The invention relates to a soapberry SmCYCD3; gene 2 and application thereof

Abstract

The invention relates to the field of plant genetic engineering, and particularly discloses a soapberry SmCYCD3; 2 gene and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO: 1, and the sequence of the protein coded by the gene is shown as SEQ ID NO: 2. Experiments show that the gene has high specificity expression in female flowers of soapberry. By heterologous overexpression of the gene in arabidopsis thaliana, typical'female promotion and male inhibition 'phenotypes such as pistil elongation, stamen number reduction and shortening, pollen viability reduction and small fruit pods of transgenic plants can be caused. Molecular mechanism studies show that: SmCYCD3; 2, performing functions by up-regulating expression of cell cycle related genes and pistil development genes AtCRC and down-regulating expression of stamen development key genes at the same time; the invention provides a key gene resource and a theoretical basis for directionally regulating and controlling the sex ratio of plant flowers through a molecular breeding means and improving the fruit yield of woody economic tree species.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to a method for obtaining plant genetic material from Sapindus mukorossi (Soapberry). Sapindus mukorossi The SmCYCD3;2 gene and its encoded protein were cloned from [the gene name missing]. Furthermore, this invention relates to the application of this gene in regulating the development of plant floral organs, particularly in increasing the ratio of male to female flowers, and its use in molecular breeding to increase the fruit yield of woody plants. Background Technology

[0002] Sex differentiation in plant flowers is one of the core issues in developmental biology, directly affecting the yield and quality of crop fruits and seeds. For monoecious plants with separate male and female flowers, the ratio of female to male flowers is a key agronomical trait influencing yield.

[0003] Soapberry ( Sapindus mukorossi Sapindus mukorossi is an important woody industrial raw material tree species. Its fruits are rich in saponins and oils, and have extremely high economic value in fields such as bioenergy, daily chemical products, and pharmaceuticals. However, in production, Sapindus mukorossi generally suffers from low fruit yield and unstable quality. The core reason is that, as a monoecious plant with separate male and female flowers, Sapindus mukorossi naturally exhibits a severely imbalanced ratio of female to male flowers (approximately 1:10), resulting in a low number of female flowers and a low fruit set rate. Therefore, how to effectively improve the ratio of female to male flowers in Sapindus mukorossi has become a key bottleneck for the development of its industry.

[0004] Currently, research on the molecular mechanisms of plant sex differentiation is mostly focused on herbaceous model plants such as cucumber and melon, and several key sex-determining genes have been identified. However, research on sex differentiation in woody plants is relatively lagging, mainly focusing on morphological, hormonal regulation, and cellular observations, lacking a complete molecular regulatory model.

[0005] Cyclins, particularly the D3 isoform cyclin (CYCD3), are key factors regulating the transition of cells from G1 to S phase, influencing not only the development of vegetative organs but also the formation of reproductive organs. Studies in Arabidopsis thaliana have shown that overexpression of AtCYCD3;1 alters leaf morphology and inhibits cell differentiation; in rice, OsCYCD3;1 participates in maintaining meristematic tissue activity to regulate branching. Furthermore, CYCD3 genes have been shown to affect female gametophyte cell division and pistil tissue differentiation. However, these functional studies have been limited to herbaceous plants; the function of CYCD3 genes in woody plants, especially their role in sex determination, remains a blank, with no reports to date.

[0006] Therefore, existing technologies cannot provide an effective solution for actively regulating the ratio of male and female flowers in Sapindus mukorossi through molecular means, thereby increasing fruit yield.

[0007] This invention aims to fill this gap by cloning and functionally analyzing the SmCYCD3;2 gene, verifying its specific role in sex differentiation, and providing key genetic resources and theoretical basis for molecular design breeding of Sapindus mukorossi. Summary of the Invention

[0008] Therefore, the primary objective of this invention is to provide a novel Sapindus mukorossi SmCYCD3;2 gene and its encoded protein.

[0009] Another objective of this invention is to provide the application of the above-mentioned genes in regulating the sexual differentiation of plant flowers, particularly by promoting the development of pistils and inhibiting the development of stamens, thereby effectively increasing the ratio of male to female flowers in plants.

[0010] Another objective of this invention is to provide a method for increasing plant fruit yield using the aforementioned gene, providing key gene resources and theoretical basis for molecular design breeding of Sapindus mukorossi and other woody economic tree species.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an isolated Sapindus mukorossi SmCYCD3;2 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0012] In a second aspect, the present invention provides a protein encoded by the above-mentioned gene, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0013] Thirdly, the present invention provides a recombinant expression vector containing the SmCYCD3;2 gene, a host cell, and a transgenic plant cell.

[0014] Fourthly, the present invention provides a specific primer set for cloning and detecting the SmCYCD3;2 gene.

[0015] The specific primer set includes at least one of the following primer pairs: a) Cloning primer pairs, the sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4; b) Cloning primer pairs containing restriction enzyme sites, the sequences of which are shown in SEQ ID NO: 5 and SEQ ID NO: 6; c) Real-time quantitative PCR primer pairs, the sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0016] Fifthly, the present invention provides a method for regulating the sex ratio of plant flowers, the method comprising increasing the expression level of the SmCYCD3;2 gene in the plant.

[0017] The regulation of flower sex ratio is manifested by one or more of the following: promoting pistil elongation, reducing the number of stamens, and decreasing pollen viability.

[0018] In a sixth aspect, the present invention provides a method for increasing the yield of plant fruits, the method being achieved by implementing the above-described method for regulating the sex ratio of flowers.

[0019] In a seventh aspect, the present invention provides the application of the SmCYCD3;2 gene or a recombinant vector containing it in the breeding of plant varieties with a high proportion of female flowers.

[0020] This application also provides a method for producing transgenic plants, comprising introducing the Sapindus mukorossi SmCYCD3;2 gene or the recombinant vector into plant cells and regenerating transgenic plants, wherein the ratio of male to female flowers in the transgenic plants is higher than that in the wild type.

[0021] Compared with the prior art, the present invention has the following significant advantages: This invention is the first to clone and functionally identify the SmCYCD3;2 gene in the woody plant Sapindus mukorossi, revealing that the gene has an unprecedented bidirectional regulatory function of "promoting females and suppressing males", providing a new perspective for the study of plant sex determination mechanisms.

[0022] This invention, through the construction of an overexpression vector and heterologous expression in Arabidopsis thaliana, combined with detailed phenotypic observation and molecular detection, systematically and powerfully verified the function of the gene, and established a complete gene function research system.

[0023] This invention provides a direct gene target and effective technical means to solve the core industrial problem of low female flower count and low yield in Sapindus mukorossi. By introducing and expressing this gene, it is possible to selectively breed new Sapindus mukorossi varieties with a higher proportion of female flowers, thereby significantly increasing fruit yield and possessing significant economic value and market potential. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is an agarose gel electrophoresis image of the CDS sequence clone of the Sapindus mukorossi SmCYCD3;2 gene of this invention.

[0026] Figure 2 This is a multiple sequence alignment diagram of CYCD3 proteins from different species.

[0027] Figure 3 This is a phylogenetic tree analysis of CYCD proteins from different species.

[0028] Figure 4 This is a graph showing the relative expression levels of the SmCYCD3;2 gene in different tissues of Sapindus mukorossi.

[0029] Figure 5 In the diagram, A is a schematic diagram of the SmCYCD3;2 gene overexpression vector constructed in this invention; B is a schematic diagram of the screening of transgenic Arabidopsis thaliana positive seedlings; C is a diagram of the RT-qPCR detection results of the relative expression level of the SmCYCD3;2 gene in transgenic Arabidopsis thaliana lines.

[0030] Figure 6 This is a phenotypic comparison between transgenic Arabidopsis thaliana overexpressing the SmCYCD3;2 gene and wild-type Arabidopsis thaliana.

[0031] Figure 7 This is a graph showing the relative expression levels of endogenous cell cycle and floral organ development-related genes in transgenic Arabidopsis thaliana overexpressing the SmCYCD3;2 gene. Detailed Implementation

[0032] The following description is based on specific embodiments.

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. In the present invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0034] Example 1 1. Experimental Materials Plant material: Sapindus mukorossi ( Sapindus mukorossi Female flower samples were collected from the Sapindus mukorossi National Germplasm Resource Bank in Jianning County, Sanming City, Fujian Province. The samples were flash-frozen in liquid nitrogen and then stored at -80℃ for later use.

[0035] Main reagents: Universal Plant Total RNA Isolation Kit (purchased from Nanjing Novizan Co., Ltd.); TianScript II RT Kit (purchased from Nanjing Novizan Co., Ltd.); high-fidelity DNA polymerase; agarose gel DNA recovery kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0036] Instruments and equipment: NanoDrop 8000 micro-volume spectrophotometer (Thermo Fisher Scientific, USA); PCR instrument; agarose gel electrophoresis system.

[0037] 2. Experimental Methods 2.1 Total RNA extraction and cDNA first-strand synthesis Accurately weigh 100 mg of Sapindus mukorossi female flower tissue and rapidly grind it into powder in liquid nitrogen. Extract total RNA according to the instructions of the Universal Plant Total RNA Isolation Kit. After extraction, use a NanoDrop 8000 to detect the concentration and purity of RNA. The A260 / A280 ratio should be between 1.8 and 2.0, and the A260 / A230 ratio should be greater than 2.0 to ensure the RNA quality meets standards.

[0038] Take 1 μg of high-quality total RNA and perform genomic DNA removal and reverse transcription strictly according to the TianScript II RT Kit instructions to synthesize the first strand of cDNA. The resulting cDNA was stored at -20℃ for subsequent gene cloning.

[0039] 2.2 Cloning of the coding region (CDS) of the SmCYCD3;2 gene Primer design and synthesis: Based on the annotation information of SmCYCD3;2 (gene ID: Samuk02G0146500) obtained from the previous transcriptome database, its predicted CDS sequence was obtained. Specific primers for cloning the complete CDS were designed using Primer3plus online software. The nucleotide sequences of the primers are SEQ ID NO: 3 and SEQ ID NO: 4. The primers were synthesized by Riboxing Biotechnology (Beijing) Co., Ltd.

[0040] PCR amplification: Using the female flower cDNA synthesized in step 2.1 as a template, PCR amplification was performed using high-fidelity DNA polymerase.

[0041] The reaction system (50 μL) is as follows: cDNA template, 2.0 μL; upstream primer (10 μM), 2.0 μL; downstream primer (10 μM), 2.0 μL; 2× high-fidelity PCR premix, 25.0 μL; ddH2O, to a final volume of 50.0 μL.

[0042] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min 30 s, for a total of 35 cycles; and finally 72℃ final extension for 5 min.

[0043] Product validation and recovery: 5 μL of PCR product was subjected to 1.2% agarose gel electrophoresis. Under UV light, a single, clear, specific amplification band was observed at approximately 1000 bp (e.g., ...). Figure 1 As shown in the image, the band was consistent with the expected size of 1092 bp. The target band was then recovered by gel cutting using an agarose gel DNA recovery kit.

[0044] Sequencing verification: The purified PCR product was sent to Riboxin Biotechnology (Beijing) Co., Ltd. for Sanger bidirectional sequencing. The sequencing results were compared with the transcriptome sequence to confirm that the cloned gene sequence was completely correct. Its nucleotide sequence is shown in SEQ ID NO: 1. This sequence encodes a protein composed of 364 amino acids, and its amino acid sequence is shown in SEQ ID NO: 2.

[0045] 3. Results and Analysis Through the above experiments, the full-length CDS of the Sapindus mukorossi SmCYCD3;2 gene was successfully cloned. Bioinformatics analysis showed that: Sequence characteristics: The gene is 1092 bp in length and encodes 364 amino acids.

[0046] Conservative structural domains: such as Figure 2 As shown, multiple sequence alignment results indicate that the SmCYCD3;2 protein contains typical Cyclin_N and Cyclin_C domains, and has a conserved LxCxE motif near the N-terminus that binds to RBR proteins, suggesting that it is a functionally typical D-type cell cycle protein.

[0047] System evolution analysis: such as Figure 3 As shown, the constructed phylogenetic tree clearly clusters SmCYCD3;2 in the CYCD3 subfamily, and also associates it with the woody model plant Populus tomentosa (…). Populus trichocarpa It is most closely related to CYCD3, which provides an evolutionary basis for the study of its function in woody plants.

[0048] This embodiment successfully obtained the correct coding sequence of the Sapindus mukorossi SmCYCD3;2 gene, and preliminarily predicted its function through bioinformatics analysis, laying the foundation for subsequent functional verification experiments.

[0049] Example 2 This embodiment describes in detail the tissue-specific expression analysis of the SmCYCD3;2 gene.

[0050] 1. Experimental Objective The expression level of the SmCYCD3;2 gene in different tissues and organs (roots, stems, leaves, female flowers, and male flowers) of Sapindus mukorossi was detected by real-time quantitative PCR (RT-qPCR) to clarify its expression specificity and provide key clues for exploring its biological function.

[0051] 2. Experimental Materials Plant materials: root, stem, leaf, female flower (FF), and male flower (MF) tissues of Sapindus mukorossi. Three biological replicates were set up for each tissue, and each biological replicate consisted of a mixture of corresponding tissues from three clones with consistent growth status.

[0052] Main reagents: Universal Plant Total RNA Isolation Kit (Nanjing Novozymes Co., Ltd.); TianScript II RT Kit (Nanjing Novozymes Co., Ltd.); SYBR Green Pro Taq HS premixed qPCR kit (Nanjing Novozymes Co., Ltd.).

[0053] Instruments and equipment: NanoDrop 8000 micro-volume spectrophotometer (Thermo Fisher Scientific, USA); Real-time quantitative PCR instrument.

[0054] 3. Experimental Methods 3.1 Total RNA extraction and cDNA synthesis Approximately 100 mg of each tissue material was taken and rapidly ground in liquid nitrogen. Total RNA was extracted from the roots, stems, leaves, female flowers, and male flowers of *Sapindus mukorossi* according to the instructions of the RNA extraction kit. After extraction, the concentration and purity of the RNA were measured using a NanoDrop 8000 to ensure that all samples met the required RNA quality (A260 / A280 ≈ 1.8-2.0).

[0055] Take 1 μg of total RNA from each tissue, remove genomic DNA using the TianScript II RT Kit, and reverse transcribe to synthesize the first strand of cDNA. Dilute all cDNA templates to the same concentration and store at -20℃ for later use.

[0056] 3.2 Real-time quantitative PCR (RT-qPCR) Primer design: Specific RT-qPCR primers were designed and synthesized targeting the CDS sequence of the SmCYCD3;2 gene. The nucleotide sequences of the primers are SEQ ID NO: 7 and SEQ ID NO: 8.

[0057] The Sapindus mukorossi SmACTIN gene (Gene ID: Samuk13G0061200) was used as an internal reference gene for standardization.

[0058] qPCR reaction: Amplification was performed using the SYBR Green Pro Taq HS premixed qPCR kit on a real-time quantitative PCR instrument.

[0059] The reaction mixture (20 μL) consisted of: 10.0 μL of 2× SYBR Green Pro Taq HS Premix; 0.4 μL of upstream primer (10 μM); 0.4 μL of downstream primer (10 μM); 2.0 μL of cDNA template; and 7.2 μL of ddH2O. Reaction procedure: 95℃ pre-denaturation for 30 s; followed by 40 cycles of 95℃ denaturation for 5 s, 60℃ annealing / extension for 30 s; finally, melting curve analysis was performed to verify the specificity of the amplified product.

[0060] Experimental design: Three technical replicates were set up for each cDNA sample, and a template-free control (NTC) was set up to exclude contamination.

[0061] 3.3 Data Analysis Use 2 -ΔΔCt The relative expression levels of the SmCYCD3;2 gene in various tissues were calculated using Student's method. t -Text test to analyze the significance of differences between groups.

[0062] 4. Results and Analysis RT-qPCR analysis results are as follows Figure 4 As shown, the expression level of the SmCYCD3;2 gene differed significantly among different tissues of Sapindus mukorossi. Its expression level was highest in female flowers (FF), significantly higher than in other tissues (P<0.01); the expression level was second highest in leaves; the expression level was lower in roots and stems; and the expression level was lowest in male flowers (MF), significantly lower than in female flowers. This expression pattern is highly consistent with the trend shown by previous transcriptome sequencing data (FPKM values), fully confirming that the SmCYCD3;2 gene has a significant female-biased expression characteristic.

[0063] This embodiment confirms that the SmCYCD3;2 gene is specifically highly expressed in the reproductive organs of Sapindus mukorossi, especially in female flowers where the expression level is significantly higher than that in male flowers.

[0064] Given its characteristics as a cell cycle protein, this result strongly suggests that the SmCYCD3;2 gene may play an important role in the development or sex differentiation process of female Sapindus mukorossi flowers, providing direct evidence for subsequent functional verification through heterologous transformation.

[0065] This embodiment reveals the pattern of high expression of the SmCYCD3;2 gene in female flowers. In the breeding population, by detecting the expression level of the SmCYCD3;2 gene or its related molecular markers (such as CAPS and SSR markers developed based on its sequence), superior individual plants with a high proportion of female flowers can be screened early and accurately, which can greatly shorten the breeding cycle and improve the selection efficiency.

[0066] Example 3 This embodiment details the construction of the SmCYCD3;2 gene overexpression vector and its genetic transformation in Arabidopsis thaliana.

[0067] 1. Experimental Objective A plant overexpression vector for the SmCYCD3;2 gene driven by the strong promoter of cauliflower mosaic virus 35S (CaMV 35S) was constructed, and transgenic plants were obtained by transforming Arabidopsis thaliana, a model plant, using the Agrobacterium-mediated flower immersion method, providing materials for subsequent functional studies.

[0068] 2. Experimental Materials Genes and vectors: The purified product of the SmCYCD3;2 gene verified by sequencing in Example 1; the plant binary expression vector pCAMBIA1301 (with a hygromycin resistance selection marker).

[0069] Strains and competent cells: Escherichia coli DH5α competent cells; Agrobacterium tumefaciens GV3101 competent cells.

[0070] Plant material: wild-type Arabidopsis thaliana ecotype Columbia-0 (Col-0).

[0071] Main reagents and culture media: restriction endonucleases NcoI and BstEII (NEB Biotechnology, USA); seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.); plasmid miniprep kit (Nanjing Novizan Biotechnology Co., Ltd.); LB liquid / solid medium; 1 / 2 MS solid medium; hygromycin B.

[0072] 3. Experimental Methods 3.1 Construction of overexpression vectors Vector linearization: The empty pCAMBIA1301 vector was linearized at the multiple cloning site by double digestion with restriction endonucleases NcoI and BstEII. The digestion products were purified by agarose gel electrophoresis and then used for further processing.

[0073] Insert preparation: Using the SmCYCD3;2 gene fragment cloned in Example 1 as a template, PCR amplification was performed using specific primers with a terminal homologous arm to the linearized pCAMBIA1301 vector. The nucleotide sequences of the primers are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0074] Homologous recombination ligation: The purified linearized vector was mixed with the PCR amplified fragment of the SmCYCD3;2 gene at the ratio recommended in the seamless cloning kit instructions, and incubated at 50℃ for 30 minutes to construct a recombinant overexpression vector named CaMV35S-pCAMBIA1301-SmCYCD3;2 (see schematic diagram of the vector structure). Figure 5 (A)

[0075] Transformation and Validation: The homologous recombination product was heat-shocked into *E. coli* DH5α competent cells and screened on LB agar plates containing kanamycin. Single colonies were picked for colony PCR validation, and positive clones were sent to a biotechnology company for sequencing. High-purity plasmids were extracted from correctly sequenced strains using a plasmid miniprep kit for later use.

[0076] 3.2 Agrobacterium-mediated transformation The correctly extracted recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells using electroporation. The plasmid was then plated on LB agar plates containing rifampin and kanamycin and incubated upside down at 28°C for 2 days. Single colonies were picked for PCR verification to confirm the presence of the target Agrobacterium engineered strain.

[0077] 3.3 Arabidopsis genetic transformation and positive seedling screening Arabidopsis thaliana culture: Wild-type Arabidopsis thaliana (Col-0) seeds were sown in nutrient soil and cultured in an artificial climate chamber (12 hours of light / 12 hours of darkness, 22°C) until bolting and flowering.

[0078] Transformation by flower immersion method: Take a healthy Agrobacterium strain and resuspend it in an infiltration solution (containing 5% sucrose and 0.03% Silwet L-77) to OD. 600 ≈ 0.8. Immerse the Arabidopsis inflorescence in the bacterial suspension and gently agitate for 30-45 seconds. After infection, place the plants horizontally in the dark for 24 hours, then resume normal photoperiod culture.

[0079] Transgenic seed screening: Mature seeds (T1 generation) are harvested after approximately 4-5 weeks. T1 generation seeds are sterilized with ethanol and sodium hypochlorite solution and then evenly sown on 1 / 2 MS solid selection medium containing hygromycin B (25 mg / L). After vertical cultivation in a light incubator for 7-10 days, resistant seedlings with normal root development and green cotyledons (e.g., ...) are observed and selected. Figure 5 As shown in Figure B), it was transplanted into nutrient soil to continue growing.

[0080] Molecular identification of transgenic plants: After the transplanted seedlings have grown new leaves, genomic DNA or total RNA is extracted from the leaves. The integration and expression of the SmCYCD3;2 gene are detected by PCR or RT-qPCR. Figure 5 As shown in Figure C, RT-qPCR analysis revealed that the expression level of the SmCYCD3;2 gene in the selected T3 generation homozygous lines was 15 to 55 times that of wild-type Arabidopsis, and transgenic Arabidopsis lines overexpressing SmCYCD3;2 were successfully obtained.

[0081] This embodiment successfully constructed a plant overexpression vector for the SmCYCD3;2 gene and obtained multiple homozygous transgenic lines that stably and efficiently express this gene in Arabidopsis thaliana through Agrobacterium-mediated genetic transformation. This lays a solid material foundation for further research on the biological function of the SmCYCD3;2 gene in model plant systems.

[0082] By directly introducing and overexpressing the SmCYCD3;2 gene, which has the function of "promoting females and suppressing males", the reproductive growth allocation of Sapindus mukorossi can be improved from the genetic root, which is an effective way to achieve high-yield breeding of Sapindus mukorossi.

[0083] Example 4 This embodiment describes in detail the phenotypic observation and analysis of transgenic Arabidopsis thaliana.

[0084] 1. Experimental Objective Based on obtaining homozygous transgenic Arabidopsis lines, we systematically observed and analyzed the effects of overexpression of the SmCYCD3;2 gene on vegetative growth, floral organ development, pollen viability and reproductive output of Arabidopsis, thereby verifying the biological function of this gene in a model plant.

[0085] 2. Experimental Materials Plant materials: Three homozygous T3 generation Arabidopsis thaliana lines (OE-1, OE-3, OE-5) with SmCYCD3;2 overexpression (OE) obtained in Example 3 and wild-type Arabidopsis thaliana (WT, Col-0).

[0086] Main reagents and instruments: Alexander staining solution (Beijing Qingke Biotechnology Co., Ltd.); pollen germination medium; scanning electron microscope; stereomicroscope; digital camera.

[0087] 3. Experimental Methods 3.1 Observation of vegetative growth phenotype Transgenic lines and wild-type Arabidopsis thaliana were cultured simultaneously under identical environmental conditions (12 hours of light / 12 hours of darkness, 22°C). The following indicators were observed and recorded periodically throughout the growth cycle: Growth rate: Observe the growth rate of rosette leaves and the rate of bolting.

[0088] Plant height: After the plant has stopped growing, measure the length from the base of the rosette leaves to the top of the main inflorescence.

[0089] Leaf and stem morphology: Carefully observe and record the shape, size, curling of the stem leaves, and whether the stem nodes are bent.

[0090] 3.2 Observation of floral organ phenotypes During the peak flowering period, the floral organs of wild-type and transgenic plants were carefully observed and compared using a stereomicroscope, with a focus on: Pistil: used to measure the length of the style.

[0091] Stamens: Count the number of stamens in a single flower, and observe the length of the filaments and the size, shape, and dehiscence of the anthers.

[0092] Classification statistics: Based on the observed abnormal phenotypes, the flowers of transgenic plants were classified (e.g., Type 1: pistil elongation with pollination impairment; Type 2: reduced stamens or low pollen viability).

[0093] 3.3 Pollen viability and morphology detection Alexandrine staining: Mature anthers about to dehisce from wild-type and transgenic plants were taken separately, placed on glass slides, and Alexandrine staining solution was added. After staining at room temperature for 10-15 minutes, the staining was observed under an optical microscope. Viable pollen was stained red, while inactive or immature pollen appeared blue or green. Pollen viability was counted by randomly selecting fields of view.

[0094] In vitro pollen germination experiment: Fresh anthers were gently smeared on the surface of pollen germination medium and incubated at 25°C in the dark for 4-6 hours. The germination rate and length of pollen tubes were then observed and counted under a microscope.

[0095] Scanning electron microscopy observation: Mature anthers were fixed with 2.5% glutaraldehyde, dehydrated by gradient ethanol, critical point drying and gold sputtering, and the surface ultrastructure of the pollen was observed using a scanning electron microscope.

[0096] 3.4 Statistical analysis of pod and seed phenotypes During the plant's maturity period, the main inflorescence pods of wild-type and transgenic plants were harvested, and the length and size of the pods were compared. The number of seeds in each pod was also counted.

[0097] 4. Results and Analysis The results are as follows Figure 6 As shown, compared with the wild type, Arabidopsis thaliana overexpressing the SmCYCD3;2 gene exhibited the following series of significant and stable phenotypic changes: 4.1 Abnormal vegetative growth: All transgenic lines exhibited stunted growth, with final plant height significantly lower than the wild type. Figure 6 (A, B). The stem leaves are significantly enlarged and exhibit an inward curling phenotype, and most plants show a curved shape at the stem nodes.

[0098] 4.2 Significant malformations of floral organs: In some transgenic flowers (Type 1), the pistil (style) is abnormally elongated, far exceeding that of the wild type; at the same time, the stamens are shortened, and the anthers are small, do not dehisce, or contain very little pollen, making self-pollination impossible. Figure 6 (C)

[0099] In another group of flowers (Type 2), the number of stamens tends to decrease (5 stamens appear), and pollen development is poor. Figure 6 (C)

[0100] 4.3 Pollen viability and morphological defects: Alexandrine staining showed that almost all wild-type pollen was stained bright red, indicating vigorous growth; while the pollen of transgenic plants was mostly stained green, indicating a loss of viability. Figure 6 (D).

[0101] In vitro germination experiments further confirmed that the pollen germination rate of transgenic plants was extremely low, and the germinated pollen tubes were short. Figure 6 (D).

[0102] Scanning electron microscopy revealed that wild-type pollen grains are plump and regularly spherical; while transgenic pollen grains are generally shrunken and collapsed, with blurred surface patterns. This morphological defect is the direct cause of their low viability. Figure 6 (D).

[0103] 4.4 Severely Impaired Reproductive Output: Due to pollination failure and poor seed development, the pods produced by transgenic plants were generally short and weak, containing significantly fewer seeds than the wild type. Figure 6 ).

[0104] This embodiment, through systematic phenotypic analysis, confirms that heterologous overexpression of the Sapindus mukorossi SmCYCD3;2 gene leads to a series of comprehensive phenotypes in Arabidopsis, including growth inhibition, pistil elongation, stamen development inhibition, pollen viability loss, and decreased seed set. These results, especially the typical phenomena of "promoting female development" (pistil elongation) and "suppressing male development" (reduced stamens and pollen abortion), strongly demonstrate that the SmCYCD3;2 gene plays a key role in regulating plant floral organ development and sex differentiation, exhibiting a clear function of promoting female development and suppressing male development.

[0105] Example 5 This embodiment describes in detail the analysis of endogenous gene expression in transgenic Arabidopsis thaliana.

[0106] 1. Experimental Objective By detecting changes in the expression of endogenous genes in transgenic Arabidopsis inflorescences, this study explores the mechanism by which the SmCYCD3;2 gene causes abnormal floral organ phenotypes at the molecular level, and analyzes whether it functions by regulating the expression of key genes in the cell cycle network and floral development.

[0107] 2. Experimental Materials Plant materials: Inflorescence tissues from the three SmCYCD3;2 overexpression (OE) homozygous T3 generation Arabidopsis thaliana lines (OE-1, OE-3, OE-5) used in Example 4 and wild-type Arabidopsis thaliana (WT). Three biological replicates were collected from each line.

[0108] Main reagents: Plant total RNA extraction kit; reverse transcription kit; SYBR Green Pro Taq HS premixed qPCR kit.

[0109] Instruments and equipment: Real-time quantitative PCR instrument.

[0110] 3. Experimental Methods 3.1 Total RNA extraction and cDNA synthesis Inflorescences of wild-type and transgenic Arabidopsis thaliana with consistent growth stages were collected and flash-frozen in liquid nitrogen. Total RNA was extracted from each sample according to the instructions of the plant total RNA extraction kit. After the RNA quality was verified by NanoDrop 8000, an equal volume (1 μg) of total RNA was used for genomic DNA removal and reverse transcription to synthesize the first strand of cDNA. All cDNA samples were uniformly diluted and stored at -20℃ for later use.

[0111] 3.2 Selection of endogenous genes and primer design Based on the phenotypes observed in Example 4 (abnormal cell division, pistil elongation, and stamen development defects), we selected two major categories of endogenous genes for expression analysis: Cell cycle regulation-related genes: AtCYCD1;1(AT1G70210), AtCYCD2;1(AT2G22490), AtCYCD3;1(AT4G34160), AtCDKA1(AT3G48750), AtKRP1(AT2G23430), AtRBR(AT3G12280).

[0112] Genes related to floral organ development: AtAP3 (AT3G54340), AtPI (AT5G20240), AtAMS (AT2G16910), AtDEL1 (AT3G48160); AtPIN1 (AT1G73590); AtCRC (AT1G69180).

[0113] The Arabidopsis thaliana AtEF1α gene (AT1G07940) was used as an internal reference gene. The qPCR primer sequences for all genes are shown in Table 1. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0114] Table 1 Primer sequences 3.3 Real-time quantitative PCR (RT-qPCR) qPCR analysis was performed using the SYBR Green method.

[0115] The reaction system (20 μL) was as follows: 2× SYBR Green Pro Taq HS Premix, 10.0 μL; upstream primer (10 μM), 0.4 μL; downstream primer (10 μM), 0.4 μL; cDNA template, 2.0 μL; ddH2O, 7.2 μL.

[0116] Reaction procedure: 95℃ for 30 s; 40 cycles (95℃ for 5 s, 60℃ for 30 s); finally, melting curve analysis was performed. Three technical replicates were set up for each sample.

[0117] 3.4 Data Analysis Use 2 -ΔΔCt The method calculates the relative expression levels of each endogenous gene in transgenic plants relative to the wild type. The wild-type expression level is used as the calibration value (set to 1). Student's... t -Text test analysis was used to determine the significant difference in gene expression levels between transgenic and wild-type plants (P<0.05 was considered statistically significant).

[0118] 4. Results and Analysis RT-qPCR results are as follows Figure 7As shown, overexpression of SmCYCD3;2 significantly altered the expression profile of endogenous genes in Arabidopsis thaliana: 4.1 Effects on the cell cycle regulatory network: Compared with the wild type, the expression of several genes positively regulating the cell cycle was significantly upregulated in transgenic plants (P<0.05), including AtCYCD1;1, AtCYCD2;1, AtCYCD3;1, and AtCDKA1. Conversely, the expression levels of the negative cell cycle regulators AtKRP1 and AtRBR were significantly downregulated (P<0.05). This indicates that overexpression of SmCYCD3;2 disrupts normal cell cycle regulation.

[0119] 4.2 Effects on key genes in floral organ development: Stamen and pollen development genes: The phenotypes of abnormal stamen development and pollen abortion in transgenic plants were completely consistent. The expression levels of all key genes for stamen development, including AtAP3, AtPI, AtAMS and AtDEL1, were significantly downregulated in transgenic plants (P<0.01).

[0120] Stem and leaf development genes: Consistent with the phenotypes of reduced plant height and curled leaves in transgenic plants, the expression of AtPIN1, a gene related to stem and leaf development, was also significantly downregulated.

[0121] Pistil development genes: Corresponding to the observed pistil elongation phenotype, the expression of the key gene AtCRC, which promotes pistil development, was significantly upregulated in transgenic plants (P<0.05).

[0122] This embodiment demonstrates at the molecular level that overexpression of the Sapindus mukorossi SmCYCD3;2 gene in Arabidopsis thaliana disrupts the original floral development programming by upregulating the expression of positive cell cycle regulators and the pistil development gene AtCRC, while downregulating the expression of negative cell cycle regulators and a series of core genes for stamen development, ultimately leading to a dominant phenotype of "promoting females and suppressing males." This result clearly reveals the molecular mechanism by which the SmCYCD3;2 gene regulates floral sex differentiation, strongly supporting its function as a key sex-regulating gene.

Claims

1. An isolated Sapindus mukorossi SmCYCD3;2 gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

2. A protein encoded by the Sapindus mukorossi SmCYCD3;2 gene as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

3. A recombinant vector containing the Sapindus mukorossi SmCYCD3;2 gene as described in claim 1.

4. A host cell containing the Sapindus mukorossi SmCYCD3;2 gene as described in claim 1 or the recombinant vector as described in claim 3.

5. A primer set for cloning or detecting the Sapindus mukorossi SmCYCD3;2 gene as described in claim 1, characterized in that, Includes at least one of the following primer pairs: a) Cloning primer pairs, the sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4; b) Cloning primer pairs containing restriction enzyme sites, the sequences of which are shown in SEQ ID NO: 5 and SEQ ID NO: 6; c) Real-time quantitative PCR primer pairs, the sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO:

8.

6. A method for regulating the sex ratio of plant flowers, characterized in that, The method includes introducing and expressing the Sapindus mukorossi SmCYCD3;2 gene of claim 1 into a plant.

7. The method according to claim 6, characterized in that, The regulation of flower sex ratio is manifested by one or more of the following: promoting pistil elongation, reducing the number of stamens, and decreasing pollen viability.

8. A method for increasing plant fruit yield, characterized in that, This is achieved by implementing the method described in claim 6 or 7.

9. The application of the Sapindus mukorossi SmCYCD3;2 gene as described in claim 1 or the recombinant vector as described in claim 3 in the cultivation of plant varieties with a high proportion of female flowers.

10. A method for producing transgenic plants, characterized in that, This includes introducing the Sapindus mukorossi SmCYCD3;2 gene as described in claim 1 or the recombinant vector as described in claim 3 into plant cells and regenerating them to obtain transgenic plants, wherein the ratio of male to female flowers in the transgenic plants is higher than that in the wild type.

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