Sapindus mume SmSOC2 gene and application thereof

Abstract

The application discloses a sapindus mume SmSOC2 gene and application thereof, the gene is expressed in plants to regulate flowering time, equal height of pistil and stamen, and stamen loss and variation, the gene is a nucleotide sequence shown in a sequence table Seq ID NO.1 or a nucleotide sequence encoding an amino acid sequence shown in a sequence table Seq ID NO.2.The gene is used for cultivating sapindus mume new varieties with different flowering times.Through whole plant observation and flowering time difference analysis of transgenic arabidopsis, overexpression of the gene can make the plant early in the bolting and flowering time, equal height of pistil and stamen, and stamen loss and variation.The flowering time, bolting days and rosette leaf number of the transgenic arabidopsis plant are significantly different from those of the wild type plant, which is helpful to accurately improve the flower structure of the plant, cultivate new varieties with higher ornamental value, speed up the sapindus mume breeding cycle and cultivate high-yield varieties.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering and relates to a gene that affects 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] Sapindus mukorossi (scientific name: Sapindus mukorossi Gaertn.), a deciduous tree belonging to the Sapindaceae family and the Sapindus genus, is widely distributed across China and is known by nicknames such as "wood soapberry," "soap tree," and "soap fruit." Known for its versatility, Sapindus mukorossi not only has important applications in daily chemicals and medicine, but also shows great potential in biomass energy and landscaping.

[0003] The soapberry life cycle encompasses several key developmental stages, from the early growth of flower buds, leaves, and stems, to the emergence of inflorescences, flowering, fruiting, and ultimately maturity and senescence. Each stage plays a decisive role in the plant's ultimate yield and quality. In spring, soapberry flower buds first appear as small yellow dots in the leaf axils. This process continues through the floral induction phase, flower initiation, flower bud differentiation, and ultimately fruit development and maturity, a process that spans the entire growing season.

[0004] Although soapberry has significant economic value, its long juvenile period limits the efficiency of breeding and the rapid realization of economic benefits. Therefore, studying the flowering regulation mechanism of soapberry and discovering genetic resources that can control flowering time have significant theoretical and practical value for cultivating new soapberry varieties with early maturing characteristics, shortening the breeding cycle, and improving economic benefits. Summary of the Invention

[0005] In view of this, the present invention aims to provide a gene that can advance the flowering time of plants and inhibit stamen development.

[0006] Through long-term exploration and experimentation, as well as numerous experiments and efforts, the inventors have continuously innovated and reformed to solve the above technical problems. The present invention provides a technical solution that provides a soapberry SmSOC2 gene that is expressed in plants to regulate the flowering time of the plant, the height of stamens and stamens, and the absence and variation of stamens. The SmSOC2 gene comprises a nucleotide sequence selected from the following group:

[0007] A. The nucleotide sequence shown in Seq ID NO.1 of the sequence listing;

[0008] B. The nucleotide sequence encoding the amino acid sequence shown in Seq ID NO.2.

[0009] The present invention also provides a protein encoded by the soapberry SmSOC2 gene, wherein the amino acid sequence of the protein is selected from the amino acid sequence shown in Seq ID NO.2 of the sequence table.

[0010] The present invention also provides a primer pair for cloning the Sapindus mukorossi SmSOC2 gene, the base sequence of the primer pair is as follows:

[0011] The first upstream primer F: 5'-TTGGAAAGAATCCTAGAACGATATG-3',

[0012] First downstream primer R: 5′-TTATTCATTC ACATGGCGAA GC-3′;

[0013] Optionally, the base sequence of the primer pair further includes an enzyme cleavage site. The base sequence of the primer pair including the enzyme cleavage site is as follows:

[0014] The second upstream primer F: 5'-AGAACACGGGGGACTCTTGACTTGGAAAGAATCCTAGAACGA TATG-3',

[0015] The second downstream primer R: 5′-GGGGAAATTCGAGCTGGTCACTTATTCATTC ACATGGCGAA GC-3′.

[0016] The present invention also provides a fluorescent quantitative primer pair for the Sapindus mukorossi SmSOC2 gene, the base sequence of the fluorescent quantitative primer pair is as follows:

[0017] The third upstream primer F: 5'-CTCGGAAGGAAAAAGCATTG-3',

[0018] The third downstream primer R: 5′-CTCTCGCCTGAAAACTGTCC-3′.

[0019] The present invention also provides an expression vector containing the soapberry SmSOC2 gene, wherein the pCAMBIA1301 vector plasmid is double-digested with restriction endonucleases BstEII-HF and Ncol-HF, and the expression vector is expressed using Ⅱ One Step Cloning Kit was used for vector ligation to obtain the overexpression vector pCAMBIA1301-SmSOC2.

[0020] The present invention also provides a use of the soapberry SmSOC2 gene for cultivating new soapberry varieties with different flowering times.

[0021] The application also provides a method for cultivating new plant varieties by regulating the flowering time of the SmSOC2 gene to obtain new varieties with desired flowering characteristics.

[0022] The application also provides a plant transgenic method comprising introducing the expression vector into plant cells to change the flowering time of the plant.

[0023] The application also provides a method for analyzing the expression pattern of a flowering time regulation-related gene by determining the expression amount of the SmSOC2 gene at different developmental stages to analyze the regulation mechanism of the flowering time of the plant.

[0024] The application also provides a method for determining the expression amount of a flowering time regulation-related gene by using the fluorescent quantitative primer pair to quantitatively analyze the expression amount of the flowering time regulation-related gene in the transgenic plant.

[0025] Compared with the prior art, one of the above technical solutions has the following advantages:

[0026] The application discloses the important role of SmSOC2 gene in regulating the flowering time of plants. By expressing the SmSOC2 gene in plants, the flowering time can be significantly advanced, and the development of stamens can be inhibited. This discovery has great significance in the field of plant breeding, as it not only accelerates the development of new varieties, but also helps to improve the yield and quality of crops. In addition, the application also provides a cloning method of the SmSOC2 gene, a construction technology of the expression vector, and a cultivation method of the transgenic plant, which provides a complete solution for plant genetic engineering.

[0027] The application is verified by transgenic Arabidopsis thaliana experiments, which proves that the up-regulation of SmSOC2 gene expression can promote flowering and affect the reproductive development stage of plants. Overexpression of this gene leads to an advance in flowering time, reducing the period from sowing to flowering, thereby accelerating the breeding process. In addition, by quantitatively analyzing the expression pattern of related genes in transgenic plants, the application also reveals the molecular mechanism of SmSOC2 gene in regulating the flowering process, providing theoretical basis and experimental data support for further gene function research and crop improvement. These technical effects not only prove the innovativeness of the application, but also demonstrate its extensive potential in practical applications.

[0028] Observation of the entire plant and analysis of flowering time differences in transgenic Arabidopsis thaliana expressing the SmSOC2 gene revealed that the SmSOC2 gene can advance flowering time to varying degrees. The flowering time, days to bolting, and number of rosette leaves at bolting in transgenic Arabidopsis plants were significantly different from those in wild-type plants. A normal Arabidopsis flower typically has six stamens. However, in transgenic plants, the number of stamens was reduced to four to five. This could help precisely refine plant floral structures, cultivate new varieties with higher ornamental value, accelerate the breeding cycle of soapberry trees, and accelerate the development of high-yielding varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is the electrophoresis diagram of the SmSOC2 gene clone.

[0031] Figure 2 This is the result of phylogenetic analysis of SOC subclass proteins and SmSOC2 proteins in different species.

[0032] Figure 3 This is the result of cis-acting element analysis of the SmSOC2 gene.

[0033] Figure 4 This is the predicted tertiary structure of SmSOC2 protein.

[0034] Figure 5 These are the results of the expression pattern analysis of SmSOC2 during the development of male and female flower buds.

[0035] Figure 6 The expression pattern analysis results of SmSOC2 in male and female floral organs

[0036] Figure 7 These are the results of expression pattern analysis of the SmSOC2 gene during eight stages of pericarp development.

[0037] Figure 8 These are the results of expression pattern analysis of the SmSOC2 gene during eight developmental stages of the plant.

[0038] Figure 9 Analysis results of the expression pattern of the SmSOC2 gene during the floral induction period.

[0039] Figure 10 This is the identification result of 35S::SmSOC2 transgenic plants.

[0040] Figure 11 This is a comparison chart of the phenotypic observations of the wild type and 35S::SmSOC2 transgenic lines.

[0041] Figure 12 This is a comparison of flowering time between the wild type and 35S::SmSOC2 transgenic lines.

[0042] Figure 13 Statistical graph of bolting time, rosette leaf number and flowering time of wild type and 35S::SmSOC2 transgenic lines.

[0043] Figure 14 This is a graph of the expression levels of genes related to regulating flowering time in Arabidopsis flower tissue. DETAILED DESCRIPTION

[0044] The following describes a specific embodiment with reference to the accompanying drawings.

[0045] 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 making 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.

[0046] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it may not be further defined or explained in subsequent drawings.

[0047] Example 1

[0048] The nucleotide sequence of the Sapindus mukorossi SmSOC2 gene described in this example is shown in Seq ID No. 1 in the sequence listing. Based on this nucleotide sequence, the amino acid sequence shown in Seq ID No. 2 in the sequence listing can be encoded. The CDS sequence is shown in Seq ID No. 9 in the sequence listing.

[0049] The Sapindus mukorossi SmSOC2 gene is expressed in plants, particularly in Sapindus mukorossi. Functional validation and transgenic Arabidopsis thaliana have demonstrated that the gene significantly regulates flowering time. Utilizing the Sapindus mukorossi SmSOC2 gene could shorten the Sapindus mukorossi breeding cycle and accelerate the development of high-yield Sapindus mukorossi varieties.

[0050] Example 2

[0051] The primer pair for cloning the SmSOC2 gene of the soapberry described in this example was used to clone the SmSOC2 gene of the soapberry described in Example 1. The base sequence of the primer pair is as follows:

[0052] The first upstream primer F: 5'-TTGGAAAGAATCCTAGAACGATATG-3',

[0053] The first downstream primer R: 5'-TTATTCATTC ACATGGCGAA GC-3'.

[0054] The first upstream primer F is shown in the sequence listing Seq ID NO.3, and the first downstream primer R is shown in the sequence listing Seq ID NO.4.

[0055] Example 3

[0056] The primer pair for cloning the SmSOC2 gene of Sapindus mukorossi described in this example is based on the primer pair described in Example 2, and a restriction enzyme cleavage site is added to obtain the second upstream primer F as shown in Seq ID NO.5 of the sequence listing and the second downstream primer R as shown in Seq ID NO.6 of the sequence listing. The base sequences of the primer pair including the restriction enzyme cleavage site are as follows:

[0057] The second upstream primer F: 5'-AGAACACGGGGGACTCTTGACTTGGAAAGAATCCTAGAACGATATG-3',

[0058] The second downstream primer R: 5′-GGGGAAATTCGAGCTGGTCACTTATTCATTC ACATGGCGAA GC-3′.

[0059] The cloning primer sequences were designed using Primer3web (4.1.0) (https: / / bioinfo.ut.ee / primer3 / ) and then sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for synthesis.

[0060] Example 4

[0061] The fluorescent quantitative primer pair for the Sapindus mukorossi SmSOC2 gene described in this example is used for quantitative analysis of the Sapindus mukorossi SmSOC2 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 listing:

[0062] The third upstream primer F: 5'-CTCGGAAGGAAAAAGCATTG-3',

[0063] The third downstream primer R: 5′-CTCTCGCCTGAAAACTGTCC-3′.

[0064] The fluorescence quantitative primer sequences were designed using Primer3web (4.1.0) (https: / / bioinfo.ut.ee / primer3 / ) and then sent to Beijing Ruiboxing Biotechnology Co., Ltd. for synthesis.

[0065] Example 5

[0066] This example is an example of verifying the nucleotide sequence and amino acid sequence described in Example 1 using the primers described in Examples 3 to 4.

[0067] In this example, three Sapindus mukorossi trees were selected as the experimental materials, and three biological replicates were performed. The sampling location was located in Jianning County, Sanming City, Fujian Province. The sampling time was fixed at 10:00 am to 12:00 pm. The upper and middle parts of the one-year-old 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.

[0068] There are four stages of floral induction and flower initiation (bud1-4) in soapberry: bud1, the dormant period of flower buds; bud2, the floral induction period; bud3, the flower initiation period; and bud4, the inflorescence differentiation period.

[0069] After floret formation, eight samples from each sex of female (FF1-8) and male (MF1-8) flowers of Sapindus mukorossi were collected during floral organ development: F1 represents the stage of complete floral primordium formation, F2 represents stamen meiosis, F3 represents microspore development, F4-6 represents rapid filament and style elongation, F7 represents anthesis development, and F8 represents late anthesis, for a total of 48 samples. Ten tissue locations of key male and female floral organs (petals, pistils, and stamens) were analyzed, covering floral organs from stages 4 (rapid filament and style elongation) to 7 (anthesis 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, and petals (PeFF) and petals (PeMF) of male flowers.

[0070] After preliminary experimental screening, this example used cDNA from flower buds during inflorescence differentiation as a template for PCR amplification. The primers described in Example 3 were used for amplification, and the TA cloning reaction system is shown in Table 1 below. The reaction procedure was 34 cycles of pre-denaturation at 98°C for 3 minutes, denaturation at 98°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 40 seconds, followed by a final extension at 72°C for 5 minutes, followed by storage at 4°C. The PCR product was added to 2 μl of 10× Loading buffer and separated by 1% agarose gel electrophoresis. A clean gel fragment of the desired fragment was excised and recovered using a DNA gel electrophoresis kit. The entire process was performed on ice.

[0071] Table 1 TA cloning PCR sequence amplification system

[0072]

[0073]

[0074] The gel recovery product was connected to the T vector. The reaction system is shown in Table 2.

[0075] Table 2T cloning and ligation vector system

[0076]

[0077] Place at room temperature (20-30°C) for 5 minutes. After the reaction, place at 4°C. Add 5 μl of the ligation solution to 50 μl of freshly melted DH10B competent cells, mix gently, place on ice for 30 minutes, heat shock in a 42°C water bath for 30 seconds, and immediately place on ice for 2 minutes. Add 300-500 μL of sterile LB medium and culture at 37°C, 200 rpm, shaking for 1 hour. Take 200 μl of the bacterial solution (containing Amp ampicillin) and culture at 37°C overnight (12-16 hours). Select a single colony for PCR molecular detection. The E. coli positive clone was commissioned to Beijing Ruibo Xingke Biotechnology Co., Ltd. for determination.

[0078] After electrophoresis detection, the extracted total RNA bands were clear and complete, meeting the experimental requirements. The cDNA of the soapberry flower tissue was used as a template to clone the SmSOC2 gene. The clone electrophoresis diagram is shown in Figure 1 After sequencing, the target fragment obtained a 549 bp coding sequence, see Seq ID NO.1 in the sequence list, encoding 182 amino acids, see Seq ID NO.2 in the sequence list.

[0079] To further study the conserved motif sequence of SmSOC2, the protein sequences of MADS genes in each species database in Phytozome were screened from the Phytozome v13 public data platform, including the MADS gene sequences of Arabidopsis thaliana TAIR10, Glycine max Wm82.a4.v1, Malus domestica v1.1, Populus trichocarpa v4.1, Vitis vinifera v2.1 and Prunus persica v2.1, combined with the SmMADS gene family of Sapindus mukorossi, and the MUSCLE alignment of the MADS protein sequences of multiple species was performed by MEGA7, the phylogenetic tree of Type I and Type II subfamily was reconstructed by NJ adjacent method, and the parameter bootstrap value was checked and set to repeat 1000 times.

[0080] The SOC amino acid sequences of each species were screened for phylogenetic development analysis, as shown in Figure 2 The number of SOC proteins of Arabidopsis, poplar, grape, soybean, peach, apple and Sapindus mukorossi was 1, 5, 3, 7, 3, 5 and 1, respectively. Evolution showed that SmSOC2 and grape SOC2 protein had close genetic relationship, and the SOC2 protein of different species had obvious difference in regulation function.

[0081] The promoter sequence of 3000bp upstream of the coding region of Type I and Type II subfamily of SmMADS gene family was taken, the cis-acting elements were predicted by online data analysis software PlantCARE, and the statistical graph was made by Execl, as shown in Figure 3 .

[0082] Figure 3 It is shown that the expression of SmSOC2 transcription factor may be involved in the response process to different biological and abiotic stresses, has the ability to perceive and respond to environmental stress, plant hormones and photoperiod, and participates in the regulation of plant growth and development. SmSOC2 has some common basic elements (TATA) and other four main categories:

[0083] (1) Related elements of environmental stress regulation: mechanical damage response element (WUN-motif);

[0084] (2) Drought-induced binding site-related elements (MYB, MBS, MBS and MYC) and anaerobic-related elements (ARE) and the like, which respond to abiotic stress of plants;

[0085] (3) Related elements of plant growth and development regulation (AAGAA-motif);

[0086] (4) A large number of circadian elements (G-box, TCT-motif and STRE, etc.) regulate the plant circadian rhythm.

[0087] The function of protein is closely related to its structure. Therefore, the three-dimensional structure of SmSOC2 protein was constructed by using the online SWISS-MODEL website, see Figure 4 .

[0088] RNA extraction and real-time fluorescence quantitative test

[0089] The centrifuge tube, gun head, mortar and other materials used in the experiment were sterile. The samples of various tissue parts of Sapindus were ground with liquid nitrogen, and the total RNA of Sapindus was extracted by using Omega RNA kit. The RNA concentration was determined by using NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The integrity of the RNA was detected by 1% agarose gel electrophoresis. The cDNA synthesis was performed by using TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR reverse transcription kit, and the obtained cDNA was diluted by adding nuclease-free water.

[0090] The qRT-PCR experiment of SmSOC2 gene was performed by using TB Green Premix Ex Taq (SYBR Green) enzyme, and SmACT was used as the internal reference gene for fluorescence quantitative experiment. The sequence of the fluorescence quantitative PCR primer of the gene is shown in Table 3.

[0091] Table 3 qRT-PCR amplification reaction system

[0092]

[0093]

[0094] The qRT-PCR reaction program was set as follows: 95℃ for 30s; 95℃ for 52s, 60℃ for 30s, 40 cycles. After the end of the cycle, the product specificity was detected by using the melting curve: slowly increasing from 60℃ to 95℃, and collecting 5 fluorescence signals for each 1℃ increase. Sapindus SmACT gene was used as the internal reference for detection, and three biological replicates, technical replicates were set, the average value was calculated, and the gene expression was calculated by using 2 -ΔΔCt method and processed for drawing.

[0095] The expression pattern of SmSOC2 in the developing organs of male and female flowers of each 8 periods, the specific results are shown in Figure 5The results show that the expression of SmSOC2 gene increases first and then decreases during the development of female flower, and the expression amount is the highest in the second stage (stamen meiosis), and then gradually decreases. The expression of SmSOC2 gene is significantly high in the fifth stage (rapid elongation of filament and style).

[0096] Figure 6 PiFF4: pistil of female flower in the rapid elongation of filament and style stage; PiFF7: pistil of female flower in the flowering development stage; PiMF4: pistil of male flower in the rapid elongation of filament and style stage; PiMF7: pistil of male flower in the flowering development stage; StFF4: stamen of female flower in the rapid elongation of filament and style stage; StFF7: stamen of female flower in the flowering development stage; StMF4: stamen of male flower in the rapid elongation of filament and style stage; StMF7: stamen of male flower in the flowering development stage; PeFF: petal of female flower; PeMF: petal of male flower. It can be seen that SmSOC2 gene is involved in the regulation of the development of stamen and pistil, and the expression is the most significant in the pistil of female and male flowers.

[0097] Figure 7 and Figure 8 The eight stages are the development of pericarp and seed. SmSOC2 gene is involved in the growth and development of pericarp in the first stage (initial fruit stage), and is involved in the development of pericarp and seed in the second stage (cotyledon growth stage), the fourth stage (fruit swelling stage) and the sixth stage (fruit color changing stage).

[0098] See Figure 9 The expression amount of SmSOC2 gradually increases in the four stages of flower bud induction and flower initiation of Jatropha curcas, which indicates that the induction process of flower bud may be regulated.

[0099] Construction of SmSOC2 gene overexpression vector

[0100] The SmSOC2 bacterial plasmid with correct sequence is extracted by using high-purity plasmid DNA extraction kit, and the pCAMBIA1301 (preserved in the laboratory) plasmid is double-digested by restriction endonuclease BstEII-HF and Ncol-HF. The enzyme digestion system is shown in Table 4, and the reaction program is 37°C, 30 min. After cutting, the vector is purified by DNA gel recovery kit, and electrophoresis experiment is performed for detection. The product after enzyme digestion is stored at -20°C.

[0101] Table 4 Enzyme digestion reaction system of 1301 expression vector

[0102]

[0103]

[0104] Using II. Vector ligation was performed using the One Step Cloning Kit. The ligation system was shown in Table 5. The pCAMBIA1301-SmSOC2 overexpression vector was obtained and 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 thaliana

[0108] Under sterile conditions, an appropriate amount of wild-type Arabidopsis seeds were placed in a 1.5ml centrifuge tube. A 1 / 50 volume of sodium hypochlorite solution was added and mixed thoroughly. The tube was shaken repeatedly for 10-15 minutes. After disinfection, the seeds were rinsed 4-5 times with sterile distilled water. The seeds were then plated onto sterilized 1 / 2 MS solid culture medium and vernalized at 4°C for 3 days. After one week of light exposure, the Arabidopsis seedlings were transplanted into sterilized culture soil (a 1:1 mixture of substrate soil and vermiculite) and cultured in a light incubator with long days (16 / 8 hours), 22°C, and a relative moisture content of 70%. Arabidopsis plants were transformed using the floral dipping method. After bolting and developing 3-4 cauline leaves, the apical inflorescences of all plants were removed simultaneously to promote lateral branch growth and flowering through apical dominance. Infect a large number of unopened flower buds with Agrobacterium, soak them for 15-30 seconds, dry them, and place the plants in a dark environment for about 24 hours. 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, Arabidopsis thaliana was mixed and harvested to obtain first-generation seeds (T0 generation). These seeds were sown on 1 / 2 MS solid medium containing 30 mg / L hygromycin. Transgenic seedlings that showed normal growth were selected. DNA from resistant plants was extracted and used as a template for PCR amplification and molecular analysis according to the Plant Direct PCR Kit instructions. Gel electrophoresis was used to observe the presence of target bands. The above process was repeated to obtain T2 generation homozygous transgenic plants. Subsequent phenotypic observation and functional analysis were performed.

[0111] DNA was extracted from leaves of WT wild-type Arabidopsis and 35S::SmSOC2 overexpressing plants and used as templates for SmSOC2 gene detection. The results are as follows: Figure 10 As shown in the figure, Marker represents DL2000 marker; WT represents wild-type Arabidopsis; OE represents 35S::SmSOC2 plants. Figure 10The wild type was used as 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 thaliana, the bolting time, flowering time, and number of rosette leaves of the plants were recorded and compared in real time. ANOVA analysis was performed using IBM SPSS Statistics 27 software, and the mean, standard deviation, etc. were obtained. Execl 2020 software was used to draw charts.

[0113] Figure 11 Figures A-D (left) show WT wild-type Arabidopsis, and the right show 35S::SmSOC2 plants. Figure A shows that overexpression of the SmSOC2 gene advances bolting and flowering in Arabidopsis. Figure B shows that flower morphology in 35S::SmSOC2 plants develops normally, but the stamens and pistils are of equal height and slightly shorter, indicating stunted development. Figure C further demonstrates that overexpression of the SmSOC2 gene shortens the pistils in three lines and leads to stamen mutation and loss. In lines 35S::SmSOC2#8-9, the Arabidopsis has only 4-5 stamens, while line 35S::SmSOC2#5 exhibits mutation or loss of stamens and anthers, but maintains normal fertility. Figure D shows that the inflorescence of 35S::SmSOC2 plants is identical to that of the wild type, with both being indeterminate. Figures E and F show that the pod length of 35S::SmSOC2 plants is slightly shorter, but this has little effect on the fruit setting rate.

[0114] Comparison of flowering time between wild type and 35S::SmSOC2 transgenic lines can be found in Figure 12 . Figure 12 In the middle, Panel G shows a WT plant on the left, followed by 35S::SmSOC2#2 and 35S::SmSOC2#4 transgenic plants from left to right. Panel H shows a WT plant on the left, followed by 35S::SmSOC2#5 and 35S::SmSOC2#8 transgenic plants from left to right. Panel I shows a WT plant, followed by 35S::SmSOC2#9 and 35S::SmSOC2#10 transgenic plants from left to right. These results indicate that the flowering time of all six 35S::SmSOC2 transgenic lines is earlier than that of WT Arabidopsis.

[0115] Figure 13In panels a–c, WT represents wild-type Arabidopsis. There are six 35S::SmSOC2 transgenic lines. Panel a: Statistical analysis of bolting time (days); Panel b: Statistical analysis of rosette leaves at bolting; Panel c: Statistical analysis of flowering time (days). The bolting days (Panel b) and flowering time (Panel c) of these six lines were significantly different from those of wild-type plants, as shown by SPSS analysis. The bolting time of wild-type Arabidopsis was 20.16 days, the number of rosette leaves at bolting was 12.83, and the flowering time was 22 days. The bolting time of the six transgenic lines was 16.83 days, the number of rosette leaves at bolting was 11.83, and the flowering time was 19.67 days. The flowering time of 35S::SmSOC2 plants was advanced to varying degrees, specifically 2.33 days earlier. In addition, the number of rosette leaves in most 35S::SmSOC2 transgenic lines was positively correlated with the bolting time. The earlier the bolting, the fewer the rosette leaves.

[0116] Study on the Expression Pattern of Endogenous Genes in Transgenic Plants

[0117] Representative transgenic lines of Arabidopsis thaliana with the aforementioned flowering time differences were selected. Flowers of positive transgenic plants and wild-type Arabidopsis thaliana were extracted. RNA was also extracted from WT wild-type plants and transgenic plants of 35S::SmSOC2#2, 35S::SmSOC2#5, and 35S::SmSOC2#8. After electrophoresis, the total RNA bands of all samples were clear and intact. Reverse transcription was performed to obtain cDNA, which served as a template for subsequent qRT-PCR experiments. Fluorescence quantitative PCR experiments were performed using the same method as described above, with AtEF1 also serving as an internal reference gene. The primer sequences for these genes are shown in Table 6.

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

[0119] Table 6 Fluorescence quantitative PCR primers

[0120]

[0121] To further investigate gene regulation in 35S::SmSOC2 transgenic plants and explain their phenotypic changes at the molecular level, the expression levels of genes involved in the upstream and downstream regulation of flowering time by SOC2 were measured in the floral tissues of Arabidopsis thaliana and compared with those in wild-type plants. Figure 14 . Figure 14In the figure, WT represents wild-type Arabidopsis plants and 35S::SmSOC2#2, 35S::SmSOC2#5, and 35S::SmSOC2#8 transgenic lines. First, the expression of SmSOC2 members in the flowers of 35S::SmSOC2 transgenic plants was extremely high, significantly different from that of the wild type, indicating that the SmSOC2 gene functions to regulate flowering time and that its overexpression causes early flowering in Arabidopsis.

[0122] In the floral tissues of 35S::SmSOC2 Arabidopsis, the expression levels of genes such as AtSOC1, AtSEP4, AtLFY, AtAG, and AtFT were higher than those of the wild type, while the expression levels of genes such as AtSVP, AtAP3, and AtTFL1 were lower than those of the wild type.

[0123] In Arabidopsis floral tissue, the expression levels of genes involved in flowering time integration (AtFT and AtSOC1), floral organ development (AtPI), and flowering meristem regulation (AtLFY) were higher than in the wild type, while the expression levels of flowering repressors (AtTFL1 and AtSVP) were lower than in the wild type. Therefore, overexpression of the SmSOC2 gene promoted the expression of flowering integrators and floral meristem regulation factors, leading to premature bolting and flowering in 35S::SmSOC2 transgenic Arabidopsis.

[0124] This example demonstrates, using the model plant Arabidopsis thaliana, that the SmSOC2 gene of soapberry can be used to cultivate new varieties of soapberry with different flowering times; the SmSOC2 gene can be used to regulate flowering time to obtain new varieties with desired flowering characteristics. This example also illustrates a method for plant transgenic engineering, in which the expression vector is introduced into plant cells to change the flowering time of a plant. This example also illustrates a method for analyzing the expression pattern of genes related to plant flowering time regulation, in which the regulatory mechanism of plant flowering time is analyzed by measuring the expression level of the SmSOC2 gene at different developmental stages. This example also illustrates a method for measuring the expression level of genes related to flowering time regulation in transgenic plants, in which the expression level of genes related to flowering time regulation is quantitatively analyzed using the fluorescent quantitative primer pair.

[0125] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kind of soapberry SmSOC2 A gene characterized by described SmSOC2 The gene is selected from the group consisting of: A. The nucleotide sequence shown in Seq ID NO.1 of the sequence listing; B. The nucleotide sequence encoding the amino acid sequence shown in Seq ID NO.

2.

2. A soapberry according to claim 1 SmSOC2 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is selected from the amino acid sequence shown in SeqIDNO.2 of the sequence table.

3. A method for cloning the soapberry according to claim 1 SmSOC2 A primer pair for a gene, characterized in that The base sequences of the primer pairs are as follows: The first upstream primer F: 5'-TTGGAAAGAATCCTAGAACGATATG-3', The first downstream primer R: 5'-TTATTCATTC ACATGGCGAA GC-3'.

4. The primer pair according to claim 3, characterized in that The base sequence of the primer pair also includes an enzyme cleavage site. The base sequence of the primer pair including the enzyme cleavage site is as follows: The second upstream primer F: 5'-AGAACACGGGGGACTCTTGACTTGGAAAGAATCCTAGAACGATATG-3', The second downstream primer R: 5′-GGGGAAATTCGAGCTGGTCACTTATTCATTC ACATGGCGAA GC-3′.

5. A soapberry according to claim 1 SmSOC2 A fluorescent quantitative primer pair for a gene, characterized in that: The base sequences of the fluorescent quantitative primer pairs are as follows: The third upstream primer F: 5'-CTCGGAAGGAAAAAGCATTG-3', The third downstream primer R: 5′-CTCTCGCCTGAAAACTGTCC-3′.

6. A kind of soapberry containing the soapberry according to claim 1 SmSOC2 A gene expression vector, characterized in that The pCAMBIA1301 vector plasmid was double-digested with restriction endonucleases BstEII-HF and Ncol-HF, and the vectors were connected using ClonExpress ® Ⅱ One Step Cloning Kit to obtain the overexpression vector pCAMBIA1301- SmSOC2.

7. A method for breeding new plant varieties, characterized in that: Overexpression of claim 1 SmSOC2 The invention relates to a gene that causes a plant to flower earlier in time, thereby obtaining a new variety having desirable flowering characteristics, the plant being Arabidopsis thaliana.

8. A plant genetic modification method, characterized in that: The method comprises introducing the expression vector according to claim 6 into a plant cell, wherein the plant is Arabidopsis thaliana.

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