CN116064564A - Cymbidium CgARF4 gene and application thereof - Google Patents

Abstract

The invention discloses a cymbidium CgARF4 gene and application thereof, wherein the nucleotide sequence of the CgARF4 gene is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2. The invention clones and obtains the gene sequence of CgARF4 from the spring orchid cultivar Song Mei, carries out expression analysis in the spring orchid, then constructs the gene sequence into an over-expression vector to be introduced into target plants to verify the functions, discovers that the flowering phase of the arabidopsis thaliana over-expressing the CgARF4 gene is advanced, but the fruit part is sterile, the fruiting rate is reduced compared with the wild plant, the rooting rate of in vitro leaves is obviously improved on an MS culture medium containing IAA, the root length is increased, the endogenous hormone IAA, GA, ABA content in the leaves is reduced, the MeJA and BR content is increased, and the gene can be widely applied to the cultivation and genetic improvement of orchid and other horticultural plants.

Description

Cymbidium CgARF4 gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a cymbidium CgARF4 gene and application thereof.

Background

Orchid (Orchidaceae) is one of the largest families in flowering plants, and 25000 species worldwide account for about 10% of all flowering plants. The cymbidium (Cymbidium goeringii) belongs to the small-flower type ground orchid category in the orchid genus of the orchid family, has peculiar flower type, elegant flower color, delicate fragrance, she Zi, elegant appearance, extremely high ornamental value and economic value, and is a typical leaf art and flower art two-whole orchid. However, the time required from seedling to flowering of the cymbidium goeringii is long, the seedlings of the wild cymbidium goeringii can bloom about ten years, and the artificial tissue culture seedlings can bloom at least three years under the proper maintenance condition. And the spring orchid has long breeding period and slow fruit development. Therefore, research on the molecular mechanism of genes acting on plant growth and development has important significance for breeding, production and application of cymbidium goeringii. The ARF gene family plays an important role in plant growth and development, and can provide important basis for genetic improvement of plants.

Auxin response factors (auxin response factor, ARF) are a family of transcription factors found in 1997 that regulate the expression of auxin response genes, which can be involved in the growth and development of a variety of plants by interacting with auxin response elements to regulate the expression of the associated genes. In Arabidopsis, atARF6 and AtARF8 can regulate the maturation of stamens and pistils, pollen of arf6/arf8 double mutants cannot be released, pistils are immature, and fertility is reduced. Okushima found that flowers of the arf2 mutant had significantly elongated pistils and sepals at early stage of flower formation, and flowers appeared sterile, but the ratio of fertile/sterile flowers increased as plants grew; and entering the flower organ abscission stage, atARF2 is found to promote stamen development, thereby delaying flower organ abscission. Another study indicated that the AtARF7 and AtARF19 genes enhanced the phenotype of arf2 in flower organ abscission. All the above studies demonstrate that ARF transcription factors can regulate flower development in other plant species. In tomato, slARF6 and SlARF8 can regulate the formation of plant flower organs; studies have also found that SlARF2 is involved in regulating the senescence process in tomato flower organs. In dendrobium candidum, the relative expression quantity of part of DnARF in flowers is obviously higher than that of other organs, which indicates that the DnARF participates in regulating and controlling the growth and development of flower organs; in fruit Mei Zhong, pmARF is not only involved in the morphogenesis of floral organs, but also plays an important regulatory role in the development of pistil thereof. All the above studies demonstrate that ARF transcription factors can regulate floral development in other plant species. Therefore, the CgARF4 gene cloned from the cymbidium is transferred into the mode plant by utilizing the genetic engineering technology, which has important significance for researching the functions of the plant and has great application prospect.

Disclosure of Invention

Aiming at the defects existing in the prior art of breeding, the invention aims to provide a cymbidium CgARF4 gene. It is another object of the present invention to provide the use of the cymbidium CgARF4 gene in plant breeding.

In order to achieve the above object, the present invention adopts the following technical scheme:

a nucleotide sequence of the cymbidium CgARF4 gene is shown in SEQ ID NO. 1.

The amino acid sequence of the expressed protein of the cymbidium CgARF4 gene is shown as SEQ ID NO. 2.

The application of the spring orchid CARF4 gene in plant growth and development, namely the application of the spring orchid CgARF4 in changing the development of the arabidopsis thaliana 'Columbia' flower, the application in promoting the rooting of the arabidopsis thaliana 'Columbia' leaves in vitro and the application in changing the endogenous hormone content of the arabidopsis thaliana 'Columbia' leaves.

The cymbidium CgARF4 gene is connected to a vector, and is transformed into wild arabidopsis thaliana 'Columbia' through agrobacterium mediation, and is screened and cultured to obtain a transgenic plant.

The beneficial effects are that: compared with the prior art, the invention verifies the functions of the arabidopsis seedlings through cloning and identifying the cymbidium CgARF4 gene, analyzing the gene expression and carrying out genetic transformation, and discovers that compared with the wild type arabidopsis seedlings over-expressing the CgARF4 gene, the flowering phase is advanced, partial flowers are sterile, and the fruit setting rate is reduced; but the rooting rate of the in-vitro leaves is increased, and the content of each endogenous hormone is changed, so that the gene can be widely applied to the growth and development of orchid and the production and breeding of other plants.

Drawings

FIG. 1A is a diagram showing the bacterial detection electrophoresis of the cloning of the CgARF4 gene of the spring orchid, wherein M is DL2000 Marker, and the length of a target band is 1179bp; panel B is a CgARF4 over-expression vector double-enzyme digestion verification electrophoresis chart;

FIG. 2A is a graph showing the expression of CgARF4 in tissues of cymbidium, where R represents roots, S represents pseudobulbs, L represents leaves, and F represents flowers; panel B shows the expression of the CgARF4 gene of cymbidium goeringii under IAA treatment;

FIG. 3 is a schematic diagram of the structure of an overexpression vector cloned and constructed from the CgARF4 gene of cymbidium;

FIG. 4 is a graph A showing the PCR results of transgenic Arabidopsis plants, wherein M represents DL2000 Marker,1 uses wild type DNA as a negative control, and 2-9 uses transgenic plant DNA templates;

FIG. 5A is a graph comparing flowering time of plants overexpressing the CgARF4 gene with wild type Arabidopsis plants; panel B shows the phenomenon of partial flower sterility of transgenic plants;

FIG. 6 is rooting of in vitro leaves of plants overexpressing the CgARF4 gene and wild type Arabidopsis plants on MS medium containing different concentrations of IAA;

FIG. 7 is a comparison of endogenous hormone levels in leaves of an overexpressed CgARF4 gene plant versus a wild type Arabidopsis plant.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Example 1

The materials used in this example were the tissue of cymbidium Song Mei' and were snap frozen in liquid nitrogen and stored in an ultra low temperature refrigerator (-80 ℃).

1) Extraction of total RNA from tissues of cymbidium goeringii

The method is carried out according to the specification of a TaKaRa plant total RNA extraction kit, and comprises the following specific operations:

rapidly transferring the tissue of the ultra-low temperature frozen cymbidium goeringii into a mortar precooled by liquid nitrogen, grinding the tissue by a pestle, and continuously adding the liquid nitrogen until the tissue is respectively ground into powder; adding the ground powder samples into 1.5mL sterilized tubes containing 450 μl Buffer PE respectively, and repeatedly blowing with a pipette until no obvious precipitate exists in the lysate; the lysate was centrifuged at 12,000 rpm at 4℃for 5 minutes; the supernatant was carefully aspirated into a fresh 1.5. 1.5mL sterilization tube. Adding Buffer NB with the volume of 1/10 of the supernatant, shaking and mixing uniformly by Vortex, centrifuging at 12,000 rpm and 4 ℃ for 5 minutes; carefully aspirate the supernatant to a new 1.5. 1.5mIn the L sterilization tube, 450 mu L of Buffer RL is added, and a pipetting gun is used for uniformly mixing the solution; adding 1/2 volume of absolute ethyl alcohol into the mixed solution, uniformly mixing the solution by using a pipetting gun, and immediately transferring the mixed solution into the RNA Spin Column; centrifugation at 12,000 rpm for 1min, discarding the filtrate and returning RNA Spin Column to 2ml Collection Tube; 500. Mu.L of Buffer RWA was added to RNA Spin Column, centrifuged at 12,000 rpm for 30 seconds, and the filtrate was discarded; 600uL of Buffer RWB was added to RNA Spin Column, centrifuged at 12,000 rpm for 30 seconds, and the filtrate was discarded; 50 mu L of DNase I reaction solution is added to the center of the RNA Spin Column membrane, and the mixture is kept stand for 15 minutes at room temperature; 350. Mu.L 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; RNA Spin Column was relocated to 2mL Collection Tube, centrifuged at 12,000 rpm for 2 minutes; RNA Spin Column was mounted on RNase Free Collection Tube of 1.5. 1.5mL, and 30. Mu.L of RNase Free dH was added to the center of the RNA Spin Column membrane ₂ O was allowed to stand at room temperature for 5 minutes, and the RNA was eluted by centrifugation at 12,000 rpm for 2 minutes. The obtained RNA is stored in a refrigerator at-80 ℃ for standby after concentration and purity detection.

The result of sucking 2 mu L of RNA and detecting by using 1% agarose gel electrophoresis shows that 28S and 18S bands are clear, the brightness of the 28S band is about twice of that of the 18S band, and the quality of the RNA is good. RNA purity, OD, was measured by a trace accounting protein meter ₂₆₀ /OD ₂₈₀ And OD (optical density) ₂₆₀ /OD ₂₃₀ The whole cell is between 1.8 and 2.1, has better integrity and can be used for reverse transcription.

2) Synthesis of first strand cDNA

The total RNA obtained was used as a template, and reverse transcription was performed using a Tiangen reverse transcription kit. The specific operation is as follows:

thawing template RNA on ice, 5 XFastking-RT Supermix and RNase-Free ddH ₂ O thawed at room temperature and rapidly placed on ice after thawing. Preparing the mixture in a centrifuge tube to give a total of 10uL containing 5 XFastking-RT Supermix 4uL,Total RNA 800ng, and adding RNase-Free ddH to the remaining volume ₂ And O is complemented. Centrifuging the centrifuge tube to make the mixed solution in the centrifuge tube sink to the bottom of the tube, slowly shaking, removing genome on PCR instrument at 42deg.C for 15min, and performingReverse transcription reaction, inactivating enzyme at 95deg.C for 3min, and standing on ice to obtain cDNA solution.

3) Design and cloning of target gene primer

According to the existing sequencing result of the cymbidium transcriptome, a CE Design is used for designing a cymbidium CgARF4 gene amplification primer, and the primer sequence is as follows:

CgARF4-F: 5’- GAGAACACGGGGGACTCTAGAATGGGGATCGATCTGAACACG -3’

CgARF4-R: 5’- ATAAGGGACTGACCACCCGGGAAAGACTGTTGGGCAAGAGACTG-3’。

cloning of the cymbidium CgARF4 gene was performed using the primestar Max high fidelity enzyme of Takara using cDNA as a template. The PCR amplification system (50. Mu.L) was: 25 μ lL PrimerStar Max,2 μL Forward Primer,2 μL Reverse Primer,2 μL Template cDNA,19 μL ddH ₂ O. The PCR procedure was: the reaction conditions were 94℃for 3min,98℃for 10s,60℃for 15s,72℃for 30s,32 cycles, 72℃for 5min total extension, and 16℃for incubation.

After the PCR reaction is completed, all PCR products are taken and detected by 1.8% agarose gel electrophoresis (the PCR amplification result is shown in FIG. 3A) and target fragments are cut, and the PCR target amplification products are recovered and purified by gel. The DNA gel recovery kit of TransGen company is adopted to purify and recover the target fragment, and the specific operation is as follows: cutting a single target strip from agarose gel, putting the cut single target strip into a clean centrifuge tube, and weighing; adding 3 times volume of solution GSB (if the gel is 0.1g, the volume can be regarded as 100 mu L, 300 mu L of GSB solution) into the gel block, placing in a water bath at 55 ℃, and continuously gently turning the centrifuge tube up and down until the gel block is completely dissolved; the melted gel solution was cooled to room temperature, 1 volume of isopropanol (100 μl isopropanol if the gel was 0.1 g) was added, and gently mixed; adding the mixed solution into a centrifugal column, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, discarding effluent, and then placing the centrifugal column into a collecting pipe; adding 650 mu L of solution WB into the centrifugal column, centrifuging at 12000rpm for 1min, and discarding effluent; centrifuging at 12000rpm for 2min, removing residual WB as far as possible, standing the adsorption column at room temperature for 5min under cover opening, and completely air drying; placing the centrifugal column into a clean centrifuge tube, and suspending the centrifugal column to the middle position of the adsorption film30 mu L ddH was added dropwise ₂ O（ddH ₂ O is placed in a water bath at 60-70 ℃ in advance for preheating, and kept stand at room temperature for 2min and centrifuged at 12000rpm for 2min to collect DNA solution. Taking 2 mu L of recovered and purified product, performing gel electrophoresis detection by using 1.5% agarose, and placing the rest of the product in a refrigerator at the temperature of minus 20 ℃, and subsequently connecting the product with a pBI121 vector to construct an overexpression vector.

3) Extraction of plasmids:

extracting plasmids according to the specification of a small and medium-amount kit of the Tiangen plasmids, wherein the specific steps are as follows:

taking 10mL of fungus liquid cultured overnight, centrifuging at 12000rpm for 1min, and removing supernatant; adding 500 mu L P1 solution (containing RNase A) into a centrifuge tube with bacterial sediment, and thoroughly suspending the bacterial sediment by using a vortex instrument; adding 500 mu L P solution into a centrifuge tube, fully cracking the thalli when the thalli are gently turned upside down, adding 700 mu L P solution into the centrifuge tube, immediately and gently turning upside down, fully mixing, and centrifuging at 12000rpm for 10min after white flocculent precipitation occurs; adding 500 μl of balance liquid BL into the adsorption column CP4, centrifuging at 12000rpm for 1min, discarding the waste liquid in the collection tube, placing the adsorption column back into the collection tube, adding the collected supernatant into the filtration column CS in batches, centrifuging at 12000rpm for 2min, carefully adding the solution collected in the collection tube into the adsorption column CP4 in batches, centrifuging at 12000rpm for 1min, discarding the waste liquid in the collection tube, and placing the adsorption column CP4 back into the collection tube; adding 500 μl deproteinized solution PD into an adsorption column CP4, centrifuging at 12000rpm for 1min, discarding the waste liquid in the collection tube, and placing the adsorption column CP4 back into the collection tube; adding 600 μl of rinsing solution PW (containing absolute ethanol) into an adsorption column CP4, centrifuging at 12000rpm for 1min, discarding the waste liquid in the collection tube, placing the adsorption column CP4 into the collection tube, centrifuging at 12000rpm for 2min, and removing residual rinsing solution in the adsorption column; the column CP4 was transferred to a new 1.5ml centrifuge tube, and 60. Mu.L of ddH was added to the middle of the adsorption film ₂ O; standing for 2min at room temperature, and centrifuging at 12000rpm for 1min to obtain the plasmid as the solution. Finally, the plasmid concentration was determined and prepared for the next experiment.

4) Double enzyme digestion reaction

The extracted pBI121 plasmid is digested with XbaI and SmaI at 37deg.C for 30min, and the linear vector is recovered by electrophoresis, and maintained at-20deg.CAnd storing for standby. The double cleavage reaction system was 50. Mu.L: pBI121 plasmid 20. Mu.L, 5 Xbuffer 5. Mu.L, xbaI 1. Mu.L, smaI 1. Mu.L, ddH ₂ O 23μL。

5) Recombination reactions

The target gene and vector pBI121 recovered after digestion are detected by agarose gel electrophoresis, and each reagent is added according to the detected purity and concentration and a connection system. Wherein, the molecular number of the target fragment: carrier molecular number=3:1 to 5:1, and the connection reaction system is as follows: 7. Mu.L of linearized pBI121 vector, 3. Mu.L of insert, 4. Mu.L of 5 XCE II buffer, 2. Mu.L of Exnase II, ddH ₂ Up to 20. Mu.L. The reaction was carried out at 37℃for 30min, and the reaction was allowed to stand at room temperature (without immediately cooling) for 10min, after which the reaction was allowed to stand for E.coli.

6) Transfer of ligation products into E.coli

The competent cell Trans5α strain was taken out from the ultra-low temperature refrigerator and thawed on ice. mu.L of recombinant product was pipetted into 100. Mu.L of competent cells; placing the centrifuge tube on ice for 10min; water bath in a water bath kettle at 42 ℃ and heat shock 90 s, wherein the water bath kettle is not required to shake; immediately placing the mixture on ice for 2 min; adding 500 mu L of liquid culture medium without antibiotics into an ultra-clean bench, shaking at 37 ℃ and 200 rpm for 25min for resuscitation; 6000 Centrifuging at rpm for 1min, and sucking 350 μL of supernatant; the precipitated cells were resuspended, plated on LB plates (Kana 50 mg/L) and incubated overnight at 37 ℃.

7) Identification of recombinants

Single colonies on the plates were picked and inoculated into LB liquid medium containing antibiotics (Kana) and shake-cultured overnight at 37℃and 200 rpm. Bacterial liquid PCR is performed by using the full-length primers of the target genes to screen positive clones, and the bacterial detection result is shown in figure 1A. The positive clones after screening were sent to Nanjing Sipu gold company for sequencing. Positive clones with correct sequencing results are amplified, plasmids are extracted by using a root plasmid extraction kit and subjected to double enzyme digestion verification, whether the sizes of fragments after enzyme digestion are consistent is judged, and the enzyme digestion results are shown in figure 1B.

According to analysis of the sequencing result, the final cloning is carried out to obtain 1 cymbidium CgARF coding gene, which is named CgARF4 gene, the nucleotide sequence of the gene is shown as SEQ ID NO.1, the coding length of the gene CgARF4 is 1179bp, the gene contains an ATG start codon and a TAG stop codon, the full length of the ORF is 1179bp, and the gene codes 392 amino acids, and the amino acid sequence is shown as SEQ ID NO. 2.

Example 2

The cloned cymbidium CgARF4 gene is used as a reference to design a fluorescent quantitative primer, and the primer sequence is as follows:

qCgARF4-F：5’- ATGCTGCTGCGTTACGATGT-3’

qCgARF4-R：5’- GCTATCCATCTCATCCACTCCT-3’

meanwhile, 18S is used as an internal reference gene, and the primer sequence is as follows:

18S-F：5’-GGTCCTATTGTGTTGGCT-3’

18S-R：5’-TCGCAGTGGTTCGTCTTT-3’

the preparation of the reaction solution was carried out using the instructions of the ChamQ ™ Universal SYBR Qpcr Master Mix kit (company Vazyme), and the PCR procedure was run on a Applied Biosystems type real-time fluorescence quantitative analyzer: 95 ℃ for 5min; cycling for 40 times at 95 ℃ for 10s and 60 ℃ for 30 s; 95℃15s,60℃1min,95℃15s. After the reaction is finished, an amplification curve is obtained, data is derived through StepOne Software v2.3, excel is used for data analysis, and 2 is used according to CT values ^-ΔΔCt The relative expression level was calculated by the relative quantification method, and the data analysis result is shown in FIG. 2.

The results of the study in this example show that the CgARF4 gene is expressed in various tissues of cymbidium (fig. 2A), but the gene is expressed in higher amounts in cymbidium roots, leaves and flowers secondary, indicating that the gene is functionally active in these three organs; analysis of expression of cymbidium IAA hormone spray-treated leaves demonstrated that the CgARF4 gene plays an important role in the IAA treatment response of cymbidium leaves (fig. 2B).

Example 3

1) Preparation and transformation of Agrobacterium competent cells

In the embodiment, agrobacterium GV3101 is utilized to prepare agrobacterium competence, and an infection experiment of arabidopsis thaliana is carried out; the preparation process of the agrobacteria competence comprises the following steps: picking an activated agrobacterium single colony, inoculating the agrobacterium single colony into 5mL of liquid LB culture medium, and culturing at 28 ℃ and 250 rpm for 20-24 h; suction pipeInoculating 2mL of the bacterial liquid into a triangular flask containing 50mL of liquid LB medium, shaking at 28 ℃ and 250 rpm to OD ₆₀₀ The value is about 0.8; placing the amplified bacterial liquid in ice bath for 30min at 4deg.C and 5000 rpm for centrifugation for 5min, and discarding supernatant; 10mL of precooled 0.1 mo1/L CaCl was added ₂ A solution for sufficiently suspending the precipitated thalli; centrifuging at 5000 rpm for 5min at 4deg.C, and discarding supernatant; 1mL of precooled 20 mmo1/L CaCl was added ₂ And fully suspending thalli by the solution to obtain GV3101 competent cells to be prepared, subpackaging the GV3101 competent cells into 100 mu L/tube by using a centrifuge tube, rapidly adding 20% sterile glycerol, and standing at-80 ℃ for preservation.

Agrobacterium transformation of recombinants: melting the agrobacteria competent cells in an ice bath, adding 600ng of the recovered and purified plasmid into 100 μl of agrobacteria competent cells, gently mixing, and carrying out ice bath for 5min; quick-freezing with liquid nitrogen for 5min, heating in 37 deg.C metal bath for 5min, and rapidly placing in ice bath for 5min; 800. Mu.l of LB medium without any antibiotics was added, and resuscitated at 28℃and 200 rpm for 2 h; centrifuging at 4000 rpm for 3min, and sucking away part of the liquid culture medium; the residual bacterial liquid is fully mixed by a liquid-transferring gun and then is coated on a solid LB culture medium added with 50 mg/L kanamycin and 200 mg/L rifampicin; and culturing for 30-48 hours at 28 ℃ in an inverted mode.

Identification of Agrobacterium recombinants: picking single colonies from the plate culture medium, and inoculating the single colonies into a liquid culture medium containing corresponding antibiotics; culturing at 28deg.C and 200 rpm overnight, performing bacterial liquid PCR, detecting PCR product by 1.5% agarose gel electrophoresis, identifying whether target fragment is contained, adding appropriate amount of sterile 50% glycerol into positive clone after identification, and preserving at-80deg.C for use.

2) Agrobacterium-mediated transformation of Arabidopsis thaliana

The inflorescence infection method is adopted to transfer the target gene into the arabidopsis, and the specific operation method is as follows: arabidopsis thaliana (Columbia type) maintains healthy growth status until flowering; activating the Agrobacterium GV3101 strain carrying the target gene. Picking single colony, inoculating in 5mL LB culture solution containing kanamycin and rifampicin, shaking at 28 ℃ and 200 rpm until the bacterial solution becomes turbid just about 8-10 h; sucking 1mL of bacterial liquid, inoculating into a triangular flask (50 mL) and shaking 24 and h until the OD value is about 0.8; centrifuging the bacterial liquid at 6000 rpm at room temperature for 5min, removing the supernatant, collecting bacterial cells, and suspending with 3% sucrose solution with pH of 5.8; before soaking, silwet L-77 (concentration: 0.03% (300. Mu.l/L)) was added and the foam shaken out; soaking aerial parts of Arabidopsis thaliana in agrobacterium suspension for 1min, and gently shaking during the soaking; placing the immersed Arabidopsis in a tray, covering with a preservative film, sealing with tinfoil paper, and placing 24 h; and (5) uncovering the tinfoil paper, culturing under normal conditions, and stopping watering when the seeds are ripe.

The 3% sucrose solution resuspension comprises the following components: MS culture medium, adding 30g/L sucrose and 300 mu L/L Silwet-77. (note: pH was adjusted to 5.8 after preparation, bacterial liquid was centrifuged and resuspended, silwet L-77 was added, and the conversion relationship between the resuspension and bacterial liquid was such that the amount of resuspension was calculated as bacterial liquid OD. Bacterial liquid volume=0.8. Resuspension).

3) Screening of transgenic plants

The collected seeds of T1 generation transgenic arabidopsis thaliana are sterilized by alcohol and sodium hypochlorite, and the steps are as follows: placing a proper amount of obtained transgenic seeds into a 1.5mL centrifuge tube, and soaking for 5min by using 8% NaClO and ethanol mixed solution (the ratio is 1:1 by volume); sterilizing with 75% alcohol for 5-6 times, each time for 2 min; washing with sterile water for 3-4 times; suspended with 0.1% agarose solution.

Sterilized transgenic Arabidopsis seeds were sown on MS solid medium containing antibiotics (kanamycin 50 mg/L and cephalosporin 100 mg/L), wrapped with tinfoil and placed in a refrigerator at 4℃for vernalization. After 2 days, the culture medium was taken out of the refrigerator and incubated at 22℃under light. After about one week, the arabidopsis thaliana which can grow normally on the culture medium is transplanted into soil and grows continuously.

4) DNA detection of transgenic plants

Taking a proper amount of tender leaves of T1 generation arabidopsis and transgenic plants, and detecting by adopting a plant DNA direct amplification kit of the family of the qing, wherein the specific operation steps are as follows: cutting a proper amount of tender leaves, placing the tender leaves into a 2mL centrifuge tube after sterilization treatment, adding 50uL Lysis Buffer A solution, heating and cracking for 10min at 95 ℃, and continuing to crack overnight at 4 ℃. The next day was centrifuged at 14000 rpm for 3min, and the supernatant was transferred to a new sterile centrifuge tube as a template for PCR reaction. PCR detection was performed using 2×T5 Direct PCR Mix and specific primers for the gene, and the results are shown in FIG. 4A.

5) Fluorescent quantitative PCR detection of transgenic plants

Total RNA was extracted from young stem and leaf of the 6 over-expressed Yucca CgARF4 gene Arabidopsis lines, and the reverse transcription and fluorescence quantitative primers, methods and procedures were the same as in example 2, and the final data analysis results are shown in FIG. 4B.

6) Obtaining of transgenic homozygous lines

Sterilizing the harvested transgenic T1 generation seeds, screening and culturing, transplanting the transgenic T1 generation seeds into nutrient soil, and culturing in 16 h light/8 h dark at the temperature of 22 ℃; after detection, retaining the preliminarily confirmed transgenic plants, harvesting the T1 generation seeds after maturation, and numbering to obtain the T2 generation; as in the T1 generation, the T2 generation seeds are sterilized and then coated on a screening culture medium containing antibiotics, and the culture medium is placed at 22 ℃ for continuous illumination; counting the survival rate of T2 generation seeds with different numbers about 10 days, selecting plants with the survival proportion of 75% to be transplanted and cultured in nutrient soil according to 22 ℃ and 16 h light/8 h darkness, and taking leaves for positive detection; numbering the positive T2 generation plants continuously, and collecting seeds to obtain T3 generation seeds; after the seeds are sterilized, screening the seeds by a screening culture medium, and placing the seeds under light for continuous illumination culture; around 10 days, different numbered T3 generation plants were observed, all survived and no segregating T3 generation homozygous plants appeared.

7) Phenotypic observation

And selecting a transgenic plant line with obvious phenotype for observation, and finding that compared with wild type arabidopsis, the flowering time of the transgenic arabidopsis plant seedling is obviously longer than that of the wild type arabidopsis, the phenomenon of stem extraction in advance exists, the plant growth vigor is obviously weakened when the transgenic arabidopsis plant seedling enters a reproduction stage, and the phenomenon of sterility of part of flowers occurs.

After being disinfected, wild type and transgenic seeds are sowed on an MS culture medium, seedlings with consistent growth vigor are selected in an ultra-clean workbench after 4-6 true leaves are cultivated, the true leaves are cut off and respectively placed on MS, MS+0.2mg/L IAA and MS+0.5mg/L IAA culture mediums, 15 leaves are selected for each gene treatment, the process is repeated for 3 times, and wild type arabidopsis thaliana is used as a control under each treatment. After inoculation, the mixture is placed in an incubator, the culture condition is that the temperature is 23 ℃, the illumination is 24 hours, the illumination intensity is 6000lx, and the relative humidity is 70%. Leaf growth changes were observed and recorded at 12d inoculation, and transgenic leaves were found to have higher rooting rate and root length on each medium than the wild type.

The endogenous hormone content of the arabidopsis leaves is measured by an enzyme-linked immunosorbent assay (ELISA). The sample was triturated in 10mL of an 80% (v/v) methanol extraction medium containing 1 mM Butyl Hydroxy Toluene (BHT) as an antioxidant. Incubating the extract at 4℃for 4h, and centrifuging at 4000 rpm for 15 min. The supernatant was passed through a C-18 column and washed successively with 80% (v/v) methanol, 100% (w/v) ether, and 100% (w/v) methanol. 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. Monoclonal antigens and antibodies against IAA, ABA, GAs (GA 1+ GA 3), meJA and BR in ELISA kits were produced by the plant hormone institute of the university of agriculture, china. Enzyme-linked immunosorbent assay was performed using 96-well microtiter plates. Each well was treated with 0.25. Mu.g/mL of coating buffer (1.5. 1.5 g/L Na ₂ CO ₃ 、2.93 g/L NaHCO ₃ 、0.02 g/L NaN ₃ ) 100. Mu.L of coating and incubation at 37℃for 30 min. After washing 4 times with PBS containing 0.1% (v/v) Tween 20, each well was filled with 50. Mu.L of sample extract and 50. Mu.L of 20. Mu.g/mL antibody, incubated and washed as described above. Each well was charged with a solution containing 1.5. 1.5 mg/mL of 0-phenylenediamine and 0.008% (v/v) H ₂ O ₂ 100. Mu.L of the color development liquid of (C). 12 mol/L H per well ₂ SO ₄ The reaction was stopped. ELISA (model EL310, bio-TEK, winioski, VT) 490, nm was used for color development. Hormone content was calculated according to Weiler et al (1981). 3 biological replicates were set for each hormone. As a result, it was found that the content of endogenous hormone IAA, GA, ABA in leaves of transgenic plants was reduced as compared with the wild type, while the content of MeJA and BR was increased.

In this example, a recombinant plasmid of the overexpressed cymbidium CgARF4 gene was transferred into a model plant, arabidopsis thaliana, and phenotypically observed and analyzed. From the results, the arabidopsis plant over-expressing the CgARF4 gene has the advantages that compared with the wild plant seedling, the flowering time is advanced, the fertility is reduced, the rooting rate of the isolated leaves on an MS culture medium containing IAA is improved, the root length is increased, the content of endogenous hormone IAA, GA, ABA in the leaves is reduced, the content of MeJA and BR is increased, and the gene has influence on the flower development, adventitious root formation and hormone level change of the plant.

SEQ ID NO.1：

ATGGGGATCGATCTGAACACGATCGAGGAGGAGGAGGAGGAAGAGCCGGCGGAGCTAAGGTCGCATCCGCCGGCGGCGGCAGTCGGCGGTGTGGGTTCGGTGTGCCTTGAACTGTGGCACGCCTGCGCGGGGCCTAGGATTTCGCTTCCAAGGAAGGGGAGCTTGGTGGTGTACTTCCCGCAAGGGCACCTCGAACATCTTGCCGGCGGGCAACGCGCCGGCGGAGGAGGGATGCTGCTGCGTTACGATGTGCCTCCTCATGTGTTCTGCCGGGTCGTGGAGGTCCAGCTTCGCGCGGAGGTAGCGACGGATGAAGTCTACGCCCAGCTCTCTGTTGTTGCGGAATGTGAGGGTTTTGAGAAGCATATTCAAGGTGGCGCTGAGGAAGAAGGGGAAGGAGTGGATGAGATGGATAGCGGAGGCCAATCTCCAATTCCTCACATGTTCTGTAAAACTCTCACGGCATCTGACACGAGCACTCATGGAGGCTTTTCCGTACCACGCCGAGCTGCTGAGGACTGCTTCCCTCCGCTGGATTACAGACAACAGAGGCCTTCCCAAGAGCTTATTGCTAAAGATTTGCATGGCGTGGAGTGGCGCTTTCGACACATTTACAGGGGTCAACCACGTCGGCACTTGCTCACAACAGGTTGGAGTGCTTTTGTGAACAAGAAGAAACTTGTCTCTGGGGATGCTGTTCTTTTTCTTCGGGGAGGTAATGGTGAGCTTAGATTGGGCATTCGGAGAGCATCTCAATTAAAGGGCAACATTTCTTATAACATTTCTGGAAGTCAGAACTTGAATGTTGGAGCTGGTGCATTAGCATCACTGGCAAATGCTGTCTCGACGAAGAAAATGTTTCAAATTAATTATAATCCAAGGGTTAACCAATCAGAGTTCATTGTACCTTATTGGAAGTTCAGGAAGAGCTGCAGTTATTCAATATCTGTTGGGACTAGGTTCAAAATGCATTTTGAAAGTGAAGATGCAGCAGAGAGAAGGTGCACAGGATTGATCACTGCAGTTAGTGACTTGGATCCAGTCCAGTGGCCGGGCTCAAAATGGAGGTGCCTTGTGGTAAGATGGGATGAGGATGATATGGACAACAGTCTACATAACAGGGTCTCTCCTTGGGAAATAGAGGCCACCGGATCAGTCTCTTGCCCAACAGTCTTTTAG

SEQ ID NO.2：

MGIDLNTIEEEEEEEPAELRSHPPAAAVGGVGSVCLELWHACAGPRISLPRKGSLVVYFPQGHLEHLAGGQRAGGGGMLLRYDVPPHVFCRVVEVQLRAEVATDEVYAQLSVVAECEGFEKHIQGGAEEEGEGVDEMDSGGQSPIPHMFCKTLTASDTSTHGGFSVPRRAAEDCFPPLDYRQQRPSQELIAKDLHGVEWRFRHIYRGQPRRHLLTTGWSAFVNKKKLVSGDAVLFLRGGNGELRLGIRRASQLKGNISYNISGSQNLNVGAGALASLANAVSTKKMFQINYNPRVNQSEFIVPYWKFRKSCSYSISVGTRFKMHFESEDAAERRCTGLITAVSDLDPVQWPGSKWRCLVVRWDEDDMDNSLHNRVSPWEIEATGSVSCPTVF-。

Claims (3)

1. The application of the cymbidium CgARF4 in changing the development of the arabidopsis 'Columbia' flower has the nucleotide sequence shown in SEQ ID NO. 1; comprising the following steps: the cymbidium CgARF4 gene is connected to a vector, and is transformed into wild arabidopsis thaliana 'Columbia' through agrobacterium mediation, and is screened and cultured to obtain a transgenic plant.

2. The application of the cymbidium CgARF4 in promoting the rooting of the in vitro leaves of the arabidopsis thaliana Columbia is shown in SEQ ID NO. 1; comprising the following steps: the cymbidium CgARF4 gene is connected to a vector, and is transformed into wild arabidopsis thaliana 'Columbia' through agrobacterium mediation, and is screened and cultured to obtain a transgenic plant.

3. The application of the cymbidium CgARF4 in changing the endogenous hormone content of leaves of the arabidopsis thaliana 'Columbia', and the nucleotide sequence of the cymbidium CgARF4 is shown as SEQ ID NO. 1; comprising the following steps: the cymbidium CgARF4 gene is connected to a vector, and is transformed into wild arabidopsis thaliana 'Columbia' through agrobacterium mediation, and is screened and cultured to obtain a transgenic plant.

Images