A Sapindus mukorossi SmSEP3 gene and its application

Abstract

This invention discloses a Sapindus mukorossi SmSEP3 gene and its application. This gene, when expressed in plants, regulates flowering time, promotes pistil development, sepal curling, and pod development. The gene is the nucleotide sequence shown in Sequence Listing Seq ID NO.1 or the nucleotide sequence encoding the amino acid sequence shown in Sequence Listing Seq ID NO.2. This gene is used to breed new Sapindus mukorossi varieties with different flowering times, flower types, and larger fruits. Analysis of transgenic Arabidopsis thaliana shows that overexpression of this gene advances bolting and flowering times, significantly lengthens pistils, curls sepals, and increases fruit size. The flowering time, bolting days, and number of rosette leaves at bolting of transgenic Arabidopsis thaliana plants are significantly different from wild-type plants. The pistils are longer, and the pods are also larger, which is beneficial for accelerating the breeding cycle of Sapindus mukorossi trees, cultivating ornamental varieties with different flower types, and accelerating the development of high-yielding new varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and relates to a gene that affects the flowering time of plants, the protein encoded by the gene, an expression vector containing the gene, a primer set for cloning the gene from plant DNA, and the use of the gene. Background Technology

[0002] In the field of plant genetic engineering, the regulation of flowering time is a crucial step in plant growth and development, playing a significant role in plant reproduction, growth cycle, and adaptation to environmental changes. Sapindus mukorossi Gaertn., as a multifunctional economic forest tree, boasts abundant germplasm resources and possesses significant economic and ecological value. However, the relatively long juvenile stage of Sapindus mukorossi limits the progress of breeding efforts and the rapid realization of economic benefits.

[0003] Traditionally, the growth and development of Sapindus mukorossi includes eight main stages: flower bud, leaf and stem development, inflorescence appearance, flowering, fruit development, fruit ripening, and fruit senescence followed by dormancy. In spring, the flowers of Sapindus mukorossi begin to develop, their first morphological feature being small "yellow spots" in the leaf axils. The stages of reproductive development and their corresponding times include dormancy (December to February of the following year), flowering induction (March to April), flower initiation (April to May), flower bud differentiation (June to July), and fruit development and ripening (August to November).

[0004] Although existing research has focused on the regulation of flowering time in plants, the mechanism of flowering regulation in the specific species Sapindus mukorossi is still not fully understood. Therefore, developing new genetic resources, especially those genes that can significantly affect flowering time, is of great theoretical and practical significance for improving Sapindus mukorossi varieties, shortening the juvenile stage, and accelerating the breeding cycle.

[0005] The challenge for those skilled in the art lies in how to identify and utilize key genes affecting the flowering time of Sapindus mukorossi through molecular biology techniques to regulate its growth and development. Furthermore, how to apply these genetic resources to the breeding and improvement of Sapindus mukorossi to cultivate new varieties with early maturity and high yield is a current research focus. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a gene that can advance the flowering time of plants, promote the development and elongation of pistils, curl sepals into a flowering shape, and increase the volume of pods.

[0007] Through long-term exploration and experimentation, and continuous reform and innovation, the inventors have provided the following technical solution to address the aforementioned technical problems: a Sapindus mukorossi SmSEP3 gene is expressed in plants to regulate flowering time, promote pistil development, sepal curling, and pod development. The SmSEP3 gene contains nucleotide sequences selected from the following group:

[0008] A. The nucleotide sequence shown in Seq ID NO.1 of the sequence listing;

[0009] B. The nucleotide sequence of the amino acid sequence shown in Seq ID NO.2 of the coding sequence listing.

[0010] The present invention also provides a protein encoded by the Sapindus mukorossi SmSEP3 gene, wherein the amino acid sequence of the protein is selected from the amino acid sequence shown in Sequence Listing Seq ID NO.2.

[0011] The present invention also provides a primer pair for cloning the Sapindus mukorossi SmSEP3 gene, the base sequence of which is as follows:

[0012] First upstream primer F: 5'-ATGTACATTGGCAAATTCAACTATT-3'.

[0013] First downstream primer R: 5'-TCAAGCCATCCACCTTGGTAT-3'.

[0014] Optionally, the base sequence of the primer pair further includes an enzyme cleavage site, and the base sequence of the primer pair including the enzyme cleavage site is as follows:

[0015] Second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGTACATTGGCAAATTCAA CTATT-3'.

[0016] Second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCAAGCCATCCACCTTGGTAT-3'.

[0017] This invention also provides a fluorescence quantitative primer pair for the Sapindus mukorossi SmSEP3 gene, the base sequence of which is as follows:

[0018] Third upstream primer F: 5'-CCAAAGCGTAACGTACAGCA-3',

[0019] Third downstream primer R: 5'-CAAGCCATCCACCTTGGTAT-3'.

[0020] This invention also provides an expression vector containing the Sapindus mukorossi SmSEP3 gene. The pCAMBIA1301 vector plasmid is double-digested with restriction endonucleases BstEII-HF and Ncol-HF. II. The vector was ligated using the One Step Cloning Kit to obtain the overexpression vector pCAMBIA1301-SmSEP3.

[0021] The present invention also provides a use for the SmSEP3 gene of Sapindus mukorossi, for breeding new varieties of Sapindus mukorossi with different flowering times.

[0022] The present invention also provides a method for breeding new plant varieties, which utilizes the SmSEP3 gene to regulate flowering time in order to obtain new varieties with desired flowering characteristics.

[0023] The present invention also provides a method for transgenic plants, comprising introducing the expression vector into plant cells to alter the flowering time of the plant.

[0024] This invention also provides a method for analyzing gene expression patterns related to the regulation of flowering time in plants. By measuring the expression level of the SmSEP3 gene at different developmental stages, the regulatory mechanism of flowering time in plants can be analyzed.

[0025] The present invention also provides a method for measuring the expression levels of genes related to flowering time regulation, using the aforementioned quantitative fluorescent primer pair to quantitatively analyze the expression levels of genes related to flowering time regulation in transgenic plants.

[0026] Compared with the prior art, one of the above technical solutions has the following advantages:

[0027] The SmSEP3 gene from Sapindus mukorossi of this invention provides an innovative mechanism for regulating flowering time in the field of plant genetic engineering. Through transgenic technology, the SmSEP3 gene can significantly advance the flowering time of plants, which is of great significance for accelerating the plant's reproductive cycle. Furthermore, the expression of this gene promotes the development of the pistil, causing the sepals to curl and form a more pronounced flowering state. This helps improve the plant's reproductive efficiency and fruit set rate, potentially increasing fruit yield.

[0028] The application of the SmSEP3 gene can shorten the breeding cycle of Sapindus mukorossi, which has significant economic value for accelerating the development and promotion of new varieties. Meanwhile, the expression of transcription factors of the SmSEP3 gene is involved in the response to various biotic and abiotic stresses, indicating that this gene not only plays a role in regulating flowering time but may also enhance the plant's adaptability to environmental changes and improve its stress resistance.

[0029] Through transgenic verification in Arabidopsis thaliana, this invention confirms the function of the SmSEP3 gene in regulating flowering time, providing a scientific basis for subsequent genetic engineering applications. Furthermore, the discovery and application of the SmSEP3 gene provides a new tool for molecular breeding of Sapindus mukorossi, helping to cultivate new varieties with early maturity and high yield. The breeding and promotion of these new varieties will directly increase the economic value of Sapindus mukorossi, promote the development of related industries, and bring higher economic benefits to growers.

[0030] Through observation of whole plants of Arabidopsis thaliana transgenic with the SmSEP3 gene and analysis of differences in flowering time, it was found that the SmSEP3 gene can advance the flowering time of plants to varying degrees. The flowering time, bolting days, and number of rosette leaves at bolting of transgenic Arabidopsis thaliana plants were significantly different from those of wild-type plants, which is beneficial for accelerating the breeding cycle of Sapindus mukorossi and developing high-yielding varieties. In addition, the SmSEP3 gene promotes pistil growth, sepal curling, and pod development and growth. These characteristics not only provide a reference for breeding new varieties with high ornamental value, but also can achieve a dual improvement in crop yield and quality through genetic improvement. Attached Figure Description

[0031] 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.

[0032] Figure 1 This is a clone electrophoresis image of the SmSEP3 gene.

[0033] Figure 2 These are the results of a phylogenetic analysis of SEP subclass proteins from different species.

[0034] Figure 3 These are the results of the cis-acting element analysis of the SmSEP3 gene.

[0035] Figure 4 This is a predicted tertiary structure diagram of the SmSEP3 protein.

[0036] Figure 5 The results are from the analysis of the expression pattern of SmSEP3 during the development of male and female flower buds.

[0037] Figure 6 The results of the expression pattern analysis of SmSEP3 in male and female floral organs.

[0038] Figure 7 This is the result of an analysis of the expression patterns of the SmSEP3 gene at eight stages of pericarp development.

[0039] Figure 8 This is the result of an analysis of the expression patterns of the SmSEP3 gene at eight stages of seed development.

[0040] Figure 9 Analysis results of the expression pattern of the SmSEP3 gene during the flowering induction period.

[0041] Figure 10 This is the identification result of the 35S::SmSEP3 transgenic plant.

[0042] Figure 11 This is a phenotypic comparison between the wild type and the 35S::SmSEP3 transgenic line.

[0043] Figure 12 This is a comparison of the flowering time between the wild type and the 35S::SmSEP3 transgenic line.

[0044] Figure 13 This is a statistical chart showing the bolting time, number of rosette leaves, and flowering time of wild-type and 35S::SmSEP3 transgenic lines.

[0045] Figure 14 This is a graph showing the expression levels of genes related to regulating flowering time in Arabidopsis thaliana flower tissue. Detailed Implementation

[0046] The following description, in conjunction with the accompanying drawings and a specific embodiment, will be provided.

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0048] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

[0049] Example 1

[0050] The Sapindus mukorossi SmSEP3 gene described in this embodiment has a nucleotide sequence as shown in Sequence Listing Seq ID NO.1. Based on this nucleotide sequence, the amino acid sequence shown in Sequence Listing Seq ID NO.2 can be obtained. The CDS sequence is shown in Sequence Listing Seq ID NO.9.

[0051] The SmSEP3 gene of Sapindus mukorossi is expressed in plants, particularly Sapindus mukorossi. Functional verification and transgenic verification in Arabidopsis thaliana show that this gene can significantly regulate the flowering time of plants. Utilizing the SmSEP3 gene of Sapindus mukorossi can shorten the breeding cycle of Sapindus mukorossi and accelerate the development of high-yield Sapindus mukorossi varieties.

[0052] Example 2

[0053] The primer pair described in this embodiment for cloning the Sapindus mukorossi SmSEP3 gene is used to clone the Sapindus mukorossi SmSEP3 gene described in Example 1. The base sequence of the primer pair is as follows:

[0054] First upstream primer F: 5'-ATGTACATTGGCAAATTCAACTATT-3'.

[0055] First downstream primer R: 5'-TCAAGCCATCCACCTTGGTAT-3'.

[0056] The first upstream primer F is shown in Seq ID NO.3 of the sequence listing, and the first downstream primer R is shown in Seq ID NO.4 of the sequence listing.

[0057] Example 3

[0058] The primer pair for cloning the Sapindus mukorossi SmSEP3 gene described in this embodiment is based on the primer pair described in Example 2 with the addition of restriction enzyme sites, resulting in the second upstream primer F as shown in Sequence Listing Seq ID NO.5 and the second downstream primer R as shown in Sequence Listing Seq ID NO.6. The base sequences of the primer pair including the restriction enzyme sites are as follows:

[0059] Second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGTACATTGGCAAATTCAAC TATT-3',

[0060] Second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCAAGCCATCCACCTTGGTAT-3'.

[0061] The cloning primer sequences were designed using Primer3web (4.1.0) (https: / / bioinfo.ut.ee / primer3 / ) and then sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for synthesis.

[0062] Example 4

[0063] The quantitative fluorescence primer pair for the Sapindus mukorossi SmSEP3 gene described in this embodiment is used for quantitative analysis of the Sapindus mukorossi SmSEP3 gene. The base sequences of the quantitative fluorescence primer pair are shown in Seq ID NO.7 and Seq ID NO.8 of the sequence listing:

[0064] Third upstream primer F: 5'-CCAAAGCGTAACGTACAGCA-3',

[0065] Third downstream primer R: 5'-CAAGCCATCCACCTTGGTAT-3'.

[0066] The primer sequences for quantitative fluorescence were designed using Primer3web (4.1.0) (https: / / bioinfo.ut.ee / primer3 / ) and then synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd.

[0067] Example 5

[0068] This embodiment is an example of verifying the nucleotide sequence and amino acid sequence described in Example 1 using the primers described in Examples 3 and 4.

[0069] In this embodiment, three Sapindus mukorossi sample trees were selected as experimental materials. Biological sampling was carried out three times. The sampling location was located in Jianning County, Sanming City, Fujian Province. The sampling time was fixed from 10:00 am to 12:00 pm. The middle and upper parts of one-year-old branches at the same height of the tree canopy were quickly placed into cryovials and frozen in liquid nitrogen. Then they were stored in a -80°C freezer for subsequent RNA extraction.

[0070] The four stages of flower induction and flower initiation (bud1-4) in Sapindus mukorossi are: flower bud dormancy period (bud1), flower induction period (bud2), flower initiation period (bud3), and inflorescence differentiation period (bud4).

[0071] After the formation of Sapindus mukorossi florets, the developmental stages of floral organs were analyzed. Eight stages were selected from the female (FF1-8) and male (MF1-8) florets of different sexes: F1 was the stage of complete formation of floral organ primordia; F2 was the stage of meiosis of stamens; F3 was the stage of microspore development; F4-6 was the stage of rapid elongation of filaments and styles; F7 was the flowering development stage; and F8 was the late flowering stage, totaling 48 samples. Ten tissue parts of the important floral organs (petals, pistils, and stamens) were analyzed, covering the various parts of floral organs in the 4th stage (rapid elongation of filaments and styles) and the 7th stage (flowering development stage): pistils of female flowers (PiFF4 and PiFF7), pistils of male flowers (PiMF4 and PiMF7), stamens of female flowers (StFF4 and StFF7), stamens of male flowers (StMF4 and StMF7), as well as petals of female flowers (PeFF) and male flowers (PeMF).

[0072] Following preliminary experimental screening, this embodiment ultimately used cDNA material from male flower buds at the 8th developmental stage as a template for PCR amplification. The primers described in Example 3 were used for amplification. The TA cloning reaction system is shown in Table 1 below. The reaction program was: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 40 s, repeated 34 times, followed by a final extension at 72℃ for 5 min, and storage at 4℃. After adding 2 μl of 10× Loading buffer, the PCR product was separated by 1% agarose gel electrophoresis. The clean target fragment was excised from the gel and recovered using a DNA gel extraction kit. The entire process was performed on ice.

[0073] Table 1. TA Cloning PCR Sequence Amplification System

[0074]

[0075] The recycled gel product was connected to the T-carrier, and the reaction system is shown in Table 2.

[0076] Table 2. T-cloning ligation vector system

[0077]

[0078] Incubate at room temperature (20-30℃) for 5 min. After the reaction, incubate at 4℃. Take 5 μl of ligation solution and add it to 50 μl of freshly thawed DH10B competent cells. Mix gently, incubate on ice for 30 min, heat shock in a 42℃ water bath for 30 s, and immediately place on ice for 2 min. Add 300-500 μL of sterile LB medium and incubate at 37℃ with shaking at 200 rpm for 1 h. Take 200 μl of the bacterial culture (containing ampicillin) and incubate overnight at 37℃ (12-16 h). Select single colonies for PCR molecular detection. Positive E. coli clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for testing.

[0079] Electrophoresis analysis showed that the extracted total RNA bands were clear and intact, meeting the experimental requirements. The SmSEP3 gene was cloned using cDNA from Sapindus mukorossi flower tissue as a template; the cloning electrophoresis image is shown below. Figure 1 After sequencing the target fragment, a 507bp coding sequence was obtained, as shown in Sequence Listing Seq ID NO.1, which encodes 168 amino acids, as shown in Sequence Listing Seq ID NO.2.

[0080] To further investigate the conserved motif sequence of Sapindus mukorossi SmSEP3, protein sequences of MADS genes from various species databases in the Phytozome v13 public database were screened, including MADS gene sequences from Arabidopsis thaliana TAIR10, soybean (Glycine max Wm82.a4.v1), apple (Malus domestica v1.1), grape (Vitis vinifera v2.1), and peach (Prunus persica v2.1). Combined with the Sapindus mukorossi SmMADS gene family, the MADS protein sequences of multiple species were MUSCLE aligned using MEGA7, and the NJ nearest neighbor method was used to reconstruct phylogenetic trees for the Type I and Type II subfamilies, respectively. The bootstrap parameter was checked and set to 1000 replicates.

[0081] SEP subclass amino acid sequences of each species were screened and phylogenetic analysis was performed. (See attached image) Figure 2 The number of SEP proteins in Arabidopsis thaliana, grape, soybean, peach, apple and Sapindus mukorossi in each group were 3, 4, 10, 4, 8 and 1, respectively. Evolution shows that SmSEP3 is closely related to the SEP3 protein in soybean and Arabidopsis thaliana, and the regulatory function of SEP3 protein in different species is significantly different.

[0082] The promoter sequences of the SmMADS gene family upstream of the coding region in Type I and Type II subclasses were taken respectively. The cis-regulatory elements were predicted using the online data analysis software PlantCARE, and statistical plotting was performed using Excel. (See figure) Figure 3 .

[0083] Figure 3 This indicates that the expression of the SmSEP3 transcription factor may be involved in the response process to different biotic and abiotic stresses, possessing the ability to sense and respond to environmental stresses, plant hormones, and photoperiod, and participating in the regulation of plant growth and development. Besides some common basic elements (TATA), SmSEP3 has four other main categories:

[0084] (1) Hormone regulation-related elements: Jasmonic acid response elements (CGTCA-motif and TGACG-motif);

[0085] (2) Components related to environmental pressure regulation: Mechanical damage response element (WUN-motif);

[0086] (3) Drought-induced binding site-related elements (MYB and MYC) and anaerobic-related elements (ARE) respond to plant abiotic stress;

[0087] (4) A large number of photoperiodic response elements (TCT-motif and G-box) regulate the diurnal rhythm of plants.

[0088] (5) Relevant elements for regulating plant growth and development (AAGAA-motif and O2-site);

[0089] Protein function and structure are closely related. Therefore, the three-dimensional structure of the SmSEP3 protein was constructed using the online SWISS-MODEL website, see [link to SmSEP3 structure]. Figure 4 .

[0090] RNA extraction and real-time fluorescence quantitative assay

[0091] All centrifuge tubes, pipette tips, and mortars used in the experiment were sterile. Samples from various tissue parts of *Sapindus mukorossi* were ground with liquid nitrogen, and total RNA was extracted using the Omega RNA kit. RNA concentration was determined using a NanoDrop 2000 spectrophotometer (ThermoScientific, USA). RNA integrity was assessed by 1% agarose gel electrophoresis. cDNA was synthesized using the TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR reverse transcription kit, and the obtained cDNA was diluted with nuclease-free water.

[0092] The SmSEP3 gene was subjected to qRT-PCR using TB Green Premix Ex Taq (SYBR Green) enzyme, while SmACT was used as an internal control gene for quantitative real-time PCR. The primer sequences for the gene's quantitative real-time PCR are shown in Table 3.

[0093] Table 3 qRT-PCR amplification reaction system

[0094]

[0095] The qRT-PCR reaction program was set as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. After the cycles, product specificity was detected using melting curve analysis: the temperature was slowly increased from 60℃ to 95℃, with 5 fluorescence signals collected for each 1℃ increase. The Sapindus mukorossi SmACT gene was used as an internal control for detection. Three biological replicates and three technical replicates were set up, and the average value was calculated. A 23 -ΔΔCt Gene expression levels were calculated and plotted using a method.

[0096] The expression patterns of SmSEP3 in trophic organs at eight stages of male and female flower development, with specific results as follows: Figure 5 As shown, the expression of the SmSEP3 gene showed a continuous upward trend during the development of male flowers, reaching its highest value at stage 8 (late flowering stage); during the development of female flowers, the expression level first increased slowly and then decreased, and was significantly expressed at stage 7 (late flowering stage).

[0097] Figure 6 In the diagram, PiFF4 represents the pistil of a female flower during the rapid elongation of the filament and style; PiFF7 represents the pistil of a female flower during the flowering and development stage; PiMF4 represents the pistil of a male flower during the rapid elongation of the filament and style; PiMF7 represents the pistil of a male flower during the flowering and development stage; StFF4 represents the stamen of a female flower during the rapid elongation of the filament and style; StFF7 represents the stamen of a female flower during the flowering and development stage; StMF4 represents the stamen of a male flower during the rapid elongation of the filament and style; StMF7 represents the stamen of a male flower during the flowering and development stage; PeFF represents the petals of a female flower; PeMF represents the petals of a male flower. It can be seen that the SmSEP3 gene participates in regulating the development of stamens, pistils, and petals, with the most significant expression in the pistils of male flowers.

[0098] Figure 7 and Figure 8 The SmSEP3 gene is involved in regulating the development of the pericarp and seed in the 5th stage (seed hardening stage), the 6th stage (fruit color change stage), and the 7th stage (fruit near ripening stage), with a particularly high expression level in the fruit near ripening stage. The SmSEP3 gene is most significantly expressed in the 4th stage (fruit enlargement stage) of the seed.

[0099] See Figure 9 The expression level of SmSEP3 was gradually upregulated in four stages of Sapindus mukorossi flower induction and flower initiation, suggesting that it may regulate the flower bud induction process.

[0100] Construction of SmSEP3 gene overexpression vector

[0101] The SmSEP3 bacterial culture plasmid with a confirmed sequence was extracted using a high-purity plasmid DNA miniprep kit. The pCAMBIA1301 plasmid (preserved in our laboratory) was double-digested with restriction endonucleases BstEII-HF and Ncol-HF. The digestion system is shown in Table 4. The reaction program was 37℃ for 30 min. After digestion, the vector was purified using a DNA gel extraction kit and detected by electrophoresis. The digested products were stored at -20℃.

[0102] Table 4. Enzyme digestion reaction system for the 1301 expression vector

[0103]

[0104] use II. The vector was ligated using the One Step Cloning Kit. The ligation system is shown in Table 5. The pCAMBIA1301-SmSEP3 overexpression vector was obtained. The recombinant was introduced into Agrobacterium GV3101 competent cells for subsequent infection of Arabidopsis thaliana.

[0105] Table 5. Ligation reaction system of 1301 expression vector

[0106]

[0107] Cultivation and Infection of Wild-type Arabidopsis

[0108] Under sterile conditions, take an appropriate amount of wild-type Arabidopsis seeds, place them in a 1.5ml centrifuge tube, add 1 / 50 volume of sodium hypochlorite solution, mix well, shake repeatedly up and down for 10-15 minutes, and after sterilization, wash 4-5 times with sterile distilled water. Spread the seeds evenly on sterilized 1 / 2MS solid medium, vernalize at 4℃ for 3 days, and then expose to light for one week. Transplant the Arabidopsis seedlings into sterilized culture soil (a mixture of substrate soil and vermiculite = 1:1) and place them in a light incubator for long-day (16 / 8h) cultivation at 22℃ and a relative moisture content of 70%. Transform Arabidopsis plants using the flower immersion method. When the Arabidopsis plants have bolted and grown 3-4 stem leaves, remove the terminal inflorescence from all plants simultaneously to utilize apical dominance to promote lateral branch growth and flowering. A large number of unopened flower buds were infected with Agrobacterium, soaked for 15-30 seconds, and then dried. The plants were then placed in a dark environment for about 24 hours. The infection was repeated 4 times during the flowering period, with an interval of about one week between each infection, to improve the conversion efficiency.

[0109] Screening and identification of transgenic Arabidopsis thaliana

[0110] After infection with Arabidopsis thaliana, seeds were mixed and harvested to obtain the first generation (T0 generation). These seeds were sown on 1 / 2 MS solid medium containing 30 mg / L hygromycin. Normally growing transgenic seedlings were selected. For resistant plants, DNA was extracted and used as a template for PCR amplification and molecular testing according to the Plant Direct PCR Kit instructions. Gel electrophoresis was used to observe the presence of the target band. The above process was repeated to obtain T2 generation homozygous transgenic plants. Subsequent phenotypic observation and functional analysis were then performed.

[0111] DNA was extracted from leaves of WT wild-type Arabidopsis thaliana and 35S::SmSEP3 overexpressing plants, respectively, and used as templates for SmSEP3 gene detection. The results are as follows: Figure 10 As shown in the figure, Marker represents the DL2000 marker; WT represents wild-type Arabidopsis thaliana; OE represents the 35S::SmSEP3 plant. According to... Figure 10 Wild-type lines served as the control group and did not show any bands. The T1 generation lines that were successfully transfected with the exogenous gene showed the target bands. A total of 6 lines were verified.

[0112] During the growth of wild-type and transgenic Arabidopsis thaliana, the bolting time, flowering time, and number of rosette leaves were recorded and compared in real time. ANOVA analysis was performed using IBM SPSS Statistics 27 software to obtain the mean, standard deviation, etc., and graphs were generated using Excel 2020 software.

[0113] Figure 11 The image (AD) shows a comparison between wild-type Arabidopsis thaliana (left) and the 35S::SmSEP3 transgenic plant (right). Image A shows that overexpression of the SmSEP3 gene in Arabidopsis affects bolting and flowering time, causing earlier flowering. Image B shows that the flower morphology is normal, with normal development of petals, sepals, pistils, and stamens. Compared to the wild type, the pistils in the 35S::SmSEP3#2-3 transgenic lines showed significant elongation, and the upper part of the sepals in the 35S::SmSEP3#4-5 transgenic lines showed an inward curling deformity, changing from a cuboid split to a flowering shape. Image C shows that at the same fold, the number and size of stamens in the 35S::SmSEP3#2-3 line are consistent with the wild type, further confirming the significant elongation of the pistils. Meanwhile, the sepal deformities did not affect the development of stamens and pistils in the 35S::SmSEP3#4-5 line, and their growth remained normal. Figure D shows that the inflorescences of the 35S::SmSEP3 line are indeterminate, just like the wild type. Figures E and F show that the pods of the 35S::SmSEP3 line are larger than those of the wild type, but the seed setting rate is the same.

[0114] The results comparing the flowering time of the wild-type and the 35S::SmSEP3 transgenic lines can be found in [link to relevant documentation]. Figure 12 . Figure 12 In Figure G: the left side shows the WT wild-type plants, and from left to right are the 35S::SmSEP3#2, 35S::SmSEP3#3, and 35S::SmSEP3#4 transgenic plants; in Figure H: the first one on the left is the WT wild-type plant, and from left to right are the 35S::SmSEP3#5, 35S::SmSEP3#7, and 35S::SmSEP3#8 transgenic plants; it can be seen that the flowering time of the six 35S::SmSEP3 transgenic lines is earlier than that of WT Arabidopsis.

[0115] Figure 13 In Figures a through c, WT represents wild-type Arabidopsis thaliana, with a total of six 35S::SmSEP3 transgenic lines. Figure a: Statistical analysis of bolting time (days); Figure b: Statistical analysis of rosette leaves (number of leaves) at bolting; Figure c: Statistical analysis of flowering time (days). The bolting time (Figure b) and flowering time (Figure c) of these six lines were significantly different from those of the wild-type plants, and SPSS analysis showed statistical significance. The bolting time of wild-type Arabidopsis thaliana was 20.16 days, with 12.83 rosette leaves at bolting, and the flowering time was 22 days; the bolting time of the six transgenic lines was 14.33 days, with 10.5 rosette leaves at bolting, and the flowering time was 15.33 days. The flowering time of 35S::SmSEP3 plants was advanced to varying degrees, specifically by 6.67 days. Furthermore, the number of rosette leaves in most 35S::SmSEP3 transgenic lines was positively correlated with the bolting time; the earlier the bolting, the fewer the number of rosette leaves.

[0116] Study on the expression patterns of endogenous genes in transgenic plants

[0117] Representative transgenic lines were selected from the Arabidopsis thaliana plants exhibiting the aforementioned differences in flowering time. RNA was extracted from WT wild-type plants and transgenic plants of 35S::SmSEP3#2, 35S::SmSEP3#5, and 35S::SmSEP3#8, and analyzed by electrophoresis. All samples showed clear and intact total RNA bands. cDNA was obtained through reverse transcription and used as a template for subsequent qRT-PCR experiments. The AtEF1 gene was used as an internal control gene. The quantitative PCR method was the same as described previously. Simultaneously, AtEF1 was used as an internal control gene for quantitative PCR experiments. The primer sequences for the quantitative PCR of the genes are shown in Table 6.

[0118] Use 2 -ΔΔCt Gene expression levels were calculated and plotted using a method.

[0119] Table 6 Primers for Quantitative Real-Time PCR

[0120]

[0121] To further investigate gene regulation in 35S::SmSEP3 transgenic plants and explain their phenotypic changes at the molecular level, the expression levels of genes upstream and downstream of SEP3 regulating flowering time were measured in the floral tissues of Arabidopsis thaliana and compared with those in wild-type plants subjected to total wheat wing (WT). (See [reference needed]). Figure 11 . Figure 11 In the study, WT represents wild-type Arabidopsis thaliana plants and transgenic lines 35S::SmSEP3#2, 35S::SmSEP3#5, and 35S::SmSEP3#8. Firstly, the expression level of the SmSEP3 gene in the floral parts of the 35S::SmSEP3 transgenic plants was extremely high, significantly different from the wild type, indicating that the SmSEP3 gene regulates flowering time, and its overexpression leads to earlier flowering in Arabidopsis thaliana.

[0122] In the tissues of 35S::SmSEP3 Arabidopsis flowers, the expression levels of genes such as AtLFY, AtPI, AtSHP1, AtSEP2, AtSEP3, AtSTK, AtSOC1, and AtFT were higher than in the wild type. In Arabidopsis flower tissues, the expression levels of genes related to flowering time (AtFT and AtSOC1), floral organ development (AtSEP3 and AtPI), and flowering meristem regulators (AtLFY) were also higher than in the wild type. Therefore, overexpression of the SmSEP3 gene promotes the expression of flowering integrons and floral meristem regulators, leading to earlier bolting and flowering in the 35S::SmSEP3 transgenic Arabidopsis.

[0123] This embodiment demonstrates, using the model plant Arabidopsis thaliana, that the Sapindus mukorossi SmSEP3 gene can be used to cultivate new Sapindus mukorossi varieties with different flowering times; the SmSEP3 gene can be used to regulate flowering time to obtain new varieties with desired flowering characteristics. This embodiment also describes a plant transgenic method for introducing the expression vector into plant cells to alter the flowering time of plants. This embodiment further describes a method for analyzing the expression patterns of genes related to flowering time regulation by measuring the expression levels of the SmSEP3 gene at different developmental stages. This embodiment also describes a method for determining the expression levels of genes related to flowering time regulation in transgenic plants using the described quantitative fluorescent primer pairs.

[0124] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of soapberry SmSEP3 Genes, characterized by, The SmSEP3 The gene is selected from the following group of nucleotide sequences: A. The nucleotide sequence shown in Seq ID NO.1 of the sequence listing; B. The nucleotide sequence of the amino acid sequence shown in Seq ID NO.2 of the coding sequence listing.

2. A soapberry variety according to claim 1 SmSEP3 Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in Sequence Listing Seq ID NO.

2.

3. A method for cloning the Sapindus mukorossi as described in claim 1 SmSEP3 A primer pair for a gene, characterized in that, The base sequences of the primer pair are as follows: First upstream primer F: 5'-ATGTACATTGGCAAATTCAACTATT-3'. First downstream primer R: 5'-TCAAGCCATCCACCTTGGTAT-3'.

4. A primer pair for cloning the Sapindus mukorossi SmSEP3 gene as described in claim 1, characterized in that, The primer pair includes restriction enzyme sites in its base sequence, and the base sequence is as follows: Second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGTACATTGGCAAATTCAACTATT-3'. Second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCAAGCCATCCACCTTGGTAT-3'.

5. A method for use with the Sapindus mukorossi described in claim 1 SmSEP3 The fluorescent quantitative primer pair for gene expression measurement is characterized by, The base sequences of the fluorescence quantitative primer pairs are as follows: Third upstream primer F: 5'-CCAAAGCGTAACGTACAGCA-3', Third downstream primer R: 5'-CAAGCCATCCACCTTGGTAT-3'.

6. A type of soapberry containing the soapberry of claim 1 SmSEP3 Gene expression vectors, characterized in that, The pCAMBIA1301 vector plasmid was double-digested with restriction endonucleases BstEII-HF and Ncol-HF, and ligation was performed using the ClonExpress® II One Step Cloning Kit to obtain the overexpression vector pCAMBIA1301- SmSEP3.

7. A soapberry according to claim 1 SmSEP3 The uses of genes, characterized by, Used to cultivate new varieties of Sapindus mukorossi with earlier flowering time.

8. A method for cultivating new plant varieties, characterized in that, Overexpression of the claim 1 SmSEP3 Genes advance the flowering time to obtain new varieties with desired flowering characteristics; the plant in question is either Sapindus mukorossi or Arabidopsis thaliana.

9. A method for transgenic plants, characterized in that, This includes introducing the expression vector of claim 6 into plant cells to advance the flowering time of the plant, wherein the plant is Sapindus mukorossi or Arabidopsis thaliana.

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