Sapindus mukorossi smap1 gene and application thereof

Abstract

The application discloses a Sapindus mume SmAP1 gene and application thereof, and the SmAP1 gene is expressed in plants to regulate the flowering time of the plants, and the SmAP1 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 application of the Sapindus mume SmAP1 gene is used for cultivating new Sapindus mume varieties with different flowering times. Through observation of the whole transgenic Arabidopsis thaliana plants of 35S::SmAP1 and analysis of the flowering time difference, overexpression of the SmAP1 gene can make the flowering time of the plants advance to different degrees, and the flowering time, days of bolting and the number of rosette leaves of the transgenic Arabidopsis thaliana plants are significantly different from those of wild type plants, which is beneficial to accelerating the breeding cycle of the Sapindus mume species and accelerating the cultivation of high-yield varieties.

Description

Technical Field

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

[0002] Soapberry (Sapindus mukorossi Gaertn.), also known as soap tree, is a deciduous tree belonging to the genus Sapindus L. of the family Sapindaceae. With abundant germplasm resources, soapberry is a multi-functional economic forest tree integrating daily chemical and pharmaceutical applications, biomass energy, and landscaping. The growth and development process of soapberry includes eight main stages: flower bud, leaf, and stem development; inflorescence appearance; flowering process; fruit development; fruit ripening; and fruit senescence and dormancy. The vegetative growth process spans three main stages: bud, leaf, and stem development. During the spring foliage growth, the first morphological feature of the flower is the appearance of small "yellow spots" in the leaf axils. The stages of reproductive development and their corresponding times are: dormancy (December to February of the following year), flowering induction period (March to April), flower initiation period (April to May), flower bud differentiation period (June to July), and fruit development and ripening period (August to November).

[0003] Currently, the long juvenile period of Sapindus mukorossi hinders breeding efforts and the rapid realization of economic benefits. Conducting research on flowering regulation in Sapindus mukorossi and developing new gene resources will be beneficial for further developing Sapindus mukorossi varieties with earlier flowering and fruiting and a shorter juvenile period, which has significant theoretical and practical implications. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a gene that can advance the flowering time of plants.

[0005] The second objective of this invention is to provide a protein encoded by the Sapindus mukorossi SmAP1 gene.

[0006] The third objective of this invention is to provide a primer pair for cloning the Sapindus mukorossi SmAP1 gene.

[0007] The fourth objective of this invention is to provide a fluorescent quantitative primer pair for the Sapindus mukorossi SmAP1 gene.

[0008] The fifth objective of this invention is to provide an expression vector containing the SmAP1 gene.

[0009] The sixth objective of this invention is to provide a use for the Sapindus mukorossi SmAP1 gene.

[0010] Through long-term exploration and experimentation, and continuous reform and innovation, the inventors have provided a technical solution to solve the above-mentioned technical problems: a Sapindus mukorossi SmAP1 gene, which is expressed in plants to regulate flowering time. The SmAP1 gene contains nucleotide sequences selected from the following group:

[0011] A. The nucleotide sequence shown in Seq ID NO.1 of the sequence listing;

[0012] B. A nucleotide sequence complementary to the sequence described in A, or a nucleotide sequence with more than 80% homology;

[0013] C. The nucleotide sequence of the amino acid sequence shown in Seq ID NO.2 of the coding sequence listing.

[0014] Preferably, the plant is a Sapindus order plant.

[0015] Preferably, the plant is a Sapindaceae plant.

[0016] Preferably, the plant is a plant of the Sapindus genus.

[0017] The present invention also provides a protein encoded by the aforementioned Sapindus mukorossi SmAP1 gene, wherein the amino acid sequence of the protein is selected from the amino acid sequence shown in Sequence Listing Seq ID NO.2.

[0018] The present invention also provides primer pairs for cloning the aforementioned Sapindus mukorossi SmAP1 gene, the base sequences of which are as follows:

[0019] First upstream primer F: 5'-ATGGGGAGAGGTAGGGTTCAGTT-3',

[0020] First downstream primer R: 5'-TCATGCAGCGAAACATCCAA-3'.

[0021] Furthermore, 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:

[0022] Second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGGGGAGAGGTAGGGTTCAG TT-3',

[0023] Second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCATGCAGCGAAACATCCAA-3'.

[0024] This invention also provides a fluorescence quantitative primer pair for the Sapindus mukorossi SmAP1 gene, the base sequence of which is as follows:

[0025] Third upstream primer F: 5'-TTAGGGCCCTGATTTTGATG-3'

[0026] Third downstream primer F: 5'-TTGGAACAGCAGCTTGACAC-3'.

[0027] This invention also provides an expression vector containing the aforementioned Sapindus mukorossi SmAP1 gene. The pCAMBIA1301 vector plasmid is double-digested using restriction endonucleases BstEII-HF and Ncol-HF. II. The vector was ligated using the One Step Cloning Kit to obtain the overexpression vector pCAMBIA1301-SmAP1.

[0028] The present invention also provides a use for the aforementioned Sapindus mukorossi SmAP1 gene, for breeding new Sapindus mukorossi varieties with different flowering times.

[0029] Compared with the prior art, one of the above technical solutions has the following advantages:

[0030] Through observation of whole plants of Arabidopsis thaliana transgenic with the SmAP1 gene and analysis of differences in flowering time, it was found that the SmAP1 gene can advance the flowering time of plants to varying degrees. The flowering time, bolting days, and number of rosette leaves at bolting of transgenic Arabidopsis thaliana plants were significantly different from those of wild-type plants, which is beneficial to accelerating the breeding cycle of Sapindus mukorossi and speeding up the cultivation of high-yield varieties. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 This is a clone electrophoresis image of the SmAP1 gene.

[0033] Figure 2 These are the results of a phylogenetic analysis of AP1 subclass genes and proteins.

[0034] Figure 3 These are the results of the cis-acting element analysis of the SmAP1 gene.

[0035] Figure 4 This is a predicted diagram of the tertiary structure of the SmAP1 protein.

[0036] Figure 5The results are the expression patterns of SmAP1 gene family members in different trophic tissues.

[0037] Figure 6 This is the result of the expression pattern analysis of the SmAP1 gene during the flowering induction period.

[0038] Figure 7 The results are from the analysis of the expression pattern of SmAP1 during the development of male and female flower buds.

[0039] Figure 8 The results are the analysis of the expression patterns of the SmAP1 gene in different floral organs.

[0040] Figure 9 This is the identification result of the 35S::SmAP1 transgenic plant.

[0041] Figure 10 This is a phenotypic comparison between the wild type and the 35S::SmAP1 transgenic line.

[0042] Figure 11 This is a comparison of the flowering time between the wild type and the 35S::SmAP1 transgenic line.

[0043] Figure 12 This is a comparative diagram of the expression of AP1 upstream and downstream related genes in Arabidopsis thaliana flower tissue. Detailed Implementation

[0044] The following description, in conjunction with the accompanying drawings and a specific embodiment, will be provided.

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0046] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

[0047] Example 1

[0048] The Sapindus mukorossi SmAP1 gene described in this embodiment has a nucleotide sequence as shown in Sequence Listing Seq ID NO.1. Based on this nucleotide sequence, the amino acid sequence shown in Sequence Listing Seq ID NO.2 can be encoded.

[0049] The SmAP1 gene of Sapindus mukorossi is expressed in plants, particularly Sapindus mukorossi. Functional verification and transgenic verification in Arabidopsis thaliana show that this gene can significantly regulate the flowering time of plants. Utilizing the SmAP1 gene of Sapindus mukorossi can shorten the breeding cycle of Sapindus mukorossi and accelerate the development of high-yield Sapindus mukorossi varieties.

[0050] Example 2

[0051] The primer pair described in this embodiment for cloning the Sapindus mukorossi SmAP1 gene is used to clone the Sapindus mukorossi SmAP1 gene described in Example 1. The base sequence of the primer pair is as follows:

[0052] First upstream primer F: 5'-ATGGGGAGAGGTAGGGTTCAGTT-3',

[0053] First downstream primer R: 5'-TCATGCAGCGAAACATCCAA-3'.

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

[0055] Example 3

[0056] The primer pair for cloning the Sapindus mukorossi SmAP1 gene described in this embodiment is based on the primer pair described in Example 2 with the addition of restriction enzyme sites, resulting in the second upstream primer F as shown in Sequence Listing Seq ID NO.5 and the second downstream primer R as shown in Sequence Listing Seq ID NO.6. The base sequences of the primer pair including the restriction enzyme sites are as follows:

[0057] Second upstream primer F: 5'-AGAACACGGGGGACTCTTGACATGGGGAGAGGTAGGGTTCAG TT-3',

[0058] Second downstream primer R: 5'-GGGGAAATTCGAGCTGGTCACTCATGCAGCGAAACATCCAA-3'.

[0059] 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.

[0060] Example 4

[0061] The quantitative fluorescence primer pair for the Sapindus mukorossi SmAP1 gene described in this embodiment is used for quantitative analysis of the Sapindus mukorossi SmAP1 gene. The base sequences of the quantitative fluorescence primer pair are shown in Seq ID NO.7 and Seq ID NO.8 of the sequence listing:

[0062] Third upstream primer F: 5'-TTAGGGCCCTGATTTTGATG-3'

[0063] Third downstream primer R: 5'-TTGGAACAGCAGCTTGACAC-3'.

[0064] 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.

[0065] Example 5

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

[0067] In this embodiment, three Sapindus mukorossi sample trees were selected as experimental materials. Biological sampling was carried out three times. The sampling location was located in Jianning County, Sanming City, Fujian Province. The sampling time was fixed from 10:00 am to 12:00 pm. The middle and upper parts of one-year-old branches at the same height of the tree canopy were quickly placed into cryovials and frozen in liquid nitrogen. Then they were stored in a -80°C freezer for subsequent RNA extraction.

[0068] 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).

[0069] After the formation of Sapindus mukorossi florets, the developmental stages of floral organs were analyzed. Eight stages were selected from the female (FF1-8) and male (MF1-8) florets of different sexes: F1 was the stage of complete formation of floral organ primordia; F2 was the stage of meiosis of stamens; F3 was the stage of microspore development; F4-6 was the stage of rapid elongation of filaments and styles; F7 was the flowering development stage; and F8 was the late flowering stage, totaling 48 samples. Ten tissue parts of the important floral organs (petals, pistils, and stamens) were analyzed, covering the various parts of floral organs in the 4th stage (rapid elongation of filaments and styles) and the 7th stage (flowering development stage): pistils of female flowers (PiFF4 and PiFF7), pistils of male flowers (PiMF4 and PiMF7), stamens of female flowers (StFF4 and StFF7), stamens of male flowers (StMF4 and StMF7), as well as petals of female flowers (PeFF) and male flowers (PeMF).

[0070] After preliminary experimental screening, this embodiment ultimately used cDNA material from the pistil at the fourth developmental stage as a template for PCR amplification. The primers described in Example 3 were used for amplification. The TA cloning reaction system is shown in Table 1 below. The reaction program was: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 40 s, repeated 34 times, followed by a final extension at 72℃ for 5 min, and storage at 4℃. After adding 2 μl of 10× Loading buffer, the PCR product was separated by 1% agarose gel electrophoresis. The clean target fragment was excised from the gel and recovered using a DNA gel extraction kit. The entire process was performed on ice.

[0071] Table 1. TA Cloning PCR Sequence Amplification System

[0072]

[0073] The recycled gel product was connected to the T-carrier, and the reaction system is shown in Table 2.

[0074] Table 2. T-cloning ligation vector system

[0075]

[0076] Incubate at room temperature (20-30℃) for 5 min. After the reaction, incubate at 4℃. Take 5 μl of ligation solution and add it to 50 μl of freshly thawed DH10B competent cells. Mix gently, incubate on ice for 30 min, heat shock in a 42℃ water bath for 30 s, and immediately place on ice for 2 min. Add 300-500 μL of sterile LB medium and incubate at 37℃ with shaking at 200 rpm for 1 h. Take 200 μl of the bacterial culture (containing ampicillin) and incubate overnight at 37℃ (12-16 h). Select single colonies for PCR molecular detection. Positive E. coli clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for testing.

[0077] Electrophoresis analysis showed that the extracted total RNA bands were clear and intact, meeting the experimental requirements. The SmAP1 gene was cloned using cDNA from Sapindus mukorossi flower tissue as a template; the cloning electrophoresis image is shown below. Figure 1 After sequencing the target fragment, a 726bp coding sequence was obtained (see Sequence Listing Seq ID NO.1), which encodes 241 amino acids (see Sequence Listing Seq ID NO.2).

[0078] To further investigate the conserved motif sequence of Sapindus mukorossi SmAP1, the amino acid sequences of AP1 from Arabidopsis thaliana, grape, soybean, apple, peach, and poplar were compared. Proteins compared with the SmAP1 amino acid sequence included AtAP1, PpAP1, VvAP1.1, VvAP1.2, VvAP1.3, VvAP1.4, MdAP1.1, MdAP1.2, GmAP1.1, GmAP1.2, GmAP1.3, PtAP1.1, and PtAP1.2, with an overall similarity of 65.55%. Comparison revealed that AP1 from different species shares common characteristics of the MADS-box family, namely, the highest conservation of the MADS-box domain, and relatively similar conserved I and K regions, although the C-terminus shows significant variation. Furthermore, compared to other species, the SmAP1 amino acid sequence from Sapindus mukorossi is shorter.

[0079] Protein sequences of MADS genes from various species databases in Phytozome v13 were screened from the Phytozome public database platform, including those of Arabidopsis thaliana TAIR10, soybean (Glycine maxWm82.a4.v1), apple (Malus domestica v1.1), poplar (Populus trichocarpa v4.1), grape (Vitis vinifera v2.1), and peach (Prunus persica v2.1). Combined with the Sapindus mukorossi SmMADS gene family, the MADS protein sequences of multiple species were MUSCLE aligned using MEGA7, and the NJ nearest neighbor method was used to reconstruct phylogenetic trees for Type I and Type II subfamilies. The bootstrap parameter was checked and set to 1000 replicates.

[0080] The AP1 amino acid sequences of each species were screened and phylogenetic analysis was performed. (See attached image) Figure 2The number of AP1 proteins in Arabidopsis thaliana, poplar, grape, soybean, peach, apple and soapberry in each group were 1, 2, 4, 3, 1, 2 and 1, respectively. Evolution shows that SmAP1 is distantly related to the AP1 proteins in Arabidopsis thaliana, grape, soybean and peach, and the regulatory functions of AP1 proteins in different species are significantly different.

[0081] The promoter sequences of the SmMADS gene family upstream of the coding region in Type I and Type II subclasses were taken respectively. The cis-regulatory elements were predicted using the online data analysis software PlantCARE, and statistical plotting was performed using Excel. (See figure) Figure 3 .

[0082] Figure 3 This indicates that the expression of the SmAP1 transcription factor may be involved in the response to various biotic and abiotic stresses, possessing the ability to sense and respond to environmental stresses, plant hormones, and photoperiod, and participating in the regulation of plant growth and development. In addition to some common basic elements (CAAT-box and TATA), SmAP1 has four other main categories:

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

[0084] (2) Relevant components for environmental pressure regulation: mechanical damage response component (WUN-motif), salt stress response component (W-box);

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

[0086] (4) A large number of photoperiodic response elements (STRE and GT1-motif, etc.) regulate the diurnal rhythm of plants.

[0087] Protein function and structure are closely related. Therefore, the three-dimensional structure of the SmAP1 protein was constructed using the online SWISS-MODEL website, see [link to SmAP1 structure]. Figure 4 .

[0088] RNA extraction and real-time quantitative PCR

[0089] 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 cDNASynthesis SuperMIX for qPCR reverse transcription kit, and the obtained cDNA was diluted with nuclease-free water.

[0090] The SmAP1 gene was subjected to qRT-PCR using TB Green Premix Ex Taq (SYBR Green) enzyme, while SmACT was used as an internal control gene for quantitative real-time PCR. The primer sequences for the gene's quantitative real-time PCR are shown in Table 3.

[0091] Table 3 qRT-PCR amplification reaction system

[0092]

[0093] The qRT-PCR reaction program was set as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. After the cycles, product specificity was detected using melting curve analysis: the temperature was slowly increased from 60℃ to 95℃, with 5 fluorescence signals collected for each 1℃ increase. The Sapindus mukorossi SmACT gene was used as an internal control for detection. Three biological replicates and three technical replicates were set up, and the average value was calculated. A 23 -ΔΔCt Gene expression levels were calculated and plotted using a method.

[0094] The expression pattern of SmAP1 in the vegetative organs of Sapindus mukorossi, such as roots, stems, and leaves, is shown in the following figures. Figure 5 As shown, the SmAP1 gene of the AP1 subclass is expressed at a high level in Sapindus mukorossi stems.

[0095] See Figure 6 SmAP1 expression is upregulated in the early stages of flower bud differentiation, and remains high during flower bud initiation and inflorescence differentiation. In female flower development stages 1-8, SmAP1 expression shows an increasing trend from stages 1-4, reaching its peak, and plays an important role during the rapid elongation period of the filament and style (M4-6), before slightly decreasing from stages 4-8. See also... Figure 7 During male flower development stages 1-8, the expression level of the SmAP1 gene is relatively stable, with higher expression levels observed during stages 2-6. (See also...) Figure 8 SmAP1 plays an important role in various floral organs, participating in the development of stamens, pistils and petals, and is most significantly expressed in pistils. Figure 8 In the diagram, PiFF4: pistil of female flowers during the rapid elongation period of filament and style; PiFF7: pistil of female flowers during the flowering and development period; PiMF4: pistil of male flowers during the rapid elongation period of filament and style; PiMF7: pistil of male flowers during the flowering and development period; StFF4: stamen of female flowers during the rapid elongation period of filament and style; StFF7: stamen of female flowers during the flowering and development period; StMF4: stamen of male flowers during the rapid elongation period of filament and style; StMF7: stamen of male flowers during the flowering and development period; PeFF: petal of female flower; PeMF: petal of male flower.

[0096] Construction of SmAP1 gene overexpression vector

[0097] The SmAP1 bacterial culture plasmid with a confirmed sequence was extracted using a high-purity plasmid DNA miniprep kit. The pCAMBIA1301 plasmid (preserved in our laboratory) was double-digested with restriction endonucleases BstEII-HF and Ncol-HF. The digestion system is shown in Table 4. The reaction program was 37℃ for 30 min. After digestion, the vector was purified using a DNA gel extraction kit and detected by electrophoresis. The digested products were stored at -20℃.

[0098] Table 4. Enzyme digestion reaction system for the 1301 expression vector

[0099]

[0100] use II. The vector was ligated using the One Step Cloning Kit. The ligation system is shown in Table 5. The pCAMBIA1301-SmAP1 overexpression vector was obtained. The recombinant was introduced into Agrobacterium GV3101 competent cells for subsequent infection of Arabidopsis thaliana.

[0101] Table 5. 1301 expression vector ligation reaction system

[0102]

[0103] Cultivation and Infection of Wild-type Arabidopsis

[0104] Under sterile conditions, take an appropriate amount of wild-type Arabidopsis seeds, place them in a 1.5ml centrifuge tube, add 1 / 50 volume of sodium hypochlorite solution, mix well, shake repeatedly up and down for 10-15 minutes, and after sterilization, wash 4-5 times with sterile distilled water. Spread the seeds evenly on sterilized 1 / 2MS solid medium, vernalize at 4℃ for 3 days, and then expose to light for one week. Transplant the Arabidopsis seedlings into sterilized culture soil (a mixture of substrate soil and vermiculite = 1:1) and place them in a light incubator for long-day (16 / 8h) cultivation at 22℃ and a relative moisture content of 70%. Transform Arabidopsis plants using the flower immersion method. When the Arabidopsis plants have bolted and grown 3-4 stem leaves, remove the terminal inflorescence from all plants simultaneously to utilize apical dominance to promote lateral branch growth and flowering. A large number of unopened flower buds were infected with Agrobacterium, soaked for 15-30 seconds, and then dried. The plants were then placed in a dark environment for about 24 hours. The infection was repeated 4 times during the flowering period, with an interval of about one week between each infection, to improve the conversion efficiency.

[0105] Screening and identification of transgenic Arabidopsis thaliana

[0106] 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.

[0107] DNA was extracted from leaves of WT wild-type Arabidopsis thaliana and 35S::SmAP1 overexpressing plants, respectively, and used as templates for SmAP1 gene detection. The results are as follows: Figure 9 As shown in the figure, Marker represents the DL2000 marker; WT represents wild-type Arabidopsis thaliana; OE represents the 35S::SmAP1 plant. According to... Figure 9 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 band. A total of 10 lines were verified.

[0108] 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.

[0109] Figure 10 The images in the middle A to I series show the WT wild-type Arabidopsis thaliana on the left and the 35S::SmAP1 plant on the right.

[0110] See Figure 10 Figure A shows that by comparing WT wild-type Arabidopsis thaliana and 35S::SmAP1 plants, it was found that overexpression of the SmAP1 gene in Arabidopsis thaliana affects the bolting and flowering time, causing it to flower earlier. When the plants flower, some transgenic lines have slightly darker leaves, smaller leaves, and slightly fewer leaves than wild-type plants.

[0111] See Figure 10 In Figure B, the flower morphology is developing normally, and the number of petals, pistils, and stamens is normal. It is worth noting that the ovary of the transgenic plant is large and long, while the stamens are slightly short.

[0112] See Figure 10 In the middle F figure, at the same magnification, the ovary of the 35S::SmAP1 plant in the field of view is larger, and its stigma pollination is significantly worse than that of the wild type, which is speculated to have a certain impact on the fruit set rate.

[0113] See Figure 10 Figures C and E show that throughout the entire life cycle of Arabidopsis thaliana, the leaves grow healthily and the overall changes are not significant.

[0114] See Figure 10 In Figure G, the inflorescence of the 35S::SmAP1 plant is the same as that of the wild type, both being indeterminate inflorescences.

[0115] Further research revealed, see Figure 10 In Figures H and I, the pod phenotype is consistent with the wild type, the pods are full, and there is no significant difference in the individual fertilization rate.

[0116] The results comparing the flowering time of the wild-type and the 35S::SmAP1 transgenic lines can be found in [link to relevant documentation]. Figure 11 . Figure 11 In the diagram, a) the left side shows the WT wild-type plant, and the right side shows the 35S::SmAP1#1 transgenic plant; b) the left side shows the first WT wild-type plant, followed by 35S::SmAP1#2, WT wild-type plant, 35S::SmAP1#6, 35S::SmAP1#8, and 35S::SmAP1#9 transgenic plants from left to right; c) the left side shows the first WT wild-type plant, followed by 35S::SmAP1#7 transgenic plant from left to right; d) the left side shows the WT wild-type plant, and 35S::SmAP1#3, 35S::SmAP1#4, and 35S::SmAP1#5 transgenic plants from left to right. In eg, WT represents wild-type Arabidopsis thaliana, and OE1-9 represent nine 35S::SmAP1 transgenic lines. Figure e: Statistical analysis of flowering time (days); Figure f: Statistical analysis of bolting time (days); Figure g: Statistical analysis of rosette leaves (pieces) at bolting.

[0117] See Figure 11The flowering time of the 35S::SmAP1 plants was advanced to varying degrees. The flowering time (e), bolting days (f), and number of rosette leaves at bolting (g) of nine transgenic lines were significantly different from those of the wild-type plants, with SPSS analysis showing a significance level of 0.1%. Specifically, the flowering time (in days) of the wild-type Arabidopsis was 33±0.246; the flowering times of the nine transgenic lines OE1-9 were 23.08±0.336, 28.67±0.31, 30.15±0.25, 30.25±0.25, 30.05±0.25, 27.75±0.305, 23±0.275, 27.41±0.398, and 27.33±0.280, respectively. Figures f and g show the statistical analysis of bolting time and the number of rosette leaves at bolting, respectively. The results show that the bolting time for the wild-type WT line was 31.33±0.342 seconds, while the bolting times for the transgenic lines were: 21.08±0.356, 26.33±0.242, 28.15±0.350, 28.25±0.219, 28.2±0.223, 21.42±0.279, 25.75±0.307, and 25.75±0.33, respectively. 05, 25.33±0.283. Furthermore, the WT for the number of rosette leaves at bolting was 11.83±0.271, and the values ​​for the nine 35S::SmAP1 lines were 7.53±0.231, 8.21±0.193, 9.33±0.225, 9.58±0.149, 9.16±0.167, 7.83±0.271, 7.66±0.225, 7.75±0.218, and 7.83±0.207, respectively. In most 35S::SmAP1 transgenic plants, the number of rosette leaves was positively correlated with the bolting time; the earlier the bolting, the fewer the rosette leaves.

[0118] Among them, the OE#1 and OE#7 strains showed the most obvious early flowering: the bolting time was 21 days, while the wild type was 31.3 days. At this time, the number of rosette leaves of the 35S::SmAP1 plant was 7.53-7.83, while that of the wild type was 11.83. The number of leaves was three fewer than that of the wild type. The transgenic plants flowered at around 23.1-32.3 days of growth, which was nearly ten days earlier than the wild type. Secondly, the OE#2, OE#6, OE#8, and OE#9 lines bolted at 25.3-26.6 days, at which time the number of rosette leaves was 7.75-8.21. The plants flowered at 27.3-28.6 days, nearly six days earlier than the wild type, which had 11.83 leaves. On the other hand, the OE#3, OE#4, and OE#5 lines bolted at 28-28.25 days, at which time the number of rosette leaves was 9.16-9.33. The plants flowered at 30-30.25 days, three days earlier.

[0119] Study on the expression patterns of endogenous genes in transgenic plants

[0120] Flowers and fruits of positive transgenic plants and wild-type Arabidopsis thaliana were collected, and RNA was extracted from WT wild-type plants and 35S::SmAP1 transgenic plants, including three lines: 35S::SmAP1#1, 35S::SmAP1#3, and 35S::SmAP1#6. cDNA was obtained by reverse transcription and used as a template for subsequent qRT-PCR experiments. The AtEF1 gene was used as an internal control gene. The quantitative PCR method was the same as described above. Simultaneously, AtEF1 was used as an internal control gene for quantitative PCR experiments. The primer sequences for quantitative PCR of the genes are shown in Table 6.

[0121] Use 2 -ΔΔCt Gene expression levels were calculated and plotted using a method.

[0122] Table 6 Primers for Quantitative Real-Time PCR

[0123]

[0124] Three representative lines, 35S::SmAP1#1, 35S::SmAP1#3, and 35S::SmAP1#6, were selected from Arabidopsis thaliana with the above-mentioned differences in flowering time. RNA was extracted from the flower and fruit tissues of WT wild-type plants and 35S::SmAP1 transgenic plants. After electrophoresis, the total RNA bands of all samples were clear and complete, which can be used for subsequent quantitative fluorescence experiments.

[0125] To further investigate gene regulation in 35S::SmAP1 transgenic plants and explain their phenotypic changes at the molecular level, the expression levels of genes upstream and downstream of AP1 regulating flowering time were measured in the floral tissues of Arabidopsis thaliana and compared with those in wild-type plants subjected to WT. (See [link to relevant documentation]). Figure 11 . Figure 11 In the diagram, WT represents wild-type Arabidopsis thaliana plants, and OE#1, OE#3, and OE#5 represent the 35S::SmAP1#1, 35S::SmAP1#3, and 35S::SmAP1#6 transgenic lines, respectively. Firstly, the expression level of the SmAP1 gene in the floral parts of the 35S::SmAP1 transgenic plants is extremely high, significantly different from the wild type, and the expression levels are OE#1>OE#3>OE#6, consistent with the phenotypic order of early flowering in Arabidopsis thaliana. This indicates that the SmAP1 gene functions to regulate flowering time, and its overexpression leads to earlier flowering in Arabidopsis thaliana. In the tissues of 35S::SmAP1 Arabidopsis thaliana flowers, the expression levels of genes such as AtSOC1, AtSVP, AtFT, AtCAL, AtPI, AtLFY, AtAP1, and AtAP3 were higher than those of the wild type. Among them, the expression levels of AtFT, AtPI, AtLFY, and AtAP3 genes were significantly upregulated, while the expression levels of genes such as AtFUL, AtTFL1, and AtAGL24 were lower than those of the wild type.

[0126] In Arabidopsis flower tissues, the expression levels of genes related to flowering time (AtFT and AtSOC1), floral organ development (AtAP1, AtAP3, and AtPI), and flowering meristem regulators (AtLFY and AtCAL) were higher than in the wild type, while the expression levels of flowering inhibitors (AtTFL1, AtAGL24, and AtSVP) were lower. Therefore, overexpression of the SmAP1 gene promoted the expression of flowering integrons and flowering meristem regulators, leading to earlier bolting and flowering in the 35S::SmAP1 transgenic Arabidopsis and promoting pistil development. In the fruit, the expression of genes promoting fruit set was slightly affected, but the fruit set rate of the 35S::SmAP1 overexpression transgenic plants was not affected.

[0127] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An application of the Sapindus mukorossi SmAP1 gene, characterized in that, Used to prepare transgenic Sapindus mukorossi or Arabidopsis thaliana plants with shortened stamens; the SmAP1 gene is selected from the following group of nucleotide sequences: A. The nucleotide sequence shown in Seq ID NO.1 of the sequence listing; B. A nucleotide sequence complementary to the sequence described in A; C. The nucleotide sequence of the amino acid sequence shown in Seq ID NO.2 of the coding sequence listing.

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