Application of a Cymbidium orchid CgARF2 gene in regulating leaf growth and development

Abstract

The present invention discloses a spring orchid CgARF2 gene and its application in regulating leaf growth and development. The nucleotide sequence of the CgARF2 gene is shown in SEQ ID NO. 1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO. 2. The present invention cloned the CgARF2 gene sequence from the spring orchid cultivar "Songmei" and performed expression analysis in the spring orchid. Subsequently, the gene sequence was constructed into an overexpression vector and introduced into target plants to verify its function. It was found that Arabidopsis plants overexpressing the CgARF2 gene showed phenotypes such as an increased number of rosette leaves, smaller leaves, and curled leaf margins compared to wild-type plants. The rooting rate and root length of detached leaves on MS medium containing IAA were increased, and the content of endogenous hormones IAA and GA in the leaves was increased, while the content of ABA, MeJA, and BR was reduced. It can be seen that the gene will have a wide range of uses in the cultivation and trait improvement of orchids and other horticultural plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of a Cymbidium orchid CgARF2 gene in regulating leaf growth and development. Background Art

[0002] The Orchidaceae family is one of the largest families of flowering plants, with over 25,000 species worldwide, accounting for approximately 10% of all flowering plants. Cymbidium goeringii, a small-flowered terrestrial orchid in the genus Orchidaceae, boasts unique flower shapes, elegant colors, a subtle fragrance, and graceful foliage, earning it both high ornamental and economic value. It exemplifies the beauty of both foliage and flowers. However, in recent years, overexploitation of this species and deteriorating ecological conditions have posed significant challenges to the maintenance of Cymbidium goeringii seed resources and its habitat. Plant leaves are crucial organs and the primary site of photosynthesis. Proper vegetative growth and leaf development are crucial for successful reproduction. Therefore, studying the molecular mechanisms by which genes influence plant growth and development is crucial for the breeding, production, and application of Cymbidium goeringii. The ARF gene family plays a crucial role in plant growth and development, providing valuable insights for plant genetic improvement.

[0003] Auxin response factors (ARFs), a family of transcription factors discovered in 1997, regulate the expression of auxin-responsive genes. They interact with auxin response elements to regulate the expression of related genes, thereby participating in the growth and development of various plants. In Arabidopsis, AtARF1, AtARF2, AtARF7, and AtARF19 are associated with leaf senescence, while AtARF3 and AtARF4 affect leaf polarity and development. In tomato, SlARF10 inhibits leaf growth and alters leaf shape; SlARF12 plays a role in early leaf development, while SlARF2 affects leaf senescence. In rice, OsARF19 is strongly expressed in leaf nodes and is involved in regulating the growth angle of rice leaves. Osarf111 mutants also exhibit a reduced flag leaf angle. These studies demonstrate that ARF transcription factors regulate leaf growth and development in other plant species. Therefore, using genetic engineering technology to transfer the CgARF2 gene cloned from Cymbidium orchid into model plants is of great significance for studying its function and has great application prospects. Summary of the Invention

[0004] In view of the shortcomings of existing breeding technologies, the present invention aims to provide a CgARF2 gene of Cymbidium orchid. Another object of the present invention is to provide the application of the CgARF2 gene of Cymbidium orchid in plant breeding, especially in leaf growth and development.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0006] A Cymbidium orchid CgARF2 gene, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0007] The amino acid sequence of the expressed protein of the Cymbidium orchid CgARF2 gene is shown in SEQ ID NO.2.

[0008] The application of the Chunlan CARF2 gene in the growth and development of plant leaves.

[0009] The application of Chunlan CARF2 gene in changing the leaf morphology of Arabidopsis thaliana "Columbia", promoting the rooting of detached leaves of Arabidopsis thaliana "Columbia", and changing the endogenous hormone content of Arabidopsis thaliana "Columbia" leaves.

[0010] The Chunlan CgARF2 gene is connected to a vector, and transformed into wild-type Arabidopsis thaliana "Columbia" through Agrobacterium-mediated transformation, and then screened and cultured to obtain transgenic plants.

[0011] Beneficial effects: Compared with the prior art, the present invention verifies the function of the CgARF2 gene of spring orchid by cloning and identifying it, analyzing its expression, and genetic transformation. It is found that compared with the wild type, the Arabidopsis thaliana overexpressing the CgARF2 gene has an increased number of rosette leaves, smaller leaves, curled leaf edges, an increased rooting rate of detached leaves, and changes in the content of various endogenous hormones. It can be seen that this gene will have a wide range of uses in the nutritional growth of orchids and other plant production and breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure A is the electrophoresis of the CgARF2 gene clone of Chunlan, where M is the DL2000 marker and the target band is 2103 bp long; Figure B is the electrophoresis of the double enzyme digestion verification of the CgARF2 overexpression vector;

[0013] Figure 2 Figure A shows the expression of CgARF2 in various tissues of Cymbidium orchid, where R represents root, S represents pseudobulb, L represents leaf, and F represents flower; Figure B shows the expression of CgARF2 gene in Cymbidium orchid under IAA treatment;

[0014] Figure 3 This is a schematic diagram of the overexpression vector structure for cloning and constructing the Chunlan CgARF2 gene;

[0015] Figure 4 Panel A shows the PCR results of transgenic Arabidopsis plants, where M represents DL2000 Marker, 1 uses wild-type DNA as a negative control template, and 2-8 uses transgenic plant DNA as templates;

[0016] Figure 5 Comparison diagrams of plants overexpressing the CgARF2 gene and wild-type Arabidopsis plants: Figure A shows the plant shape; Figure B shows the leaf shape; Figure C shows the fruit shape.

[0017] Figure 6 The figure shows the rooting of detached leaves of plants overexpressing the CgARF2 gene and wild-type Arabidopsis plants on MS medium containing different concentrations of IAA.

[0018] Figure 7 Comparison of endogenous hormone levels in leaves of plants overexpressing the CgARF2 gene and wild-type Arabidopsis plants. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to specific embodiments.

[0020] Example 1

[0021] The materials used in this example were various tissues of Cymbidium 'Songmei', which were quickly frozen in liquid nitrogen and stored in an ultra-low temperature freezer (-80°C).

[0022] 1) Extraction of total RNA from various tissues of Cymbidium orchid

[0023] Follow the instructions of the TaKaRa Plant Total RNA Extraction Kit. The specific steps are as follows:

[0024] Transfer the cryopreserved Cymbidium orchid tissues quickly to a mortar pre-cooled with liquid nitrogen and grind the tissues with a pestle while continuously adding liquid nitrogen until they are ground into powder. Add the powdered samples to 1.5 mL sterile tubes containing 450 μl Buffer PE and repeatedly pipette until there is no obvious precipitation in the lysate. Centrifuge the lysate at 12,000 rpm and 4°C for 5 minutes. Carefully pipette the supernatant into a new 1.5 mL sterile tube. Add 1 / 10 volume of Buffer NB to the supernatant, Vortex to mix, and centrifuge at 12,000 rpm at 4°C for 5 minutes. Carefully transfer the supernatant to a new 1.5 mL sterile tube, add 450 μL of Buffer RL, and mix the solution evenly with a pipette. Add 1 / 2 volume of anhydrous ethanol to the mixture, mix the solution evenly with a pipette, and immediately transfer the entire mixture to the RNA Spin Column. Centrifuge at 12,000 rpm for 1 minute, discard the filtrate, and return the RNA Spin Column to the 2 mL Collection Tube. Add 500 μL of Buffer RWA to the RNA Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add 600 μL of Buffer RWB to the RNA Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add 50 μL of DNase I to the center of the RNA Spin Column membrane. The reaction mixture was allowed to stand at room temperature for 15 minutes. 350 μL of Buffer RWB was added to the center of the RNA Spin Column membrane and centrifuged at 12,000 rpm for 30 seconds. The filtrate was discarded. The RNA Spin Column was reinstalled in a 2 mL Collection Tube and centrifuged at 12,000 rpm for 2 minutes. The RNA Spin Column was then placed in a 1.5 mL RNase-free Collection Tube. 30 μL of RNase-free dH2O was added to the center of the RNA Spin Column membrane and allowed to stand at room temperature for 5 minutes. The RNA was then centrifuged at 12,000 rpm for 2 minutes to elute the RNA. The resulting RNA was tested for concentration and purity and stored at -80°C until further use.

[0025] 2 μL RNA was aspirated and tested by 1% agarose gel electrophoresis. The results showed that the 28S and 18S bands were relatively clear, and the brightness of the 28S band was about twice that of the 18S band, indicating that the RNA quality was good. The RNA purity was tested by a micronucleoprotein analyzer, and the OD 260 / OD 280 and OD260 / OD 230 All of them are between 1.8 and 2.1, with good integrity and can be used for reverse transcription.

[0026] 2) Synthesis of first-strand cDNA

[0027] The total RNA was used as a template for reverse transcription using the Tiangen Reverse Transcription Kit. The specific steps are as follows:

[0028] Thaw the template RNA on ice, and thaw 5× Fastking-RT SuperMix and RNase-Free ddH2O at room temperature. Immediately place on ice after thawing. Prepare a 10µL mixture in a centrifuge tube containing 4µL of 5× Fastking-RT SuperMix and 800ng of total RNA. Fill the remaining volume with RNase-Free ddH2O. Centrifuge the tube until the mixture settles to the bottom. Gently shake to mix. Incubate in a PCR machine at 42°C for 15 minutes to remove the genome and perform the reverse transcription reaction. Inactivate the enzyme at 95°C for 3 minutes, and place on ice to obtain the cDNA solution.

[0029] 3) Design and cloning of target gene primers

[0030] Based on the existing transcriptome sequencing results of Chunlan, CE Design was used to design the Chunlan CgARF2 gene amplification primers. The primer sequences are as follows:

[0031] CgARF2-F: 5'-GAGAACACGGGGGACTCTAGAATGTTTTTGGGTTTGGAGATTGA -3'

[0032] CgARF2-R: 5'-ATAAGGGACTGACCACCCGGGGATTCCAGTTTCTGAAAAGAGAGGTG-3'.

[0033] The CgARF2 gene from Cymbidium orchid was cloned using cDNA as a template using Takara's PrimerStar Max high-fidelity enzyme. The PCR amplification system (50 μL) consisted of 25 μL PrimerStar Max, 2 μL Forward Primer, 2 μL Reverse Primer, 2 μL Template cDNA, and 19 μL ddH2O. The PCR program was as follows: 32 cycles of initial denaturation at 94°C for 3 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds, followed by a total extension at 72°C for 5 minutes, followed by a 16°C incubation period.

[0034] After the PCR reaction was completed, all PCR products were detected by 1.8% agarose gel electrophoresis (PCR amplification results were as follows Figure 3 ) and cut the target fragment, gel recovery PCR target amplification product. Use TransGen's DNA gel recovery kit to purify the target fragment. The specific operation is as follows: cut the single target band from the agarose gel, put it into a clean centrifuge tube, and weigh it; add 3 times the volume of GSB solution to the gel (if the gel is 0.1g, its volume can be regarded as 100μL, then add 300μL 1 min at room temperature, centrifuge at 12,000 rpm for 1 min, discard the flow-through, and return the column to the collection tube. Add 650 μL of WB solution to the column and centrifuge at 12,000 rpm for 1 min. Discard the flow-through and centrifuge at 12,000 rpm for 2 min to remove as much residual WB as possible. Place the adsorption column at room temperature with the lid open for 5 min to dry thoroughly. Place the column in a clean centrifuge tube and drop 30 μL of ddH2O (ddH2O must be preheated in a 60-70°C water bath) onto the center of the adsorption membrane. Let it stand at room temperature for 2 min and centrifuge at 12,000 rpm for 2 min to collect the DNA solution. Take 2 μL of the recovered purified product and perform gel electrophoresis on it using 1.5% agarose gel. The rest is placed in a -20°C refrigerator and will be used to connect with the pBI121 vector to construct an overexpression vector.

[0035] 3) Plasmid extraction:

[0036] Extract the plasmid according to the instructions of the Tiangen Plasmid Mini-Prep Kit. The specific steps are as follows:

[0037] Take 10mL of overnight cultured bacterial solution, centrifuge at 12000 rpm for 1min, and remove the supernatant; take 500μL of P1 solution (containing RNase A) and add it to the centrifuge tube with bacterial pellet, and use a vortex instrument to thoroughly suspend the bacterial pellet; take 500μL of P2 solution and add it to the centrifuge tube, gently turn it upside down to fully lyse the bacteria, and take 700μL Add P3 solution to the centrifuge tube, immediately turn it upside down and mix it thoroughly. When a white flocculent precipitate appears, centrifuge it at 12000rpm for 10min. Take 500μL of equilibrium solution BL and add it to the adsorption column CP4. Centrifuge it at 12000rpm for 1min. Discard the waste liquid in the collection tube. Put the adsorption column back into the collection tube. Add the collected supernatant to the filter column CS in batches. Centrifuge it at 12000rpm for 2min. Carefully add the solution collected in the collection tube to the adsorption column CP4 in batches. Centrifuge it at 12000rpm for 1min. Discard the waste liquid in the collection tube. Put the adsorption column CP4 back into the collection tube. Take 500μL of deproteinized solution PD and add it to the adsorption column CP4. Centrifuge it at 12000rpm for 1min. Discard the waste liquid in the collection tube. Put the adsorption column CP4 back into the collection tube. Take 600μl Add the rinse solution (PW) (containing anhydrous ethanol) to the adsorption column CP4 and centrifuge at 12,000 rpm for 1 minute. Discard the waste solution in the collection tube and return the adsorption column CP4 to the collection tube. Centrifuge at 12,000 rpm for 2 minutes to remove any remaining rinse solution. Transfer the adsorption column CP4 to a new 1.5 ml centrifuge tube and add 60 μL of ddH2O to the center of the adsorption membrane. Let it stand at room temperature for 2 minutes and centrifuge at 12,000 rpm for 1 minute. The solution collected in the centrifuge tube is the plasmid. Finally, determine the plasmid concentration to prepare for the next step of the experiment.

[0038] 4) Double enzyme digestion reaction

[0039] The extracted pBI121 plasmid was digested with XbaI and SmaI at 37°C for 30 min. The linear vector was recovered by electrophoresis and stored at -20°C until use. The double enzyme digestion reaction system (50 μL) consisted of: 20 μL pBI121 plasmid, 5 μL 5× buffer, 1 μL XbaI, 1 μL SmaI, and 23 μL ddH2O.

[0040] 5) Recombination reaction

[0041] After digestion, verify the target gene and vector pBI121 by agarose gel electrophoresis. Based on the purity and concentration, add the appropriate reagents according to the ligation system. The target fragment: vector molecule ratio is 3:1 to 5:1. The ligation reaction system is: 7 μL of linearized pBI121 vector, 3 μL of insert, 4 μL of 5× CE II buffer, 2 μL of Exnase II, and 20 μL of ddH2O. Incubate at 37°C for 30 minutes, then allow to stand at room temperature (do not immediately cool). After 10 minutes, transform into competent E. coli.

[0042] 6) Transform the ligation product into E. coli

[0043] Remove competent cells of the Trans5α strain from the ultra-low temperature freezer and thaw on ice. Pipette 10 μL of the recombinant product into 100 μL of competent cells. Place the centrifuge tube on ice for 10 minutes. Heat shock the tube in a 42°C water bath for 90 seconds without shaking. Immediately place the tube on ice for 2 minutes. Add 500 μL of antibiotic-free liquid culture medium in a clean hood and shake at 37°C, 200 rpm, for 25 minutes to revive the tube. Centrifuge at 6000 rpm for 1 minute, aspirate 350 μL of supernatant, resuspend the pellet, spread it on an LB plate (Kana concentration is 50 mg / L), and culture at 37°C overnight.

[0044] 7) Identification of recombinants

[0045] Pick a single colony from the plate and inoculate it into LB liquid medium containing antibiotics (Kana), and culture it at 37℃ and 200 rpm overnight. Use the full-length primers of the target gene to perform PCR on the bacterial liquid to screen for positive clones. The bacterial test results are as follows: Figure 1 As shown in A. The positive clones after screening were sent to Nanjing Sipujin Company for sequencing. After the positive clones with correct sequencing results were expanded and cultured, the plasmids were extracted using the Tiangen Plasmid Extraction Kit and double enzyme digestion was performed to determine whether the fragment sizes after enzyme digestion were consistent. The enzyme digestion results were as shown in Figure 1 As shown in B.

[0046] Based on the analysis of the sequencing results, it was finally determined that a CgARF encoding gene of Cymbidium orchid was cloned and named CgARF2 gene. Its nucleotide sequence is shown in SEQ ID NO.1. The coding length of the CgARF2 gene is 2103bp, containing an ATG start codon and a TGA stop codon. The full length of the ORF is 2103bp, encoding 700 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0047] Example 2

[0048] Fluorescence quantitative primers were designed based on the cloned CgARF2 gene of Cymbidium orchid. The primer sequences are as follows:

[0049] qCgARF2-F: 5'-ATCCCTTAGCGTCCACTGCC-3'

[0050] qCgARF2-R: 5'-ATTATTGGTATTTCCCTTATCTGCCT-3'

[0051] At the same time, 18S was used as the internal reference gene, and the primer sequences were:

[0052] 18S-F: 5'-GGTCCTATTGTGTTGGCT-3'

[0053] 18S-R: 5'-TCGCAGTGGTTCGTCTTT-3'

[0054] The reaction solution was prepared using the instructions for the ChamQ™ Universal SYBR qPCR Master Mix Kit (Vazyme). The PCR protocol was run on an Applied Biosystems real-time fluorescence quantitative analyzer: 95°C for 5 minutes; 95°C for 10 seconds, 60°C for 30 seconds, 40 cycles; 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. After the reaction was complete, an amplification curve was obtained and data was exported using StepOne Software v2.3. Data analysis was performed using Excel. Relative expression levels were calculated using the 2-ΔΔCt relative quantification method based on the Ct values. The data analysis results are shown in Figure 2. Figure 2 shown.

[0055] The results of this study showed that the CgARF2 gene was expressed in all tissues of Cymbidium orchid ( Figure 2 A), but the gene is expressed at the highest level in spring orchid leaves, indicating that the gene is functionally active in the leaves; expression analysis of spring orchid leaves treated with IAA hormone spraying proved that the CgARF2 gene plays an important role in the response of spring orchid leaves to IAA treatment ( Figure 2 B).

[0056] Example 3

[0057] 1) Preparation and transformation of Agrobacterium competent cells

[0058] In this example, Agrobacterium GV3101 was used to prepare competent Agrobacterium for infection experiments with Arabidopsis thaliana. The preparation process for competent Agrobacterium was as follows: a single colony of activated Agrobacterium was picked and inoculated into 5 mL of liquid LB medium, and cultured at 28°C and 250 rpm for 20-24 h; 2 mL of the bacterial solution was aspirated and inoculated into a flask containing 50 mL of liquid LB medium, and cultured at 28°C and 250 rpm until the OD reached 0. 600 The value is about 0.8; place the expanded bacterial liquid on ice for 30 minutes, centrifuge at 4℃ and 5000rpm for 5 minutes, and discard the supernatant; add 10mL of pre-cooled 0.1 mol / L CaCl2 solution to fully suspend the precipitated bacteria; centrifuge at 4℃ and 5000rpm for 5 minutes, and discard the supernatant; add 1mL of pre-cooled 20 mmol / L CaCl2 solution to fully suspend the bacteria, and the GV3101 competent cells to be prepared are obtained. Divide them into 100µL / tube using centrifuge tubes, quickly add 20% sterile glycerol, and store at -80℃.

[0059] Agrobacterium transformation of recombinants: Thaw the competent Agrobacterium cells in an ice bath, add 600 ng of the recovered and purified plasmid to 100 μl of competent Agrobacterium, mix gently, and place on ice for 5 min; quick-freeze with liquid nitrogen for 5 min, heat shock in a 37°C metal bath for 5 min, and quickly place on ice for 5 min; add 800 μl of LB medium without any antibiotics and resuscitate at 28°C, 200 rpm for 2 h; centrifuge at 4000 rpm for 3 min, and aspirate some of the liquid medium; use a pipette to thoroughly mix the remaining bacterial liquid, and then spread it on solid LB medium supplemented with 50 mg / L kanamycin and 200 mg / L rifampicin; and culture inverted at 28°C for 30-48 h.

[0060] Identification of Agrobacterium recombinants: Single colonies grown on the plate culture medium were picked and inoculated into liquid culture medium containing the corresponding antibiotics. After overnight culture at 28°C and 200 rpm, PCR was performed on the culture medium. PCR products were detected by 1.5% agarose gel electrophoresis to determine whether they contained the target fragment. Positive clones were added with an appropriate amount of sterile 50% glycerol and stored at -80°C until further use.

[0061] 2) Agrobacterium-mediated transformation of Arabidopsis thaliana

[0062] The target gene was transferred into Arabidopsis thaliana using the inflorescence infection method. The specific operation method is as follows: Arabidopsis thaliana (Columbia type) is kept in a healthy growth state until flowering; and the Agrobacterium GV3101 strain carrying the target gene is activated. Pick a single colony and inoculate it into 5 mL of LB culture medium containing kanamycin and rifampicin. Shake the culture at 28°C and 200 rpm until the culture liquid just becomes turbid, about 8-10 hours; aspirate 1 mL of the culture liquid and inoculate it into a 50 mL Erlenmeyer flask and shake it for 24 hours until the OD value is about 0.8; centrifuge the culture liquid at 6000 rpm at room temperature for 5 minutes, remove the supernatant and collect the bacteria, and suspend them in a 3% sucrose solution with a pH of 5.8; before soaking, add Silwet L-77 at a concentration of 0.03% (300 μl / L) and shake out the foam; soak the aerial part of Arabidopsis in the Agrobacterium suspension solution for 1 minute, shaking gently during the process; lay the soaked Arabidopsis flat on a tray, cover it with plastic wrap, seal it with tin foil to avoid light, and place it for 24 hours; remove the tin foil, culture under normal conditions, and stop watering when the seeds are mature.

[0063] The composition of the 3% sucrose resuspension is as follows: MS medium supplemented with 30 g / L sucrose and 300 µl / L Silwet-77. (Note: After preparation, the pH is adjusted to 5.8. The Silwet L-77 is added after centrifugation and resuspension of the culture. The conversion relationship between the resuspension solution and the culture medium is: resuspension solution volume: culture medium OD x culture medium volume = 0.8 x resuspension solution volume).

[0064] 3) Screening of transgenic plants

[0065] The collected T1 generation transgenic Arabidopsis seeds were sterilized with alcohol and sodium hypochlorite as follows: an appropriate amount of transgenic seeds were placed in a 1.5 mL centrifuge tube and soaked in a mixture of 8% NaClO and ethanol (freshly prepared, with a volume ratio of 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 0.1% agarose solution.

[0066] Sterilized transgenic Arabidopsis seeds were sown on MS solid medium supplemented with antibiotics (kanamycin 50 mg / L and cephalosporin 100 mg / L), wrapped in tin foil, and placed in a 4°C refrigerator for vernalization. After two days, the seeds were removed from the refrigerator and incubated at 22°C under light. Approximately one week later, Arabidopsis plants that were growing normally on the medium were transplanted to soil for further growth.

[0067] 4) DNA testing of transgenic plants

[0068] Appropriate amounts of young leaves from T1 generation Arabidopsis and transgenic plants were tested using the Qingke Plant DNA Direct Amplification Kit. The specific steps were as follows: An appropriate amount of young leaves were minced and placed in a sterilized 2mL centrifuge tube. 50uL of Lysis Buffer A was added and lysed by heating at 95°C for 10 minutes, followed by an overnight lysis at 4°C. The next day, the leaves were centrifuged at 14,000 rpm for 3 minutes. The supernatant was transferred to a new sterile centrifuge tube and used as a template for the PCR reaction. PCR detection was performed using 2×T5 Direct PCR Mix and gene-specific primers. The results were as follows: Figure 4 As shown in A.

[0069] 5) Fluorescence quantitative PCR detection of transgenic plants

[0070] Total RNA was extracted from the young stem leaves of the five Arabidopsis thaliana strains overexpressing the CgARF2 gene of Cynanchum thaliana. The reverse transcription and fluorescence quantitative primers, methods and procedures were the same as those in Example 2. The final data analysis results are shown in FIG. Figure 4 As shown in B.

[0071] 6) Obtaining homozygous transgenic lines

[0072] The harvested transgenic T1 generation seeds were sterilized, screened and cultured, and then transplanted into nutrient soil and cultured at 22°C, 16 h light / 8 h dark; after testing, the preliminarily confirmed transgenic plants were retained, and the T1 generation seeds were harvested after maturity and numbered to obtain the T2 generation; as with the T1 generation, the T2 generation seeds were sterilized and spread on a screening medium containing antibiotics, and placed at 22°C and continuous light; after about 10 days, the survival rate of the T2 generation seeds with different numbers was counted, and plants with a survival rate of 75% were selected and transplanted into nutrient soil and cultured at 22°C, 16 h light / 8 h dark, and leaves were taken for positive detection; the positive T2 generation plants were further numbered, and the seeds were collected to obtain T3 generation seeds; after the seeds were sterilized, they were screened with a screening medium and cultured under light and continuous light; after about 10 days, the T3 generation plants with different numbers were observed, and those that all survived and did not separate were T3 generation homozygous plants.

[0073] 7) Phenotypic observation

[0074] Transgenic strains with obvious phenotypes were selected for observation. The results showed that compared with wild-type Arabidopsis, the transgenic Arabidopsis plants had more stems and rosette leaves, and the leaves of the whole plant became significantly smaller. At the same time, the leaf edges curled, the plants grew weakly, and were accompanied by dwarfism. The fruits became shorter and the fruit set rate was reduced.

[0075] Wild-type and transgenic seeds were sterilized and sown on MS medium. After culturing to 4-6 true leaves, seedlings of uniform growth were selected in a clean bench. True leaves were excised and plated on MS, MS + 0.2 mg / L IAA, or MS + 0.5 mg / L IAA medium. Fifteen leaves were selected for each gene and treatment, replicated three times. Wild-type Arabidopsis served as a control for each treatment. After inoculation, the seedlings were incubated in an incubator at 23°C, 24 h light intensity at 6000 lx, and 70% relative humidity. Leaf growth was observed and recorded 12 days after inoculation. The transgenic leaves exhibited higher rooting rates and root lengths than the wild-type leaves on all media. Wild-type leaves on media containing high IAA concentrations exhibited shriveled and yellowed growth.

[0076] Endogenous hormone content in Arabidopsis leaves was measured using an 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 hours and centrifuged at 4000 rpm for 15 minutes. 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 fractions were then dried under nitrogen and dissolved in phosphate-buffered saline (PBS) containing 0.1% (v / v) Tween 20 and 0.1% (w / v) gelatin for analysis. The 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 in 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 of 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 color development solution containing 1.5 mg / mL 0-phenylenediamine and 0.008% (v / v) H₂O₂ was added to each well. The reaction was stopped with 12 mol / L H₂SO₄ per well. The color was developed 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 contents of endogenous hormones IAA and GA in the leaves of transgenic plants were higher than those in the wild type, while the contents of ABA, MeJA and BR were lower.

[0077] In this example, a recombinant plasmid overexpressing the CgARF2 gene from Chunlan was introduced into the model plant Arabidopsis thaliana, and phenotypic observations and analyses were performed. The results indicate that, compared to wild-type plants, Arabidopsis plants overexpressing the CgARF2 gene exhibited phenotypes such as an increased number of rosette leaves, smaller leaves, and curled leaf margins. Detached leaves exhibited increased rooting rates and root length on MS medium containing IAA. The leaf contents of the endogenous hormones IAA and GA increased, while the contents of ABA, MeJA, and BR decreased. This indicates that the gene affects both leaf growth and development and hormone levels.

[0078] SEQ ID NO.1:

[0079]

[0080] SEQ ID NO.2:

[0081] MFLGLEIDGRVEHLRRMVGDALYEELWRACAGPLVEIPRVGERVYYFSQGHMEQLEASTNQEVDQRIPLFNLPCGRSKILCRVLNVELRAEHETDEVYAQITLLPDADQSELNTPDPCLPEPKRPVVNSFSKILTASDTSTHGGFSVLRRHANECLPPLDMLQPTPTQELCAKDLHGYEWRFKHIFRGQPRRHLLTTGWSTFVTSKRLVAGDAFVFLRGENGELRVGVKRASRQQSTIPSSVISCQSMHLGVLATASHAVTTQTLFTVCYKPRVSQFIVVVNKYLEAINNKFSVGMRFTMRLEGEDTPEKRYNGTIVGVGDLSSQWEGSKWRQLKVQWDEQSNILRPDRVSPWEIEPYNPLASTASVPQILSAKSKRPRPSSDLTDPLNSESSPPFWYSATTRTCDLGPLTTPVIWPPKPKADKGNTIMKASNFHSCDKWLKEHHSSFMSSTSSYNDASLKLFQDAALDNKVVVTNLHSTPGNIVEDPNSKASTGTDELKKSDCAYRLFGINLVNLINPSTSSIDKKPETAAMEQPQVQIITSVEESDQLSELSKTSKCSKQVVQVSPKEIQSRTSSSTRTRIKVHMHGVAVGRAVDLMNLGGYDDLISELEKMFEISGELCQREKWELVFTDDEGDMMLVGDDPWLEFCDMVRKIYIYPSEEVKKLNPRSKLPDPPPNNLKINQEREITLPPLFSRTGI-。

Claims

1. Application of Chunlan CgARF2 in promoting rooting of detached leaves of Arabidopsis thaliana "Columbia", characterized in that: The nucleotide sequence of CgARF2 is shown in SEQ ID NO. 1; the application includes: connecting the Chunlan CgARF2 gene to a vector, transforming it into wild-type Arabidopsis thaliana "Columbia" through Agrobacterium-mediated transformation, screening, culturing, and obtaining transgenic plants.

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