Sapindus mume smsoc3 gene and application thereof

Abstract

The application discloses a sapindus mume SmSOC3 gene and application thereof, the gene is expressed in plants to regulate flowering time, pistil and stamen length and calyx shape, 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 time and different flower types.Through whole plant observation and flowering time difference analysis of transgenic arabidopsis, overexpression of the gene can make the plant bolting and flowering time in advance, the development height of pistil and stamen is equal but both slightly short, and the calyx shape is changed into conical shape.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 beneficial to accelerating sapindus mume breeding period, cultivating ornamental varieties with different flower types and accelerating cultivation of new varieties with high yield.

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] Soapberry (scientific name: Sapindus mukorossi) Sapindus mukorossi Gaertn As a deciduous tree, it belongs to the genus *Sapindaceae*. Sapindus (L.), has wide applications in landscaping, daily chemical products, pharmaceuticals, and biomass energy industries. The life cycle of Sapindus mukorossi includes two main stages: vegetative growth and reproductive development. The reproductive development stage involves a complex flowering process, from the dormancy period to the flowering induction period, the flower initiation period, the flower bud differentiation period, and finally the development and ripening of the fruit.

[0003] Although Sapindus mukorossi has significant economic and ecological value, its long juvenile period limits breeding efficiency and delays the realization of economic benefits. Therefore, a deeper understanding of the flowering regulation mechanism of Sapindus mukorossi is of great theoretical and practical significance for shortening the juvenile period and improving breeding efficiency. Currently, some progress has been made in genetic research on plant flowering regulation, but research on flowering regulation genes in the specific species Sapindus mukorossi is still insufficient. This limits the possibility of improving Sapindus mukorossi varieties and achieving early flowering and fruiting through genetic engineering.

[0004] In the field of plant genetic engineering, discovering and studying the flowering-regulating genes specific to Sapindus mukorossi is of great theoretical and practical significance for promoting the breeding improvement and industrial development of this tree species. Summary of the Invention

[0005] Therefore, the present invention aims to provide a gene that can regulate the flowering time of plants, and the protein encoded by the gene, thereby providing new strategies and methods for the breeding of Sapindus mukorossi.

[0006] Through long-term exploration and experimentation, and continuous innovation, the inventor has provided a technical solution to solve the above-mentioned technical problems: a soapberry variety. SmSOC3 Genes, expressed in plants, regulate flowering time, stamen and pistil length, and sepal shape. SmSOC3 Genes contain nucleotide sequences 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 of the amino acid sequence shown in Seq ID NO.2 of the coding sequence listing.

[0009] The present application also provides a protein encoded by the Sapindus SmSOC3 gene, wherein the amino acid sequence of the protein is selected from the amino acid sequence shown in the sequence listing Seq ID NO. 2.

[0010] The present application also provides a primer pair for cloning the Sapindus SmSOC3 gene, wherein the base sequence of the primer pair is as follows:

[0011] a first upstream primer F: 5'-ATGGTGAGGGGAAAAACTCAGA-3',

[0012] a first downstream primer R: 5'-TCAAGCACTTCGTCTCTCAGGT-3'.

[0013] Alternatively, the base sequence of the primer pair further comprises a restriction site, and the base sequence of the primer pair comprising the restriction site is as follows:

[0014] a second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGGTGAGGGGAAAAACTCAGA-3',

[0015] a second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCAAGCACTTCGTCTCTCAGGT-3'.

[0016] The present application also provides a fluorescent quantitative primer pair of the Sapindus SmSOC3 gene, wherein the base sequence of the fluorescent quantitative primer pair is as follows:

[0017] a third upstream primer F: 5'-AGGAGAGCCAAAGACGATGA-3',

[0018] a third downstream primer R: 5'-ATTGAGCATCGTTCCAGTCC-3'.

[0019] The present application also provides an expression vector containing the Sapindus SmSOC3 gene, wherein the pCAMBIA1301 vector plasmid is double-digested by using restriction endonucleases BstEII-HF and Ncol-HF, and the vector is connected by using ClonExpress II OneStep Cloning Kit to obtain an overexpression vector pCAMBIA1301- SmSOC3 .

[0020] The present application also provides a use of the Sapindus SmSOC3 gene for cultivating new varieties of Sapindus with different flowering times.

[0021] The present application also provides a method for cultivating new varieties of plants, using the expression vector. SmSOC3 The gene regulates flowering time to obtain new varieties with desired flowering characteristics.

[0022] The present application also provides a method for transgenically modifying plants, comprising introducing the expression vector into plant cells to change the flowering time of plants.

[0023] The present application also provides a method for analyzing the expression pattern of genes related to the regulation of flowering time in plants, by measuring the expression levels of genes at different developmental stages to analyze the regulatory mechanisms of flowering time in plants. SmSOC3 The present application also provides a method for measuring the expression levels of genes related to the regulation of flowering time, using the fluorescent quantitative primer pair to quantitatively analyze the expression levels of genes related to the regulation of flowering time in transgenic plants.

[0024] The present application also provides a method for measuring the expression levels of genes related to the regulation of flowering time, using the fluorescent quantitative primer pair to quantitatively analyze the expression levels of genes related to the regulation of flowering time in transgenic plants.

[0025] The present application provides a soapberry SmSOC3 gene and its applications, which have the following significant technical effects:

[0026] The core technology of the present application lies in identifying and applying the SmSOC3 gene in soapberry, which can effectively regulate flowering time when expressed in plants. Through the application of transgenic technology, SmSOC3 the gene achieves the advancement of flowering time in plants, which not only provides a new perspective for plant biology research, but also provides an effective means for accelerating flowering and shortening the breeding cycle for commercial breeding. In addition, SmSOC3 the verification of the function of the gene in the model plant Arabidopsis thaliana further confirms its significant role in regulating flowering time.

[0027] SmSOC3 The application of the gene not only accelerates the flowering process, but also helps to cultivate new varieties with specific flowering characteristics, enhancing the adaptability of plants to different environmental conditions. Through the analysis of the expression pattern of the SmSOC3 gene, we understand its key role in the development of plant floral organs and fruit ripening, providing a molecular basis for improving plant growth and development and enhancing stress resistance. In addition, the expression response of the gene under different biological and abiotic stresses provides a new strategy for plant stress biology research and molecular design breeding.

[0028] The SmSOC3The gene provides new gene resources for molecular design breeding, so that it is possible to improve the variety of soapberry and other plants by precise gene editing technology. This molecular level breeding method not only improves the accuracy and efficiency of breeding, but also helps to realize the directional improvement of plant traits. SmSOC3 The discovery and application of the gene enrich our understanding of the regulation mechanism of plant flowering in theory, and provide innovative tools and methods for agricultural production and plant biotechnology in practice, which has important scientific significance and application prospect.

[0029] After transformation SmSOC3 Whole plant observation of the gene Arabidopsis thaliana and flowering time difference analysis, SmSOC3 The gene can make the flowering time of plants appear different degrees of advance, and the flowering time, days to bolting and rosette leaf number of transgenic Arabidopsis thaliana plants are significantly different from those of wild type plants, which is beneficial to speed up the breeding cycle of soapberry species and accelerate the cultivation of high-yield varieties. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0031] Figure 1 is SmSOC3 Cloning electropherogram of the gene.

[0032] Figure 2 is the result of phylogenetic analysis of different species SOC sub-class proteins.

[0033] Figure 3 is SmSOC3 Analysis result of cis-acting elements of the gene.

[0034] Figure 4 is SmSOC3 Protein tertiary structure prediction diagram.

[0035] Figure 5 is the expression pattern analysis result of the gene in the development process of female and male flower buds. SmSOC3

[0036] Figure 6 is the expression pattern analysis result of the gene in female and male flower organs SmSOC3

[0037] Figure 7 is the expression pattern analysis result of the gene in 8 periods of pericarp development. SmSOC3 ​​​

[0038] Figure 8 is a development of 8 periods SmSOC3 The results of the expression pattern analysis of the genes.

[0039] Figure 9 Flower induction period SmSOC3 The results of the expression pattern analysis of the genes.

[0040] Figure 10 is 35S: SmSOC3 The results of the identification of the transgenic plants.

[0041] Figure 11 is wild type and 35S: SmSOC3 The comparison chart of the phenotype observation of the transgenic lines.

[0042] Figure 12 is wild type and 35S: SmSOC3 The comparison chart of the flowering time of the transgenic lines.

[0043] Figure 13 is wild type and 35S: SmSOC3 The statistical chart of the bolting time, rosette leaf number and flowering time of the transgenic lines.

[0044] Figure 14 is the expression amount chart of the genes related to the regulation of flowering time in the flower tissue of Arabidopsis thaliana. DETAILED DESCRIPTION

[0045] The present application will be described below in conjunction with the accompanying drawings and a specific embodiment.

[0046] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.

[0047] It should be noted that: similar reference numerals and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it can not be further defined and explained in the subsequent drawings.

[0048] Example 1

[0049] The Sapium sebiferum described in the present embodiment SmSOC3A gene with a nucleotide sequence as shown in the sequence listing Seq ID NO. 1, based on which an amino acid sequence as shown in the sequence listing Seq ID NO. 2 can be encoded. The CDS sequence is shown in the sequence listing Seq ID NO. 9.

[0050] Soapberry SmSOC3 The gene is expressed in plants, particularly in soapberry, and through functional verification and Arabidopsis thaliana transgenic verification, it is shown that the gene can significantly regulate the flowering time of plants. By using the soapberry SmSOC3 gene, the breeding cycle of soapberry can be shortened, and the development of high-yield soapberry varieties can be accelerated.

[0051] Example 2

[0052] The primer pair described in this embodiment for cloning the soapberry SmSOC3 gene of Example 1 is as follows: SmSOC3 The base sequence of the primer pair is as follows:

[0053] First upstream primer F: 5'-ATGGTGAGGGGAAAAACTCAGA-3',

[0054] First downstream primer R: 5'-TCAAGCACTTCGTCTCTCAGGT-3'.

[0055] 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.

[0056] Example 3

[0057] The primer pair described in this embodiment for cloning the soapberry SmSOC3 gene is based on the primer pair described in Example 2, and an enzyme cutting site is added to obtain a second upstream primer F as shown in the sequence listing Seq ID NO. 5 and a second downstream primer R as shown in the sequence listing Seq ID NO. 6. The base sequence of the primer pair including the enzyme cutting site is as follows:

[0058] Second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGGTGAGGGGAAAAACTCAGA-3',

[0059] Second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCAAGCACTTCGTCTCTCAGGT-3'.

[0060] 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.

[0061] Example 4

[0062] The soapberry described in this embodiment SmSOC3 A quantitative fluorescent primer pair for gene sequencing, used to detect Sapindus mukorossi. SmSOC3 For the quantitative analysis of the gene, the base sequences of the fluorescent quantitative primer pairs are shown in Seq ID NO.7 and Seq ID NO.8 of the sequence listing:

[0063] Third upstream primer F: 5'-AGGAGAGCCAAAGACGATGA-3',

[0064] Third downstream primer R: 5'-ATTGAGCATCGTTCCAGTCC-3'.

[0065] 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.

[0066] Example 5

[0067] This embodiment is an example of using the primers described in Examples 3-4 to verify the nucleotide sequence and amino acid sequence described in Example 1.

[0068] In this embodiment, the experimental material selected was 3 Sapindus mukorossi plants ( Sapindus mukorossi Sample trees were collected three times, with sampling conducted at Jianning County, Sanming City, Fujian Province, at a fixed time of 10:00 AM to 12:00 PM. The middle and upper parts of one-year-old branches at the same height on the outer edge of the tree canopy were quickly placed into cryovials and frozen in liquid nitrogen, and then stored in a -80°C freezer for subsequent RNA extraction.

[0069] 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).

[0070] After the small flower of Sapindus saponaria L. was formed, 8 female flower buds (FF1-8) and 8 male flower buds (MF1-8) were selected at different development stages of the flower organs: F1 is the stage of complete formation of flower organ primordia, F2 is the stage of meiosis of stamens, F3 is the stage of microspore development, F4-6 is the stage of rapid elongation of filaments and style, F7 is the stage of flowering development, and F8 is the stage of post-flowering. A total of 48 groups of samples were obtained. Ten tissue parts of important flower organs (petals, pistils and stamens) were obtained at the 4th stage (rapid elongation of filaments and style) and the 7th stage (flowering development) of flower development: pistil of female flower (PiFF4 and PiFF7), pistil of male flower (PiMF4 and PiMF7), stamen of female flower (StFF4 and StFF7), stamen of male flower (StMF4 and StMF7), petal of female flower (PeFF) and petal of male flower (PeMF).

[0071] After preliminary experiment screening, the cDNA material of flower bud at the flower induction stage was finally used as a template for PCR amplification in this example. The primers used were the primers described in Example 3. The TA cloning reaction system is shown in Table 1. The reaction procedure was as follows: 98 ℃ pre-denaturation for 3 min, 98 ℃ denaturation for 30 s, 56 ℃ annealing for 30 s, 72 ℃ extension for 40 s, 34 cycles, and finally 72 ℃ extension for 5 min, and storage at 4 ℃. After 2 μl of 10×Loading buffer was added to the PCR product, 1% agarose electrophoresis separation was performed. The clean target fragment gel block was cut and recovered using a DNA gel kit. The whole process was performed on ice.

[0072] Table 1 TA cloning PCR sequence amplification system

[0073]

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

[0075] Table 2 T cloning connection vector system

[0076]

[0077] After being placed at room temperature (20-30 ℃) for 5 min, the reaction was completed. After being placed at 4 ℃, 5 μl of the connection liquid was taken into 50 μl of just melted DH10B competent cells, gently mixed, and then placed on ice for 30 min. The cells were heat shocked at 42 ℃ for 30 s, and then immediately placed on ice for 2 min. 300-500 μL of sterile LB medium was added, and the cells were cultured at 37 ℃ with 200 rpm shaking for 1 h. 200 μl of bacterial liquid was plated (containing Amp ampicillin), and the plate was incubated at 37 ℃ overnight (12-16 h). Single colonies were selected for PCR molecular detection. The positive E. coli clones were determined by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0078] The total RNA band was clear and complete after electrophoresis detection, meeting the experimental requirements. The cDNA of the flower tissue of Sapindus delavayi was used as a template to clone the gene SmSOC3 The electropherogram of the cloned gene is shown in Figure 1 After sequencing of the target fragment, a 624 bp coding sequence was obtained, as shown in the sequence listing Seq ID NO. 1, which encodes 207 amino acids, as shown in the sequence listing Seq ID NO. 2.

[0079] In order to further study the Sapindus delavayi SmSOC3 Conserved motif sequences, the protein sequences of the SOC genes of various species in Phytozome were screened from the Phytozome v13 public data platform, including Arabidopsis thaliana (TAIR10), Glycine max (Wm82.a4.v1), Malus domestica (M17.v1.1), Populus trichocarpa (Ptitrogen v4.1), Vitis vinifera (Vv2.1) and Prunus persica (Pp2.1). MADS Arabidopsis thaliana TAIR10), Glycine max (Wm82.a4.v1), Malus domestica (M17.v1.1), Populus trichocarpa (Ptitrogen v4.1), Vitis vinifera (Vv2.1) and Prunus persica (Pp2.1). Glycine max Malus domestica Populus trichocarpa Vitis vinifera Prunus persica MADS The MADS protein sequences of multiple species were subjected to MUSCLE alignment, NJ neighbor method, and the phylogenetic trees of Type I and Type II subfamilies were reconstructed by using MEGA7, and the parameter bootstrap value was checked and set to repeat 1000 times. SmMADS The SOC amino acid sequences of various species were screened for phylogenetic development analysis, as shown in

[0080] The number of SOC proteins of Arabidopsis, poplar, grape, soybean, peach, apple and Sapindus delavayi was 1, 5, 3, 7, 3, 5 and 1 respectively, and the evolution showed that Figure 2 The SOC3 proteins of Arabidopsis and poplar were closely related, and the SOC3 proteins of different species had obvious differences in regulatory functions. SmSOC3 The promoter sequences of 3000 bp upstream of the coding region of the Type I and Type II subfamily genes were taken respectively, and the cis-acting elements were predicted by online data analysis software PlantCARE, and statistical mapping was performed by means of Execl, as shown in

[0081] . SmMADS Figure 3

[0082] Figure 3 SmSOC3 ​​​​​​​​​The expression of transcription factors may be involved in the response process to different biotic and abiotic stresses, and have the ability to sense and respond to environmental stresses, plant hormones and photoperiod, and participate in the regulation of plant growth and development. SmSOC3 In addition to some common basic components (TATA), there are four other main categories:

[0083] (1) Hormone regulation related elements: Jasmonic acid response elements (CGTCA-motif and TGACG-motif).

[0084] (2) Components related to environmental pressure regulation: mechanical damage response element (WUN-motif);

[0085] (3) Drought-induced binding site-related elements (MYB, MBS and MYC) respond to plant abiotic stress;

[0086] (4) A large number of photoperiodic response elements (TCT-motif and G-box) regulate the diurnal rhythm of plants;

[0087] (5) Related elements for plant growth and development regulation (AAGAA-motif);

[0088] Protein function is closely related to its structure. Therefore, a model was constructed using the online SWISS-MODEL website. SmSOC3 Protein three-dimensional structure, see Figure 4 .

[0089] RNA extraction and real-time quantitative PCR

[0090] 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.

[0091] Using TB Green Premix Ex Taq (SYBR Green) enzyme pair SmSOC3 Genes were subjected to qRT-PCR experiments, and at the same time, SmACT This gene was used as an internal reference for quantitative real-time PCR. The primer sequences for quantitative real-time PCR of the gene are shown in Table 3.

[0092] Table 3 qRT-PCR amplification reaction system

[0093]

[0094] The qRT-PCR reaction program was set as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 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 the fluorescence signal 5 times for each 1℃ increase. The expression of the gene was detected as an internal reference, and three biological replicates, technical replicates were set, the average value was calculated, and the expression of the gene was calculated by 2 SmACT -∆∆Ct method and processed for plotting.

[0095] SmSOC3 The expression patterns in the organs of 8 periods of male and female flower development are shown in Table 3, and the specific results are shown in Table 3: in the process of male flower development Figure 5 SmSOC3 The expression of the gene showed a trend of first increasing and then slowly decreasing, and the expression reached the highest value at the 2nd period (stamen meiosis); in the process of female flower development, the expression first slowly increased, then decreased, and then increased, and the expression was significantly expressed at the 4th period (rapid elongation period of filament and style) and the 8th period (post-flowering period).

[0096] Figure 6 PiFF4: pistil of female flower in the rapid elongation period of filament and style; PiFF7: pistil of female flower in the flowering development period; PiMF4: pistil of male flower in the rapid elongation period of filament and style; PiMF7: pistil of male flower in the flowering development period; StFF4: stamen of female flower in the rapid elongation period of filament and style; StFF7: stamen of female flower in the flowering development period; StMF4: stamen of male flower in the rapid elongation period of filament and style; StMF7: stamen of male flower in the flowering development period; PeFF: petal of female flower; PeMF: petal of male flower. It can be seen that SmSOC3 The gene participates in the regulation of the development of stamen, pistil and petal, especially the most significant expression in the stamen of female flower and the pistil in the flowering development period.

[0097] Figure 7 and Figure 8 are 8 periods of pericarp and seed development, SmSOC3 The gene is expressed in all 8 periods of pericarp and seed development, especially the highest expression in the 1st period of pericarp (initial fruit period), and SmSOC3 The gene has a relatively high expression in the 1st period (initial fruit period) and the 5th period (seed hard shell period) of the seed.

[0098] See Figure 9 , SmSOC3 ​​The expression level was high in bud1-3 period (flower bud dormancy period, flower induction period and flower initiation period), and the expression level was low in bud4 period (inflorescence differentiation period).

[0099] SmSOC3 Construction of gene overexpression vector

[0100] Using high-purity plasmid DNA extraction kit to extract the correct sequence of the determination SmSOC3 Bacterial plasmid, restriction endonuclease BstEII-HF and Ncol-HF were used to double enzyme cut pCAMBIA1301 (laboratory preserved) plasmid, the enzyme cutting system was shown in Table 4, the reaction program was 37℃, 30 min, after cutting, the vector was purified by DNA gel recovery kit, electrophoresis experiment was performed for detection, and the products after enzyme cutting were stored at -20℃.

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

[0102]

[0103] Cloning Kit was used for vector ligation, the ligation system was shown in Table 5, and pCAMBIA1301- overexpression vector was obtained. SmSOC3 The recombinant was introduced into the competent Agrobacterium GV3101 for subsequent infection of Arabidopsis.

[0104] Table 5 Ligation reaction system of 1301 expression vector

[0105]

[0106] Culture and infection of wild type Arabidopsis

[0107] Under sterile environment, an appropriate amount of wild type Arabidopsis seeds were taken and placed in a 1.5 ml centrifuge tube, 1 / 50 volume of sodium hypochlorite solution was added and mixed, and then shaken up and down for 10-15 min, after sterilization, the seeds were washed with sterile distilled water for 4-5 times, and then spread on sterilized 1 / 2MS solid medium, and then cultured at 4℃ for 3 days, then lighted for a week, and then transplanted to sterilized culture soil (substrate soil, vermiculite mixture = 1:1) in a light incubator, and then cultured under long day (16 / 8 h), 22℃, and relative humidity 70%. The Arabidopsis plants were transformed by flower soaking method, after the Arabidopsis plants were elongated, 3-4 stem leaves were grown, all plants were removed from the top inflorescence synchronously, and then the apical dominance was used to promote the growth of lateral branches and flowering. A large number of unopened flower buds were infected with Agrobacterium, soaked for 15-30 s, and then dried, and then placed in dark environment for about 24 h, and then repeatedly infected for 4 times during flowering period, and then interval about a week, and then improved the transformation efficiency.

[0108] Screening and identification of transgenic Arabidopsis thaliana

[0109] 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.

[0110] Extracts were extracted from WT wild-type Arabidopsis thaliana and 35S:: SmSOC3 Overexpressing DNA from plant leaves and using it as a template for further processing SmSOC3 Genetic testing, results as follows Figure 10 As shown in the figure, "Marker" represents the DL2000 marker; "WT" represents wild-type Arabidopsis thaliana; and "OE" represents 35S:: SmSOC3 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.

[0111] 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.

[0112] Figure 11 Image A through D, left image is WT wild-type Arabidopsis thaliana, right image is 35S:: SmSOC3 Plant. Figure A shows, SmSOC2 Overexpression of the gene advanced the bolting and flowering time of Arabidopsis thaliana. Figure B shows that 35S:: SmSOC3 The plant's flower morphology is normal, but the stamens and pistils are of equal height and both slightly shorter, indicating poor development, although fertility is normal. Compared to the wild type, 35S:: SmSOC3 The plant's calyx changes from a cuboid to a cone. Figure C further shows that... SmSOC3 Overexpression of the gene resulted in shorter pistils in both lines. Figure D shows that 35S:: SmSOC2 The inflorescences of the plants are identical to those of the wild type, both being indeterminate inflorescences. Figures E and F show that 35S:: SmSOC3 The pod length of the plant is the same as that of the wild type, but the seed density is lower and the seed setting rate is slightly lower. (35S::) SmSOC3 The average ratio of single fruit to seeded fruit between the plant and wild type is approximately 36:40.

[0113] Wild type and 35S:: SmSOC3 See the comparison results of flowering time of transgenic lines. Figure 12 . Figure 12 In the middle, G image: the left side shows the WT wild-type plant, and from left to right are the 35S:: SmSOC3#1 and 35S:: SmSOC3#3 Transgenic plants; H diagram: The first one on the left is the WT wild-type plant, and from left to right are 35S:: SmSOC3#4 and 35S:: SmSOC3#5 Transgenic plants; Figure I: From left to right, WT wild-type plants, 35S:: SmSOC3#6 and 35S:: SmSOC3#8 Transgenic plant. This shows six 35S:: SmSOC3 The flowering time of the transgenic lines was earlier than that of WT Arabidopsis thaliana.

[0114] Figure 13 In diagrams a through c, WT represents wild-type Arabidopsis thaliana, and there are a total of 6 35S:: SmSOC3 Figure a: Statistical analysis of bolting time (days) for transgenic lines; Figure b: Statistical analysis of rosette leaves (number of leaves) at bolting; Figure c: Statistical analysis of flowering time (days) for transgenic lines. 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 the SPSS analysis results showed significant differences. The bolting time of the 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 18.83 days, with 12.17 rosette leaves at bolting, and the flowering time was 20.5 days. SmSOC3 The flowering time of the plants has been advanced to varying degrees, specifically by 1.5 days, and this is true for most strains. 35S::SmSOC3 The number of rosette leaves in genetically modified plants is positively correlated with the bolting time; the earlier the bolting, the fewer the rosette leaves.

[0115] Study on the expression patterns of endogenous genes in transgenic plants

[0116] Representative transgenic lines were selected from Arabidopsis thaliana exhibiting the aforementioned differences in flowering time. Flowers were extracted from positive transgenic plants and wild-type Arabidopsis thaliana. Flowers were also extracted from WT wild-type plants and 35S:: SmSOC3#3, 35S:: SmSOC3#5 and 35S:: SmSOC3#8 RNA from transgenic plants was collected and detected by electrophoresis. All samples showed clear and intact total RNA bands. cDNA was then reverse transcribed and used as a template for subsequent qRT-PCR experiments. AtEF1 The gene was used as an internal reference gene, and the quantitative fluorescence assay method was the same as described above. Meanwhile, AtEF1As an internal reference gene for fluorescence quantitative experiment. The sequence of the fluorescence quantitative PCR primer of the gene is shown in Table 6.

[0117] The 2 -∆∆Ct method was used to calculate the gene expression level and plot.

[0118] Table 6 Fluorescence quantitative PCR primer

[0119]

[0120] To further study the gene regulation of 35S: SmSOC3 transgenic plants, explain the changes of the phenotype at the molecular level, in the flower tissue of Arabidopsis, the expression level of the genes related to the flowering time was determined, and compared with the WT wild type plants, see SOC3 . Figure 14 . Figure 14 In the present embodiment, WT is the wild type Arabidopsis plant 35S: SmSOC3#3, 35S: SmSOC3#5 and 35S: SmSOC3#8 transgenic lines. First, the expression level of 35S: SmSOC3 in the flower tissue of the transgenic plants is extremely high, which is significantly different from the wild type, indicating that the function of the gene is to regulate the flowering time, and the overexpression of the gene makes Arabidopsis bloom early. SmSOC3 SmSOC3

[0121] In the flower tissue of Arabidopsis, the expression level of the genes such as SmSOC3 , AtAG, AtSOC1, AtSOC3, AtLFY , AtFT, AtSEP4 and AtSEP2 is higher than that of the wild type. AtSVP, AtAP3 The expression level of the genes such as AtTFL1 and is lower than that of the wild type.

[0122] In the flower tissue of Arabidopsis, the expression level of the flowering time related integrator genes AtFT and AtSOC1 and the flowering meristem regulator genes AtLFY is higher than that of the wild type, while the expression level of the flowering inhibitor genes AtTFL1 and AtSVP is lower than that of the wild type. Therefore, SmAP3 the overexpression of the genes promotes the expression of the flowering integrator and the flower meristem regulator, so that the 35S: SmSOC3 transgenic Arabidopsis blooms and flowers early.

[0123] The present embodiment proves that the SmSOC3 gene can be used to cultivate new varieties of Sapindus with different flowering times by using the​SmSOC3 The gene can regulate the flowering time to obtain new varieties with desired flowering characteristics. This embodiment also illustrates a plant transgenic method for introducing the expression vector into plant cells to change the flowering time of plants. This embodiment also illustrates a gene expression pattern analysis method related to the regulation of plant flowering time by determining the expression of genes at different developmental stages to analyze the regulation mechanism of plant flowering time. This embodiment also illustrates a method for determining the expression amount of the flowering time regulation related gene by using the fluorescence quantitative primer pair to quantitatively analyze the expression amount of the flowering time regulation related gene in the transgenic plant. SmSOC3 The gene can regulate the flowering time to obtain new varieties with desired flowering characteristics. This embodiment also illustrates a plant transgenic method for introducing the expression vector into plant cells to change the flowering time of plants. This embodiment also illustrates a gene expression pattern analysis method related to the regulation of plant flowering time by determining the expression of genes at different developmental stages to analyze the regulation mechanism of plant flowering time. This embodiment also illustrates a method for determining the expression amount of the flowering time regulation related gene by using the fluorescence quantitative primer pair to quantitatively analyze the expression amount of the flowering time regulation related gene in the transgenic plant.

[0124] The above is only a preferred embodiment of the present application, and it should be noted that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be subject to the defined range. For ordinary skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A soapnut SmSOC3 gene, characterized in that, The SmSOC3 gene is the nucleotide sequence shown in the sequence listing Seq ID NO.

1.

2. A Sapindus mucus-nigra SmSOC3 gene, characterized in that, The SmSOC3 gene is a nucleotide sequence encoding the amino acid sequence shown in the sequence listing Seq ID NO.

2.

3. A fruit of the plant of claim 1 or 2. SmSOC3 A protein encoded by the gene, characterized in that, The amino acid sequence of the protein is the amino acid sequence shown in the sequence listing Seq ID NO.

2.

4. A primer pair for cloning the gene of Sapindus mukorossi as claimed in claim 1 or 2, characterized in that, SmSOC3 The base sequence of the primer pair is as follows: ​ First upstream primer F: 5'-ATGGTGAGGGGAAAAACTCAGA-3', First downstream primer R: 5'-TCAAGCACTTCGTCTCTCAGGT-3'.

5. The primer pair according to claim 4, wherein, The base sequence of the primer pair further comprises a restriction site, and the base sequence of the primer pair comprising the restriction site is as follows: Second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGGTGAGGGGAAAAACTCAGA-3', Second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCAAGCACTTCGTCTCTCAGGT-3'.

6. A plant based on the plant of claim 1 or 2 SmSOC3 The present application provides a pair of fluorescent quantitative primers for the gene of claim 1 or 2, wherein the pair of fluorescent quantitative primers comprises a forward primer and a reverse primer. The base sequence of the fluorescent quantitative primer pair is as follows: Third upstream primer F: 5'-AGGAGAGCCAAAGACGATGA-3', Third downstream primer R: 5'-ATTGAGCATCGTTCCAGTCC-3'.

7. A composition comprising the expression vector of claim 1 or 2 and the Euphorbia lathyris seed of claim 6. SmSOC3 An expression vector containing the gene of claim 1 or 2, 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- SmSOC3.

8. A method for breeding a new variety of a plant, characterized by, Overexpression of the gene of claim 1 or 2 SmSOC3 genes causes the flowering time of plants to be advanced, in order to obtain new varieties with desired flowering characteristics, said plants being Arabidopsis thaliana.

9. A method for plant transformation comprising the steps of, The method comprises introducing the expression vector of claim 7 into a plant cell to advance the flowering time of a plant, and the plant is Arabidopsis thaliana.

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