CN116064564B

CN116064564B - A CgARF4 gene of Cymbidium orchid and its application

Info

Publication number: CN116064564B
Application number: CN202210786625.XA
Authority: CN
Inventors: 胡凤荣; 徐子涵; 朱丽华
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-07-06
Filing date: 2022-07-06
Publication date: 2025-02-11
Anticipated expiration: 2042-07-06
Also published as: CN116064564A

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

Cymbidium CgARF gene and application thereof

Technical Field

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

Background

Orchid (Orchidaceae) is one of the largest families in flowering plants, more than 25000 being found worldwide, accounting for about 10% of all flowering plants. The cymbidium (Cymbidium goeringii) belongs to the small-flower type ground orchid species in the orchid genus of the orchid family, has peculiar flower type, elegant flower color, delicate flower fragrance, beautiful She Zi, extremely high ornamental value and economic value, and is a typical leaf art and flower art double 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, and 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, atARF and AtARF are able to regulate the maturation of stamens and pistils, pollen from the arf6/arf8 double mutant cannot be released, pistil development is immature, and fertility is reduced. Okushima found that flowers of arf2 mutants had a significantly longer pistil and sepals than wild type in early stage of flower formation, the flowers appeared sterile but the proportion of fertile/sterile flowers increased as the plants grew, but entered the flower organ abscission stage, atARF2 was found to promote stamen development and thus delay flower organ abscission. Another study indicated that AtARF 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 SlARF and SlARF8 can regulate the formation of plant flower organs, and SlARF2 is also found to be involved in regulating the senescence process of tomato flower organs. In Dendrobium officinale, the relative expression level of part DnARF in flowers is significantly higher than that of other organs, which indicates that these DnARF are involved in regulating the growth and development of flower organs, while in fruit Mei Zhong PmARF is not only involved in the morphogenesis of flower organs, but also plays an important role in regulating the development of pistils. All the above studies demonstrate that ARF transcription factors can regulate floral development in other plant species. Therefore, by utilizing the genetic engineering technology, cgARF gene cloned from cymbidium is transferred into a model plant, which has important significance for researching the functions of the cymbidium 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 CgARF gene. It is another object of the present invention to provide the use of the cymbidium CgARF gene in plant breeding.

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

a gene of cymbidium CgARF with the nucleotide sequence shown in SEQ ID NO. 1.

The amino acid sequence of the expressed protein of the cymbidium CgARF 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 CgARF 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 CgARF gene is connected to a carrier, and is transformed into wild arabidopsis thaliana 'Columbia' through agrobacterium mediation, screening and culturing to obtain a transgenic plant.

Compared with the prior art, the invention has the beneficial effects that the functions of the arabidopsis seedlings over-expressing the CgARF gene are verified through cloning and identification of the CgARF gene of the cymbidium, expression analysis and genetic transformation of the gene, and the arabidopsis seedlings over-expressing the CgARF gene are found to be early in flowering phase, partial flowers are sterile and the fruit setting rate is reduced compared with the wild type seedlings, but the rooting rate of in vitro leaves is increased, the content of endogenous hormones is changed, and the gene can be widely applied to the growth and development of orchids and the production and breeding of other plants.

Drawings

FIG. 1A is a bacterial detection electrophoresis chart of the cymbidium CgARF gene clone, wherein M is DL2000 Marker, the target band length is 1179bp, and B is a CgARF4 over-expression vector double enzyme digestion verification electrophoresis chart;

FIG. 2A shows CgARF4 expressed in tissues of cymbidium, where R represents roots, S represents pseudobulbs, L represents leaves, F represents flowers, and B shows the gene of cymbidium CgARF under IAA treatment;

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

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

FIG. 5A is a graph showing the comparison of flowering time of a plant over-expressing CgARF gene with a wild type Arabidopsis plant, and B is a transgenic plant part of the phenomenon of flower sterility;

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

FIG. 7 is a comparison of endogenous hormone levels in leaves of an overexpressed CgARF 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 tissues of cymbidium Song Mei, which 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:

The tissue of the ultra-low temperature frozen cymbidium is rapidly transferred to a mortar precooled by liquid nitrogen, the tissue is ground by a pestle, liquid nitrogen is continuously added until the tissue is respectively ground into powder, the ground powder samples are respectively added into 1.5 mL sterilization tubes containing 450 mu l of Buffer PE, the mixture is repeatedly blown by a pipette until no obvious precipitation exists in the lysate, the lysate is centrifuged for 5 minutes at 12,000 rpm and 4 ℃, and the supernatant is carefully sucked into a new 1.5 mL sterilization tube. Adding 1/10 volume of Buffer NB, shaking and mixing the supernatant, centrifuging at 12,000 rpm and 4 ℃ for 5 minutes, carefully sucking the supernatant into a fresh 1.5 mL sterilization Tube, adding 450 uL of Buffer RL, mixing the solution uniformly by using a pipette, adding 1/2 volume of absolute ethanol into the mixture, immediately after mixing the solution uniformly by using a pipette, transferring all the mixed solution into RNA Spin Column, centrifuging at 12,000 rpm for 1 minute, discarding the filtrate, putting RNA Spin Column back into 2ml Collection Tube, adding 500 uL of Buffer RWA into RNA Spin Column, centrifuging at 12,000 rpm for 30 seconds, discarding the filtrate, adding 600uL of Buffer RWB into RNA Spin Column, centrifuging at 12,000 rpm for 30 seconds, discarding the filtrate, adding 50 uL DNase I reaction solution into the center of RNA Spin Column membrane, standing for 15 minutes, adding all the mixed solution into RNA Spin Column membrane at 350 uL, centrifuging at 2,000 rpm for 2,37 minutes, centrifuging at 350 uL of RNA Spin Column for 2.37 minutes, and centrifuging at 35 minutes, and placing 500 uL of RNA Spin Column at 2,000 rpm for 2.35 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 is detected by a trace accounting protein tester, and OD260/OD280 and OD260/OD230 are both 1.8-2.1, so that the integrity is good, and the method 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:

The template RNA was thawed on ice, and 5X Fastking-RT Supermix and RNase-Free ddH2O were thawed at room temperature and rapidly placed on ice after thawing. The mixture was prepared in a centrifuge tube to a total volume of 10uL containing 5X Fastking-RT Supermix 4uL,Total RNA 800ng, the remaining volume being made up with RNase-Free ddH 2O. Centrifuging the centrifuge tube, allowing the mixed solution in the centrifuge tube to sink at the bottom of the tube, slowly shaking, removing genome at a temperature of 42 ℃ for 15min on a PCR instrument, performing reverse transcription reaction, inactivating enzyme at a temperature of 95 ℃ 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 CgARF gene amplification primer, and the primer sequence is as follows:

CgARF4-F: 5’- GAGAACACGGGGGACTCTAGAATGGGGATCGATCTGAACACG -3’

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

Cloning of the cymbidium CgARF gene was performed using PRIMERSTAR MAX Hi-Fi enzyme of Takara using cDNA as a template. The PCR amplification system (50. Mu.L) was 25. Mu. LL PRIMERSTAR Max, 2. Mu.L Forward Primer, 2. Mu. L REVERSE PRIMER, 2. Mu. L TEMPLATE CDNA, 19. Mu.L ddH2O. The PCR procedure was performed with a reaction condition of 94℃pre-denaturation for 3min,98℃denaturation for 10s,60℃annealing for 15s,72℃extension for 30s,32 cycles, 72℃total extension for 5min,16℃incubation.

After the PCR reaction is completed, all PCR products are taken, the target fragments are detected and cut through 1.8% agarose gel electrophoresis, and the PCR target amplified products are recovered and purified through gel. A DNA gel recovery kit of TransGen company is adopted for purifying and recovering target fragments, the specific operation is that a single target strip is cut from agarose gel and put into a clean centrifuge tube, the weight is weighed, 3 times of volume solution GSB (300 mu L of GSB solution is added if the gel is 0.1g and the volume of the gel can be regarded as 100 mu L) is added into a gel block, the solution is placed in a 55 ℃ water bath, the centrifuge tube is continuously and gently turned up and down until the gel block is completely dissolved, the melted gel solution is cooled to room temperature, 1 time of volume isopropanol (100 mu L of isopropanol is added if the gel is 0.1 g) is added, the mixture is gently mixed, the mixture is added into a centrifuge column, 1min is placed at room temperature, the effluent is discarded, the centrifuge column is put back into a collecting tube, 650 mu L of solution WB is added into the centrifuge column, 1min is discarded at 12000rpm, the effluent is discarded, 12000rpm is centrifuged for 2min, the residual WB is removed as much as possible, the centrifuge column is placed in a cover for 5min, the centrifuge column is thoroughly dried, the centrifuge column is placed in a clean centrifuge tube until the gel block is completely dissolved, 100 mu L of isopropanol is added into a suspended middle membrane, and the solution is required to be cooled down and placed in advance at room temperature for 2 ℃ for cooling for 30 mu L of 2h (30 ℃ for 2m, and water bath is required to be cooled for cooling and placed in water bath for 2h at room temperature for 2 h). Taking 2 mu L of the recovered and purified product, performing gel electrophoresis detection by using 1.5% agarose, and placing the rest in a refrigerator at-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 bacterial liquid cultured overnight, centrifuging for 1min 12000rpm, removing supernatant, adding 500 mu L P1 solution (containing RNase A) into a centrifuge tube with bacterial precipitation left, thoroughly suspending bacterial precipitation by using a vortex meter, adding 500 mu L P2 solution into the centrifuge tube, fully lysing bacterial cells when the bacterial cells are gently turned upside down, adding 700 mu L P3 solution into the centrifuge tube, immediately and gently turning upside down, fully mixing, centrifuging for 10min 12000rpm after white flocculent precipitation occurs, adding 500 mu L of balancing liquid BL into an adsorption column CP4, centrifuging for 1min 12000, discarding waste liquid in a collecting tube, placing the adsorption column back into a collecting tube, adding the collected supernatant into a filter column CS in batches, centrifuging for 2min 12000rpm, carefully adding the solution collected in the collecting tube into the adsorption column CP4, centrifuging for 1min 12000rpm, discarding waste liquid in the collecting tube, placing the adsorption column CP4 into the collecting tube, immediately and gently turning upside down, adding 500 mu L of deproteinized liquid PD into the adsorption column CP4, centrifuging for 1min 12000rpm, removing the balancing liquid BL into the filter column, centrifuging for 1min 12000rpm, removing residual liquid in the filter column, and removing the residual liquid in the filter column by removing the filter column by centrifuging for 120 mu L of the filter column, and centrifuging for 1min after the filter column is placed into the filter column, and the filter column is placed into the filter column for 1min, and the filter column is placed for 1min after the filter column is placed for 1min, and the filter column is placed into the filter column is left for 1min, and the filter column is placed for the filter column and placed. Finally, the plasmid concentration was determined and prepared for the next experiment.

4) Double enzyme digestion reaction

The extracted pBI121 plasmid was digested with XbaI and SmaI at 37℃for 30min, and the linear vector was recovered by electrophoresis and stored at-20℃for further use. The double cleavage reaction system was 50. Mu.L of pBI121 plasmid 20. Mu.L, 5 Xbuffer 5. Mu.L, xbaI 1. Mu.L, smaI 1. Mu.L, ddH2O 23. Mu.L.

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 target fragment number is 7 mu L of the linearized pBI121 vector, 3 mu L of the inserted fragment, 4 mu L of the 5 XCE II buffer, exnase II mu L of the carrier molecule number=3:1-5:1, and 20 mu L of the ddH2O Up to. The reaction was carried out at 37℃for 30min, left at normal temperature (without immediate cooling), and transformed to E.coli competence after 10 min.

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. Absorbing 10 mu L of recombinant product, adding into 100 mu L of competent cells, placing a centrifuge tube on ice for 10min, placing in a water bath at 42 ℃ for water bath, heat shock 90 s without shaking, immediately placing in ice for 2min, adding 500 mu L of liquid culture medium without antibiotics into a super clean bench, shaking for 25min for resuscitation at 37 ℃ and 200rpm, centrifuging 6000 rpm for 1 min, absorbing 350 mu L of supernatant, re-suspending the precipitated bacteria on an LB plate (Kana concentration is 50 mg/L), and culturing at 37 ℃ overnight.

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 cloning is finally determined to obtain 1 cymbidium CgARF coding genes, which are named CgARF genes, the nucleotide sequence of the genes is shown as SEQ ID NO.1, the coding length of the genes CgARF is 1179bp, the genes contain an ATG start codon and a TAG stop codon, the full length of the ORFs is 1179bp, and the genes code 392 amino acids, and the amino acid sequence is shown as SEQ ID NO. 2.

Example 2

The cloned cymbidium CgARF 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 ChamQTM Universal SYBR QPCR MASTER Mix kit (Vazyme company) and the PCR procedure was run on a Applied Biosystems real-time fluorescence quantitative analyzer at 95℃for 5min, 95℃for 10s,60℃for 30s, and 40 cycles, 95℃for 15s,60℃for 1min, and 95℃for 15s. After the reaction is finished, an amplification curve is obtained, data is derived through StepOne Software v 2.3.3, excel is used for data analysis, the relative expression quantity is calculated by a relative quantification method of 2-delta Ct according to CT values, and the data analysis result is shown in figure 2.

The results of the study in this example show that CgARF4 gene is expressed in various tissues of cymbidium (FIG. 2A), but the expression level of the gene in cymbidium roots is higher, leaves and flowers are secondary, which shows that the gene is active in the three organs, and the analysis of the expression of the leaves treated by spraying cymbidium IAA hormone proves that CgARF gene plays an important role in the IAA treatment response of the leaves of cymbidium (FIG. 2B).

Example 3

1) Preparation and transformation of Agrobacterium competent cells

The method comprises the steps of preparing agrobacterium competence by using agrobacterium GV3101, carrying out an infection experiment of arabidopsis thaliana, selecting single colony of activated agrobacterium, inoculating the single colony into 5mL of liquid LB culture medium, carrying out shaking culture at 28 ℃ and 250 rpm for 20-24 h, absorbing 2mL of bacterial liquid, inoculating the bacterial liquid into a triangular flask containing 50mL of liquid LB culture medium, carrying out shaking culture at 28 ℃ and 250 rpm until the OD600 value is about 0.8, placing the bacterial liquid subjected to expansion propagation on ice for 30 min, centrifuging at 4 ℃ and 5000 rpm for 5 min, discarding supernatant, adding 10mL of precooled 0.1 m/L CaCl2 solution, fully suspending precipitated thalli, centrifuging at 4 ℃ and 5000 rpm for 5 min, discarding supernatant, adding 1mL of precooled 20 mm o1/L CaCl2 solution, fully suspending thalli, obtaining GV3101 competent cells to be prepared, packaging into a centrifuge tube with 100 mu L/tube, rapidly adding 20% glycerol, and carrying out aseptic storage at-80 ℃.

The recombinant agrobacterium transformation comprises the steps of ice bath, melting agrobacterium competent cells, adding 600ng of recovered and purified plasmids into 100 mu L of agrobacterium competent cells, lightly mixing, ice bath 5min, quick freezing 5min by using liquid nitrogen, heat shock 5min in 37 ℃ metal bath, rapidly placing on ice bath 5min, adding 800 mu L of LB culture medium without any antibiotics, resuscitating 2h at 28 ℃,200 rpm, centrifuging 3 min at 4000 rpm, sucking off part of liquid culture medium, fully mixing the residual bacterial liquid by using a pipetting gun, coating on solid LB culture medium added with 50 mg/L kanamycin and 200 mg/L rifampicin, and culturing for 30-48 h at 28 ℃ in an inverted mode.

The identification of agrobacterium recombinants includes picking single colony from plate culture medium, inoculating to liquid culture medium containing corresponding antibiotic, culturing at 28 deg.c and 200 rpm deg.c overnight, PCR, detecting the PCR product with 1.5% agarose gel electrophoresis, identifying whether to contain target segment, adding proper amount of sterile 50% glycerin into the positive clone, and storing at-80 deg.c for use.

2) Agrobacterium-mediated transformation of Arabidopsis thaliana

The inflorescence infection method is adopted to transfer the target gene into the arabidopsis thaliana, and the specific operation method comprises the steps of keeping the healthy growth state of the arabidopsis thaliana (Columbia type) until the arabidopsis thaliana flowers, and activating the agrobacterium GV3101 strain carrying the target gene. Picking single colony, inoculating in 5mL LB culture solution containing kanamycin and rifampicin, shaking at 28deg.C and 200 rpm until the bacterial solution just becomes turbid, about 8-10 h, sucking 1mL bacterial solution, inoculating in a triangular flask (50 mL) shaking 24 and h until OD value is about 0.8, centrifuging the bacterial solution 6000 rpm at room temperature for 5 min, removing supernatant, suspending with 3% sucrose solution with pH of 5.8, adding Silwet L-77 with concentration of 0.03% (300 μl/L) before soaking, shaking out foam, soaking aerial parts of Arabidopsis in agrobacterium suspension solution for 1min, slightly shaking, lying the soaked Arabidopsis in a tray, covering with fresh-keeping film, sealing with tinfoil paper, keeping away from light, standing for 24 h, uncovering tinfoil paper, culturing under normal condition, and stopping watering when seeds mature.

The 3% sucrose solution heavy suspension comprises MS culture medium, sucrose 30g/L, silwet-77. Mu.l/L. (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 T1 generation transgenic arabidopsis seeds are sterilized by alcohol and sodium hypochlorite, and the steps are that a proper amount of obtained transgenic seeds are placed in a 1.5mL centrifuge tube, soaked in 8% NaClO and alcohol mixed solution (the proportion is 1:1 in terms of volume ratio) for 5min, sterilized by 75% alcohol for 5-6 times, each time is 2 min, washed by sterile water for 3-4 times, and suspended by 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 a4℃refrigerator for vernalization. After 2 days, the medium was taken out of the refrigerator and placed at 22℃for light cultivation. 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

The method comprises the specific operation steps of cutting a proper amount of tender leaves of T1 generation Arabidopsis and transgenic plants, placing the cut tender leaves into a 2mL centrifuge tube after sterilization treatment, adding 50uL Lysis Buffer A solution, heating and cracking at 95 ℃ for 10min, and continuing to crack at 4 ℃ overnight. The next day 14000 rpm was centrifuged for 3min and the supernatant was transferred to a new sterile centrifuge tube as a template for the PCR reaction. PCR was performed using 2×T5 DIRECT PCR Mix and gene specific primers, 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, method and procedure 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, culturing at 22 ℃ in 16 h light/8 h in the dark, reserving the initially confirmed transgenic plants after detection, harvesting the T1 generation seeds after maturation, numbering to obtain the T2 generation seeds, sterilizing the T2 generation seeds as the T1 generation seeds, coating the T2 generation seeds on a screening culture medium containing antibiotics, placing the T2 generation seeds at 22 ℃ for continuous light, culturing the T2 generation seeds with different numbers in the dark according to 22 ℃ and 16 h light/8 h in the nutrient soil after about 10 days, selecting plants with the survival proportion of 75% for transplanting, carrying out positive detection on leaves, continuing numbering the positive T2 generation plants, collecting the seeds to obtain the T3 generation seeds, screening the T2 generation seeds with the screening culture medium after sterilization, continuously culturing the T2 generation seeds under light, observing the T3 generation plants with different numbers for about 10 days, and completely surviving and not appearing separated into T3 generation homozygous plants.

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, and the culture condition is that the temperature is 23 ℃, the illumination is carried out for 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 10 mL of an 80% (v/v) methanol extraction medium containing 1mM Butyl Hydroxy Toluene (BHT) as an antioxidant. The extract was incubated for 4h at 4℃and centrifuged 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 coated with 100. Mu.L of coating buffer (1.5 g/L Na2CO3, 2.93 g/L NaHCO3, 0.02 g/L NaN 3) containing anti-hormonal antigen at 0.25. Mu.g/mL, incubated 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 100. Mu.L of a color-developing solution containing 1.5 mg/mL of 0-phenylenediamine and 0.008% (v/v) H2O 2. Each well 12 mol/L H SO4 stopped the reaction. 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 endogenous hormone IAA, GA, ABA content in leaves of the transgenic plants was decreased compared with the wild type, while the MeJA and BR contents were increased.

In this example, a recombinant plasmid of the overexpressed cymbidium CgARF gene was transferred into the model plant arabidopsis thaliana, and phenotypic observation and analysis were performed. From the results, the arabidopsis plant over-expressing CgARF gene has earlier flowering time and lower fertility than wild plant seedling, while the in vitro leaf has higher rooting rate and root length on MS culture medium containing IAA, the endogenous hormone IAA, GA, ABA content in the leaf is reduced, the MeJA and BR content are increased, and the gene has influence on the flower development, adventitious root formation and hormone level change of the plant.

Claims

1. The application of the cymbidium CgARF in changing the development of the arabidopsis thaliana Columbia flowers is characterized in that the nucleotide sequence of the CgARF is shown as SEQ ID NO.1, and the application comprises the steps of connecting a cymbidium CgARF gene to a vector, transforming the cymbidium CgARF gene into the wild arabidopsis thaliana Columbia through agrobacterium mediation, screening and culturing to obtain a transgenic plant.

2. The application of the cymbidium CgARF in promoting the rooting of the in vitro leaves of the arabidopsis thaliana Columbia is characterized in that the nucleotide sequence of the CgARF is shown as SEQ ID NO. 1, and the application comprises the steps of connecting a cymbidium CgARF gene to a vector, carrying out mediated transformation to the wild arabidopsis thaliana Columbia through agrobacterium, screening, and culturing to obtain a transgenic plant.

3. The application of the cymbidium CgARF4 in reducing the endogenous hormone IAA, GA, ABA content of arabidopsis thaliana 'Columbia' leaves or increasing the content of MeJA and BR is characterized in that the nucleotide sequence of the CgARF4 is shown as SEQ ID NO. 1, the application comprises the steps of connecting a cymbidium CgARF4 gene to a carrier, transforming the gene into wild arabidopsis thaliana 'Columbia' through agrobacterium mediation, screening and culturing to obtain a transgenic plant.