A CgARF4 gene of Cymbidium orchid and its application

Abstract

The invention discloses a spring orchid CgARF4 gene and its application. The nucleotide sequence of the CgARF4 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 invention clones the gene sequence of CgARF4 from the spring orchid cultivar "Songmei", performs expression analysis in the spring orchid, then constructs it into an overexpression vector and introduces it into a target plant to verify its function. It is found that the Arabidopsis thaliana overexpressing the CgARF4 gene has an early flowering period, but the fruit is partially sterile, and the fruit setting rate is reduced compared with the wild-type plant. The rooting rate of the detached leaves on the MS medium containing IAA is significantly improved, the root length is increased, the content of endogenous hormones IAA, GA, and ABA in the leaves is reduced, and the content of MeJA and BR is increased. It can be seen that the gene will have a wide range of uses in the cultivation and genetic improvement of orchids and other horticultural plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a Cymbidium orchid CgARF4 gene and an application thereof. Background Art

[0002] Orchidaceae is one of the largest families of flowering plants, with more than 25,000 species worldwide, accounting for about 10% of all flowering plants. Cymbidium goeringii is a small-flowered terrestrial orchid in the genus Orchidaceae. It has a unique flower shape, elegant color, delicate fragrance, beautiful leaves, and high ornamental and economic value. It is a typical orchid with both leaf and flower art. However, it takes a long time for Cymbidium goeringii to bloom from seedlings. Wild Cymbidium goeringii seedlings need about ten years to bloom, and artificial tissue culture seedlings need at least three years to bloom under proper maintenance. In addition, the breeding cycle of Cymbidium goeringii is long and the fruit development is slow. Therefore, studying the molecular mechanism of gene action on plant growth and development is of great significance to the breeding, production and application of Cymbidium goeringii. The ARF gene family plays an important role in plant growth and development, and can provide an important basis for the genetic improvement of plants.

[0003] The auxin response factor (ARF) is a family of transcription factors that was discovered in 1997 and regulates the expression of auxin-responsive genes. It can regulate the expression of related genes by interacting with auxin response elements, thus participating in the growth and development of a variety of plants. In Arabidopsis, AtARF6 and AtARF8 can regulate the maturation of stamens and pistils. The pollen of the arf6 / arf8 double mutant cannot be released, the pistils are immature, and the fertility is reduced. Okushima found that in the early stage of flower formation, compared with the wild type, the flowers of the arf2 mutant had significantly elongated pistils and sepals, and the flowers were sterile. However, as the plant grew, the ratio of fertile flowers to sterile flowers increased; and when entering the flower organ abscission stage, AtARF2 was found to promote stamen development and thus delay the abscission of flower organs. Other studies have shown that the AtARF7 and AtARF19 genes can enhance the phenotype of arf2 in flower organ abscission. The above studies have proved that ARF transcription factors can regulate flower development in other plant species. In tomatoes, SlARF6 and SlARF8 can regulate the formation of floral organs of plants; the study also found that SlARF2 is involved in regulating the aging process of floral organs of tomatoes. In Dendrobium officinale, the relative expression levels of some DnARFs in flowers are significantly higher than in other organs, indicating that these DnARFs are involved in regulating the growth and development of floral organs; in Prunus armeniaca, PmARF is not only involved in the morphological construction of floral organs, but also plays an important regulatory role in the development of its pistil. The above studies have proved that ARF transcription factors can regulate the flower development process in other plant species. Therefore, using genetic engineering technology to transfer the CgARF4 gene cloned from Chunlan 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 the existing breeding technology, the object of the present invention is to provide a CgARF4 gene of Cymbidium orchid. Another object of the present invention is to provide the application of the CgARF4 gene of Cymbidium orchid in plant breeding.

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

[0006] A Cymbidium orchid CgARF4 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 CgARF4 gene is shown in SEQ ID NO.2.

[0008] The application of the Chunlan CARF4 gene in plant growth and development, namely, the application of Chunlan CgARF4 in changing the flower development of Arabidopsis thaliana "Columbia", in promoting the rooting of Arabidopsis thaliana "Columbia" detached leaves, and in changing the endogenous hormone content of Arabidopsis thaliana "Columbia" leaves.

[0009] The Chunlan CgARF4 gene is connected to a vector, transformed into wild-type Arabidopsis thaliana "Columbia" through Agrobacterium-mediated transformation, and screened and cultured to obtain transgenic plants.

[0010] Beneficial effects: Compared with the prior art, the present invention verifies the function of the CgARF4 gene of spring orchid by cloning and identifying it, analyzing the expression of the gene, and genetic transformation. It is found that compared with the wild type, the Arabidopsis seedlings overexpressing the CgARF4 gene have an earlier flowering period, some flowers are sterile, and the fruit setting rate is reduced; however, the rooting rate of detached leaves is increased, and the contents of various endogenous hormones are changed. It can be seen that this gene will have a wide range of uses in the growth and development of orchids and the production and breeding of other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figure A is the electrophoresis of the cloned CgARF4 gene of Chunlan, where M is DL2000 Marker and the target band length is 1179bp; Figure B is the electrophoresis of the double enzyme digestion verification of the CgARF4 overexpression vector;

[0012] Figure 2 Figure A shows the expression of CgARF4 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 CgARF4 gene in Cymbidium orchid under IAA treatment;

[0013] Figure 3 This is a schematic diagram of the overexpression vector structure of the cloning and construction of the Chunlan CgARF4 gene;

[0014] Figure 4 Figure A is the PCR result of transgenic Arabidopsis plants, where M represents DL2000 Marker, 1 uses wild-type DNA as template as negative control, and 2-9 uses transgenic plant DNA as template;

[0015] Figure 5 Figure A is a comparison of flowering time between plants overexpressing the CgARF4 gene and wild-type Arabidopsis plants; Figure B shows the phenomenon of partial flower sterility in transgenic plants;

[0016] Figure 6 The figure shows the rooting of detached leaves of plants overexpressing the CgARF4 gene and wild-type Arabidopsis plants on MS medium containing different concentrations of IAA.

[0017] Figure 7 Comparison of endogenous hormone content in leaves of plants overexpressing CgARF4 gene and wild-type Arabidopsis plants. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific embodiments.

[0019] Example 1

[0020] The materials used in this example are various tissues of Cymbidium 'Songmei', which were quickly frozen in liquid nitrogen after being harvested and stored in an ultra-low temperature refrigerator (-80°C).

[0021] 1) Extraction of total RNA from various tissues of Cymbidium orchid

[0022] Follow the instructions of the TaKaRa Plant Total RNA Extraction Kit. The specific steps are as follows:

[0023] Quickly transfer the ultra-low temperature frozen Chunlan tissues to a mortar pre-cooled with liquid nitrogen, and grind the tissues with a pestle, adding liquid nitrogen continuously until they are ground into powder; add the ground samples into 1.5 mL sterile tubes containing 450 μl Buffer PE, and repeatedly blow with a 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℃ for 5 minutes; carefully pipette the supernatant into a new 1.5 mL sterile tube, add 450 μL of Buffer RL, and use a pipette to mix the solution evenly; add 1 / 2 volume of anhydrous ethanol to the mixture, use a pipette to mix the solution evenly, and immediately transfer all the mixture to the RNA Spin Column; centrifuge at 12,000 rpm for 1 minute, discard the filtrate, and put the RNA Spin Column back into the 2 ml CollectionTube; add 500 μL of Buffer RWA to the RNA Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate; add 600uL of Buffer RWB to the RNA Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate; add 50 μL DNase I to the center of the RNA Spin Column membrane The reaction solution 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, centrifuged at 12,000 rpm for 30 seconds, and the filtrate was discarded; the RNA Spin Column was re-placed on a 2 mL Collection Tube, and centrifuged at 12,000 rpm for 2 minutes; the RNA Spin Column was placed on 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, allowed to stand at room temperature for 5 minutes, and centrifuged at 12,000 rpm for 2 minutes to elute the RNA. The obtained RNA was tested for concentration and purity and stored in a -80°C refrigerator for future use.

[0024] 2 μL RNA was taken 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. The OD260 / OD280 and OD260 / OD230 were both between 1.8 and 2.1, indicating good integrity and could be used for reverse transcription.

[0025] 2) Synthesis of first-strand cDNA

[0026] The total RNA obtained was used as a template and reverse transcription was performed using the Tiangen Reverse Transcription Kit. The specific operation is as follows:

[0027] Thaw the template RNA on ice, thaw 5×Fastking-RT SuperMix and RNase-Free ddH2O at room temperature, and quickly place on ice after thawing. Prepare a mixed solution in a centrifuge tube with a total volume of 10uL, containing 5×Fastking-RTSuperMix 4uL, Total RNA 800ng, and make up the remaining volume with RNase-Free ddH2O. Centrifuge the centrifuge tube to make the mixed solution in the centrifuge tube sink to the bottom of the tube, shake it slowly, remove the genome and perform reverse transcription reaction on the PCR instrument at 42℃ for 15min, inactivate the enzyme at 95℃ for 3min, and place it on ice to obtain a cDNA solution.

[0028] 3) Design and cloning of target gene primers

[0029] According to the existing transcriptome sequencing results of Chunlan, CE Design was used to design the Chunlan CgARF4 gene amplification primers. The primer sequences are:

[0030] CgARF4-F: 5'-GAGAACACGGGGGACTCTAGAATGGGGATCGATCTGAACACG -3'

[0031] CgARF4-R: 5'-ATAAGGGACTGACCACCCGGGAAAGACTGTTGGGCAAGAGACTG-3'.

[0032] Using cDNA as template, Takara's PrimerStar Max high-fidelity enzyme was used to clone the CgARF4 gene of Chunlan. The PCR amplification system (50μL) was: 25μlL PrimerStar Max, 2μL Forward Primer, 2μL ReversePrimer, 2μL Template cDNA, 19μL ddH2O. The PCR program was: reaction conditions were 94℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 60℃ annealing for 15s, 72℃ extension for 30s, 32 cycles, 72℃ total extension for 5min, and 16℃ insulation.

[0033] After the PCR reaction is completed, all PCR products are taken for detection and cutting of the target fragments through 1.8% agarose gel electrophoresis, and the target PCR amplification products are purified by gel recovery. The DNA gel recovery kit of TransGen is used to purify and recover the target fragments. 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 volumes of GSB solution to the gel block (if the gel is 0.1g, its volume can be regarded as 100μL, then add 300μL GSB solution), place it in a 55℃ water bath, and constantly and gently turn the centrifuge tube up and down until the gel block is completely dissolved; cool the melted gel solution to room temperature, add 1 volume of isopropanol (if the gel is 0.1g, then add 100μL isopropanol), gently mix; add the mixture to the centrifuge column, place at room temperature for 1 minute, centrifuge at 12000rpm for 1 minute, discard the effluent, and then put the centrifuge column back into the collection tube; add 650μL solution WB to the centrifuge column, centrifuge at 12000rpm for 1 minute, discard the effluent; centrifuge at 12000rpm for 2 minutes to remove as much residual WB as possible, place the adsorption column at room temperature with the cover open for 5 minutes, and dry it thoroughly; place the centrifuge column in a clean centrifuge tube, add 30μL ddH2O (ddH2O needs to be preheated in a 60~70℃ water bath in advance) to the middle of the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12000rpm for 2 minutes to collect the DNA solution. Take 2μL to recover the purified product, use 1.5% agarose for gel electrophoresis detection, and place the rest in a -20℃ refrigerator, which will be used to connect with the pBI121 vector to construct an overexpression vector.

[0034] 3) Plasmid extraction:

[0035] Extract the plasmid according to the instructions of Tiangen Plasmid Mini-Extraction Kit. The specific steps are as follows:

[0036] 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 vortexer 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 white flocculent precipitate appears, centrifuge it at 12000rpm for 10min. Take 500μL of balance solution BL and add it to adsorption column CP4, centrifuge it at 12000rpm for 1min, discard the waste liquid in the collection tube, put the adsorption column back to 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, and put the adsorption column CP4 back to 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, and put the adsorption column CP4 back to the collection tube. Take 600μl Add the rinse solution PW (containing anhydrous ethanol) to the adsorption column CP4, centrifuge at 12000rpm for 1min, discard the waste liquid in the collection tube, put the adsorption column CP4 back into the collection tube, centrifuge at 12000rpm for 2min, remove the residual rinse solution in the adsorption column; move the adsorption column CP4 to a new 1.5ml centrifuge tube, add 60μL ddH2O to the middle of the adsorption membrane; let it stand at room temperature for 2min, centrifuge at 12000rpm for 1min, and the solution collected in the centrifuge tube is the plasmid. Finally, measure the plasmid concentration to prepare for the next experiment.

[0037] 4) Double enzyme digestion reaction

[0038] The extracted pBI121 plasmid was digested with XbaI and SmaI at 37°C for 30 min, and the linear vector was recovered by electrophoresis and stored at -20°C for later use. The double enzyme digestion reaction system was 50 μL: 20 μL pBI121 plasmid, 5 μL 5× buffer, 1 μL XbaI, 1 μL SmaI, and 23 μL ddH2O.

[0039] 5) Recombination reaction

[0040] Agarose gel electrophoresis was used to detect the target gene and vector pBI121 recovered after enzyme digestion. According to the detected purity and concentration, each reagent was added according to the ligation system. Among them, the number of target fragment molecules: the number of vector molecules = 3:1~5:1, and the ligation reaction system was: 7μL of linearized pBI121 vector, 3μL of inserted fragment, 4μl of 5×CE II buffer, 2μl of Exnase II, and 20μL of ddH2O. React at 37℃ for 30min, place at room temperature (do not cool immediately), and transform into E. coli competent state after 10min.

[0041] 6) Transform the ligation product into E. coli

[0042] Take out the competent cell Trans5α strain from the ultra-low temperature refrigerator and place it on ice to melt. Pipette 10μL of the recombinant product and add it to 100μL of competent cells; place the centrifuge tube on ice for 10 min; heat shock in a 42℃ water bath for 90 s without shaking during the period; immediately place it on ice for 2 min; add 500μL of antibiotic-free liquid culture medium in the clean bench, shake at 37℃ and 200 rpm for 25min to revive; centrifuge at 6000 rpm for 1 min, and remove 350μL of supernatant; resuspend the precipitated bacteria, spread on LB plates (Kana concentration is 50 mg / L), and culture at 37℃ overnight.

[0043] 7) Identification of recombinants

[0044] Pick a single colony from the plate and inoculate it into LB liquid medium containing antibiotics (Kana), and culture it overnight at 37°C and 200 rpm. Use the full-length primers of the target gene to perform PCR on the bacterial liquid to screen 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.

[0045] According to the analysis of the sequencing results, it was finally determined that a CgARF encoding gene of Cymbidium orchid was cloned and named CgARF4 gene, whose nucleotide sequence is shown in SEQ ID NO.1. The coding length of CgARF4 gene is 1179bp, containing ATG start codon and TAG stop codon, of which the full length of ORF is 1179bp, encoding 392 amino acids, and the amino acid sequence is shown in SEQID NO.2.

[0046] Example 2

[0047] Fluorescence quantitative primers were designed based on the cloned Chunlan CgARF4 gene. The primer sequences are as follows:

[0048] qCgARF4-F: 5'-ATGCTGCTGCGTTACGATGT-3'

[0049] qCgARF4-R: 5'-GCTATCCATCTCATCCACTCCT-3'

[0050] At the same time, 18S was used as the internal reference gene, and the primer sequences were:

[0051] 18S-F: 5'-GGTCCTATTGTGTTGGCT-3'

[0052] 18S-R: 5'-TCGCAGTGGTTCGTCTTT-3'

[0053] The reaction solution was prepared using the instructions of the ChamQ™ Universal SYBR QPCR Master Mix Kit (Vazyme), and the PCR program was run on an Applied Biosystems real-time fluorescence quantitative analyzer: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, 40 cycles; 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. After the reaction was completed, the amplification curve was obtained, and the data was exported using StepOne Software v2.3. The data was analyzed using Excel, and the relative expression was calculated using the 2-ΔΔCt relative quantitative method based on the CT value. The data analysis results are shown in the figure. Figure 2 shown.

[0054] The results of this study show that the CgARF4 gene is expressed in all tissues of Cymbidium orchid ( Figure 2 A), but the gene was highly expressed in the roots of spring orchids, followed by leaves and flowers, indicating that the gene was active in these three organs; expression analysis of leaves of spring orchids treated with IAA hormone spraying proved that the CgARF4 gene played an important role in the response of spring orchid leaves to IAA treatment ( Figure 2 B).

[0055] Example 3

[0056] 1) Preparation and transformation of Agrobacterium competent cells

[0057] In this embodiment, Agrobacterium GV3101 was used to prepare Agrobacterium competent cells and conduct infection experiments on Arabidopsis thaliana. The process of preparing Agrobacterium competent cells is as follows: a single colony of Agrobacterium that has been activated was picked up and inoculated into 5 mL of liquid LB medium, and cultured at 28°C and 250 rpm for 20-24 h; 2 mL of bacterial solution was aspirated and inoculated into a triangular flask containing 50 mL of liquid LB medium, and cultured at 28°C and 250 rpm until the OD600 value was about 0.8; the expanded bacterial solution was placed on ice for 30 min, centrifuged at 4°C and 5000 rpm for 5 min, and the supernatant was discarded; 10 mL of pre-cooled 0.1 mol / L CaCl2 solution was added to fully suspend the precipitated bacteria; centrifuged at 4°C and 5000 rpm for 5 min, and the supernatant was discarded; 1 mL of pre-cooled 20 mmol / L The CaCl2 solution was used to fully suspend the bacterial cells to obtain the GV3101 competent cells to be prepared. The cells were divided into 100 µL / tube using centrifuge tubes, 20% sterile glycerol was quickly added, and the cells were stored at -80°C.

[0058] Agrobacterium transformation of recombinants: Place in an ice bath to thaw the competent Agrobacterium cells, add 600 ng of the recovered and purified plasmid to 100 μl of the competent Agrobacterium cells, mix gently, and place in an ice bath for 5 min; use liquid nitrogen to quickly freeze 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 fully mix the remaining bacterial liquid, and then spread it on a solid LB medium supplemented with 50 mg / L kanamycin and 200 mg / L rifampicin; incubate at 28°C for 30-48 h.

[0059] Identification of Agrobacterium recombinants: Pick a single colony grown on the plate culture medium and inoculate it into a liquid culture medium containing the corresponding antibiotics; culture it overnight at 28°C, 200 rpm, and then perform PCR on the bacterial solution. Detect the PCR product by 1.5% agarose gel electrophoresis to determine whether it contains the target fragment. Add an appropriate amount of sterile 50% glycerol to the positive clones after identification and store them at -80°C for later use.

[0060] 2) Agrobacterium-mediated transformation of Arabidopsis thaliana

[0061] The inflorescence infection method was used to transfer the target gene into Arabidopsis thaliana. The specific operation method was as follows: Arabidopsis thaliana (Columbia type) was kept in a healthy growth state until flowering; and the Agrobacterium GV3101 strain carrying the target gene was activated. Pick a single colony and inoculate it into 5mL LB culture medium containing kanamycin and rifampicin, shake the culture at 28℃ and 200 rpm until the culture solution just becomes turbid, about 8-10 hours; take 1mL of the culture solution, inoculate it into a triangular flask (50mL) and shake it for 24 hours until the OD value is about 0.8; centrifuge the culture solution at 6000rpm for 5 minutes at room temperature, remove the supernatant and collect the bacteria, and suspend them in a 3% sucrose solution with a pH of 5.8; before soaking, add SilwetL-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 period; 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; uncover the tin foil, culture under normal conditions, and stop watering when the seeds are mature.

[0062] The components of the 3% sucrose solution resuspension are as follows: MS medium, sucrose 30g / L, Silwet-77 300µl / L. (Note: pH is adjusted to 5.8 after preparation, and SilwetL-77 is added after the bacterial solution is centrifuged and resuspended; the conversion relationship between the resuspension and the bacterial solution is: the amount of resuspension: bacterial solution OD* bacterial solution volume = 0.8* resuspension).

[0063] 3) Screening of transgenic plants

[0064] The collected T1 transgenic Arabidopsis seeds were sterilized with alcohol and sodium hypochlorite, and the steps were 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 to 6 times, 2 min each time; rinsed with sterile water 3 to 4 times; and suspended in 0.1% agarose solution.

[0065] The sterilized transgenic Arabidopsis seeds were sown on MS solid medium containing antibiotics (kanamycin 50 mg / L and cephalosporin 100 mg / L), wrapped in tin foil and placed in a 4°C refrigerator for vernalization. After 2 days, the seeds were taken out of the refrigerator and the medium was placed at 22°C under light. After about a week, the Arabidopsis that could grow normally on the medium were transplanted into the soil and continued to grow.

[0066] 4) DNA detection of transgenic plants

[0067] Take appropriate amount of young leaves of T1 generation Arabidopsis and transgenic plants, and use Qingke's plant DNA direct amplification kit for detection. The specific operation steps are as follows: cut appropriate amount of young leaves into pieces and place them in a sterilized 2mL centrifuge tube, add 50uL Lysis Buffer A solution, heat and lyse at 95℃ for 10 min, and continue to lyse at 4℃ overnight. Centrifuge at 14000 rpm for 3 min the next day, take the supernatant and transfer it to a new sterile centrifuge tube as a PCR reaction template. Use 2×T5 Direct PCR Mix and gene-specific primers for PCR detection, the results are as follows Figure 4 As shown in A.

[0068] 5) Fluorescence quantitative PCR detection of transgenic plants

[0069] Total RNA was extracted from the young stem leaves of the six Arabidopsis strains overexpressing the CgARF4 gene of C. cylindrica. 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.

[0070] 6) Obtaining homozygous transgenic lines

[0071] 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; the transgenic plants preliminarily confirmed were retained after testing, and the T1 generation seeds were harvested after maturity, numbered, and the T2 generation was obtained; like the T1 generation, the T2 generation seeds were sterilized and spread on the screening medium containing antibiotics, and placed at 22°C and continuously illuminated; about 10 days, the survival rate of 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 continued to be numbered, and the seeds were collected to obtain T3 generation seeds; after the seeds were sterilized, they were screened with screening medium and placed under light for continuous light culture; about 10 days, the T3 generation plants with different numbers were observed, and all the plants that survived and did not separate were T3 generation homozygous plants.

[0072] 7) Phenotypic observation

[0073] The transgenic strains with obvious phenotypes were selected for observation. The results showed that compared with the wild-type Arabidopsis, the flowering time of the transgenic Arabidopsis seedlings was significantly earlier than that of the wild-type, and there was a phenomenon of premature stem emergence. In addition, the growth of the plants significantly weakened when entering the reproductive stage, and some flowers became sterile.

[0074] After sterilization, wild-type and transgenic seeds were sown on MS medium. After 4-6 true leaves were cultivated, seedlings with consistent growth were selected in the clean bench, and the true leaves were cut off and placed on MS, MS+0.2mg / L IAA, and MS+0.5mg / LIAA medium, respectively. 15 leaves were selected for each gene and each treatment, and repeated 3 times. Wild-type Arabidopsis was used as a control in each treatment. After inoculation, they were placed in an incubator under the conditions of 23°C, 24h light, 6000lx light intensity, and 70% relative humidity. On the 12th day of inoculation, the growth changes of leaves were observed and recorded, and it was found that the rooting rate and root length of transgenic leaves on each culture medium were higher than those of wild type.

[0075] Enzyme-linked immunosorbent assay (ELISA) was used to determine the endogenous hormone content in Arabidopsis leaves. 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 with 80% (v / v) methanol, 100% (w / v) methanol, 100% (w / v) ether, and 100% (w / v) methanol in sequence. The hormone fractions were 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. 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 was performed using a 96-well microtiter plate. 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, incubated and washed as 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 in each well. The color was developed using an ELISA instrument (model EL310, Bio-TEK, Winooski, VT) at 490 nm. The hormone content was calculated according to Weiler et al. (1981). Three biological replicates were set for each hormone. The results showed that the contents of endogenous hormones IAA, GA, and ABA in the leaves of transgenic plants were lower than those of the wild type, while the contents of MeJA and BR were higher.

[0076] In this example, the recombinant plasmid of the overexpressed CgARF4 gene of Chunlan was transferred into the model plant Arabidopsis, and phenotypic observation and analysis were performed. From the results, it can be seen that the Arabidopsis plants overexpressing the CgARF4 gene have an earlier flowering time and decreased fertility compared with wild-type plant seedlings, while the rooting rate and root length of the detached leaves on the MS medium containing IAA are improved, the content of endogenous hormones IAA, GA, and ABA in the leaves is reduced, and the content of MeJA and BR is increased. It can be seen that the gene has an effect on the flower development, adventitious root formation and hormone level changes of the plant.

Claims

1. Application of Chunlan CgARF4 in changing the flower development of Arabidopsis thaliana "Columbia", characterized in that: The CgARF4 nucleotide sequence is shown in SEQ ID NO. 1; the application includes: connecting the Chunlan CgARF4 gene to a vector, transforming it into wild-type Arabidopsis thaliana "Columbia" through Agrobacterium-mediated transformation, screening, culturing, and obtaining transgenic plants.

2. Application of Chunlan CgARF4 in promoting rooting of Arabidopsis thaliana "Columbia" detached leaves, characterized in that: The CgARF4 nucleotide sequence is shown in SEQ ID NO. 1; the application includes: connecting the Chunlan CgARF4 gene to a vector, transforming it into wild-type Arabidopsis thaliana "Columbia" through Agrobacterium-mediated transformation, screening, culturing, and obtaining transgenic plants.

3. Application of Chunlan CgARF4 in reducing the content of endogenous hormones IAA, GA, ABA or increasing the content of MeJA and BR in leaves of Arabidopsis thaliana "Columbia", characterized in that: The nucleotide sequence of the CgARF4 is shown in SEQ ID NO. 1; the application includes: connecting the Chunlan CgARF4 gene to a vector, transforming it into the wild-type Arabidopsis thaliana "Columbia" through Agrobacterium-mediated transformation, screening, culturing, and obtaining transgenic plants.

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