Application of the peony PsPYL4 gene in regulating root development

Abstract

This invention discloses a peony PsPYL4 Genes and their application in regulating root development PsPYL4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. This invention was cloned from the peony cultivar 'Zhengwu'. PsPYL4 The gene sequence was obtained and its expression was analyzed in peony. Subsequently, an overexpression vector was constructed and introduced into the target plant to verify its function. It was found that overexpression... PsPYL4 Compared to wild-type Arabidopsis seedlings, the genetically modified Arabidopsis seedlings... PsPYL4 The overexpression lines showed faster growth, larger rosette leaves, significantly shorter taproot length, and significantly increased lateral root number. The levels of endogenous hormones IAA, ABA, ZR, and GAs in the leaves were increased. The average number of roots in the detached leaves was significantly increased on MS medium containing IAA, and the average root length was significantly increased on MS medium containing IBA. It is evident that this gene will have wide applications in the cultivation and genetic improvement of peonies and other horticultural plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a peony. PsPYL4 The application of genes in regulating root development. Background Technology

[0002] peony( Paeonia sect. Moutan (This belongs to the genus Paeonia of the family Paeoniaceae) Paeonia Peony Group (Sect.) Moutan Peony, a deciduous subshrub, boasts a rich variety of flower colors and beautiful flower shapes, making it a unique and precious plant in China, valued for its ornamental, medicinal, edible, and oil-producing uses. Peony propagation primarily employs traditional methods such as sowing, division, and grafting, but these methods suffer from drawbacks such as low propagation coefficients and long cycles. In recent years, micropropagation techniques have enabled the mass propagation of peonies in a short period, but problems remain, including difficulty in adventitious root development in test-tube seedlings and low rooting quality. Therefore, researching the molecular mechanisms by which genes act on plant root development is of great significance for peony breeding, production, and application.

[0003] Previous studies have found that endogenous abscisic acid (ABA) inhibits adventitious root development, and the endogenous auxin (IAA / ABA) ratio can be used as a measure of plant rooting ability. The abscisic acid receptor protein PYL, as a core member of the ABA signal transduction pathway, is a major component of ABA signal perception. Arabidopsis thaliana has 14 ABA receptors, including PYR1 and PYL1-13, and all PYR / PYL (except PYL13) can activate the expression of ABA-responsive genes. Among them, PYL8 plays a role in auxin-mediated lateral root growth through interaction with MYB77, MYB44, and MYB73. pyl8 Lateral root growth in mutant seedlings is more sensitive to ABA; and pyl8 compared to, pyl8-1pyl9 The double mutant exhibits a weakened ability of exogenous ABA to inhibit taproot growth and lateral root formation; ABA has a reduced effect on... pyl1 / 2 / 4 The mutant showed a four-fold reduction in the inhibition of lateral root growth; while PYL9 controls the expression of auxin-responsive genes in lateral root primordia by directly regulating the transcriptional activity of MYB77 and MYB44, independent of ABA signaling. These studies indicate that PYL not only regulates adventitious root formation through the abscisic acid signaling pathway, but also participates in the regulation of important transcription factors in the auxin pathway, thereby affecting adventitious root formation.

[0004] In summary, this demonstrates that PYL can regulate root growth and development as well as plant morphogenesis in other plant species, but this gene has not yet been reported in peony. Therefore, using genetic engineering techniques, the gene cloned from peony... PsPYL4The transfer of genes into model plants is of great significance for studying their functions and has great application potential. Summary of the Invention

[0005] To address the shortcomings of existing breeding technologies, the purpose of this invention is to provide a peony... PsPYL4 Genes. Another object of the present invention is to provide peony. PsPYL4 The application of genes in plant breeding.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A type of peony PsPYL4 The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] The peony mentioned PsPYL4 The expressed protein of the gene has the amino acid sequence shown in SEQ ID NO.2.

[0009] The peony mentioned PsPYL4 The application of genes in plant growth and development.

[0010] Peony PsPYL4 Applications in altering leaf growth of Arabidopsis thaliana 'Columbia' seedlings, in promoting vertical rooting of Arabidopsis thaliana 'Columbia', and in altering the endogenous hormone content in leaves of Arabidopsis thaliana 'Columbia'.

[0011] The peony PsPYL4 The gene was linked to a vector and transformed into wild-type Arabidopsis thaliana 'Columbia' via Agrobacterium-mediated transformation. After screening and cultivation, transgenic plants were obtained.

[0012] Beneficial effects: Compared with the prior art, the present invention improves upon the peony... PsPYL4 Gene cloning and identification, gene expression analysis, and genetic transformation; verification of gene function; and detection of overexpression. PsPYL4 Compared to wild-type Arabidopsis seedlings, the genetically modified Arabidopsis seedlings... PsPYL4 The overexpression lines showed faster growth, larger rosette leaves, significantly shorter taproot length, and significantly increased lateral root number. The content of endogenous hormones IAA, ABA, ZR, and GAs in the leaves was increased. The average number of roots in the detached leaves was significantly increased on MS medium containing IAA, and the average root length was significantly increased on MS medium containing IBA. It is evident that this gene will have wide applications in the development of adventitious roots and breeding of peony. Attached Figure Description

[0013] Figure 1 Image A is a peony. PsPYL4Electrophoresis images of the gene clones, where M is the DL2000 Marker and the target band length is 642 bp; Image B is... PsPYL4 Electrophoresis image to verify the overexpression vector after double enzyme digestion;

[0014] Figure 2 yes PsPYL4 The expression of peony's rooting stage;

[0015] Figure 3 It is a peony PsPYL4 Schematic diagram of the structure of the overexpression vector for gene cloning and construction;

[0016] Figure 4 Figure A shows the PCR results of transgenic Arabidopsis plants, where M represents the DL2000 Marker, WT uses wild-type DNA, (-) uses ddH2O as a template as a negative control, and 1-10 use transgenic plant DNA as a template; Figure B shows the relative expression levels of transgenic Arabidopsis plants and transgenic lines; Note: WT is wild-type Arabidopsis, and OE is a transgenic line.

[0017] Figure 5 It is an overexpression PsPYL4 Comparison of transgenic and wild-type Arabidopsis plants: Figure A shows the seedlings; Figure B shows the stems; Figure C shows the mature plants; Unless otherwise specified, the scale is 1cm; WT represents wild-type Arabidopsis, and OE represents the transgenic line;

[0018] Figure 6 It is an overexpression PsPYL4 A comparison of root growth between genetically modified and wild-type Arabidopsis thaliana plants, and a statistical diagram of taproot and lateral roots; WT represents wild-type Arabidopsis thaliana, and OE represents transgenic lines;

[0019] Figure 7 It is an overexpression PsPYL4 Comparison of endogenous hormone content in leaves of genetically modified Arabidopsis thaliana plants and wild-type Arabidopsis thaliana plants; WT represents wild-type Arabidopsis thaliana.

[0020] Figure 8 It is overexpression under IAA treatment PsPYL4 Comparison of rooting indicators between genetically modified plants and wild-type Arabidopsis thaliana plants;

[0021] Figure 9 It is overexpression under IBA treatment PsPYL4 Comparison of rooting indicators between genetically modified plants and wild-type Arabidopsis thaliana plants. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments.

[0023] Example 1

[0024] The material used in this embodiment is the root and stem tissue of peony 'Zhengwu', which was quick-frozen in liquid nitrogen after harvesting and stored in an ultra-low temperature freezer (-80℃).

[0025] 1) Extraction of total RNA from various tissues of peony

[0026] The procedure was performed according to the instructions for the OMEGA Plant RNA Mini Extraction Kit.

[0027] The cryopreserved peony tissues were rapidly transferred to a mortar pre-cooled with liquid nitrogen and ground with a pestle, with liquid nitrogen added continuously until they were ground into powder. The powdered samples were then added to a 1.5 mL sterile tube containing 5000 μl RB Buffer and vortexed until there was no obvious precipitation in the lysis buffer. The lysis buffer was then transferred to a 2 mL collection tube containing a gDNA filter column and centrifuged at 14,000 rpm and 4°C for 5 minutes. Carefully aspirate the filtrate into a new 1.5 mL sterile tube and measure the volume. Add half a volume of anhydrous ethanol to the supernatant, vortex for 20 s, and then transfer to a 2 mL collection tube containing the Hibind RNA Mini Column. Centrifuge at 12,000 rpm for 1 minute at 4°C and discard the filtrate. Add 400 μL of RWF Buffer to the Hibind RNA Mini Column and centrifuge at 10,000 rpm for 30 seconds. Discard the filtrate and collection tube. Place the Hibind RNA Mini Column into a new 2 mL collection tube. Add 500 μL of RNA WashBuffer II to the Hibind RNA Mini Column and centrifuge at 10,000 rpm for 30 seconds. Discard the filtrate. Add 500 μL of RNA WashBuffer II to the Hibind RNA Mini Column and centrifuge at 10,000 rpm for 30 seconds. Discard the filtrate. Replace the Hibind RNA Mini Column into a 2 mL collection tube. Centrifuge at the highest speed for 2 minutes on a tube, discarding the filtrate and collection tube. Place the Hibind RNA Mini Column onto a 1.5 mL RNase-Free Collection Tube, add 50 μL of DEPC Water at 65 °C to the center of the Hibind RNA Mini Column membrane, incubate at room temperature for 5 minutes, and then centrifuge at the highest speed for 1 minute to elute the RNA. After testing the concentration and purity of the obtained RNA, store it at -80 °C for later use.

[0028] 2 μL of RNA was analyzed by 1% agarose gel electrophoresis. The results showed that the 28S and 18S bands were relatively clear, with the 28S band being approximately twice as bright as the 18S band, indicating good RNA quality. RNA purity and OD were then determined using a micro-volume nucleic acid and protein analyzer. 260 / OD 280 and OD 260 / OD 230 All values ​​were between 1.8 and 2.1, indicating good integrity, and they can be used for reverse transcription.

[0029] 2) Synthesis of first-strand cDNA

[0030] Using the obtained total RNA as a template, reverse transcription was performed according to the Tiangen Reverse Transcription Kit. The specific procedures are as follows:

[0031] Thaw the template RNA and 5×Fastking-RT SuperMix on ice. Thaw the RNase-Free ddH2O at room temperature and then immediately place it on ice. Vortex the reagents before use and briefly centrifuge. Prepare a 20 μl reaction mixture in a centrifuge tube, including 4 μL of 5×Fastking-RT SuperMix, 800 ng of Total RNA, and make up the remaining volume with RNase-Free ddH2O. Perform the entire reaction on ice. After centrifugation, place the tube in a PCR instrument, incubate at 42°C for 15 min to remove the genome and perform reverse transcription, inactivate the enzymes at 95°C for 3 min, and place on ice to obtain the cDNA solution.

[0032] 3) Design and cloning of primers for the target gene

[0033] Based on existing peony transcriptome sequencing results, peony designs were created using CE Design. PsPYL4 Gene amplification primers, primer sequences are as follows:

[0034] PsPYL4-F:

[0035] 5'-acgggggactctagaggatccATGTTTTCAAATCCTCCAAAATCA-3'

[0036] PsPYL4-R:

[0037] 5'-ataagggactgaccacccgggTCATGAGTTGTTGCGGTTTCG-3'

[0038] Using cDNA as a template, Takara's PrimerStar Max high-fidelity enzyme was used to process peony. PsPYL4Gene cloning. Prepare 50 μl of PCR reaction solution, including 25 μL PrimerSTAR Max, 2.5 μL Forward Primer, 2.5 μL Reverse Primer, and 2.5 μL Template. Make up the remainder with ddH2O and centrifuge to mix thoroughly. PCR reaction conditions: 94℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 30 s, 32 cycles, 72℃ final extension for 5 min, and incubation at 16℃.

[0039] After the PCR reaction was completed, all PCR products were examined by 1.8% agarose gel electrophoresis. Correct bands were cut and purified to recover the target PCR amplification product. The target fragment was purified and recovered using the TransGen DNA gel extraction kit. The specific procedures were as follows:

[0040] Cut the correct band from the agarose gel and weigh it in a clean centrifuge tube. Add 3 times the volume of GSB solution to the gel block (if the gel weighs 0.1g, its volume can be considered as 100μL, add 300μL of GSB solution), and incubate in a 55℃ water bath, turning the centrifuge tube up and down every 2-3 minutes until the gel block is completely melted. After the gel solution cools to room temperature, add 1 volume of isopropanol and mix well. Add the mixture to the centrifuge column, let it stand for 1 min, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Add 650μL of WB solution, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Centrifuge at 12000 rpm for 2 min to remove as much residual WB as possible, and place the adsorption column at room temperature with the cap open for 5 min to dry it. Place the centrifuge column in a clean centrifuge tube, add 30μL of ddH2O at 60~70℃ above the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and elute the DNA. Take 2 μL of the purified product and perform gel electrophoresis with 1.5% agarose gel. Store the remainder in a -20°C freezer for later use in ligation with the pBI121 vector to construct an overexpression vector.

[0041] 4) Plasmid extraction:

[0042] Plasmids were extracted according to the instructions of the Tiangen plasmid mini-extraction medium-volume preparation kit. The specific steps are as follows:

[0043] Add 20 mL of the shaken bacterial culture to a centrifuge tube and centrifuge at 8000 rpm for 10 min. Discard the supernatant. Add 500 μL of P1 solution (containing RNase A) to the centrifuge tube containing the bacterial precipitate, mix thoroughly, and then transfer to a new 1.5 mL centrifuge tube. Add 500 μL of P2 solution to the centrifuge tube, gently invert 6-8 times to ensure complete lysis of the bacteria, and then add 700 μL of P2 solution to the centrifuge tube. Immediately and gently invert the P3 solution 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12000 rpm for 10 min. Add 500 μL of equilibration buffer BL to the adsorption column CP4, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and return the adsorption column to the collection tube. Transfer the supernatant collected in the previous step to the filter column CS, centrifuge at 12000 rpm for 2 min, carefully add the solution from the collection tube to the adsorption column CP4 in batches, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and return the adsorption column CP4 to the collection tube. Add 500 μL of protein removal solution PD to the adsorption column CP4, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and return the adsorption column CP4 to the collection tube again. Take 600 μL... Wash buffer PW (containing anhydrous ethanol) was added to adsorption column CP4, centrifuged at 12000 rpm for 1 min, and the waste liquid was discarded. Adsorption column CP4 was returned to the collection tube and centrifuged at 12000 rpm for 2 min to remove residual wash buffer. Adsorption column CP4 was transferred to a new 1.5 ml centrifuge tube, and 60 μL of ddH2O was added to the center of the adsorption membrane. The mixture was allowed to stand at room temperature for 2 min, then centrifuged at 12000 rpm for 1 min. The solution collected in the centrifuge tube was the plasmid. The plasmid concentration was determined to prepare for the next experiment.

[0044] 5) Double enzyme digestion reaction

[0045] The extracted pBI121 plasmid was digested with BamHI and SmaI at 37℃ for 30 min, and detected by gel electrophoresis. The linear vector was purified and recovered, and stored at -20℃ for later use. The double digestion reaction system was 50 μL: 20 μL pBI121 plasmid, 5 μL 5× buffer, 1 μL BamHI, 1 μL SmaI, and 23 μL ddH2O.

[0046] 6) Recombination reaction

[0047] Agarose gel electrophoresis was used to detect the purified target gene and vector pBI121 after enzyme digestion, and to determine their purity and concentration. The ligation reaction system consisted of: 3 μL of insert fragment, 7 μL of linearized pBI121 vector, 4 μL of 5×CE II buffer, 2 μL of Exnase II, and up to 20 μL of ddH2O. The reaction was carried out at 37°C for 30 min, then incubated at room temperature (do not immediately cool), and transformed into competent E. coli cells after 10 min.

[0048] 7) The ligation product is transferred into E. coli.

[0049] Thaw competent Trans5α cells (stored at -80℃) on ice. Add 10 μL of recombinant product to 100 μL of competent cells; place the centrifuge tube on ice for 10 min; heat shock in a 42℃ metal bath for 90 s; immediately place on ice for 2 min; add 500 μL of antibiotic-free LB liquid medium in a clean bench; incubate at 37℃ and 200 rpm for 25 min to recover; centrifuge at 6000 rpm for 1 min, and aspirate 350 μL of supernatant; resuspend the precipitated bacterial cells and spread them on LB solid medium plates (Kan concentration of 50 mg / L), and incubate overnight at 37℃.

[0050] 8) Identification of recombinants

[0051] Single colonies from the plate were transferred to LB broth containing antibiotics (Kan) and incubated overnight at 37°C with shaking at 200 rpm. Colony PCR was performed using primers for the full-length target gene to screen for positive clones. The bacterial test results are as follows: Figure 1 As shown in Figure A. The screened positive clones were sent to Nanjing Qingke Company for sequencing. Positive clones with correct sequencing results were expanded and cultured, and plasmids were extracted using the Tiangen plasmid extraction kit and subjected to double enzyme digestion verification to determine if the fragment sizes after digestion were consistent. The digestion results are shown in Figure A. Figure 1 As shown in B.

[0052] Based on the sequencing results analysis, it was finally determined that one peony was cloned. PsPYL4 The encoding gene is named PsPYL4 The gene, whose nucleotide sequence is shown in SEQ ID NO.1, PsPYL4 The gene has a coding length of 1642 bp, contains an ATG start codon and a TAA stop codon, and encodes 213 amino acids. The amino acid sequence is shown in SEQ ID NO.2.

[0053] Example 2

[0054] Peonies obtained through cloning PsPYL4 The gene was used as a reference to design quantitative fluorescent primers. The primer sequences are as follows:

[0055] qPsPYL4-F: 5'-ACGCCCCTCCCCTATAACAT-3'

[0056] qPsPYL4-R: 5'-AGGCTTGAGGGTTGTCGAAG-3'

[0057] Meanwhile, UBIQUITIN was used as an internal reference gene, and the primer sequence was:

[0058] UBIQUITIN-F:5'-GACCTATACCAAGCCGAAG-3'

[0059] UBIQUITIN-R: 5'-CGTTCCAGCACCACAATC-3'

[0060] Utilize TB Green ® The reaction solution was prepared according to the instructions of the Premix Ex Taq™ kit (TAKARA). The reaction was run on an Applied Biosystems real-time quantitative PCR instrument. The PCR program was: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles; 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s. After the reaction was complete, the amplification curve was obtained. The data was exported using StepOne Software v2.3 and analyzed using Excel. The CT value was used as a 2... -ΔΔCt The relative expression level was calculated using the relative quantification method, and the data analysis results are as follows: Figure 2 As shown.

[0061] The results of this embodiment indicate that... PsPYL4 The gene is expressed throughout the adventitious root development process of peony. Its expression level is initially downregulated and then upregulated during the dedifferentiation stage, downregulated again during the induction stage, and continues to downregulate during the differentiation stage, reaching its highest value at 25 days. Subsequently, it is downregulated to its lowest value at the end of root induction, 30 days later. This indicates the important role of this gene in adventitious root development. Figure 2 ).

[0062] Example 3

[0063] 1) Preparation and transformation of Agrobacterium competent cells

[0064] This embodiment utilizes Agrobacterium GV3101 competent cells for Arabidopsis infection experiments: Agrobacterium GV3101 transformation with recombinant cells: The competent Agrobacterium cells were thawed on ice. 1000 ng of the purified plasmid was added to 100 μl of competent Agrobacterium cells, gently mixed, and incubated on ice for 5 min; then flash-frozen in liquid nitrogen for 5 min; heat-shocked in a 37°C metal bath for 5 min, and immediately placed on ice for 5 min; 800 μl of antibiotic-free LB medium was added, and the cells were incubated at 28°C and 200 rpm for 2 h; centrifuged at 4000 rpm for 3 min, and 350 μL of supernatant was removed; the remaining bacterial culture was thoroughly mixed and then spread onto LB solid medium supplemented with 50 mg / L kanamycin and 200 mg / L rifampin; and incubated upside down at 28°C for 30–48 h.

[0065] Identification of Agrobacterium recombinants: Single colonies grown from agar plates were picked and inoculated into liquid culture medium containing the corresponding antibiotics; after incubation at 28°C and 200 rpm overnight, bacterial PCR was performed. The PCR products were detected by 1.5% agarose gel electrophoresis to identify whether they contained the target fragment. Positive clones were added with an appropriate amount of sterile 50% glycerol and stored at -80°C for later use.

[0066] 2) Agrobacterium-mediated transformation of Arabidopsis thaliana

[0067] The target gene was transferred into Arabidopsis thaliana using the inflorescence infection method. The specific operation method was as follows: Arabidopsis thaliana (Columbia type) was kept in a healthy growth state until flowering; Agrobacterium GV3101 strain carrying the target gene was activated. Pick a single colony and inoculate it into 50 mL of LB broth containing kanamycin and rifampin. Shake at 28°C and 200 rpm until the bacterial solution just becomes turbid, about 8-10 h. Take 1 mL of the bacterial solution and inoculate it into an Erlenmeyer flask (50 mL). Shake for 24 h until the OD value is about 0.8-1.0. Centrifuge the bacterial solution at 6000 rpm at room temperature for 10 min, remove the supernatant, and suspend it in 3% sucrose solution at pH 5.8. Before soaking, add Silwet L-77 at a concentration of 0.03% (300 μl / L) and mix thoroughly. Soak the aerial parts of Arabidopsis thaliana in Agrobacterium suspension for 1 min, gently shaking during the process. Seal the soaked Arabidopsis thaliana with aluminum foil to protect it from light and place it for 24 h. Remove the aluminum foil and culture under normal conditions.

[0068] The components of the 3% sucrose solution resuspension are as follows: MS medium, with added sucrose 30 g / L, and Silwet-77 300 µl / L. (Note: After preparation, adjust the pH to 5.8, centrifuge and resuspend the bacterial solution before adding Silwet-77; the conversion between resuspension and bacterial solution is: resuspension volume: bacterial solution OD * bacterial solution volume = 0.8 * resuspension).

[0069] 3) Screening of transgenic plants

[0070] The collected T0 generation transgenic Arabidopsis seeds were sterilized with alcohol and sodium hypochlorite. The steps were as follows: an appropriate amount of the obtained transgenic seeds were placed in a 1.5 mL centrifuge tube and soaked in a 10% NaClO and ethanol mixture (freshly prepared, volume ratio 1:1) for 5 min; sterilized with 75% alcohol 5-6 times, 2 min each time; rinsed with sterile water 3-4 times; and suspended in sterile water.

[0071] Sterilized transgenic Arabidopsis seeds were sown on 1 / 2 MS solid medium containing antibiotics (kanamycin 50 mg / L), wrapped in aluminum foil, and placed in a 4°C refrigerator for vernalization. After 2 days, the seeds were removed from the refrigerator, and the medium was placed at 22°C under light. After about a week, the Arabidopsis seeds that grew normally on the medium were transplanted into soil to continue growing.

[0072] 4) DNA detection of transgenic plants

[0073] Take appropriate amounts of young leaves from T1 generation Arabidopsis thaliana and transgenic plants, and perform detection using the plant DNA kit from AG Biotech. The specific steps are as follows: weigh 0.1 mg of plant sample and grind it in liquid nitrogen. Then, quickly add 500 uL of Buffer LS-3 and 10 uL of 50×DTT Buffer to the ground sample powder, and then add 10 uL of RNase A. Shake thoroughly to mix. Place the centrifuge tube in a 56℃ water bath for 10 min (inverting and mixing is allowed during this time). Add 1 / 8 volume of lysis buffer PA and mix thoroughly. Place on ice for 5 min, centrifuge at 12,000 rpm for 5 min at room temperature; collect the supernatant, add an equal volume of Buffer BS-2, and mix thoroughly; transfer the above solution to a Plant DNA Mini Column (for larger solutions, it is generally necessary to pass through the column twice, with each pass not exceeding 750 uL), let stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate; add 500 uL of Buffer WA to the Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate; add 750 uL of Buffer WB (pre-added with 63 ml of anhydrous ethanol) to the Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate, repeating this operation once; place the Mini Column on a new 2 ml Collection Tube, centrifuge at 12,000 rpm for 2 min; place the Mini Column on a new 1.5 ml centrifuge tube, and add 50 μL of Buffer WB to the center of the Mini Column membrane. The DNA was eluted by centrifugation at 12,000 rpm for 2 minutes at room temperature in 65℃ ddH2O solution. The DNA was then used for PCR detection, and the results are as follows: Figure 4 As shown in Figure A.

[0074] 5) Quantitative real-time PCR detection of transgenic plants

[0075] From the above overexpression of peony PsPYL4Total RNA was extracted from the young stems and leaves of the Arabidopsis thaliana strain. Reverse transcription and quantitative fluorescence primers, methods, and procedures were the same as in Example 2. The final data analysis results are as follows: Figure 4 As shown in B.

[0076] 6) Obtaining homozygous transgenic lines

[0077] The harvested transgenic T1 generation seeds were sterilized, screened, and then transplanted into nutrient soil and cultured at 22℃ under 16 h light / 8 h dark conditions. After testing, the preliminarily confirmed transgenic plants were retained, and their mature T1 generation seeds were harvested and numbered to obtain the T2 generation. Similar to the T1 generation, the T2 generation seeds were sterilized and spread onto a screening medium containing antibiotics, and placed at 22℃ under continuous light. After approximately 10 days, the survival rate of the T2 generation seeds with different numbers was statistically analyzed. Plants with a survival rate of 75% were selected and transplanted into nutrient soil under 22℃ under 16 h light / 8 h dark conditions, and leaves were taken for positive detection. Positive T2 generation plants were further numbered, and their seeds were collected to obtain the T3 generation seeds. These seeds were sterilized, screened using a screening medium, and cultured under continuous light. After approximately 10 days, the T3 generation plants with different numbers were observed; those that survived without segregation were considered homozygous T3 generation plants.

[0078] 7) Phenotypic observation

[0079] Transgenic lines with distinct phenotypes were selected for observation. The results showed that compared with wild-type Arabidopsis thaliana, transgenic Arabidopsis thaliana plants grew faster, had larger rosette leaves, significantly shorter taproot length, and significantly increased number of lateral roots.

[0080] Transgenic and wild-type Arabidopsis thaliana of generation T3 were sown on 1 / 2 MS medium containing kan. After 5 days of culture, positive seedlings were transferred to 1 / 2 MS medium for vertical culture. Root growth changes were observed and recorded for 7 days. It was found that the transgenic plants had significantly shorter taproots and significantly more lateral roots compared with wild-type plants.

[0081] The endogenous hormone content in Arabidopsis thaliana leaves was determined by enzyme-linked immunosorbent assay (ELISA). Samples were ground in 10 mL of 80% (v / v) methanol extraction medium containing 1 mM butylated hydroxytoluene (BHT) as an antioxidant. The extract was incubated at 4°C for 4 h and centrifuged at 4000 rpm for 15 min. The supernatant was passed through a C-18 column and washed sequentially with 80% (v / v) methanol, 100% (w / v) methanol, 100% (w / v) ether, and 100% (w / v) methanol. The hormone fraction was then dried under N2 and dissolved in phosphate-buffered saline (PBS) containing 0.1% (v / v) Tween 20 and 0.1% (w / v) gelatin for analysis. Monoclonal antigens and antibodies against IAA, ABA, GAs (GA1+GA3), MeJA, and BR in the ELISA kit were produced by the Institute of Plant Hormones, China Agricultural University. ELISA assays were performed using 96-well microtiter plates. Each well was coated with 100 μL of coating buffer (1.5 g / L Na₂CO₃, 2.93 g / L NaHCO₃, 0.02 g / L NaN₃) containing 0.25 μg / mL anti-hormone antigen and incubated at 37°C for 30 min. After washing four times with PBS containing 0.1% (v / v) Tween 20, each well was filled with 50 μL of sample extract and 50 μL of 20 μg / mL antibody, and incubated and washed as described above. 100 μL of chromogenic solution containing 1.5 mg / mL O-phenylenediamine and 0.008% (v / v) H₂O₂ was added to each well. The reaction was stopped with 12 mol / L H₂SO₄ per well. Developing was performed at 490 nm using an ELISA instrument (model EL310, Bio-TEK, Winooski, VT). Hormone content was calculated according to Weiler et al. (1981). Three biological replicates were performed for each hormone. The results showed that the levels of endogenous hormones IAA, ABA, ZR, and GAs in the leaves of transgenic plants were all increased.

[0082] This embodiment will overexpress peony PsPYL4 The recombinant plasmid of the gene was transferred into the model plant Arabidopsis thaliana, and phenotypic observation and analysis were performed. The results showed that overexpression was detected. PsPYL4 Compared to wild-type Arabidopsis seedlings, the genetically modified Arabidopsis seedlings... PsPYL4 The overexpressing lines showed faster growth, larger rosette leaves, significantly shorter taproot length, and significantly increased lateral root number. The levels of endogenous hormones IAA, ABA, ZR, and GAs in the leaves were elevated. The average number of roots in the detached leaves was significantly increased on MS medium containing IAA, and the average root length was significantly increased on MS medium containing IBA. This indicates that the gene affects plant growth and development, adventitious root formation, and hormone level changes.

Claims

1. Peony PsPYL4 Its application in increasing the rosette leaves of Arabidopsis thaliana Columbia type, shortening its taproot length, and increasing the number of its lateral roots is characterized by... The PsPYL4 The nucleotide sequence is shown in SEQ ID NO. 1; the application includes: using the peony PsPYL4 The gene was linked to a vector and transformed into wild-type Arabidopsis thaliana Columbia type via Agrobacterium-mediated transformation. After screening and cultivation, transgenic plants were obtained.

2. Peony PsPYL4 Its application in increasing the content of endogenous hormones IAA, ABA, ZR, and GAs in leaves of Arabidopsis thaliana Columbia type is characterized by, The PsPYL4 The nucleotide sequence is shown in SEQ ID NO. 1; the application includes: using the peony PsPYL4 The gene was linked to a vector and transformed into wild-type Arabidopsis thaliana Columbia type via Agrobacterium-mediated transformation. After screening and cultivation, transgenic plants were obtained.

3. Peony PsPYL4 The application of this study in increasing the average number of roots from detached Arabidopsis thaliana Columbia leaves on MS medium containing IAA is characterized by... The PsPYL4 The nucleotide sequence is shown in SEQ ID NO. 1; The application includes: using the peony PsPYL4 The gene was linked to a vector and transformed into wild-type Arabidopsis thaliana Columbia type via Agrobacterium-mediated transformation. After screening and cultivation, transgenic plants were obtained.

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