving transgenic Arabidopsis thaliana positive plants on 1 / 2 MS solid culture dishes containing kanamycin, they were transplanted into small square pots for growth. Rosette leaves of Arabidopsis thaliana were collected for analysis; crude DNA was extracted, and PCR was used to determine band size. Analysis was performed at each generation until the T3 generation, after which subsequent experiments were conducted. The test results are as follows: Figure 10 .

[0145] The detection of the Arabidopsis thaliana atga2ox8 mutant used primers atga2ox8LP and atga2ox8RP, and primers atga2ox8LB and atga2ox8RP. Using water as a template, no band was observed. Using wild-type DNA as a template, primers atga2ox8LP and atga2ox8RP produced a band approximately 1088 bp in size; primers atga2ox8LB and atga2ox8RP also produced no band. Using mutant DNA as a template, primers atga2ox8LP and atga2ox8RP produced no band, but primers atga2ox8LB and atga2ox8RP produced a band between 544-844 bp, and the band was single, consistent with the band size requirements for mutant detection. Figure 10 (A). The results show that the atga2ox8 mutant is homozygous and can be directly used for subsequent experiments.

[0146] Wild-type Arabidopsis (WT) overexpression positive lines and mutant complementation positive lines were transplanted into small square pots after surviving on 1 / 2 MS solid culture dishes containing kanamycin resistance. Rosette leaves of Arabidopsis were collected for analysis; crude DNA was extracted, and band size was determined by PCR. Analysis was performed at each generation until the T3 generation for subsequent experiments. The T3 generation test results of positive lines are shown below. Figure 10 In section B, no band was observed when water was used as a template; when cotton cDNA was used as a template, the band was approximately 1083 bp; no band was observed when wild-type and atga2ox8 mutant DNA were used as templates; and when OE1, OE2, OE3, atga2ox8-OE1, atga2ox8-OE2, and atga2ox8-OE3 strain DNA were used as templates, the band size was approximately 1083 bp, and the band was uniform, indicating that the transgenic strains met the experimental requirements.

[0147] 2.2.2 Phenotypic analysis of transgenic Arabidopsis thaliana

[0148] Phenotypic differences were observed in wild-type, overexpressing Arabidopsis thaliana, mutant, and overexpressing Arabidopsis thaliana under mutant backgrounds under the same culture conditions. Bolting was assessed by bolting time and the number of rosette leaves before bolting; flowering was assessed by flowering time and the total number of leaves at flowering. qRT-PCR analysis of expression levels in different lines revealed that among the overexpressing lines, the OE3 line had the highest relative expression level, and the OE1 line had the lowest relative expression level; among the overexpressing Arabidopsis thaliana lines under mutant backgrounds, the atga2ox8-OE1 line had the highest expression level, and the atga2ox8-OE3 line had the lowest expression level. Figure 11 (D). Gibberellin content determination revealed that OE3 had the lowest GA1 content, significantly lower than other strains, while the mutant, WT, and atga2ox8-OE3 had significantly higher GA1 content than other strains. Figure 11 The GA4 content was lowest in OE3 and atga2ox8-OE1, while the GA4 content in mutants and WT was significantly higher than in other lines. Figure 11 In the OE3 strain, the total content of GA1 and GA4 was the lowest, significantly lower than other lines. The total content of GA1 and GA4 in the mutant strain was significantly higher than in other lines. The total content of GA1 and GA4 in the WT strain was also significantly higher than in other transgenic lines. Figure 11 The results for the total active gibberellin content and relative expression level of each strain were consistent (G);

[0149] The mutants had shorter bolting time and fewer rosette leaves; the overexpression lines and the reintroduced lines had faster bolting time and more rosette leaves; the WT bolting time was also significantly faster than OE2, OE3, atga2ox8-OE1, and atga2ox8-OE2, while the number of rosette leaves was significantly less than that of OE1, OE2, OE3, atga2ox8-OE1, and atga2ox8-OE2. Figure 11 China A, Figure 12 (A and C); the phenotype was completely restored after spraying GA3. Figure 11 China A, Figure 12 (B and D). The mutant had the shortest flowering time and the fewest total leaves; the reintroduced line atga2ox8-OE1 had the longest flowering time; OE2, OE3, atga2ox8-OE1, and atga2ox8-OE2 had the most total leaves; WT had a significantly faster flowering time than OE2, OE3, atga2ox8-OE1, and atga2ox8-OE2, and also had fewer total leaves than OE2, OE3, atga2ox8-OE1, and atga2ox8-OE2. Figure 11 B, Figure 12 (E and G). Spraying with GA3 partially restored the flowering phenotype ( Figure 11 B, Figure 12(F and H); at flowering time, the mutant and WT were significantly higher than other lines, and at 7 weeks of age, the mutant was the highest, with WT significantly higher than the overexpression lines. After spraying with GA3, the phenotype was completely restored. Figure 11 C in the middle Figure 12 (Intra-IL). The results showed that GhGA2ox3 overexpression in Arabidopsis thaliana delayed development and dwarfed plant height, and exogenous GA spraying could partially or completely restore its phenotype. The expression levels and gibberellin contents of each strain were consistent with the bolting, flowering, and plant height phenotypes, indicating that GhGA2ox3 affects the Arabidopsis thaliana phenotype by reducing the content of active gibberellins GA1 and GA4 in Arabidopsis thaliana.

[0150] Besides delaying development and reducing plant height, transgenic Arabidopsis also affects root length and germination. Mutant Arabidopsis have longer roots, while overexpression results in shorter roots. Figure 13 (A and B). The germination rate of transgenic Arabidopsis thaliana was significantly reduced after 7 days. Figure 13 (C and D). The thousand-grain weight showed no significant difference among the transgenic lines OE1, OE2, atga2ox8-OE2, and atga2ox8-OE3. Figure 13 (E and F in the middle). The results show that GhGA2ox3 reduces plant height and also affects root length and germination in Arabidopsis thaliana, but does not change the thousand-seed weight of Arabidopsis thaliana.

[0151] 2.2.3 Determination of expression levels of gibberellin-related genes in transgenic Arabidopsis thaliana

[0152] Previous studies have reported that in Arabidopsis thaliana, the gene atga2ox8, which is involved in the gibberellin metabolic pathway, can reduce the height and gibberellin content of Arabidopsis thaliana; atga20ox1, atga20ox2, atga3ox1, and atga3ox2 can increase the height and gibberellin content of Arabidopsis thaliana; genes related to gibberellin signal transduction include AtGID1A, AtGAI, AtRGA, and AtRGL1, whose proteins can regulate gibberellin homeostasis in plants and are subject to feedback regulation by gibberellin content. Therefore, the expression of these genes in transgenic Arabidopsis thaliana was analyzed. Among them, the relative expression level of atga2ox8 was significantly reduced in overexpression lines and almost undetectable in overexpression lines with mutants and mutant backgrounds; the expression levels of atga20ox1, atga20ox2, atga3ox1, and atga3ox2 were increased; AtGID1A, AtGAI, AtRGA, and RGL1 ( Figure 14 The expression level was reduced. The results are consistent with the in vivo homeostasis of gibberellins and the phenotype, further verifying that GhGA2ox3 overexpression in Arabidopsis thaliana can reduce the content of active gibberellins in vivo.

[0153] 2.3 Functional verification of GhGA2ox3 in rice

[0154] 2.3.1 Detection of positive rice plants and qRT-PCR analysis

[0155] Rice lines that tested positive in the T0 generation were cultured on hygromycin-free 1 / 2 MS solid medium until tillering. Rice leaves were collected for testing, crude DNA was extracted, and band size was determined by PCR. The T1 generation test results are as follows: Figure 15 In the B-mode, no bands were observed when water was used as a template; when cotton cDNA was used as a template, a band of approximately 1083 bp was observed; when wild-type rice DNA was used as a template, no bands were observed; when OE1, OE2, and OE3 line DNA were used as templates, bands of approximately 1083 bp were observed, and the bands were uniform, indicating that the transgenic lines met the experimental requirements.

[0156] Leaf samples were collected for qRT-PCR analysis. Compared with the wild type, OE1 had the highest relative expression level, while OE2 had the lowest relative expression level, consistent with the observed phenotypic results. Figure 15 (A and C in the middle).

[0157] 2.3.2 Phenotypic Analysis of Transgenic Rice

[0158] Positive lines were cultured under the same conditions as wild-type, and phenotypic differences were observed. The length of the aboveground and underground parts and plant height of wild-type and transgenic rice at tillering were statistically analyzed. Seed length, width, and 100-seed weight of the T0 generation were also statistically analyzed. The aboveground and underground part lengths of transgenic rice at the seedling stage were significantly shorter than those of the wild type. Figure 15 China A, Figure 16 (A and B); plant height was significantly lower than that of the wild type ( Figure 15 China A, Figure 16 (C); T0 partially recovered the dwarf phenotype in the later stages of growth. OE2-positive lines showed no significant differences in grain length, grain width, and 100-grain weight compared to the wild type. Figure 16 (Df). The results showed that heterologous transformation of GhGA2ox3 in rice reduced rice plant height without changing rice yield.

[0159] 2.4VIGS inhibits the expression of GhGA2ox3

[0160] TRV:GhCLA1 plants begin to exhibit an albino phenotype at the two-leaf stage. Figure 17 (A) The albinism phenotype is obvious at the three-leaf and four-leaf stages. Figure 17 The results (B and C) indicate that the VIGS system is functioning. At the two-leaf, three-leaf, and four-leaf stages, the relative expression levels of TRV:GhGA2ox3 plants were significantly lower than those of the TRV:00 empty vector plants. Figure 17 (middle D); during the two-leaf stage ( Figure 17 (Middle A and E), three-leaf stage ( Figure 17 (Middle B and E), four-leaf stage ( Figure 17The expression of GhGA2ox3 in cotton plants (C and E) was significantly higher than that in cotton plants without the TRV:00 vector, indicating that inhibiting the expression of GhGA2ox3 in cotton can increase cotton plant height.

[0161] This invention detected gibberellin content in the stems of VIGS cotton at the four-leaf stage and found that the contents of active gibberellins GA1 and GA4, as well as the total contents of GA1 and GA4, in TRV:GhGA2ox3 plants at the four-leaf stage were significantly higher than those in TRV:00 unloaded plants. Figure 18 This indicates that inhibiting GhGA2ox3 expression can increase the content of active gibberellins in cotton, thereby promoting cotton stem elongation. Conversely, this demonstrates that GhGA2ox3 can reduce cotton plant height by decreasing the content of active gibberellins in cotton.

[0162] 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. A regulatory gene GhGA2ox3 Its application in reducing the height of upland cotton plants is characterized by, The regulatory gene GhGA2ox3 The nucleotide sequence is shown in SEQ ID NO.

1.

2. An application of a recombinant expression vector in reducing the plant height of upland cotton, characterized in that, The recombinant expression vector includes the regulatory gene described in claim 1. GhGA2ox3 .

3. The application of a recombinant microbial strain in reducing the plant height of upland cotton, characterized in that, The recombinant microbial strain includes the recombinant expression vector described in claim 2.

4. A method for reducing the height of upland cotton plants, characterized in that, Including upregulation of the regulatory genes as described in claim 1 GhGA2ox3 Steps for measuring expression levels in upland cotton.

5. The method according to claim 4, characterized in that, By the regulatory gene GhGA2ox3 Genetic transformation was introduced into upland cotton plants to obtain transgenic plants, which then upregulated the aforementioned regulatory genes. GhGA2ox3 Expression levels in upland cotton.

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