Soapberry SmSEP3 gene and application thereof

Abstract

The invention discloses a soapberry SmSEP3 gene and application thereof, the gene is expressed in a plant to regulate and control the flowering time of the plant and promote pistil development, sepal curling and pod development, and the gene is a nucleotide sequence as shown in a sequence table Seq ID NO.1 or a nucleotide sequence of an amino acid sequence as shown in a coding sequence table Seq ID NO.2. The gene is used for cultivating new soapberry varieties with different flowering times, different flower types and larger fruits. Through the analysis of transgenic arabidopsis thaliana, the bolting and flowering time of the plant can be advanced through the overexpression of the arabidopsis thaliana, the pistil is obviously lengthened, sepals are curled, and the fruit volume is larger. The flowering time, bolting days and rosette leaf number of the transgenic arabidopsis thaliana plant are obviously different from those of a wild plant, pistil development is longer, fruit pods are increased, the breeding cycle of the sapindus tree species is accelerated, ornamental varieties of different flower types are cultivated, and cultivation of high-yield new varieties is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and relates to a gene affecting the flowering time of plants, a 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 Art

[0002] In the field of plant genetic engineering, the regulation of flowering time is a key link in the growth and development of plants, and is of great significance to plant reproduction, growth cycle and adaptation to environmental changes. As a multifunctional economic forest tree, Sapindus mukorossiGaertn. has rich germplasm resources and important economic and ecological value. However, the juvenile period of Sapindus mukorossi is long, which limits the progress of breeding work and the rapid realization of economic benefits.

[0003] Traditionally, the growth and development process of soapberry includes eight main stages, including the development of flower buds, leaves and stems, the appearance of inflorescences, the flowering process, the development of fruits, the ripening of fruits, the senescence of fruits and the entry into dormancy. In spring, the flowers of soapberry begin to develop, and their first morphological characteristics are small "yellow spots" in the axils of leaves. 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 previous studies have focused on the regulation of flowering time in plants, the flowering regulation mechanism of this particular species is still not fully understood. Therefore, the development of new genetic resources, especially those that can significantly affect flowering time, has important theoretical and practical significance for improving soapberry varieties, shortening the juvenile period, and accelerating the breeding cycle.

[0005] The challenge faced by technicians in this field is how to identify and utilize key genes that affect the flowering time of soapberry through molecular biological methods to achieve regulation of its growth and development process. In addition, how to apply these gene resources to the breeding and improvement of soapberry to cultivate new varieties with characteristics such as early maturity and high yield is the focus of current research. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a gene that can advance the flowering time of plants, promote the development and lengthening of pistils, curl the sepals into a flowering shape and increase the volume of fruit pods.

[0007] The inventors have continuously innovated through long-term exploration and attempts, as well as multiple experiments and efforts, to solve the above technical problems. The technical solution provided by the present invention is to provide a soapberry SmSEP3 gene, which is expressed in plants to regulate the flowering time of plants, promote pistil development, sepal curling and fruit pod development, and the SmSEP3 gene comprises a nucleotide sequence selected from the following group:

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

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

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

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

[0012] The first upstream primer F: 5'-ATGTACATTGGCAAATTCAACTATT-3',

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

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

[0015] The second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGTACATTGGCAAATTCAA CTATT-3',

[0016] The second downstream primer R: 5′-GGGGAAATTCGAGCTGGTCACTCAAGCCATCCACCTTGGTAT-3′.

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

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

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

[0020] The present invention also provides an expression vector containing the soapberry SmSEP3 gene, wherein the pCAMBIA1301 vector plasmid is double-digested with restriction endonucleases BstEII-HF and Ncol-HF, and the expression vector is expressed by using ⅡOne Step Cloning Kit was used for vector ligation to obtain the overexpression vector pCAMBIA1301-SmSEP3.

[0021] The present invention also provides a use of the soapberry SmSEP3 gene for cultivating new soapberry varieties with different flowering times.

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

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

[0024] The present invention also provides a method for analyzing gene expression patterns related to the regulation of plant flowering time, and analyzes the regulation mechanism of plant flowering time by measuring the expression levels of the SmSEP3 gene at different developmental stages.

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

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

[0027] The soapberry SmSEP3 gene of the present invention provides an innovative flowering time regulation mechanism in the field of plant genetic engineering. Through the application of transgenic technology, the SmSEP3 gene can significantly advance the flowering time of plants, which is of great significance for accelerating the reproductive cycle of plants. In addition, the expression of the gene also promotes the development of pistils, causing sepals to curl to form a more obvious flowering state, which helps to improve the reproductive efficiency and fruit setting rate of plants, thereby possibly increasing fruit yield.

[0028] The application of the SmSEP3 gene can shorten the breeding cycle of soapberry, which has significant economic value for accelerating the development and promotion of new varieties. At the same time, the transcription factor expression of the SmSEP3 gene is involved in the response to a variety of biological and abiotic stresses, which indicates that the gene not only plays a role in regulating flowering time, but also may enhance the adaptability of plants to environmental changes and improve their stress resistance.

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

[0030] After observation of the whole plant of transgenic Arabidopsis thaliana and analysis of flowering time differences, 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 are significantly different from those of wild-type plants, which is conducive to accelerating the breeding cycle of soapberry trees and accelerating the cultivation of high-yield varieties. In addition, the SmSEP3 gene promotes the growth of pistils, the curling of sepals and the growth of fruit pod development. These characteristics not only provide a reference for the cultivation of new varieties with high ornamental value, but also can achieve a dual increase in crop yield and quality through genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is the electrophoresis diagram of the cloning of SmSEP3 gene.

[0033] Figure 2 It is the result of systematic evolutionary analysis of SEP subclass proteins in different species.

[0034] Figure 3 This is the result of the cis-acting element analysis of the SmSEP3 gene.

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

[0036] Figure 5 These are the results of the expression pattern analysis 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 These are the results of expression pattern analysis of the SmSEP3 gene during eight stages of pericarp development.

[0039] Figure 8 These are the results of the expression pattern analysis of the SmSEP3 gene at eight developmental stages of the plant.

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

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

[0042] Fig.11 This is a comparison chart of the phenotypic observations of the wild type and 35S::SmSEP3 transgenic lines.

[0043] Fig.12 It is a comparison of flowering time between wild type and 35S::SmSEP3 transgenic lines.

[0044] Fig.13 It is a statistical graph of bolting time, rosette leaf number and flowering time of wild type and 35S::SmSEP3 transgenic lines.

[0045] Fig.14 This is a graph of the expression levels of genes related to regulating flowering time in Arabidopsis flower tissues. DETAILED DESCRIPTION

[0046] The following is a description with reference to the accompanying drawings and a specific embodiment.

[0047] In order to make the purpose, 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 drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0048] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and thus, once an item is defined in one drawing, it may not be further defined or explained in the subsequent drawings.

[0049] Example 1

[0050] The nucleotide sequence of the Sapindus mukorossi SmSEP3 gene described in this embodiment is shown in Seq ID NO.1 of the sequence table, and based on the nucleotide sequence, the amino acid sequence shown in Seq ID NO.2 of the sequence table can be encoded. The CDS sequence is shown in Seq ID NO.9 of the sequence table.

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

[0052] Example 2

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

[0054] The first upstream primer F: 5'-ATGTACATTGGCAAATTCAACTATT-3',

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

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

[0057] Example 3

[0058] The primer pair for cloning the Sapindus mukorossi SmSEP3 gene described in this example is a primer pair described in Example 2 with restriction sites added thereto, to obtain a second upstream primer F as shown in Seq ID NO.5 of the sequence table and a second downstream primer R as shown in Seq ID NO.6 of the sequence table. The base sequence of the primer pair including the restriction sites is as follows:

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

[0060] The second downstream primer R: 5′-GGGGAAATTCGAGCTGGTCACTCAAGCCATCCACCTTGGTAT-3′.

[0061] The cloning primer sequences were designed by 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 fluorescent quantitative primer pair of 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 fluorescent quantitative primer pair are shown in Seq ID NO.7 and Seq ID NO.8 in the sequence table:

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

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

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

[0067] Example 5

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

[0069] In this embodiment, three Sapindus mukorossi sample trees were selected as the test materials, and the biological samples were sampled three times respectively. The sampling site was located in Jianning County, Sanming City, Fujian Province. The sampling time was fixed at 10-12 o'clock in the morning. The middle and upper parts of the annual branches outside the crown of the same height were quickly placed in cryovials and frozen in liquid nitrogen, and then stored in a -80°C refrigerator for subsequent RNA extraction.

[0070] There are four periods of floral induction and flower initiation (bud1-4) in Sapindus mukorossi: bud1, the dormant period of flower buds; bud2, the floral induction period; bud3, the flower initiation period; and bud4, the inflorescence differentiation period.

[0071] After the formation of florets, during the development of floral organs, 8 periods of different sexes of female flowers (FF1-8) and male flowers (MF1-8) of Sapindus were selected: F1 is the stage of complete formation of floral organ primordium, F2 is the meiosis of stamens, F3 is the stage of microspore development, F4-6 is the stage of rapid elongation of filaments and styles, F7 is the flowering development stage, and F8 is the late flowering stage, for a total of 48 groups of samples. A total of 10 tissue parts of male and female important floral organs (petals, pistils and stamens) were selected, covering various floral organs in the 4th period (rapid elongation period of filaments and styles) and the 7th period (flowering development stage) of floral development: pistils (PiFF4 and PiFF7) of female flowers, pistils (PiMF4 and PiMF7) of male flowers, stamens (StFF4 and StFF7) of female flowers, stamens (StMF4 and StMF7) of male flowers, as well as petals (PeFF) of female flowers and petals (PeMF) of male flowers.

[0072] After preliminary experimental screening, the cDNA material of the male flower buds at the 8th developmental stage was used as a template for PCR amplification in this embodiment. The amplification primers used were the primers described in Example 3, and the TA cloning reaction system was shown in Table 1 below. The reaction procedure was 98°C pre-denaturation for 3min, 98°C denaturation for 30s, 56°C annealing for 30s, 72°C extension for 40s, 34 cycles, and finally 72°C extension for 5min, and stored at 4°C. After adding 2μl 10× Loading buffer, the PCR product was separated by 1% agarose electrophoresis, and the clean target fragment gel block was cut off and recovered with a DNA gel kit, and the whole process was carried out on ice.

[0073] Table 1 TA cloning PCR sequence amplification system

[0074]

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

[0076] Table 2T cloning and ligation vector system

[0077]

[0078] Place at room temperature (20-30°C) for 5 minutes, place at 4°C after the reaction, take 5μl of the connection solution to 50μl of the thawed DH10B competent cells, mix gently, ice bath for 30 minutes, heat shock in a 42°C water bath for 30 seconds, and immediately place on ice for 2 minutes. Add 300-500μL of sterile LB medium, 37°C, 200rpm shaking culture for 1 hour, take 200μl of bacterial solution plate (containing Amp ampicillin), culture at 37°C overnight (12-16h), select single colonies for PCR molecular detection, and entrust Beijing Ruibo Xingke Biotechnology Co., Ltd. to determine the positive clones of E. coli.

[0079] After electrophoresis, the extracted total RNA bands were clear and complete, meeting the experimental requirements. The cDNA of the tissue part of Sapindus mukorossi was used as a template to clone the SmSEP3 gene. The electrophoresis pattern of the clone is shown in Figure 1 After sequencing the target fragment, a 507 bp coding sequence was obtained, see Seq ID NO.1 in the sequence list, encoding 168 amino acids, see Seq ID NO.2 in the sequence list.

[0080] In order to further study the conserved motif sequence of SmSEP3 in Sapindus mukorossi, the protein sequences of MADS genes in various species databases in Phytozome were screened from the Phytozome v13 public data platform, including the MADS gene sequences of 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 SmMADS gene family in Sapindus mukorossi, the MADS protein sequences of multiple species were aligned by MUSCLE using MEGA7, and the phylogenetic trees of Type I and Type II subfamilies were reconstructed using the NJ neighbor method. The parameter bootstrap value was checked and set to be repeated 1000 times.

[0081] The amino acid sequences of the SEP subclasses of each species were screened and phylogenetic analysis was performed. Figure 2 The numbers of SEP proteins in each group of Arabidopsis, grape, soybean, peach, apple and soapberry were 3, 4, 10, 4, 8 and 1, respectively. Evolution showed that SmSEP3 was closely related to the SEP3 proteins of soybean and Arabidopsis, and the regulatory functions of SEP3 proteins in different species were significantly different.

[0082] The 3000 bp promoter sequences upstream of the coding regions of the SmMADS gene families of the Type I and Type II subclasses were respectively taken, and the cis-acting elements were predicted by the online data analysis software PlantCARE, and statistical plots were performed with the help of Execl, see Figure 3 .

[0083] Figure 3 This indicates that the expression of SmSEP3 transcription factors may be involved in the response to different biotic and abiotic stresses, have the ability to perceive and respond to environmental pressures, plant hormones and photoperiods, and participate in regulating plant growth and development. In addition to 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) Elements related to environmental stress regulation: mechanical damage response element (WUN-motif);

[0086] (3) drought-induced binding site-associated elements (MYB and MYC) and anaerobic-associated elements (ARE), etc., in response to plant abiotic stress;

[0087] (4) A large number of photoperiod-responsive elements (TCT-motif and G-box) regulate plant circadian rhythms.

[0088] (5) Elements related to plant growth and development regulation (AAGAA-motif and O2-site);

[0089] The function of a protein is closely related to its structure. Therefore, the three-dimensional structure of SmSEP3 protein was constructed using the online SWISS-MODEL website. Figure 4 .

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

[0091] The centrifuge tubes, pipette tips, mortars, etc. used in the experiment were all sterile. Liquid nitrogen was used to grind the samples of various tissue parts of the soapberry, and the total RNA of the soapberry was extracted using the Omega RNA kit. The RNA concentration was determined using a NanoDrop 2000 spectrophotometer (ThermoScientific, USA). The integrity of the RNA was detected by 1% agarose gel electrophoresis. The cDNA was synthesized using the TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR reverse transcription kit, and the resulting cDNA was diluted with nuclease-free water.

[0092] TB Green Premix Ex Taq (SYBR Green) enzyme was used to perform qRT-PCR experiments on the SmSEP3 gene, and SmACT was used as an internal reference gene for fluorescence quantitative experiments. The fluorescence quantitative PCR primer sequences of the genes 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°C for 30s; 95°C for 5s, 60°C for 30s, and 40 cycles. After the cycle, the melting curve was used to detect the specificity of the product: the temperature was slowly raised from 60°C to 95°C, and the fluorescence signal was collected 5 times for every 1°C increase. The SmACT gene of Sapindus mukorossi was used as an internal reference for detection. Three biological replicates and technical replicates were set, and the average value was calculated. -ΔΔCt Gene expression was calculated and processed for graphing.

[0096] The expression patterns of SmSEP3 in the nutrient organs of male and female flowers at eight stages of development were as follows: Figure 5 As shown: During the development of male flowers, the expression of SmSEP3 gene showed a rising trend, and the expression level reached the highest value in the 8th period (late flowering period); during the development of female flowers, the expression level first rose slowly and then fell, and was significantly expressed in the 7th period (late flowering period).

[0097] Figure 6 In the figure, PiFF4: pistil of female flowers during the period of rapid elongation of filaments and styles; PiFF7: pistil of female flowers during the flowering and development period; PiMF4: pistil of male flowers during the period of rapid elongation of filaments and styles; PiMF7: pistil of male flowers during the flowering and development period; StFF4: stamen of female flowers during the period of rapid elongation of filaments and styles; StFF7: stamen of female flowers during the flowering and development period; StMF4: stamen of male flowers during the period of rapid elongation of filaments and styles; StMF7: stamen of male flowers during the flowering and development period; PeFF: petals of female flowers; PeMF: petals of male flowers. It can be seen that the SmSEP3 gene is involved in regulating the development of stamens, pistils and petals, and is most significantly expressed in the pistil of male flowers.

[0098] Figure 7 and Figure 8 There are 8 stages of pericarp and seed development. The SmSEP3 gene is involved in regulating the development of pericarp in the 5th stage (seed hard shell stage), the 6th stage (fruit color change stage) and the 7th stage (fruit near maturity stage). The expression level is particularly high in the fruit near maturity stage. The expression of the SmSEP3 gene is most significant in the 4th seed stage (fruit expansion stage).

[0099] See also Fig. 9 The expression level of SmSEP3 was gradually upregulated in the four stages of floral induction and flower initiation of flower buds, indicating that it may regulate the induction process of flower buds.

[0100] Construction of SmSEP3 gene overexpression vector

[0101] The SmSEP3 bacterial solution plasmid with correct sequence was extracted using a high-purity plasmid DNA extraction kit. The pCAMBIA1301 plasmid (stored in this laboratory) was double-digested with restriction endonucleases BstEII-HF and Ncol-HF. The enzyme digestion system is shown in Table 4. The reaction procedure was 37°C for 30 min. After cutting, the vector was purified using a DNA gel recovery kit and detected by electrophoresis. The digested products were stored at -20°C.

[0102] Table 4 Enzyme digestion reaction system of 1301 expression vector

[0103]

[0104] use Ⅱ One Step Cloning Kit was used for vector ligation. The ligation system was 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 1301 expression vector ligation reaction system

[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 1.5 ml centrifuge tube, add 1 / 50 volume of sodium hypochlorite solution and mix, shake up and down repeatedly for 10-15 minutes, wash with sterile distilled water 4-5 times after disinfection, spread the seeds on the sterilized 1 / 2MS solid culture medium, culture at low temperature 4°C for 3 days, illuminate for one week, transplant the Arabidopsis seedlings into sterilized culture soil (substrate soil, vermiculite mixture = 1:1), and culture in a light incubator with long daylight (16 / 8h), 22°C, and a relative water content of 70%. Arabidopsis plants were transformed by the floral immersion method. When Arabidopsis plants grew 3-4 stem leaves after bolting, all plants removed the apical inflorescence synchronously, and the apical dominance was used to promote the growth and flowering of lateral branches. Infect a large number of unopened flower buds with Agrobacterium, soak them for 15-30 seconds, place the plants in a dark environment for about 24 hours after drying, and repeat the infection 4 times during the flowering period, with an interval of about one week each time, to improve the transformation efficiency.

[0109] Screening and identification of transgenic Arabidopsis thaliana

[0110] After infection of Arabidopsis, mixed seeds were harvested to obtain the first generation seeds (T0 generation), which were sown on 1 / 2MS solid medium containing 30 mg / L hygromycin, and transgenic seedlings with normal growth were screened. DNA of resistant plants was extracted as a template, and PCR amplification and molecular testing were performed according to the instructions of the Plant Direct PCR Kit. Gel electrophoresis was used to observe whether the target bands were produced. The above process was repeated to obtain T2 generation transgenic homozygous plants. Subsequent phenotypic observations and functional analyses were performed.

[0111] DNA from leaves of WT wild-type Arabidopsis and 35S::SmSEP3 overexpressing plants were extracted and used as templates for SmSEP3 gene detection. The results are shown in Fig.10 As shown in the figure, Marker represents DL2000 marker; WT represents wild-type Arabidopsis; OE represents 35S::SmSEP3 plants. Fig.10 The wild type was the control group and no bands appeared. The T1 generation strains that had successfully been transformed with exogenous genes showed the target bands. A total of 6 strains were verified.

[0112] During the growth process of wild-type and transgenic Arabidopsis, the bolting time, flowering time, number of rosette leaves, etc. of the plants were recorded and compared in real time. IBM SPSS Statistics 27 software was used for ANOVA analysis, and the mean, standard deviation, etc. were obtained. Execl 2020 software was used to draw charts.

[0113] Fig.11 Figures AD show the comparison between WT wild-type Arabidopsis (left) and 35S::SmSEP3 transgenic plants (right). Figure A shows that overexpression of the SmSEP3 gene in Arabidopsis affects the bolting and flowering time of Arabidopsis, causing it to flower earlier. Figure B shows that the morphological development of the flowers is normal, and the number of petals, sepals, pistils and stamens are all normal. Compared with the wild type, the pistils in the two transgenic lines 35S::SmSEP3#2-3 showed significant elongation, and the upper part of the calyx of the two transgenic lines 35S::SmSEP3#4-5 showed an inward curling deformity, changing from a cuboid split to a flowering shape. Figure C shows that at the same multiple, the number and size of stamens in the 35S::SmSEP3#2-3 line are consistent with the wild type, further confirming the significant growth of its pistils. Meanwhile, the deformity of sepals did not affect the development of stamens and pistils of 35S::SmSEP3#4-5 plants, and their growth was still normal. Figure D shows that the inflorescence of 35S::SmSEP3 plants and wild type are both indefinite inflorescences. Figures E and F show that the fruit pods of 35S::SmSEP3 plants are larger than those of wild type, but the seed setting rate is the same.

[0114] The results of the comparison of flowering time between wild type and 35S::SmSEP3 transgenic lines can be found in Fig.12 . Fig.12 In the middle, Figure G: The left side is the WT wild-type plant, and from left to right are the 35S::SmSEP3#2, 35S::SmSEP3#3 and 35S::SmSEP3#4 transgenic plants; 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] Fig.13 In a to c, WT represents wild-type Arabidopsis, and there are 6 35S::SmSEP3 transgenic lines. A: Statistical analysis of bolting time (days); B: Statistical analysis of rosette leaves (pieces) at bolting; C: Statistical analysis of flowering time (days). The bolting days (B) and flowering time (C) of these 6 lines are extremely significantly different from those of wild-type plants, and the SPSS analysis results are all significant. The bolting time of wild-type Arabidopsis is 20.16 days, the number of rosette leaves at bolting is 12.83 pieces, and the flowering time is 22 days; the bolting time of the 6 transgenic lines is 14.33 days, the number of rosette leaves at bolting is 10.5 pieces, and the flowering time is 15.33 days. The flowering time of 35S::SmSEP3 plants was advanced to varying degrees, specifically 6.67 days earlier. In addition, 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 Pattern of Endogenous Genes in Transgenic Plants

[0117] Representative transgenic lines were selected from the above-mentioned Arabidopsis with different flowering time, and RNA was extracted from WT wild-type plants and 35S::SmSEP3#2, 35S::SmSEP3#5 and 35S::SmSEP3#8 transgenic plants, respectively. After electrophoresis detection, the total RNA bands of all samples were clear and complete, and cDNA was obtained by reverse transcription as a template for subsequent qRT-PCR experiments. The AtEF1 gene was used as an internal reference gene. The fluorescence quantitative experimental method was the same as described above. At the same time, AtEF1 was used as an internal reference gene for fluorescence quantitative experiments. The fluorescence quantitative PCR primer sequences of the genes are shown in Table 6.

[0118] Adoption 2 -ΔΔCt Gene expression levels were calculated and plotted.

[0119] Table 6 Fluorescence quantitative PCR primers

[0120]

[0121] To further study the gene regulation of 35S::SmSEP3 transgenic plants and explain their phenotypic changes at the molecular level, the expression levels of genes related to flowering time regulation upstream and downstream of SEP3 were measured in the floral tissues of Arabidopsis thaliana and compared with those of WT wild-type plants. Fig.11 . Fig.11 In the figure, WT is the wild-type Arabidopsis plant and 35S::SmSEP3#2, 35S::SmSEP3#5 and 35S::SmSEP3#8 transgenic lines. First, the expression level of SmSEP3 members in the flower parts of 35S::SmSEP3 transgenic plants is extremely high, which is significantly different from the wild type, indicating that the function of the SmSEP3 gene is to regulate flowering time, and its overexpression causes early flowering in Arabidopsis.

[0122] In the tissue parts of 35S::SmSEP3 Arabidopsis flowers, the expression levels of genes such as AtLFY, AtPI, AtSHP1, AtSEP2, AtSEP3, AtSTK, AtSOC1 and AtFT were higher than those of wild type. In Arabidopsis flower tissues, the expression levels of genes such as flowering time-related integrators (AtFT and AtSOC1), floral organ development-related (AtSEP3 and AtPI) and flowering meristem regulatory factors (AtLFY) were higher than those of wild type. Therefore, overexpression of the SmSEP3 gene promoted the expression of flowering integrators and floral meristem regulatory factors, resulting in early bolting and flowering of 35S::SmSEP3 transgenic Arabidopsis.

[0123] This example demonstrates that the soapberry SmSEP3 gene can be used to cultivate new soapberry varieties with different flowering times through the model plant Arabidopsis; the SmSEP3 gene can be used to regulate the flowering time to obtain new varieties with desired flowering characteristics. This example also illustrates a plant transgenic method for introducing the expression vector into plant cells to change the flowering time of the plant. This example also illustrates a method for analyzing the gene expression pattern related to the regulation of the flowering time of the plant by measuring the expression level of the SmSEP3 gene at different developmental stages and analyzing the regulatory mechanism of the flowering time of the plant. This example also illustrates a method for measuring the expression level of the flowering time regulation-related gene in the transgenic plant by using the fluorescent quantitative primer pair to quantitatively analyze the expression level of the flowering time regulation-related gene.

[0124] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 regarded as the protection scope of the present invention.

Claims

1. A Sapindus mukorossi SmSEP3 gene, characterized in that: The SmSEP3 gene is expressed in plants to regulate the flowering time of the plants, promote the development of pistils, sepal curling and fruit pod development, and comprises a nucleotide sequence selected from the following group: A, nucleotide sequence shown in Seq ID NO.1 in the sequence listing; B. The nucleotide sequence encoding the amino acid sequence shown in Seq ID NO.

2.

2. A protein encoded by the Sapindus mukorossi SmSEP3 gene according to claim 1, characterized in that: The amino acid sequence of the protein is selected from the amino acid sequence shown in Seq ID NO.2 in the sequence table.

3. A primer pair for cloning the Sapindus mukorossi SmSEP3 gene according to claim 1, characterized in that: The base sequences of the primer pairs are as follows: The first upstream primer F: 5'-ATGTACATTGGCAAATTCAACTATT-3', First downstream primer R: 5′-TCAAGCCATCCACCTTGGTAT-3′; Optionally, the base sequence of the primer pair also includes an enzyme cleavage site, and the base sequence of the primer pair including the enzyme cleavage site is as follows: The second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGTACATTGGCAAATTCAACTATT-3', The second downstream primer R: 5′-GGGGAAATTCGAGCTGGTCACTCAAGCCATCCACCTTGGTAT-3′.

4. A fluorescent quantitative primer pair for the SmSEP3 gene of Sapindus mukorossi, characterized in that: The base sequence of the fluorescent quantitative primer pair is as follows: The third upstream primer F: 5'-CCAAAGCGTAACGTACAGCA-3', The third downstream primer R: 5'-CAAGCCATCCACCTTGGTAT-3'.

5. An expression vector containing the Sapindus mukorossi SmSEP3 gene according to claim 1, characterized in that: The pCAMBIA1301 vector plasmid was double-digested with restriction endonucleases BstEII-HF and Ncol-HF. ⅡOneStep Cloning Kit was used for vector ligation to obtain the overexpression vector pCAMBIA1301-SmSEP3.

6. A use of the Sapindus mukorossi SmAP3 gene according to claim 1, characterized in that: Used to cultivate new varieties of soapberry with different flowering times.

7. A method for breeding new plant varieties, characterized in that: The SmAP3 gene of claim 1 is used to regulate flowering time to obtain a new variety with desired flowering characteristics.

8. A plant genetic modification method, characterized in that: The method comprises introducing the expression vector of claim 5 into plant cells to change the flowering time of the plant.

9. A method for analyzing gene expression patterns related to plant flowering time regulation, characterized in that: By measuring the expression level of SmAP3 gene at different developmental stages, the regulatory mechanism of plant flowering time was analyzed.

10. A method for measuring the expression level of genes related to flowering time regulation, characterized in that: The fluorescent quantitative primer pair according to claim 4 is used to quantitatively analyze the expression levels of genes related to flowering time regulation in transgenic plants.

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