CaTAM, a cell cycle gene in peppers, and its applications

Abstract

This invention discloses a chili pepper cell cycle gene CatAM Its application and its DNA sequence are shown in SEQ ID No. 1. This gene was transformed into *Arabidopsis thaliana* using the Agrobacterium-mediated flower-dipping transformation method, yielding... CatAM Heterologous expression of Arabidopsis thaliana lines indicates that... CatAM Heterologous expression of these substances leads to earlier bolting time, increased leaf number, decreased leaf length and width, and longer root length in Arabidopsis thaliana. Silencing peppers using VIGS technology... CatAM The expression, obtained CatAM In plants with decreased expression, it can be inferred that... CatAM The silence of pollen cells prematurely exits meiosis, failing to undergo a second meiotic division and forming larger pollen grains. This indicates... CatAM It plays an important regulatory role in meiosis, bolting time regulation, leaf growth and primary root development. This gene can be applied to the breeding of solanaceous vegetables and other horticultural plants, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and particularly relates to a chili pepper cell cycle gene. CatAM And its applications. Background Technology

[0002] Meiosis is a special type of cell division that occurs during sexual reproduction. It involves one DNA replication and two cell divisions, resulting in gametes with half the number of chromosomes. In the first meiotic division, homologous chromosomes pair, recombine, and separate, while in the second meiotic division, sister chromatids separate. The transition from meiosis I to meiosis II... OSD1 and SO MUCH Genes play a crucial role.

[0003] Through analysis of different species OSD1 Gene studies have revealed their important functions in regulating the meiotic process. In Arabidopsis thaliana, OSD1 Genes can negatively regulate the activity of the anaphase-promoting complex / cycle body (APC / C) to control the meiotic process; loss of function leads to skipping the second meiotic division and directly forming diploid gametes. However, in watermelons and tomatoes... OSD1 Genes not only participate in meiosis in germ cells but also regulate mitosis in somatic cells. TAM, a type A cyclin highly expressed during meiosis, can directly promote CDK activity to control the meiotic process. Mutations in Arabidopsis thaliana... AtTAM The genes and somatic cell development showed no obvious defects, and the first meiotic division was unaffected. However, meiosis ended prematurely, and a second meiotic division was not performed, resulting in diploid gametes. Similarly, in tomatoes, SlTAM Mutants of this gene also exhibit a phenotype of premature meiosis termination and failure to undergo a second meiotic division, and this can be further demonstrated by editing other meiosis-related genes (such as...). SlSPO11-1 and SlREC8 Genes) constructed SlMiMe The mutant exhibited typical characteristics of synthetic apomixis, namely a phenotype in which mitosis replaces meiosis.

[0004] Chili peppers are an important spice crop and one of the world's most important vegetable crops. Due to their phylogenetic similarity to the model plant, tomato, chili peppers hold significant academic value in basic plant research. Chili pepper breeding primarily relies on traditional hybridization methods, but these methods require substantial annual investment of human and material resources. The application of engineered apomixis in fixing heterosis can significantly reduce seed production costs. Given the biological similarities between chili peppers and tomatoes, CatAMGenes have enormous potential applications in achieving synthetic apomixis and ploidy breeding in peppers. Furthermore, primary roots, as the fundamental components of the root system, play a crucial role in the early growth of plants. Leaves are the site of photosynthesis and are extremely important for vegetative growth. Therefore, in pepper breeding, CatAM Genes have broad application prospects and are beneficial to... CatAM In-depth research and application of genes can provide a more efficient technical approach for chili pepper breeding. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a chili pepper cell cycle gene. CatAM And its applications.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a chili pepper cell cycle gene. CatAM This gene CatAM The gene cloned from the chili pepper 'Hangjiao 9' has the following characteristics: (1) The nucleotide sequence shown in SEQ ID No. 1; or (2) The nucleotide sequence shown in SEQ ID No.1 has been substituted, deleted and / or added one or more nucleotides.

[0007] This invention provides a chili pepper cell cycle gene containing the above-mentioned gene. CatAM The biological material is an expression vector, expression cassette, host cell, or engineered bacteria.

[0008] This invention provides the above-mentioned chili pepper cell cycle gene. CatAM Applications in regulating plant meiosis, bolting time, leaf growth, and primary root development.

[0009] Furthermore, the application specifically includes: In Arabidopsis, heterologous overexpression resulted in earlier bolting, increased leaf number, decreased leaf length and width, and longer root length; silencing the expression in peppers... CatAM During gene generation, some pollen cells prematurely exit meiosis without undergoing a second meiotic division, resulting in the production of some large pollen grains.

[0010] This invention provides the above-mentioned chili pepper cell cycle gene. CatAM Application in the preparation of transgenic plants.

[0011] This invention provides the above-mentioned chili pepper cell cycle gene. CatAM Application in the improvement of plant germplasm resources.

[0012] The beneficial effects of this invention are: the pepper cell cycle gene provided by this invention CatAM The sequence is shown in SEQ ID No. 1; the gene was introduced into Arabidopsis thaliana using Agrobacterium-mediated transformation to obtain the pepper cell cycle gene. CatAM Heterologously expressed transgenic Arabidopsis thaliana lines were found to contain pepper cell cycle genes. CatAM Heterologous overexpression of [a specific gene] leads to earlier bolting time, increased leaf number, decreased leaf length and width, and longer root length in Arabidopsis thaliana; silencing gene silencing in peppers using virus-induced gene silencing (VIGS) technology [is also mentioned]. CatAM The expression, obtained CatAM In plants with reduced expression of this gene, some pollen cells prematurely exit meiosis, failing to undergo a second meiotic division, resulting in larger pollen grains. This indicates a deficiency in pepper cell cycle genes. CatAM Pepper cell cycle genes are closely related to the timing of bolting, leaf growth, primary root development, and meiosis in plants. CatAM It plays an important regulatory role in meiosis, bolting time regulation, leaf growth and primary root development. Applying this gene to the breeding of peppers or other solanaceous vegetables has good application prospects. Attached Figure Description

[0013] Figure 1 Chili pepper cell cycle genes CatAM CDS clone PCR electrophoresis image; Figure 2 A schematic diagram of the CaTAM subcellular localization vector; Figure 3 This is a diagram showing the subcellular localization results of CaTAM; among them, Figure 3 (a) shows the localization results of the CaTAM target gene protein and the nuclear marker in the control group; Figure 3 (b) shows the localization results of the CaTAM target gene protein and the control group membrane marker; Figure 4 for CatAM A schematic diagram of a VIGS gene silencing vector; Figure 5 for pTRV2 empty control plants and CatAM qPCR identification results of silent plants; Figure 6 for pTRV2 Empty control plants, pTRV2-CaPDS Positive control plants pTRV2-CaTAM Phenotype of silent plants; Figure 7 for pTRV2 empty control plants and pTRV2-CaTAM Alexandrine staining results of pollen from silent plants; Figure 8 for pTRV2 empty control plants and pTRV2-CaTAM Results of observations on chromosome behavior during meiosis in silent plants; among which, Figure 8 (a) in the text refers to chili peppers. pTRV2 Observation results of meiosis process in empty control plants; Figure 8 (b) in the text refers to chili peppers. pTRV2-CaTAM Observational results of meiosis in silent plants; Figure 9 for CatAM Schematic diagram of the overexpression vector; Figure 10 pFGC-1008 unloaded control group and CatAM Results of relative expression levels in heterologous Arabidopsis thaliana plants; Figure 11 pFGC-1008 unloaded control group and CatAM Root growth of Arabidopsis thaliana plants heterologously overexpressing the gene on days 7 and 14; among which, Figure 11 (a) in the figure represents the pFGC-1008 unloaded control group and CatAM Root growth phenotype of Arabidopsis thaliana plants on day 7 after heterologous overexpression; Figure 11 (b) in the figure represents the pFGC-1008 unloaded control group and CatAM Root growth phenotype of Arabidopsis thaliana plants on day 14 after heterologous overexpression; Figure 11 (c) in the figure represents the pFGC-1008 unloaded control group and CatAM Statistical graph of primary root length at 7 days in Arabidopsis thaliana plants that were heterologously overexpressed; Figure 11 In the figure, (d) represents the pFGC-1008 unloaded control group and CatAM Statistical graph of primary root length at 14 days in Arabidopsis thaliana plants that were heterologously overexpressed; Figure 12 pFGC-1008 unloaded control group and CatAM Leaf growth in Arabidopsis thaliana after fourth week of heterologous overexpression; among which, Figure 12 (a) in the figure represents the pFGC-1008 unloaded control group and CatAM Leaf growth phenotype of Arabidopsis thaliana in the fourth week after heterologous overexpression; Figure 12 (b) in the figure represents the pFGC-1008 unloaded control group and CatAM A statistical chart of leaf counts in Arabidopsis thaliana after the fourth week of heterologous overexpression; Figure 12 (c) in the figure represents the pFGC-1008 unloaded control group and CatAM Leaf length statistics of Arabidopsis thaliana with heterologous overexpression in the fourth week; Figure 12 In the figure, (d) represents the pFGC-1008 unloaded control group and CatAM Leaf width statistics of Arabidopsis thaliana with heterologous overexpression in the fourth week; Figure 13 pFGC-1008 unloaded control group and CatAM Leaf growth in Arabidopsis thaliana after six weeks of heterologous overexpression; among which, Figure 13 (a) in the figure represents the pFGC-1008 unloaded control group and CatAM Leaf growth phenotypes in Arabidopsis thaliana at week 6 after heterologous overexpression; Figure 13 (b) in the figure represents the pFGC-1008 unloaded control group and CatAM Bolting in Arabidopsis thaliana after six weeks of heterologous overexpression; Figure 13 (c) in the figure represents the pFGC-1008 unloaded control group and CatAM A statistical chart of leaf counts in Arabidopsis thaliana after six weeks of heterologous overexpression; Figure 13 In the figure, (d) represents the pFGC-1008 unloaded control group and CatAM Leaf length statistics of Arabidopsis thaliana with heterologous overexpression at week 6; Figure 13 (e) in the figure represents the pFGC-1008 unloaded control group and CatAM Statistical chart of leaf width in Arabidopsis thaliana after six weeks of heterologous overexpression. Detailed Implementation

[0014] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the invention as detailed in the appended claims.

[0015] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0016] It should be understood that although the terms first, second, third, etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, first information may also be referred to as second information without departing from the scope of this invention, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to a determination."

[0017] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.

[0018] This invention provides a chili pepper cell cycle gene CatAM The gene is a gene cloned from a chili pepper variety called 'Hangjiao 9', and its gene sequence is shown in SEQ ID No. 1.

[0019] The present invention also provides the above-mentioned chili pepper cell cycle gene. CatAM Its applications in regulating bolting time, leaf growth, primary root development, and meiosis are described in detail below.

[0020] Example 1: Construction of a subcellular localization vector for the pepper cell cycle gene CaTAM 1. RNA extraction using the Trizol method: Samples were taken from the leaves of 'Hangjiao No. 9' pepper, and RNA was extracted using the Trizol method. The number of samples was calculated, and appropriate mortars, pestles, and spatulas were prepared. Samples were wrapped in aluminum foil and baked at 180℃ for 4-5 hours. Centrifuge tubes and pipette tips (RNase-free) were then autoclaved at 121℃ for 40 minutes using liquid nitrogen and centrifuge plates. The appropriate number of centrifuge tubes were taken and numbered. These tubes were placed in a fume hood, with 1 ml of Trizol added to each tube, and placed on ice. Samples were transferred from liquid nitrogen to a pre-cooled mortar, and ground 3-5 times with liquid nitrogen until powder was formed, followed by thorough vortexing. 200 μl of chloroform was added to the fume hood, and the mixture was gently vortexed 15 times. The mixture was placed on ice for 5 minutes. Centrifuged at 4℃ and 12000 rpm for 10 minutes, and 500 μl of the powder was collected. Transfer the supernatant to a new 1.5 ml centrifuge tube, add 1 volume of isopropanol and mix by inversion. Incubate at 25°C for 10 min; centrifuge at 12000 rpm for 10 min at 4°C, and discard the supernatant; add 1 ml of pre-cooled diethyl pyrocarbonate (DEPC) dissolved in 75% ethanol, centrifuge at 12000 rpm for 5 min at 4°C, and discard the supernatant; repeat the washing once; centrifuge for 20 s, aspirate the liquid, and air dry in a fume hood. Dissolve the RNA precipitate in 50 μl of DEPC water and determine the concentration for later use.

[0021] 2. cDNA synthesis: ABclonal reverse transcription kit: The system contains 4.0 μl 5x ABScript III RT Mix, 1.0 μl 20x gDNA Remover Mix, 1.0 μg / μl Tatal RNA, and 14.0 μl Nuclease-free H2O. Place the kit in a PCR instrument and perform reverse transcription under the following conditions: 37℃ for 2 min, 55℃ for 15 min, and 85℃ for 5 min to obtain cDNA. Store the cDNA at -20℃ for later use.

[0022] 3. Obtaining the target gene and linearized vector: Specific primers as shown in Table 1 were designed, and the target gene and linearized vector were obtained through high-fidelity enzyme amplification. CatAM The target gene fragment is then recovered from the gel after electrophoresis, such as... Figure 1 As shown, M represents the DNA marker, and lanes 1, 2, 3, 4, and 5 represent the amplified products of the target fragment. The pFGC5941 vector was used... Bam HI and Xba The linearized vector fragment was recovered by double enzyme digestion, electrophoresis, and gel extraction.

[0023] Table 1: Primers used for the construction and detection of subcellular localization vectors

[0024] 4. Homologous recombination method for vector construction: The Novozymes ClonExpress II One Step Cloning Kit was used. The specific steps are as follows: Take 4 μL of 5×CEII Buffer, 2 μL of Exnase II, 200 ng of linearized vector, 60 ng of the CaTAM gene target fragment, and add ddH2O to 20 μL; react in a metal bath at 37°C for 30 min to obtain the homologous recombination product.

[0025] 5. Freeze-thaw transformation of *E. coli* competent cells DH5α: Thaw DH5α on ice, add homologous recombination product, and incubate on ice for 30 min; heat shock in a 42°C metal bath for 90 s, then cool on ice for 5 min; add 1 ml LB liquid medium, and incubate at 37°C for 1.5 h with shaking; centrifuge at 5000 rpm for 1 min, discard the filtrate, and mix the remaining approximately 100 μL of bacterial culture by aspirating and plating onto LB solid medium containing kanamycin; incubate the medium upside down in a 37°C incubator overnight, and the next day, pick single colonies for bacterial PCR detection (primers are shown in Table 1). After successful sequencing, extract the plasmid and store at -20°C for later use. To verify the correct alignment of the subcellular localization vector plasmid and the pFGC5941 empty vector plasmid, transform *Agrobacterium* competent cells GV3101 using the freeze-thaw method. The CaTAM subcellular localization vector is shown in Table 1. Figure 2As shown, after single bacteria grow, spots are picked for bacterial culture PCR detection (primers are shown in Table 1). The bacterial culture that is verified to be correct is preserved and the mother liquor is stored at 4°C for later use.

[0026] 6. Transient expression experiment in tobacco to observe subcellular localization: Add 100 μL of activated Agrobacterium bacterial suspension to 30 mL of liquid LB medium containing 50 mg / mL rifampicin (Rif), streptomycin (Str), and kanamycin (Kan), and incubate overnight at 28°C with shaking. Wait until the bacterial suspension reaches OD... 600 The concentration was approximately 1.0. Centrifuge at 5000 rpm for 15 min and discard the supernatant. Resuspend Agrobacterium in an equal volume of resuspension buffer (10 mmol / L MES, 10 mmol / L MgCl2, 150 μmol / L acetosyringone) and incubate at 25 °C for 3 h.

[0027] Select healthy, four-week-old tobacco plants, and choose three relatively flat leaves from each plant. Using a disposable 1mL syringe, inject the bacterial solution into the leaf on the underside, avoiding the veins, allowing the solution to spread to two-thirds of the leaf. Mark the injection site. After culturing the injected tobacco for 36 hours, cut a 1cm square section of leaf near the injection site, prepare a slide with the underside facing up, and observe the fluorescence signal and distribution under a laser confocal microscope.

[0028] The results of the CaTAM subcellular localization experiment are as follows: Figure 3 As shown, the localization results of the nuclear markers of the CaTAM target gene protein (GFP-CaTAM) and the control group (eGFP) are as follows: Figure 3 As shown in (a), the localization results of the membrane markers for the CaTAM target gene protein (GFP-CaTAM) and the control group (eGFP) are as follows: Figure 3 As shown in (b), green fluorescent protein (GFP) is expressed on the cell membrane, and red fluorescent protein (RFP) is expressed in the cell nucleus. The synthesis of these three fluorescent channels (GFP, RFP, and FFP) yields the corresponding combined image. This localization result indicates that CaTAM protein is expressed on both the cell membrane and nucleus of tobacco.

[0029] Example 2: Chili peppers CatAM Construction of VIGS silencing vector for genes A specific gene fragment was amplified using pepper cDNA as a template and the fragment was recovered by gel electrophoresis. The primers used are shown in Table 2. Homologous recombination was used to ligate the gene fragment into a gene that had been isolated from chili pepper cDNA. Bam H Ⅰ and SmaI. The pTRV2 vector, digested with double enzymes, was transformed into competent E. coli DH5α cells. After verification by bacterial PCR and sequencing confirming the correct gene fragment and ligation, the vector plasmid was extracted and stored at -20°C for later use. Specific steps are detailed in Example 1, "Freeze-thaw transformation of competent E. coli DH5α cells." The final constructed... CatAM VIGS silencing vectors for genes, such as Figure 4 As shown.

[0030] Table 2: Primers used for constructing the VIGS silencing vector

[0031] The plasmids of the heterologous expression vectors that were successfully verified were transformed into Agrobacterium competent cells GV3101 using the freeze-thaw method. After single colonies grew, plaques were picked for PCR amplification of the bacterial culture. The primers used for PCR amplification are shown in Table 3. The bacterial cultures that were successfully verified were preserved and the mother liquor was stored at 4°C for future use.

[0032] Table 3: Primers used for VIGS silencing vector detection and transgenic Arabidopsis thaliana PCR detection

[0033] Example 3: VIGS technology for silencing pepper genes and screening of silenced plants 1. VIGS technology to silence pepper genes: The above plasmids, verified by sequencing, pTRV1 , pTRV2 Empty vector plasmids and pTRV2-CaPDS The positive control plasmid was transformed into Agrobacterium GV3101 and silenced using VIGS technology. CatAM Gene inoculation, the steps are as follows: Add 50 μl of Agrobacterium tumefaciens bacterial suspension containing the target vector to 15 ml of liquid LB containing kanamycin and rifampin (50 mg / L), and incubate at 28°C and 200 rpm for about 30 hours until OD is reached. 600 1.2; Centrifuge at 5000 rpm for 10 min to obtain Agrobacterium precipitate; Resuspend the bacterial cells in 10 mM MgCl2 solution, centrifuge at 5000 rpm for 10 min, and repeat once; Prepare resuspension: 10 mM MgCl2, 10 mM MES (morpholine ethanesulfonic acid), 150 μM acetylsuccinone; Resuspend the bacterial cells in 20 ml of resuspension solution, and incubate at 28℃ in the dark for 3 h; [The text abruptly ends here, likely due to an incomplete translation or missing information.] pTRV2 No load or pTRV2 - The resuspension of the target gene was mixed with the pTRV1 Agrobacterium suspension at a ratio of 1:1 (v / v), and Silwet-77 was added to a final concentration of 0.05%. When the pepper seedlings were cultured to 2 to 3 weeks old, the mixed bacterial solution was injected into the cotyledons of the peppers using a syringe. The mixture was then incubated in the dark at 22°C for 24 hours, followed by normal culture.

[0034] 2. Selection of silent plants: Take samples one month later. pTRV2Empty control plants and 23 plants pTRV2-CaTAM Total RNA was extracted from leaves of the silent group plants and reverse transcribed into cDNA for qRT-PCR detection. Primers used for qPCR analysis were designed using Primer Premier 6, as shown in Table 4. The reaction mixture consisted of 15 μL: 7.5 μL SYBR Green Master Mix, 0.3 μL each of forward and reverse primers, 1 μL template, and 5.9 μL double-distilled water. The qPCR reaction procedure was: 95℃: 30 s, 50 cycles (95℃: 5 s, 55℃: 45 s). The specificity of the reaction was determined by melt curve analysis. The internal reference gene was [missing information]. CaUBI-3 The relative expression level of genes is determined by 2 -ΔΔCt Method calculation (three biological replicates were set at the time of sampling).

[0035] The results are as follows Figure 5 As shown, by Figure 5 show, CatAM Genes in silent plants and transferred pTRV2 Expression was downregulated in unloaded pepper plants compared to those without the control group.

[0036] Table 4: Primers used for qRT-PCR analysis of pepper

[0037] Example 4: CatAM Observation of pollen size and chromosome behavior during meiosis in silent plants 1. Alexandrite staining method for pollen size detection: Collect newly opened pepper flowers, use tweezers to shake the pollen attached to the anthers onto a glass slide with Alexandrite staining solution, cover with a coverslip, place in the dark for 10 minutes, and observe under a microscope.

[0038] 2. DAPI staining method for observing chromosome behavior: Flower buds during meiosis were taken and fixed in Carnoy's fixative for 24 hours, with the fixative changed continuously until the buds decolorized. The buds were then removed and rinsed three times with sterile water. The buds were then placed in an antibody enzyme solution (3% pectinase and 3% cellulase, dissolved in 0.01 mol·L⁻¹). -1 Incubate the enzyme solution in a citrate buffer (pH 4.5, diluted 1-fold before use) at 37°C for 15 min. Aspirate the enzyme solution and rinse three times with sterile water. Remove the sepals and petals under a dissecting microscope, leaving only the anthers. Add one drop of sterile water to keep the anthers moist, puncture the anthers with a dissecting needle, and squeeze out the pollen. Heat the slide at 45°C until only a thin water film remains, add 20 μl of 60% glacial acetic acid, pipette a few times, and let stand at room temperature for 1 min. Add 20 μl of pre-cooled Carnoy's fixative to the center of the glacial acetic acid; the fragments will spread out in a wavy pattern. After drying the slide at 45°C, add 20 μl of DAPI staining solution to the sample area, incubate in the dark for 10 min, and then observe under a microscope. The final result will be: pTRV2Empty control plants, pTRV2-CaPDS Positive control plants pTRV2-CaTAM Phenotypes of silent plants, such as Figure 6 As shown; pTRV2 empty control plants and pTRV2-CaTAM Alexandrine staining results of pollen from silent plants are as follows Figure 7 As shown, the first row of the scale is 200 μm, and the second row of the scale is 100 μm; pTRV2 The results of observation on the meiotic process of empty control plants are as follows: Figure 8 As shown in (a) in the figure, from left to right are the leptotene stage, pachytene stage, diplotene stage, diagenesis stage, metaphase I of meiosis, anaphase I of meiosis, telophase I of meiosis, and tetrad. The scale bar in the figure is 10 μm. pTRV2-CaTAM Observation results of meiosis in silent plants are as follows Figure 8 As shown in (b) in the figure, from left to right are the leptotene stage, pachytene stage, dipterygium stage, diagenesis stage, metaphase I of meiosis I, anaphase I of meiosis I, tetrad, and tetrad. The scale bar in the figure is 10 μm.

[0039] Depend on Figure 6 The results shown indicate that CatAM Gene silencing has little effect on vegetative growth, but it significantly affects pollen development, resulting in the formation of some larger pollen grains, such as... Figure 7 As shown. Further analysis indicates that... CatAM Gene silencing disrupts the normal process of meiosis, causing some cells to fail to enter the second meiotic division, thus directly forming a dichotomy, such as... Figure 8 As shown.

[0040] Example 5: Chili peppers CatAM Construction of heterologous expression vectors Gene fragments were amplified using chili cDNA as a template and recovered via gel electrophoresis. The primers used are shown in Table 5. Homologous recombination was then used to ligate the gene fragments into the target gene. Kpn I and Salt The pFGC1008 vector, which was digested with enzyme I, was transformed into competent E. coli DH5α cells. After verification by bacterial PCR and sequencing to confirm that the gene fragment and ligation were correct, the vector plasmid was extracted and stored at -20°C for later use. For specific steps, please refer to "Transformation of competent E. coli DH5α cells by freeze-thaw method" in Example 1.

[0041] Table 5: Primers used for heterologous expression vector construction

[0042] The plasmids of the heterologous expression vectors that were successfully verified were transformed into Agrobacterium competent cells GV3101 using the freeze-thaw method. After single colonies grew, plaques were picked for bacterial PCR. The primers used are shown in Table 6. The bacterial cultures that were successfully verified were preserved and the mother liquor was stored at 4°C for future use.

[0043] Table 6: Primers used for heterologous expression vector detection and transgenic Arabidopsis thaliana PCR detection

[0044] Example 6: Transformation of Arabidopsis thaliana by flower immersion method and screening of positive transformants 1. Transformation of Arabidopsis thaliana by flower soaking: such as Figure 9 As shown, 100 μL of activated Agrobacterium tumefaciens culture containing the heterologous expression vector plasmid and the pFGC-1008 empty vector plasmid was added to 30 mL of liquid LB medium containing 50 mg / mL Rifampicin, Streptomycin, and Chloramphenicol, respectively, and incubated overnight at 28°C with shaking. The culture was then incubated until the bacterial culture reached OD200. 600 When the concentration is around 1.0, centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend in an equal volume of resuspension (5% sucrose, 200 μL / L Silwet L-77), stirring thoroughly for 2 min. Remove the siliques and open flowers from the wild-type Arabidopsis thaliana, immerse the inflorescence in the bacterial solution for about 30 s, remove it, blot off excess bacterial solution with absorbent paper, and incubate in the dark with moisture for 24 h before placing it in an incubator for normal culture. One week later, repeat the flower immersion process once more to obtain more transgenic seeds.

[0045] 2. Preparation of hygromycin screening medium: Dissolve 2.215g MS519 dry powder and 10g sucrose (analytical grade) in water. Adjust the pH to 5.8 with 2M NaOH, add 4g agar powder (purified biochemical reagent), and bring the volume to 500mL. After autoclaving at 121℃ for 20min, cool to 50-60℃ in a clean bench, add hygromycin to a final concentration of 90mg / L, and pour into solid agar plates.

[0046] 3. Initial screening of positive transformants: Arabidopsis seeds obtained from flower immersion transformation were placed in 1.5 mL centrifuge tubes and sterilized by washing the seeds in a clean bench. The seeds were washed once with 1 mL ddH2O, once with 1 mL 75% ethanol, and three times with 1 mL ddH2O, repeating the entire washing process once. The washed seeds were then evenly spread on hygromycin selection medium. After approximately two weeks of normal culture, the normally growing plants were removed from the medium.

[0047] 4. Real-time quantitative PCR detection of transgenic Arabidopsis thaliana plants CatAMRelative gene expression levels: Sampling was performed from individual plants, selecting the third leaf from each plant. After labeling, the leaves were fixed in liquid nitrogen. Total RNA was extracted using the Omega Plant RNA Kit, and cDNA was synthesized using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser for qRT-PCR analysis. Real-time quantitative PCR system: 7.5 μL SYBR Green Master Mix, 0.3 μL each of forward and reverse primers, 1 μL template cDNA, and 5.9 μL ddH2O; the forward and reverse primers are shown in SEQ ID No. 10-SEQ ID No. 11. qRT-PCR reaction program: 95°C: 30 s; (95°C: 5 s; 57°C: 45 s) 40 cycles. Internal control gene selection... AtActin7 The primers used for qRT-PCR are shown in Table 7.

[0048] Table 7: CatAM Primers for positive detection of transgenic plants using real-time PCR

[0049] CatAM The results of the relative expression level detection in heterologous Arabidopsis thaliana plants are as follows: Figure 10 As shown, 23 plants CatAM Heterologous expression of Arabidopsis thaliana plants ( CatAM The relative expression levels of -OE were compared with those of the pFGC-1008 empty vector control group, indicating that all 23 Arabidopsis thaliana plants screened in the hygromycin selection medium were... CatAM The heterologous expression of positive transformants was high.

[0050] Example 7: CatAM Observation and statistics of bolting time, leaf growth, and primary root length of heterologous expression plants Filtered CatAM Overexpression-positive plants and empty-vector-transformed positive plants (control plants) were cultured in an incubator (temperature 25°C / 22°C; photoperiod L / D: 16h / 8h). Seeds were harvested from T1 positive plants. The harvested seeds were sown, and the T2 generation was observed at 4 and 6 weeks post-sowing. CatAM The growth differences between overexpression-positive plants and empty-transformation-positive plants were observed. Furthermore, the T2 generation... CatAM Overexpressing plants and control plants transformed without vector were sown on MS sowing medium containing 90 mg / L hygromycin for root system differences comparison. The results were photographed, recorded, and statistically analyzed.

[0051] CatAMThe root growth of heterologous overexpressing Arabidopsis plants and the pFGC-1008 empty control group on day 7 is as follows: Figure 11 As shown in (a) in the figure, CatAM The root growth of heterologous overexpressing Arabidopsis plants and the pFGC-1008 empty control group on day 14 is as follows: Figure 11 As shown in (b) in the figure, CatAM The 7-day primary root length of heterologous overexpressing Arabidopsis plants and the pFGC-1008 empty control group is as follows: Figure 11 As shown in (c), CatAM The 14-day primary root length of heterologous overexpressing Arabidopsis plants and the pFGC-1008 empty control group is as follows: Figure 11 As shown in (d) in the figure, by Figure 11 The results shown indicate that on day 7, CatAM Heterologous overexpression of Arabidopsis thaliana plants ( CatAM The primary root length (-OE) showed no significant difference from the pFGC-1008 empty control group, but by day 14, the root length had increased significantly. Figure 11 As shown, the numerical values ​​represent the mean ± standard error (n≥15), and the asterisk indicates the student's... t -Significance after test (***) p <0.001, ns indicates no significant difference).

[0052] In the fourth week CatAM Leaf growth of heterologous overexpressing Arabidopsis plants and the pFGC-1008 empty control group at week four is as follows: Figure 12 As shown in (a) in the figure, CatAM The number of leaves in Arabidopsis thaliana plants overexpressing the heterologous gene and the pFGC-1008 empty control group at week four is as follows: Figure 12 As shown in (b) in the figure, CatAM Leaf lengths of heterologously overexpressed Arabidopsis plants and the pFGC-1008 empty control group at week four are as follows: Figure 12 As shown in (c), CatAM Leaf width at week 4 between heterologous overexpression Arabidopsis plants and the pFGC-1008 empty control group is as follows: Figure 12 As shown in (d) in the figure, by Figure 12 The results shown indicate that CatAM Heterologously overexpressed Arabidopsis thaliana showed a significant increase in leaf number compared to the control group, while leaf length and width were significantly reduced. Figure 12 As shown, the numerical values ​​represent the mean ± standard error (n≥15), and the asterisk indicates the student's... t -Significance after test (*) p <0.05, **** p <0.0001).

[0053] In the sixth week, CatAM Leaf growth of heterologous overexpressing Arabidopsis plants and the pFGC-1008 empty control group at week 6 is as follows: Figure 13 As shown in (a) in the figure, CatAM The number of leaves in Arabidopsis thaliana plants overexpressing the heterologous gene and the pFGC-1008 empty control group at week 6 is as follows: Figure 13 As shown in (c), CatAM Leaf lengths at week 6 in heterologous overexpression Arabidopsis plants and the pFGC-1008 empty control group were as follows: Figure 13 As shown in (d) in the figure, CatAM Leaf width at week 6 between heterologous overexpression Arabidopsis plants and the pFGC-1008 empty control group is as follows: Figure 13 As shown in (e), by Figure 13 The results shown indicate that CatAM The number of leaves in heterologously overexpressed Arabidopsis thaliana was not significantly different from that in the control group, but the leaf length and leaf width continued the trend of the fourth week, showing a significant decrease. Figure 13 As shown, the numerical values ​​represent the mean ± standard error (n≥45), and the asterisk indicates the student's... t -significance after test (**) p <0.01, **** p <0.0001, ns indicates no significant difference); at the same time, compared with the control group, CatAM Heterologous overexpression advances the bolting time in Arabidopsis thaliana, such as... Figure 13 As shown in (b) of the diagram.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chili pepper cell cycle gene CatAM The application of genes in regulating bolting time, leaf growth, and primary root development in plants is characterized by, The CatAM The gene sequence is the nucleotide sequence shown in SEQ ID No. 1; the plant is a chili pepper.

2. The application according to claim 1, characterized in that, The regulation is achieved through heterologous overexpression of the sequence shown in SEQ ID No.

1. CatAM Genetic manipulation leads to earlier bolting time, increased leaf number, decreased leaf length and width, and increased root length in plants; or The regulation is achieved by silencing the endogenous substances in chili peppers. CatAM Genetic activation causes some pollen cells to prematurely exit meiosis, failing to undergo a second meiotic division and forming larger pollen grains.

3. A chili pepper cell cycle gene as described in claim 1 CatAM The application of genes in the improvement of plant germplasm resources is characterized by, Through overexpression or silence CatAM Genes that regulate bolting time, leaf size, root development, or meiosis; the plant in question is a chili pepper.

Images