CN110295174A - Transgenic Arabidopsis line with overexpression of FIPV gene and construction method thereof - Google Patents

Abstract

The invention relates to a transgenic arabidopsis thaliana strain with over-expressed FIPV gene and a construction method thereof, belonging to the technical field of plant transgenosis, wherein the construction method comprises the following steps: constructing an expression vector of FIPV overexpression, transforming agrobacterium tumefaciens, infecting arabidopsis by using agrobacterium containing the expression vector through a floral dip method to obtain a transgenic arabidopsis strain of FIPV gene overexpression; transgenic Arabidopsis lines with over-expression of the FIPV gene are OE31 and OE 51. The FIPV gene overexpression arabidopsis transgenic strain constructed by the invention is reported for the first time, and is directly subjected to genetic transformation by using an agrobacterium-mediated floral dip method to obtain a novel FIPV overexpression germplasm, so that a basis is provided for identifying the function identification of the gene.

Description

Transgenic Arabidopsis line with overexpression of FIPV gene and construction method thereof

technical field

The invention belongs to the technical field of plant transgenesis, and in particular relates to a transgenic Arabidopsis strain with overexpression of FIPV gene and a construction method thereof.

Background technique

Nitrogen plays a very important role in the growth and development of plants. Nitrate nitrogen (NO ₃ ^- ) is the main nitrogen absorbed by most terrestrial plants. A large number of studies have shown that NO ₃ ^- is not only a nutrient, but also a signal molecule, which plays a regulatory role in the short-term and long-term growth of plants. The short-term effect is mainly reflected in the primary response of nitrate nitrogen, that is, the expression of more than 1000 genes in plants changes rapidly when NO ₃ ^- is applied, such as NRTs, NIAs and NiR are induced within a few minutes of NO ₃ ^- treatment. The long ^- term effects of NO ₃ -affect plant root morphogenesis, seed dormancy, flowering, circadian clock, stomatal closure and auxin transport independent of ABA. In the past ten years, some important regulatory genes involved in the NO ₃ ^- primary response process and affecting the NO ₃ ^-metabolism process have been identified. These regulatory genes include transporters, such as NRT1.1; protein kinases, such as CIPK8 and CIPK23; transcription factors, such as NLP7, TGA1, TGA4, TCP20, NRG2, etc.; microRNAs, such as micR167. Recent studies have shown that the polyadenylation complex (CPSF), involved in the processing of the 3' end of eukaryotic pre-mRNA, plays an important role in regulating the nitrate nitrogen signaling pathway. FIPV is a key member of CPSF, which is very likely to regulate the processes of nitrate nitrogen sensing, uptake, transport and assimilation in plants. Therefore, identifying the function of this gene is very important for studying the process of plant nitrate nitrogen metabolism and improving plant nitrogen utilization efficiency.

Identification of gene function first requires the acquisition of mutants and overexpression lines of the gene. Due to the deletion of the gene in the mutant, corresponding molecular, biochemical or physiological phenotypes will be produced, from which the function of the gene can be deduced. At the same time, it is also necessary to study the molecular, biochemical or physiological phenotype changes when the gene is expressed in large quantities. Therefore, constructing an overexpression line of the target gene is a necessary step in identifying the function of the gene. Generally, the Agrobacterium tumefaciens-mediated flower dipping method is used to construct a target gene overexpression line using Arabidopsis thaliana. First, extract the total RNA of the plant, and use it as a template to perform reverse transcription to obtain the first-strand cDNA, and perform PCR reaction to clone the cDNA or coding sequence (CDS) of the target gene. Then, the cloned cDNA or CDS is linked into a strong promoter (such as 35S promoter) downstream to construct a plant expression vector, and the expression vector is transformed into Agrobacterium tumefaciens. Finally, the transformed Agrobacterium is cultivated to a suitable concentration, and the inflorescence of Arabidopsis is invaded, and the Agrobacterium can enter the plant receptor through the pollen tube, and integrate the cDNA or CDS sequence containing the strong promoter fusion target gene into the host plant in the genome. Plant expression vectors generally contain antibiotic marker genes (such as anti-Kana, ampicillin or hygromycin genes) for screening transgenic plants. The seeds of the transgenic plants can grow on the solid medium containing the corresponding antibiotics and germinate and grow, while the non-transgenic seeds can only germinate but not grow.

Researchers generally hope that the expression level of the target gene in the overexpression line is as high as possible and can be stably inherited. Therefore, studying the function of FIPV gene and constructing an overexpression line with high expression and stable inheritance are the key joints to identify the function of this gene in regulating the NO ₃ ^- metabolic pathway.

Contents of the invention

In order to achieve the purpose of high-efficiency expression and stable inheritance of the target gene, the invention provides a transgenic Arabidopsis strain with overexpression of the FIPV gene and a construction method thereof.

In order to achieve the above object, the technical scheme adopted in the present invention is:

The construction method of the transgenic Arabidopsis strain with FIPV gene overexpression of the present invention, the construction method of the transgenic Arabidopsis strain with FIPV gene overexpression comprises: using Agrobacterium tumefaciens to obtain the transgene with FIPV gene overexpression through flower dipping method Arabidopsis strains.

The construction method of the transgenic Arabidopsis strain of described FIPV gene overexpression comprises the following steps:

Step 1, Arabidopsis culture, sow Arabidopsis seeds treated at 4°C low temperature and dark for 3 days on vermiculite, add 1/2MS culture solution, place at 70% relative humidity, temperature 23°C, light and dark time respectively Cultivate in a plant-specific incubator for 16h and /8h;

Step 2, preparing Escherichia coli DH5α competent cells;

Step 3, using Gateway technology to construct the expression vector Pmdc83-FIPV for overexpression of FIPV;

Step 4, using the kit method to extract the PMDC83-FIPV plasmid;

Step 5, preparing Agrobacterium GV3101 competent cells;

Step 7, transforming the PMDC83-FIPV expression vector into Agrobacterium;

Step 8, screening bacteria to identify positive Agrobacterium transformants;

Step 9, genetic transformation mediated by Agrobacterium;

Step 10, obtaining the T1 generation of transgenic plants and identifying the segregation ratio;

Step 11, screening homozygous FIPV overexpression transgenic lines in the T3 generation of transgenic plants;

Step eleven, identifying the DNA level, RNA level and protein level of the FIPV overexpression transgenic line.

The step three specifically includes: extracting Arabidopsis total RNA, and then performing reverse transcription to obtain the first-strand cDNA, using this as a template to design a forward primer F and a reverse primer for specifically amplifying the FIPV coding region sequence (CDS) R, the sequences of the two primers are as follows:

F (forward primer): 5'-CGGGGTACCATGGAAGAGGACGATGAG-3';

R (reverse primer): 5'-CCGCTCGAG TCATCCCACACATACTCT-3';

Use F and R to carry out PCR reaction. After the product is recovered from the gel, it is ligated into the Gateway-compatible vector pENTR3C by enzyme digestion to construct the cloning vector pENTR 3C-FIPV, and then combine pENTR 3C-FIPV with the 35S strong promoter and hygromycin marker The expression vector pMDC83 was subjected to LR reaction to obtain the pMDC83-FIPV expression vector overexpressing FIPV. Use this expression vector to transform competent escherichia coli DH5α, obtain Arabidopsis thaliana FIPV gene CDS sequence as shown in after the pMDC83-FIPV sequence is determined:

The PCR product and the Gateway-compatible vector pENTR3C were double-digested with restriction endonucleases Kpn I and Xho I respectively, and the resulting FIPV CDS containing cohesive ends were ligated into pENTR3C cut by enzymes to construct the cloning vector pENTR3C-FIPV. Then pENTR 3C-FIPV was subjected to LR reaction with expression vector pMDC83 containing 35S strong promoter and hygromycin marker gene to obtain pMDC83-FIPV expression vector overexpressing FIPV.

For the first time, the sequence of the coding region of the Arabidopsis FIPV gene was cloned, and the specific sequence is as follows:

The transgenic Arabidopsis lines with overexpression of FIPV gene in the present invention are OE31 and OE51.

The beneficial effects of the present invention are:

1. The FIPV gene overexpression Arabidopsis transgenic strain constructed by the present invention is reported for the first time, and can be directly transformed by the flower dipping method mediated by Agrobacterium to obtain a new germplasm with FIPV overexpression, which is the basis for the identification of the gene. Functional identification provides the basis.

2. The plant expression vector used to construct the overexpression line is fused with a GFP marker gene downstream of the target gene. Therefore, in addition to achieving high-efficiency expression of the target gene in the overexpression strain, the GFP protein can also be used for the subcellular localization of the FIPV gene in plants. At the same time, the FIPV fusion GFP protein in the overexpression strain can be extracted by using the GFP antibody through the immune reaction, so as to achieve the purpose of purifying the FIPV protein.

3. The antibiotic Kana was used in the screen-positive transformed Agrobacterium clones, and the antibiotic hygromycin was used in the screen-positive transgenic plants. Using two different antibiotics as the most screening conditions can greatly reduce the chance of false positives and ensure that the positive clones obtained have high specificity.

Description of drawings

Fig. 1 is a schematic diagram of the construction method of the plant expression vector pMDC 83-FIPV;

Fig. 2 is the electrophoresis diagram of cloning the FIPV gene CDS using Arabidopsis cDNA as a template, wherein the band at 3792bp in lanes 1-4 is the FIPV gene CDS;

Fig. 3 is the electrophoresis figure of cloning FIPV gene CDS using the cloning vector pENTR 3C-FIPV (recombinant plasmid) in E. negative control;

Fig. 4 is the electrophoresis figure of the cloned FIPV gene CDS using the expression vector pMDC 83-FIPV (recombinant plasmid) in Escherichia coli as a template, wherein, the band at 3792bp in the 1-4 swimming lanes is the FIPV gene CDS, and the 5 swimming lanes are without template negative control;

Figure 5 is an electrophoresis image of the cloned FIPV gene CDS using the expression vector pMDC 83-FIPV (recombinant plasmid) in Agrobacterium tumefaciens as a template, wherein the band at 3792 bp in lanes 1-4 is the CDS of the FIPV gene, and lane 5 is without template negative control;

Fig. 6 is the detection of FIPV expression levels in FIPV overexpressed transgenic lines OE31 and OE51 by fluorescent real-time quantitative PCR.

Detailed ways

Below in conjunction with example the present invention will be further described.

Example 1

Cloning of Arabidopsis thaliana FIPV gene CDS sequence, the plant material is Arabidopsis thaliana.

1. Extraction of Arabidopsis total RNA (kit method)

Utilize the kit method (column total RNA extraction kit) to extract the total RNA of corresponding Arabidopsis plants, the specific method is as follows:

(1) Weigh about 100mg of the material into a 1.5mL Eppendorf tube, and grind it fully in liquid nitrogen;

(2) Add 1mL Buffer RLT (the volume of the material is less than or equal to 10% of the volume of Buffer RLT), shake and mix well, and let stand at room temperature for 5 minutes;

(3) In order to fully remove cell wall residues, proteins, fats, polysaccharides, etc., centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a new centrifuge tube;

(4) Phase separation:

①Add 0.2mL chloroform, shake vigorously for 15s, and let stand at room temperature for 2min;

② Centrifuge (4°C, 12000rpm, do not exceed 12000rpm) for 10min;

(5) Precipitation and removal of polysaccharides:

① Take the colorless aqueous phase (about 50% of the volume of the original Buffer RLT, about 500 μL) into a new Eppendorf tube, do not suck into the middle layer;

②Add 0.25mL of 70% ethanol, mix by inverting;

(6) Add the solution obtained in the previous step into the collection tube that has been loaded into the adsorption column (if the liquid volume exceeds 700μL, transfer it twice), centrifuge at 12000rpm for 20s at 4°C, pour off the waste liquid in the collection tube, and Put the adsorption column back into the collection tube;

(7) Add 700 μL Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 20 seconds at 4°C, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

(8) Add 500 μL Buffer RW2 to the adsorption column, centrifuge at 12,000 rpm for 20 seconds at 4°C, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

(9) Repeat step (8)

(10) 4°C, 12000rpm air separation for 2min;

(11) Place the empty adsorption column at room temperature for several minutes to dry completely;

(12) Put the adsorption column in a new RNase-free centrifuge tube, add 30 μL RNase-free water, place at room temperature for 1 min, centrifuge at 12000 rpm at 4 °C for 1 min, collect the RNA solution, and store at -70 °C.

2. Reverse transcription

Take 1 μg of total RNA, add 0.5 μL of Oligd(T), and supplement RNase-free water to a total volume of 6 μL.

Place in a water bath at 65°C for 5 minutes, centrifuge slightly and prepare the following reverse transcription reaction solution in the tube.

After incubation at 42°C for 1 hour, treat at 70°C for 15 minutes and cool on ice.

3. PCR amplification using Phusion high-fidelity DNA polymerase

(1) Take a 0.2mL PCR tube and add the following ingredients in sequence:

(2) Close the PCR tube and put it into the PCR machine. The reaction conditions were: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 20 s, annealing for 20 s according to the annealing temperature of the primers, extension at 72°C for 30 s/Kb, 30 cycles, and extension at 72°C for 10 min.

(3) After the reaction is completed, perform electrophoresis on a 1% agarose gel to check the amplification effect. The results of PCR product agarose gel electrophoresis are shown in Figure 2, and a clear target band can be observed at 3792bp.

Example 2

Construction of Cloning Vector pENTR 3C-FIPV

1. PCR amplification of FIPV gene CDS and connection with pENTR 3C

F (forward primer): 5'-CGGGGTACCATGGAAGAGGACGATGAG-3';

R (reverse primer): 5'-CCGCTCGAG TCATCCCACACATACTCT-3';

Using the extracted cDNA as a template, PCR amplification was performed with specific primers F and R, and Kpn I and Xho I restriction sites were introduced upstream and downstream of the target gene, respectively. Use the Tiangen (Beijing) Gel Recovery Kit to recover the PCR product, and perform enzyme digestion and ligation of the PCR product with Gateway-compatible pENTR 3C. The operation steps are as follows.

Element Dosage Target fragment recovery product 0.5-10ng pENT3C 0.5μL Salt solution 0.5μL H₂O Up to 3μL

After mixing, react at room temperature (25°C) for 30 minutes, then ice-bath for 1 minute to transform Escherichia coli DH5α, coat the surface with an LB culture plate containing kanamycin, and incubate at 37°C for 12 hours. Then, a single white clone was picked and spread on a new LB culture plate containing kanamycin, cultured at 37° C. for 12 hours, and colony PCR amplification was performed, as shown in FIG. 3 . At the same time, the correct bacterial strains were sent to Shanghai Sangong Company for DNA sequencing. The DNAMAN software compared the sequencing sequence with the promoter sequence of the Arabidopsis FIPV gene. After the comparison was correct, it indicated that the cloning vector pENTR 3C-FIPV was successfully constructed, and the positive plasmid was extracted for future use. The extraction of the plasmid was carried out according to the instruction manual using kits from Beijing Kangwei Company.

(1) Take 1-5 mL of overnight cultured bacterial solution, add it to a centrifuge tube, centrifuge at 12,000 rpm for 1 min, and discard the supernatant as much as possible.

(2) Add 250 μL Buffer P1 to the centrifuge tube with the bacterial pellet left, and use a pipette or a vortex shaker to thoroughly suspend the bacterial pellet. Note: If the bacterial block is not thoroughly mixed, the lysis effect will be affected, resulting in low extraction volume and purity.

(3) Add 250 μL Buffer P2 to the centrifuge tube, and gently mix it up and down 4-6 times to fully lyse the bacteria. At this time, the bacteria solution should become clear and viscous. The time used should not exceed 5 minutes to avoid damage to the plasmid.

(4) Add 350 μL Buffer N3 to the centrifuge tube, and immediately mix it up and down gently for 4-6 times. At this time, a white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 minutes, and a precipitate will form at the bottom of the centrifuge tube.

(5) Column equilibration: Add 200 μL Buffer PS to the collection tube loaded with the spin column, centrifuge at 12000 rpm for 2 min, discard the waste liquid in the collection tube, and put the spin column back into the collection tube.

(6) Add the supernatant obtained in step 4 into the collection tube that has been loaded into the adsorption column, be careful not to suck out the precipitate, centrifuge at 12000rpm for 1min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

(7) Add 600 μL Buffer PW to the collection tube, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.

(8) Repeat step 7.

(9) Put the adsorption column back into the collection tube, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube. Allow the column to dry completely at room temperature for several minutes.

(10) Put the adsorption column in a new centrifuge tube, add 50-100μL Buffer EB dropwise to the middle of the adsorption membrane, let it stand at room temperature for several minutes, centrifuge at 12000rpm for 1min, and collect the plasmid solution into the centrifuge tube. Store plasmids at -20°C.

Example 3

Construction of Plant Expression Vector pMDC 83-FIPV

The sequenced correct cloning vector pENTR 3C-FIPV was subjected to LR reaction with pMDC83 containing 35S promoter and GFP reporter gene to obtain a plant expression vector of 35S promoter-driven FIPV gene fused with GFP reporter gene. The LR reaction system is as follows: 1 μL (about 100 ng) of the cloning vector, 1 μL (about 100 ng) of the pMDC83 vector, and 0.5 μL of LR cloning enzyme. The above substances were mixed and then placed at room temperature (25° C.) to react for 1-2 hours to transform Escherichia coli DH5α. The surface of the transformed strain was coated with an LB culture plate containing kanamycin, and cultured at 37°C for 12 hours. Then, a single white clone was picked and spread on a new LB culture plate containing kanamycin, cultured at 37° C. for 12 hours, and colony PCR amplification was performed, as shown in FIG. 4 . It shows that the expression vector pMDC 83-FIPV is constructed successfully, and the positive plasmid is extracted for future use. The extraction method is as described in Example 2.

Example 4

Construction of transgenic lines of Arabidopsis thaliana overexpressed with FIPV

1. Preparation and transformation of Agrobacterium tumefaciens GV3101 competent cells

The preparation method of Agrobacterium competent cells is as follows:

(1) Pick a single colony GV3101, inoculate it in 5 mL of YEP liquid medium, and cultivate overnight at 28°C and 200 rpm.

(2) Take 2 mL of the culture into 50 mL of liquid YEP and continue culturing until the OD600 is about 0.5.

(3) Place the culture in an ice bath for 30 min, centrifuge at 5000 rpm at 4°C for 5 min.

(4) Discard the supernatant, and suspend the bacteria in 10 mL of 0.1 mol/L cold NaCl; centrifuge at 5000 rpm at 4°C for 5 min.

(5) Discard the supernatant, suspend the precipitate with 1 mL of 20 mmol/L cold CaCl2, aliquot into 200 μL/tube, freeze in liquid nitrogen and store at -70 °C.

The expression vector was transformed into Agrobacterium by freeze-thaw method, and the transformation process was as follows:

(1) Take out GV3101 competent cells (250 μL) from -80°C, thaw on ice, and immediately add 1-2 μL of plasmid DNA.

(2) Stand on ice for 5 minutes.

(3) Freeze in liquid nitrogen for 5 minutes.

(4) Heat shock in a water bath at 37°C for 5 minutes.

(5) Add 1 mL of YEP liquid medium without antibiotics, and incubate on a shaker at 28°C for 2-4 hours.

(6) Centrifuge at room temperature for 5 minutes at 4000 rpm, collect the cells, and resuspend the cells with 80-100 μL of YEP solution.

(7) Evenly spread the bacterial liquid on the YEP solid selection medium, and culture it statically at 28°C for 2-3 days.

(8) After the colony grows, pick a single colony and re-streak it on the YEP solid plate containing antibiotics, and perform colony PCR identification after 36 hours, and the results are shown in Figure 5.

2. Transformation of Arabidopsis

The genetic transformation of Arabidopsis thaliana adopts the inflorescence dipping method, and the method is as follows:

(1) The main inflorescence of Arabidopsis was cut off to induce the formation of side inflorescences, and the side inflorescences bloomed at the same time to facilitate transformation. Water the nutrient solution thoroughly before transformation, and control watering or nutrient solution after transformation, so that the seeds can mature in time.

(2) Prepare the Agrobacterium needed for transformation one day in advance, take 2-5 mL of overnight cultured bacterial solution and add it to 250 mL medium for shaking culture, so that it can be used for transformation of Arabidopsis the next day.

(3) Collect the Agrobacterium cells cultivated overnight, and resuspend them in 50-100mL dipping solution.

(4) After all the elongated seed pods and bloomed flowers of Arabidopsis thaliana were cut off, the flocs were immersed in the liquid for dyeing, and the flocs were shaken up and down to facilitate the entry of the liquid for dyeing. The dipping time generally lasts about 1 hour. Put the soaked Arabidopsis thaliana flat into a large pot, cover with a film to keep moisture, and at the same time cover with a black film to avoid light. After the dark treatment, the Arabidopsis thaliana was taken out and placed in the light, and the seeds were harvested after maturity.

The Arabidopsis thaliana liquid formulation is as follows:

Adjust the pH to 5.7 with 1M KOH.

3. Screening of transgenic lines

Collect the Arabidopsis thaliana T0 generation seeds after infection, and carry out disinfection treatment, the method is as follows:

(1) Take an appropriate amount of seeds and place them in a 1.5mL Eppendorf tube, and indicate the relevant information;

(2) Prepare 70% ethanol in advance, use a pipette to take 1mL into the tube, pipette and mix well, so that the seeds can fully contact the ethanol solution, and let it stand for 5 minutes (note that the seeds on the tube wall and the tube cover are all Rinse down to ensure complete sterilization), and then use a 1mL pipette to suck out the ethanol in the centrifuge tube (be careful not to suck out the seeds);

(3) Rinse once with Tween water;

(4) Use a pipette to take 1mL of 2.6% sodium hypochlorite solution in the tube, mix it upside down, and let it stand for 10 minutes;

(5) Rinse with Tween water five times;

(6) Store at 4°C until use.

Screening of transgenic plants

The sterilized T0 generation seeds were inoculated on MS solid medium containing 50 μg/mL hygromycin, and grown for 7 days in a plant-specific cultivation room under the conditions of 22°C and long daylight (16h light/8h dark). Select the plants that can germinate and grow on the hygromycin MS plate in the T1 generation, and transfer them to the vermiculite for culture, and the culture conditions are the same as above.

Collect T1 generation seeds, sterilize according to the above method, sow on MS solid medium containing 50 μg/mL hygromycin, and grow for 7 days in a special cultivation room for plants. . Select the plants that can germinate and grow on the hygromycin MS plate in the T2 generation, and transfer them to the vermiculite for culture, and the culture conditions are the same as above.

Collect T2 generation seeds, sterilize according to the above method, sow on MS solid medium containing 50 μg/mL hygromycin, and grow in a plant-specific cultivation room for 7 days. . In the T3 generation, the plants that could germinate and grow completely on the hygromycin MS plate without segregation were selected to obtain the overexpression lines OE31 and OE51.

Example 5

Detection of FIPV expression in Arabidopsis transgenic lines with FIPV overexpression

The expression of FIPV in transgenic Arabidopsis lines OE31 and OE51 with FIPV overexpression was detected by real-time fluorescent quantitative PCR (qRT-PCR).

Dilute the cDNA samples obtained after reverse transcription of the RNA extracted in OE31 and OE51 to 0.5-2ng/μL, and the reaction system is shown in the following table:

Close the PCR tube and put it into a fluorescent quantitative PCR instrument (ABI7500FAST). The reaction conditions are: pre-denaturation at 50°C for 20s, then pre-denaturation at 95°C for 10s, denaturation at 95°C for 15s, annealing at 60°C for 1min, and then plate read, cycle 40 times from step 3, and the melting curve is 95°C for 15s, 60°C 1min at ℃, 30s at 95℃, 15s at 60℃.

The reaction used tublin2 as an internal reference gene to ensure the accuracy of the results. Each treatment had three biological repetitions and three systematic repetitions. The expression levels of FIPV in the FIPV overexpression lines OE31 and OE51 are shown in Figure 6. The results showed that the expression levels of FIPV in OE31 and OE51 were significantly higher than those in the wild type and mutants, and FIPV could be highly expressed in the overexpression lines.

Claims (5)

1. a kind of construction method of the transgenic arabidopsis strain of FIPV gene overexpression, which is characterized in that the FIPV base Because the construction method of the transgenic arabidopsis strain of overexpression includes: to obtain FIPV gene mistake by flower-dipping method with agrobacterium tumefaciens Measure the transgenic arabidopsis strain of expression.

2. the construction method of the transgenic arabidopsis strain of FIPV gene overexpression as described in claim 1, feature exist In the construction method of the transgenic arabidopsis strain of the FIPV gene overexpression is the following steps are included: step 1, arabidopsis 4 DEG C of low temperature and the arabidopsis seed for being protected from light processing 3d are seeded on vermiculite, 1/2MS culture solution are added, is placed in relatively wet by culture Degree 70%, 23 DEG C of temperature, illumination and interlunation be respectively 16h and/8h plant dedicated incubator in cultivate；Step 2, system Standby bacillus coli DH 5 alpha competent cell；Step 3, the expression vector being overexpressed using Gateway technology building FIPV Pmdc83-FIPV；Step 4 extracts PMDC83-FIPV plasmid using RNA isolation kit；Step 5, preparation Agrobacterium GV3101 sense By state cell；Step 7, PMDC83-FIPV expression vector convert Agrobacterium；Step 8 sieves dientification of bacteria positive Agrobacterium-mediated Transformation Son；Step 9, Agrobacterium-mediated genetic transformation；Step 10, the acquisition in transgenic plant T1 generation and the identification of segregation ratio；

Step 11, transgenic plant screen homozygous FIPV in T3 generation and are overexpressed transgenic line；Step 11, FIPV cross table Up to the identification of the DNA level of transgenic line, rna level and protein level.

3. the construction method of the transgenic arabidopsis strain of FIPV gene overexpression as claimed in claim 2, feature exist In the step 3 specifically includes: extracting arabidopsis total serum IgE, then carry out reverse transcription and obtain the first chain cDNA, as mould Plate, forward primer F and reverse primer R, the sequence of two primers for designing specific amplified FIPV coding region sequence (CDS) are as follows:

F(forward primer): 5 '-CGGGGTACCATGGAAGAGGACGATGAG-3 '；

R(reverse primer): 5 '-CCGCTCGAG TCATCCCACACATACTCT-3 '；

PCR reaction is carried out using F and R, connects the carrier pENTR3C compatible into Gateway after product gel recycling by digestion, Construct cloning vector pENTR 3C-FIPV, then by pENTR 3C-FIPV with contain 35S strong promoter and Hygromycin marker base The expression vector pMDC83 of cause carries out LR reaction, obtains the pMDC83-FIPV expression vector of FIPV overexpression.With the expression vector Transformed competence colibacillus bacillus coli DH 5 alpha, to being obtained after pMDC83-FIPV sequencing shown in arabidopsis FIPV gene C DS sequence:

ATGGAAGAGGACGATGAGTTCGGAGATCTATATTCCGACGTTCTCCAGCCGTTTCAACCT 60

CCCGTTGTTCTCCCTCCTCCGCCTCCTCTTCCTCACCGTTCAATCGACCTCAACCTCCGA 120

TCCCAAGATCAAGATGTCTCAGAACCTAATTCAGCTCCAATCTCTAGGGTTTCGGACAAC 180

GATGCCGTAAAATTATCTACTCAGGACGCGACTCGTCAAGCAATTGTCGATGGTGGCGGC 240

GACGATAAGGATATGAGCTTTGATATCGAAGAACCCGATGCCGATTCTACACCTACGATT 300

CCTGGTCTTTTCGTTACTGGAGCGTTACCTGGTTTGGCTACAGATCGAGGCGTTTCGCAA 360

GTTACGACAAGAATTGAGCAGCAGGTTGGTGGTGGTGGCGATGGAGGCTATGGAGGACAA 420

GGAGAAGGAGATGATTGGGATAGCGACAGTGAAGATGATTTGCAGATAGTGTTGAATGAT 480

AGTAGCCGTAACGTCATGATCGGAGGAGCTGATAGAAGATCAAGGATGGGAGATAATGAA 540

GATGACGATGATGAAGATGATGAAGACCCACTTGTTATAGTGGCCGACACGGATCCAAAT 600

CAACCTATGGAGGAGCAGATGTGGGGAGAAGATGGTCTTCAAGGGATTGAAGGAGATGGC 660

AAAGACGGAGGAGAAGCTGGCAAGGGAAGTGGACCAGGAGGTGCTACTGGACCGCCCAAA 720

GCAGGGTATAGCAGTCATGGGTATCATCCGTTTCATTCTCAGTTTAAGTATGTAAGACCG 780

GGGGCAGCTCCCATTCCTGGAGGTGCTGCATCTGTTGGTGGACCCTCCTCAGGTCAAGTT 840

CGTCCACCCGCCAACCTTGGTCCTATGGCTGGTCGTGGCAGAGGAGATTGGCGTCCACTG 900

GGAATGAGGAATGCTTCTGCTGCACAGAAAGGGTTCCACCAGCCTTGGGGTAGTAATACA 960

GCAGGGCGTGGACTGGACTTCACTCTTCCCTCTCACAAGACTATATTTGAGGTCGACATA 1020

GATAGTTTTGAAGAAAAGCCCTGGAGATATCCAGGAGTTGAGATGACAGACTACTTCAAC 1080

TTTGGACTAAATGAGGAGAGCTGGAAAGACTATTGCAAACAGCTGGACCAACACCGTATA 1140

CAGACTACGATGCAAAGCAGAATACGTGTTTATGAAAGCGGTAGAACGGATCAGGGTTAT 1200

GATCCAGATCTACCCCCAGAGTTAGCTGCAGCAACAGGGGCACAGGGTGTTCCCGTTGAT 1260

TCTTCAAATTTAGTGAAGCCAGACTCTGTTCAAGGTGATTCAGCGAAAGTGCCAGCCAAT 1320

GTTAGACCGACACTACCCCCTGGAAGACCAATACCTGTGGAGACTGGTTCTGGTGAACGT 1380

CTTCCGTCCATTGATACACGTGCTCCTCGGATGCGTGATCTAGATGCTATCATTGAGATT 1440

GTATGTCAGGATTCACATGAGGATGAACCCTCGGGTGAAAATGGCACAGATCAAGCTGAT 1500

AGTAGCCTTCCTGGAGAAAATGTACCAGTTGAGACTAGTTATGTTAACAACAAAAGACCT 1560

GACACGGAATCTGCTGAACATAGTCCTGCACAGGATGAGCCACATAAAAATCTTCTCAAA 1620

AAGCAAGACGATGAGATCTCTAGAAGCACAGATAGTGGCCAGAGTTTTCGTTCATCGTCT 1680

CCTGTTGGAGACAGAGGCACAAGGTCATCAAGTGTTGACCGCGAAGATGTGGGAGGTGAA 1740

GCTGGCAAAGATGCTGAGATGGGGGAGGAGCTTAAAATGAGTTTTACATCCCCTCAGTCA 1800

GCAGTGCAAGAAGATGATGGAGGGGAGTCAAAGACGGAGAGGAGTAGTGAAAGCAGCAAA 1860

GCAAGATCTGGAAGTCACAGAGATTTTCAGCAAGAAGAGGACGTTATTCAAGATAAGCAT 1920

TCTTCTCGACCAGCTAACAATAGGAAACAGTACGATAACAATGCACCTCATCAGAGCAGA 1980

AAGAATCAGGACAGAGGGAAGGAAATGGAAAGAACACGAGCGGCGAGCAAAGGTGGTAGA 2040

GAGAACTCTAATCCACATATGGAGCTTGATTCTACTTATATCTACTCAATTGCAAGTCGC 2100

GAGGATTTTGATAAAAGAAAAGAGCGAGATGTTGATGGCGCAGTCTGGCGCAGGAAAGAA 2160

GATGACCCATACAGTAGAAGAGGTGGGGATGAAGGGTCTAGAAAAAGGGATCGTGAAGAT 2220

GATCCAGGCTTTAGGCAGAGGGGTAAAATGCGCGAGAATGAAATACGCAGCAAAGATGAT 2280

CAGGTTCCTTCCAGAAAACATATGGATGATGCTGGTATGAGAAATATTTATGAACCGGAT 2340

GATCACATTAACAAGAGGAGGAAGGATGAAGAATACTTGAGAAGAAGCCGGCCTGAAAAA 2400

AATGAAATCTCATATGGTCAAAGGGAATCAATGAGCCGCGTGAAACGAGAACGTGATGAT 2460

AGGTTGGAGCATCAAAAGAGAGATGTCCAACATAAGATCAGAGATGATTTTGACGACCAC 2520

GGTTCTCTCAGGCAGAGAGATGATATCTATATGCAGAGGGATGGAAACGAGAGGTTGAGG 2580

GAGCGTGATGTTTTGGATAAATTGAAGCTGCCTCACGAGGATGGTATATCAGCACGAGGA 2640

AGAGAGAGGCAGGTGGCAGTAAGGGGCCACAGAGGTTCCGAAGATCGATCATCAAGGATG 2700

AAGGATGAGTATAAAGCTTCTGACAAAGAGCATGTCACGAAAGATACATTAAGGCATGCT 2760

AAACAGACAAAGAGAAGGGACTACCCTGGTGAAGAAAGTTCTTCCCATCATAGAGGACAT 2820

GAAGACTTCTCTGCACGGACAGACAACATAGTTAACAATGAGAAAAAACCAAGGCAGGAG 2880

AGGACAGGTGCTAAAATTGATAAGTTTATTGATACTTTGGATGGCCAGCGATTGCAAGAC 2940

AGAAAACATAAAGATTCTAGACGAAAGATTAAAGAACAGCGAGAGGGCACAGAATCACTT 3000

AGCAAGCAAGGGGAGCAAAATGGCAGTTCCGTAGTGACAGGATCAAAAGGAACCAACGAC 3060

GCAAGGAATTGCAGGAGTGAGATCCCACATCAGCCTAACACCGCCAAAAGACACAAGGAA 3120

AATGCATCCTCTGGTGATGAGATACACGATTCAAAGAGAGGACGTACAAAACTGGAGCGT 3180

TGGGCAAGCCACAAAGAGAGAGAAGATGCTGTCTCTGCCAAGTCATCATCCATTTCCTCA 3240

AAACTAGAAGAAAAGGAAAACAACACTAATGGCCGTCTTAGTGAACCTGTTCATGGTTCT 3300

ATTGGAAAGAGCCGGGATGTAACTGAAGAGAAAATTGGCCATGATCTTGCAGACACAAAA 3360

GATGGAAGCGAGAAGGGACCAGGAGACCGGCACTTGGATACGGTTGAGAAACTCAAGAAA 3420

CGCAGTGAAAGGTTCAAGCTTCCAATGCCCACGGAGAAAGACACCACGGGAGTAAAGAAA 3480

ATGGAGTCTGAGACACTGCCCTCCGCAAAAATTGAAGGCCCTGTGGATTCAGAGGTGAAA 3540

GCAGAGCGGCCAGCAAGGAAAAGACGGTGGACGAGTAGCTGAGAGTCATAAACTGGAGTT 3600

GAAATAAAAAGCGATGAACTGTGGCCCTCTTTGGCTGGGTTGAGGAAAGTTTGATTCTCA 3660

ATGTAGCTTACGATGCTTAAACGGATGACATGAGATTCTCCCATCCCACGGAGCAGTTGT 3720

AGCAGTAATGCGATGAAAGATCAAGCTCAATGGAGATTATTTAGGTGTTTAGGGAGAGTA 3780

TGTGTGGGATGA 3792。

4. the construction method of the transgenic arabidopsis strain of FIPV gene overexpression according to claim 1, feature It is, be cloned into the coding region sequence of arabidopsis FIPV gene for the first time, particular sequence is as follows:

ATGGAAGAGGACGATGAGTTCGGAGATCTATATTCCGACGTTCTCCAGCCGTTTCAACCT 60

CCCGTTGTTCTCCCTCCTCCGCCTCCTCTTCCTCACCGTTCAATCGACCTCAACCTCCGA 120

TCCCAAGATCAAGATGTCTCAGAACCTAATTCAGCTCCAATCTCTAGGGTTTCGGACAAC 180

GATGCCGTAAAATTATCTACTCAGGACGCGACTCGTCAAGCAATTGTCGATGGTGGCGGC 240

GACGATAAGGATATGAGCTTTGATATCGAAGAACCCGATGCCGATTCTACACCTACGATT 300

CCTGGTCTTTTCGTTACTGGAGCGTTACCTGGTTTGGCTACAGATCGAGGCGTTTCGCAA 360

GTTACGACAAGAATTGAGCAGCAGGTTGGTGGTGGTGGCGATGGAGGCTATGGAGGACAA 420

GGAGAAGGAGATGATTGGGATAGCGACAGTGAAGATGATTTGCAGATAGTGTTGAATGAT 480

AGTAGCCGTAACGTCATGATCGGAGGAGCTGATAGAAGATCAAGGATGGGAGATAATGAA 540

GATGACGATGATGAAGATGATGAAGACCCACTTGTTATAGTGGCCGACACGGATCCAAAT 600

CAACCTATGGAGGAGCAGATGTGGGGAGAAGATGGTCTTCAAGGGATTGAAGGAGATGGC 660

AAAGACGGAGGAGAAGCTGGCAAGGGAAGTGGACCAGGAGGTGCTACTGGACCGCCCAAA 720

GCAGGGTATAGCAGTCATGGGTATCATCCGTTTCATTCTCAGTTTAAGTATGTAAGACCG 780

GGGGCAGCTCCCATTCCTGGAGGTGCTGCATCTGTTGGTGGACCCTCCTCAGGTCAAGTT 840

CGTCCACCCGCCAACCTTGGTCCTATGGCTGGTCGTGGCAGAGGAGATTGGCGTCCACTG 900

GGAATGAGGAATGCTTCTGCTGCACAGAAAGGGTTCCACCAGCCTTGGGGTAGTAATACA 960

GCAGGGCGTGGACTGGACTTCACTCTTCCCTCTCACAAGACTATATTTGAGGTCGACATA 1020

GATAGTTTTGAAGAAAAGCCCTGGAGATATCCAGGAGTTGAGATGACAGACTACTTCAAC 1080

TTTGGACTAAATGAGGAGAGCTGGAAAGACTATTGCAAACAGCTGGACCAACACCGTATA 1140

CAGACTACGATGCAAAGCAGAATACGTGTTTATGAAAGCGGTAGAACGGATCAGGGTTAT 1200

GATCCAGATCTACCCCCAGAGTTAGCTGCAGCAACAGGGGCACAGGGTGTTCCCGTTGAT 1260

TCTTCAAATTTAGTGAAGCCAGACTCTGTTCAAGGTGATTCAGCGAAAGTGCCAGCCAAT 1320

GTTAGACCGACACTACCCCCTGGAAGACCAATACCTGTGGAGACTGGTTCTGGTGAACGT 1380

CTTCCGTCCATTGATACACGTGCTCCTCGGATGCGTGATCTAGATGCTATCATTGAGATT 1440

GTATGTCAGGATTCACATGAGGATGAACCCTCGGGTGAAAATGGCACAGATCAAGCTGAT 1500

AGTAGCCTTCCTGGAGAAAATGTACCAGTTGAGACTAGTTATGTTAACAACAAAAGACCT 1560

GACACGGAATCTGCTGAACATAGTCCTGCACAGGATGAGCCACATAAAAATCTTCTCAAA 1620

AAGCAAGACGATGAGATCTCTAGAAGCACAGATAGTGGCCAGAGTTTTCGTTCATCGTCT 1680

CCTGTTGGAGACAGAGGCACAAGGTCATCAAGTGTTGACCGCGAAGATGTGGGAGGTGAA 1740

GCTGGCAAAGATGCTGAGATGGGGGAGGAGCTTAAAATGAGTTTTACATCCCCTCAGTCA 1800

GCAGTGCAAGAAGATGATGGAGGGGAGTCAAAGACGGAGAGGAGTAGTGAAAGCAGCAAA 1860

GCAAGATCTGGAAGTCACAGAGATTTTCAGCAAGAAGAGGACGTTATTCAAGATAAGCAT 1920

TCTTCTCGACCAGCTAACAATAGGAAACAGTACGATAACAATGCACCTCATCAGAGCAGA 1980

AAGAATCAGGACAGAGGGAAGGAAATGGAAAGAACACGAGCGGCGAGCAAAGGTGGTAGA 2040

GAGAACTCTAATCCACATATGGAGCTTGATTCTACTTATATCTACTCAATTGCAAGTCGC 2100

GAGGATTTTGATAAAAGAAAAGAGCGAGATGTTGATGGCGCAGTCTGGCGCAGGAAAGAA 2160

GATGACCCATACAGTAGAAGAGGTGGGGATGAAGGGTCTAGAAAAAGGGATCGTGAAGAT 2220

GATCCAGGCTTTAGGCAGAGGGGTAAAATGCGCGAGAATGAAATACGCAGCAAAGATGAT 2280

CAGGTTCCTTCCAGAAAACATATGGATGATGCTGGTATGAGAAATATTTATGAACCGGAT 2340

GATCACATTAACAAGAGGAGGAAGGATGAAGAATACTTGAGAAGAAGCCGGCCTGAAAAA 2400

AATGAAATCTCATATGGTCAAAGGGAATCAATGAGCCGCGTGAAACGAGAACGTGATGAT 2460

AGGTTGGAGCATCAAAAGAGAGATGTCCAACATAAGATCAGAGATGATTTTGACGACCAC 2520

GGTTCTCTCAGGCAGAGAGATGATATCTATATGCAGAGGGATGGAAACGAGAGGTTGAGG 2580

GAGCGTGATGTTTTGGATAAATTGAAGCTGCCTCACGAGGATGGTATATCAGCACGAGGA 2640

AGAGAGAGGCAGGTGGCAGTAAGGGGCCACAGAGGTTCCGAAGATCGATCATCAAGGATG 2700

AAGGATGAGTATAAAGCTTCTGACAAAGAGCATGTCACGAAAGATACATTAAGGCATGCT 2760

AAACAGACAAAGAGAAGGGACTACCCTGGTGAAGAAAGTTCTTCCCATCATAGAGGACAT 2820

GAAGACTTCTCTGCACGGACAGACAACATAGTTAACAATGAGAAAAAACCAAGGCAGGAG 2880

AGGACAGGTGCTAAAATTGATAAGTTTATTGATACTTTGGATGGCCAGCGATTGCAAGAC 2940

AGAAAACATAAAGATTCTAGACGAAAGATTAAAGAACAGCGAGAGGGCACAGAATCACTT 3000

AGCAAGCAAGGGGAGCAAAATGGCAGTTCCGTAGTGACAGGATCAAAAGGAACCAACGAC 3060

GCAAGGAATTGCAGGAGTGAGATCCCACATCAGCCTAACACCGCCAAAAGACACAAGGAA 3120

AATGCATCCTCTGGTGATGAGATACACGATTCAAAGAGAGGACGTACAAAACTGGAGCGT 3180

TGGGCAAGCCACAAAGAGAGAGAAGATGCTGTCTCTGCCAAGTCATCATCCATTTCCTCA 3240

AAACTAGAAGAAAAGGAAAACAACACTAATGGCCGTCTTAGTGAACCTGTTCATGGTTCT 3300

ATTGGAAAGAGCCGGGATGTAACTGAAGAGAAAATTGGCCATGATCTTGCAGACACAAAA 3360

GATGGAAGCGAGAAGGGACCAGGAGACCGGCACTTGGATACGGTTGAGAAACTCAAGAAA 3420

CGCAGTGAAAGGTTCAAGCTTCCAATGCCCACGGAGAAAGACACCACGGGAGTAAAGAAA 3480

ATGGAGTCTGAGACACTGCCCTCCGCAAAAATTGAAGGCCCTGTGGATTCAGAGGTGAAA 3540

GCAGAGCGGCCAGCAAGGAAAAGACGGTGGACGAGTAGCTGAGAGTCATAAACTGGAGTT 3600

GAAATAAAAAGCGATGAACTGTGGCCCTCTTTGGCTGGGTTGAGGAAAGTTTGATTCTCA 3660

ATGTAGCTTACGATGCTTAAACGGATGACATGAGATTCTCCCATCCCACGGAGCAGTTGT 3720

AGCAGTAATGCGATGAAAGATCAAGCTCAATGGAGATTATTTAGGTGTTTAGGGAGAGTA 3780

TGTGTGGGATGA 3792。

What 5. a kind of construction method of transgenic arabidopsis strain of the FIPV gene overexpression as described in claim 1 constructed The transgenic arabidopsis strain of FIPV gene overexpression, which is characterized in that the transgenosis of the FIPV gene overexpression is quasi- Southern mustard strain is OE31 and OE51.