CN117230111A - Construction method and application of passion fruit virus mediated gene silencing system - Google Patents

Abstract

The application discloses a construction method and application of passion fruit virus mediated gene silencing system, wherein the construction method comprises the following steps: (1) extracting RNA of tender leaves of passion fruits, and carrying out reverse transcription to obtain cDNA; (2) selecting passion fruit PePDS gene as an interference fragment; (3) Designing specific upstream and downstream primers for PePDS amplification, carrying out cDNA fragment PCR amplification, and purifying to obtain a PePDS gene interference fragment; (4) The purified PePDS gene interference fragment is synchronously connected to a plant virus pTRV2 vector through a recombination reaction, and a passion fruit PePDS gene silencing system (pTRV 1, pTRV 2-PePDS) is constructed and obtained and named as TRV-PePDS. The TRV-PePDS constructed by the application can be applied to the processes of agrobacterium transformation and passion fruit leaf dip-dyeing, and has obvious silencing efficiency. The method can quickly and efficiently obtain the transgenic plant, and has strong stability and simple operation. The aim of quickly reducing the expression level of the target gene is fulfilled, the effect of knocking out the target gene is achieved, and the research progress of passion fruit functional genomics is quickened.

Description

Construction method and application of passion fruit virus-mediated gene silencing system

Technical field

The invention belongs to the field of plant genetic engineering technology, and is specifically a virus-mediated passion fruit gene silencing technology.

Background technique

Passion fruit (Passiflora edulis Sims), whose scientific name is Passiflora, belongs to the Passiflora family and the genus Passiflora. It is a tropical and subtropical perennial evergreen vine berry fruit tree. It is widely loved by consumers because of its rich and special aroma. In 2021, Song and Zhang et al. completed the sequencing of the entire passion fruit genome, providing an important reference for the study of passion fruit functional genomics. However, due to the high heterozygosity and complexity of the passion fruit genome, a genetic transformation system for passion fruit has not yet been established, which greatly limits the study of passion fruit gene functions.

It is difficult to obtain stable genetic mutants of plants, the cycle is long and the efficiency is low. Gene silencing is ubiquitous in organisms, manifested in resisting the invasion of foreign nucleic acids such as viruses and transposons, identifying and inhibiting the expression of foreign genes, and maintaining the stability of biological genomes. Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing technology. As an effective reverse genetics technology, it has been widely used in plant genetic engineering research. Its principle of action is that after the virus carrying the target gene fragment infects the plant, as the virus replicates and transcribes, it specifically induces sequence homologous gene mRNA degradation or modification such as methylation, thereby silencing the plant's endogenous genes and causing Changes in phenotype or physiological indicators, and then study the function of the target gene based on the phenotypic variation. Compared with traditional gene function research methods, VIGS can silence and functionally analyze target genes in the present day of infected plants without the need to develop stable transformants, and has the potential to silence single or multiple gene family members.

Because VIGS technology is simple to operate and takes a short time, once it was established, it was regarded as a powerful tool for studying plant gene functions. It has been deeply studied and widely used, and has been used for disease resistance in tobacco, tomatoes, wheat, rice and other plants. Functional gene studies on reaction, growth and development, and metabolism regulation. Among them, gene silencing induced by artificially modified tobacco rattle virus (TRV) is characterized by its high silencing efficiency and long duration (about 30-40 days). The host plant virus symptoms are mild and the silencing phenotype will not be masked. and other advantages, it has become the most widely used gene silencing system at present. However, so far, there have been no reports on establishing a virus-induced gene silencing system using Passiflora and Passiflora plants as experimental subjects.

As a tropical and subtropical perennial evergreen plant, passion fruit has a relatively short growth cycle and a small genome, which has great scientific research value. Therefore, there is a need to develop a fast, accurate and efficient gene silencing technology induced by passion fruit virus. Factors such as virus selection (TRV), target gene fragment selection, activity in mediating transformation of Agrobacterium, preparation of infection solution, virus vector inoculation technology, inoculation period, and light and temperature conditions of cultured plant materials after inoculation all affect the virus. The key to the successful establishment of an induced gene silencing system.

Contents of the invention

In order to solve the above technical problems, the present invention provides a construction method and application of a passion fruit virus-mediated gene silencing system, which can quickly and efficiently obtain transgenic plants with strong stability and simple operation. The purpose of quickly obtaining gene silencing was achieved and the research process of passion fruit functional genomics was accelerated.

In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

The construction method of the passion fruit virus-mediated gene silencing system includes the following steps:

(1) Extract RNA from young passion fruit leaves and reverse-transcribe to obtain cDNA;

(2) Select the passion fruit phytoene dehydrogenase gene PePDS as a conservative interference fragment, as shown in SEQ ID NO.1;

SEQ ID NO.1：

ATGCCCTTCCACAACCAAATTTTCCAAGTGAAGTAATTCTTGCTTCCAAGAAATGATTCTT

CATGCGAGTGTTCCTGCTTTGAATTTTAGCCGGCAAAGCAATGCCTTGGATGTTCGAAAC

TGCCTTTCTTCTTCCCTCAGATGCGGTGCTCATCCTTCTTCTTTAAAAATTCAATCTGCAAATCCCCGTAAAGCAAGTCTCAGGAGTGTCT.

(3) Design of specific primers for amplification of passion fruit phytoene dehydrogenase gene PePDS, upstream primer: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCCCTTCCACACCAAA-3', downstream primer: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAGACACTCCTGAGACTTG-3', and perform cDNA fragment PCR After amplification and purification, the PePDS gene interference fragment is obtained;

PCR amplification was carried out using a 25 μL system, including: 18.5 μL double-distilled water; 1 μL cDNA; 2.5 μL dNTP; 1 μL heat-resistant Taq enzyme; 1 μL upstream primer and 1 μL downstream primer.

The PCR amplification program was pre-denaturation at 98°C for 3 min; 34 cycles of denaturation at 98°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s; post-extension at 72°C for 5 min, and storage at 12°C for later use after purification.

(4) The purified PePDS gene interference fragment was synchronously connected to the plant virus pTRV2 vector through a recombination reaction to construct the passion fruit phytoene dehydrogenase gene PePDS silencing system. After successful transformation, it is pTRV2-PePDS.

The step (4) includes BP reaction and LR reaction; the BP reaction is to combine 1 μL of the purified PePDS gene interference fragment, 1 μL of pDONR 207 plasmid, 0.5 μL of Gateway BP Clonase, and ligate overnight at 22°C; the ligated product is transformed into E. coli DH5α competent cells were spread on LB plate culture medium containing 100 mg/L gentamicin sulfate antibiotic and cultured overnight at 37°C; single clones were picked into liquid LB culture medium containing 100 mg/L gentamicin sulfate antibiotic. Medium, 37℃, 200rpm/min shaking culture for 12h, extract the plasmid, and sequence it correctly for later use;

The LR reaction is 1 μL of transformed pDONR 207 plasmid containing the PePDS gene fragment, 1 μL of pTRV2 plasmid, and 0.5 μL of Gateway LR Clonase, placed at 22°C, and ligated in the dark for 3-5 h; the ligation product was transformed into E. coli DH5α competent cells, and coated Distribute on LB plate culture medium containing 100mg/L kanamycin antibiotic, incubate overnight at 37℃, pick single clones into liquid LB culture medium containing 100mg/L kanamycin antibiotic, 37℃, 250rpm/min After shaking culture for 12 hours, PCR identification was performed, the plasmid was extracted, and the successfully connected vector was named pTRV2-PePDS.

The construction method of the passion fruit virus-mediated gene silencing system of the present invention is applied in identifying the function of the passion fruit phytoene dehydrogenase gene PePDS.

The present invention is applied in Agrobacterium transformation. The specific operation is to use electroporation method to transform pTRV1, pTRV2 and the constructed pTRV2-PePDS plasmid into Agrobacterium. The specific operation is: take pTRV1, pTRV2 and the constructed pTRV2-PePDS plasmid and add it to Agrobacterium tumefaciens GV3101 competent cells, mix well and transfer to a pre-cooled electroporation cup, and then put it into an electroporation instrument for electroporation transformation; after completing the electroporation, quickly add blank LB medium to the electroporation cup, resuspend the cells and mix Transfer the liquid to a sterilized centrifuge tube, then put it into a shaker at 28°C and resuscitate at 180-220 rpm/min for 2 hours; centrifuge, discard the supernatant, leave the bacterial liquid, and apply it to Kan ⁺ antibiotics and rifampicin antibiotics (Rif ) LB plate culture medium, culture it upside down at 28°C for 48 hours, grow a single colony, pick the single clone into a liquid LB medium containing 100mg/L Kan ⁺ antibiotics and Rif ⁺ antibiotics, shake at 28°C at 180-220rpm/min After culturing for 12 hours, PCR identification was carried out to obtain a positive Agrobacterium GV3101 strain containing pTRV1, pTRV2 and pTRV2-PePDS plasmids. The virus composed of pTRV1 and pTRV2 was named: TRV-00, and the virus composed of pTRV1 and pTRV2-PePDS was named: TRV. -PePDS. Use 50% glycerin to preserve bacteria and place it at -80°C for later use.

The application of the present invention in infecting passion fruit leaves includes the following specific operations: (1) Prepare the infection solution, mix the Agrobacterium GV3101 bacterial solution containing pTRV1, pTRV2 and pTRV2-PePDS with an LB plate containing antibiotics, and culture at 28°C ; Pick a single clone, add it to LB liquid culture medium, and culture it with shaking and in the dark for 10-12 hours at 28°C to achieve single clone amplification; add LB containing antibiotics to the amplified single clone liquid at a ratio of 1:100 Liquid culture medium, conduct expanded culture, culture at 28°C in the dark for 12-24 hours; centrifuge, discard the LB culture medium, and collect the bacterial cells; use the infection solution to resuspend the bacterial cells, add an appropriate amount of infection buffer, and adjust the bacterial liquid OD (600) to 1.8; mix pTRV1 and pTRV2 (TRV-00), pTRV1 and pTRV2-PePDS (TRV-PePDS) with OD (600) = 1.8 at a volume of 1:1, and incubate at room temperature in the dark for 3 hours to obtain Agrobacterium Infectious fluid.

The infection buffer is 10mM morpholinoethanesulfonic acid, 100μM acetosyringone and 10mM MgC1 ₂ .

(2) Inject the TRV-00 and TRV-PePDS Agrobacterium infection solutions into the veinless area on the back of young passion fruit leaves, allowing the infection solution to spread throughout the leaves. After infection, the passion fruit seedlings are cultured in the dark for 48 hours, and then cultured normally. , 25℃, 16/8h day and night cycle, and then conduct observation and identification.

Beneficial effects of the present invention:

(1) The passion fruit virus-mediated gene silencing system of the present invention can quickly and efficiently obtain transgenic plants, has strong stability, and is simple to operate. It can quickly reduce the expression level of the target gene, achieve the effect of knocking out the target gene, and accelerate the growth of passion fruit. Research progress in functional genomics.

(2) During the infection experiment of passion fruit leaves, after infection with the TRV-PePDS vector constructed in the present invention, the passion fruit leaves showed obvious whitening phenomenon; indicating that the method of the present invention exerted a silencing effect on the passion fruit PePDS gene. And in the fluorescence quantitative PCR detection of the expression level of the PePDS gene in passion fruit, the expression level of the PePDS gene in the TRV-PePDS infected group of the present invention was only 0.38 of that of the control group, which was significantly lower than that of the control group. After infection by TRV-PePDS of the present invention, the expression level of the passion fruit PePDS gene is significantly reduced. It shows that the method of the present invention can effectively verify the expression of passion fruit genes.

Description of drawings

Figure 1 shows the electrophoresis detection chart of passion fruit PePDS gene PCR product;

Figure 2 is a comparison chart of passion fruit PePDS gene silencing efficiency;

Figure 3 is a comparison chart of PePDS gene expression detection 20 days after plant infection;

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only for the purpose of this application. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts should fall within the scope of protection of this application.

Example

1. Construction of passion fruit virus-mediated gene silencing system

1. Seed sowing: Take passion fruit seeds, first soak them in 20mM GA3 solution, and then place them in a 30°C incubator overnight; then sow them in loose nutrient soil, cover them with a seedling cover, and close the vents to maintain soil temperature and humidity. ; Place the sown seedling trays in a greenhouse at 30°C with a 16/8h day and night cycle. Two cotyledons and two true leaves will grow after one month.

2. Vector construction: Use genetic engineering methods to connect the amplified passion fruit phytoene dehydrogenase gene (hereinafter referred to as: PePDS) fragment into the TRV virus-induced gene silencing vector pTRV2 to obtain the pTRV2-PePDS vector. . The specific operations are as follows:

(1) RNA extraction and cDNA synthesis: According to the instructions, use the Trizol method to extract total RNA from young passion fruit leaves; according to the instructions of the reverse transcription kit, use 1 μg RNA as a template for cDNA synthesis. After completion, dilute 5 times for later use.

(2) Select the passion fruit phytoene dehydrogenase gene PePDS as a conservative interference fragment, as shown in SEQ ID NO.1.

PDS is the conserved interference fragment sequence:

ATGCCCTTCCACAACCAAATTTTCCAAGTGAAGTAATTCTTGCTTCCAAGAAATGATTCTT

CATGCGAGTGTTCCTGCTTTGAATTTTAGCCGGCAAAGCAATGCCTTGGATGTTCGAAAC

TGCCTTTCTTCTTCCCTCAGATGCGGTGCTCATCCTTCTTCTTTAAAAATTCAATCTGCAA

ATCCCCGTAAAGCAAGTCTCAGGAGTGTCT

Design primers for PDS gene for PCR amplification:

Upstream primer:

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCCCTTCCACACCAAA-3’,

Downstream primer: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGACACTCCTGAGACTTG-3’, the PCR product is 269 bp in size.

Use the above-synthesized passion fruit cDNA as a template and use a 25 μL system for amplification; including: 18.5 μL of double distilled water; 1 μL of cDNA; 2.5 μL of dNTP; 1 μL of heat-resistant Taq enzyme; 1 μL of upstream primer and 1 μL of downstream primer;

The amplification program is pre-denaturation at 98°C for 3 minutes; 34 cycles of denaturation at 98°C for 30 seconds, annealing at 62°C for 30 seconds, extension at 72°C for 30 seconds; post-extension at 72°C for 5 minutes, and storage at 12°C; the PCR product is detected as a single band by electrophoresis, such as As shown in Figure 1, it can be seen from Figure 1 that the size of the PePDS gene fragment is 269 bp and will be used after purification.

(3) BP reaction, combine 1 μL of the PePDS gene fragment purified in the above step (2), 1 μL of pDONR 207 plasmid, and 0.5 μL of Gateway BP Clonase, and ligate at 22°C overnight. Transform the ligation product into Escherichia coli DH5α competent cells, spread on LB plate medium containing 100 mg/L gentamicin sulfate (Gen ⁺ ) antibiotic, and incubate overnight at 37°C; single clones are picked until they contain 100 mg/L Gen ⁺ Antibiotic liquid LB culture medium, 37℃, 200rpm/min shaking culture for 12 hours, extract the plasmid, and sequence it correctly for later use;

(4) LR reaction, add 1 μL of pDONR 207 plasmid (containing the PePDS gene fragment) transformed in step (3), 1 μL of pTRV2 plasmid, and 0.5 μL of Gateway LR Clonase, place at 22°C, and ligate in the dark for 3h-5h; the ligation product is transformed into the large intestine Bacillus DH5α competent cells were spread on LB plate culture medium containing 100 mg/L kanamycin (Kan ⁺ ) antibiotic, incubated overnight at 37°C, and single clones were picked into liquid LB culture medium containing 100 mg/L Kan antibiotic. Medium, 37°C, 250 rpm shaking culture for 12 hours, PCR identification, extract plasmid, set aside. The successfully connected vector was named pTRV2-PePDS.

2. Agrobacterium transformation of pTRV2-PePDS vector

pTRV1, pTRV2 and the constructed pTRV2-PePDS plasmids were transformed into Agrobacterium GV3101 competent cells by electroporation respectively. After bacterial liquid PCR identification, Agrobacterium GV3101 containing plasmids pTRV1, pTRV2 and pTRV2-PePDS respectively was obtained. For positive strains, the virus composed of pTRV1 and pTRV2 is named: TRV-00; the virus composed of pTRV1 and pTRV2-PePDS is named: TRV-PePDS. Use 50% glycerol to preserve bacteria and place it at -80°C for later use;

Specific steps of the electroporation method: slowly add 5 μL of pTRV1, pTRV2, and pTRV2-PePDS plasmids to 50 μL of Agrobacterium GV3101 competent cells, mix well, transfer to a pre-cooled electroporation cup, and then put it into an electroporation machine at a voltage of 1.2 kV. Electroporation transformation; complete the electroporation, quickly add 600 μL of blank LB culture medium to the electroporation cup, resuspend the cells, transfer the mixture to a sterilized 1.5 mL centrifuge tube, and then place it in a shaker at 28°C and resuscitate at 180-220 rpm/min. 2h; centrifuge at 4000rpm/min for 5min, discard the supernatant, and the remaining 100μL bacterial liquid is spread on the LB plate medium containing 100mg/L Kan ⁺ antibiotics and Rif ⁺ , and incubate upside down at 28°C for 48h. Single clones grow and are picked. Single clone into liquid LB medium containing 100 mg/L Kan ⁺ antibiotic and Rif ⁺ antibiotic, culture at 28°C with shaking at 180-220 rpm/min for 12 hours, identify by PCR, and store at -80°C for later use.

3. Plant infection

1. Prepare infection solution

(1) Streak the stored pTRV1, pTRV2 and pTRV2-PePDS Agrobacterium GV3101 bacterial solution on an LB plate containing antibiotics and culture it at 28°C; then pick a single clone and add it to 1.5 mL of LB liquid culture medium at 28°C. Next, shake at 180-200rpm/min and culture in the dark for 10-12h to achieve monoclonal amplification;

(2) Add the amplified monoclonal bacterial liquid to LB liquid culture medium containing antibiotics at a ratio of 1:100, and conduct expanded culture at 28°C, 200 rpm/min, and culture in the dark for 12-24 hours until OD (600) = Around 1.2.

(3) 4000rpm, centrifuge for 15 minutes, discard the LB culture medium, and collect the bacteria;

(4) Use the infection solution to resuspend the bacteria, add an appropriate amount of infection buffer, and adjust the OD (600) of the bacterial solution to 1.8. Mix pTRV1 with OD(600)=1.8 with pTRV2 and pTRV2-PePDS at a volume of 1:1 respectively, and incubate in the dark at room temperature for 3 hours to form two Agrobacterium infection solutions, TRV-00 and TRV-PePDS.

Infection buffer: 10mM morpholinoethanesulfonic acid (MES), 100μM acetosyringone (AS), 10mM MgC1 ₂ .

2. Infection by injection

(1) Select passion fruit seedlings that are in good growth condition and of suitable size (including two true leaves and two cotyledons) and mark them;

(2) Take out the Agrobacterium infection solution that has been placed in the dark, mix it well, use a 1mL syringe to absorb a small amount of the infection solution, pull out the needle, put the tip of the syringe to the back of the leaf without veins, and gently push the tail of the syringe to infect The liquid spreads on the back of the leaves, and the operation is repeated several times until the infection liquid spreads throughout the leaves. For passion fruit leaves that are difficult to spread, you can use a syringe needle to gently prick a few small holes on the back of the leaf without veins. It is better not to penetrate the leaf. After pricking the holes, use a syringe to inject, which can spread quickly.

(3) After the infection is completed, first, control and infected passion fruit seedlings are cultured in the dark for 48 hours, and then cultured normally at 25°C with a 16/8 hour day and night cycle.

4. Gene silencing efficiency test

After 10 days of infection, comparative observations were made, as shown in Figure 2. Figure 2(a) shows that the passion fruit leaves in the TRV-00 control group develop normally; Figure 2(b) shows that after infection by TRV-PePDS of the present invention, the passion fruit leaves appear albino; indicating that the method of the present invention enables the passion fruit PePDS gene to exert its functions Silence effect.

After 20 days of infection, fluorescence quantitative PCR was used to detect the expression of the PePDS gene in passion fruit, and Figure 3 was obtained. As can be seen from Figure 3, compared with the PePDS gene expression in the control group (TRV-00), the PePDS gene expression in the TRV-PePDS infection group of the present invention is only 0.38, which is significantly lower than the control group. After infection by TRV-PePDS of the present invention, the expression level of the passion fruit PePDS gene is significantly reduced. It shows that the method of the present invention can effectively verify the expression of passion fruit genes.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (8)

1. The construction method of passion fruit virus mediated gene silencing system is characterized by comprising the following steps: the method comprises the following steps:

(1) Extracting RNA of passion fruit tender leaves, and carrying out reverse transcription to obtain cDNA;

(2) Selecting passion fruit phytoene dehydrogenase gene PePDS as a conserved interference fragment, as shown in SEQ ID NO. 1;

(3) Specific primer design of passion fruit phytoene dehydrogenase gene PePDS amplification, and upstream primer: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCCCTTCCACACCAAA-3', downstream primer: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAGACACTCCTGAGACTTG-3', performing cDNA fragment PCR amplification and purification to obtain a PePDS gene interference fragment;

(4) The purified PePDS gene interference fragment is synchronously connected to a plant virus pTRV2 carrier through a recombination reaction, and a passion fruit phytoene dehydrogenase gene PePDS silencing system which is pTRV1, pTRV2-PePDS is constructed, and after the transformation is successful, the TRV-PePDS is obtained.

2. The method for constructing passion fruit virus mediated gene silencing system according to claim 1, wherein: the step (3) of PCR amplification is carried out by adopting a 25 mu L system, and comprises the following steps: 18.5 mu L of double distilled water; 1 μl of cDNA; dNTP 2.5. Mu.L; 1 mu L of thermostable Taq enzyme; 1. Mu.L of the upstream primer and 1. Mu.L of the downstream primer.

3. The method for constructing passion fruit virus mediated gene silencing system according to claim 2, wherein: the PCR amplification procedure is that the PCR amplification is performed for 3min at 98 ℃;34 cycles of denaturation at 98℃for 30s, annealing at 62℃for 30s, and extension at 72℃for 30s; extending for 5min at 72deg.C, preserving at 12deg.C, and purifying.

4. The method for constructing passion fruit virus mediated gene silencing system according to claim 1, wherein: the step (4) comprises a BP reaction and an LR reaction; the BP reaction is to obtain 1 mu L of PePDS gene interference fragment after purification, 1 mu L of pDONR 207 plasmid, gateway BP Clonase 0.5.5 mu L and overnight connection at 22 ℃; e.coli DH5 alpha competent cells are transformed from the product after connection, and the cells are coated on LB plate medium containing 100mg/L gentamycin sulfate antibiotics, and are cultured in an inverted way at 37 ℃ overnight; selecting a monoclonal to a liquid LB culture medium containing 100mg/L gentamycin sulfate antibiotics, performing shake culture at 37 ℃ for 12 hours at 200 revolutions per minute, extracting plasmids, and sequencing correctly for later use;

the LR reaction is that 1 mu L of the transformed pDONR 207 plasmid containing the PePDS gene fragment, 1 mu L of the pTRV2 plasmid and Gateway LR Clonase 0.5.5 mu L are placed at 22 ℃ and connected in darkness for 3-5 hours; e.coli DH5 alpha competent cells are transformed by the connection product, the cells are coated on LB plate medium containing 100mg/L of the calicheamicin antibiotics, the cells are cultured in an inverted way at 37 ℃ overnight, the monoclonal cells are picked up to be cultured in liquid LB medium containing 100mg/L of the calicheamicin antibiotics by shaking at 37 ℃ for 12 hours at 250 revolutions per minute, the plasmids are identified by PCR, and the successfully connected vector is named pTRV2-PePDS.

5. Use of the method for constructing passion fruit virus mediated gene silencing system according to any of claims 1-4 for identifying passion fruit phytoene dehydrogenase gene PePDS gene function.

6. The use according to claim 5, characterized in that: the pTRV1, pTRV2 and the constructed pTRV2-PePDS plasmid are subjected to agrobacterium transformation by an electrotransformation method, and the specific operations are as follows: adding pTRV1, pTRV2 and constructed pTRV2-PePDS plasmid into competent cells of agrobacterium GV3101, mixing, transferring to a precooled electric rotating cup, and then placing into an electric rotating instrument for electric shock conversion; after the electrotransformation is finished, a blank LB culture medium is rapidly added into an electrotransformation cup, after the cells are resuspended, the mixed solution is transferred into a sterilized centrifuge tube, and then the centrifuge tube is placed into a shaking table at 28 ℃ for resuscitation at 180-220rpm for 2 hours; centrifuging, discarding the supernatant, leaving bacterial liquid, coating on LB plate culture medium of Kan antibiotics and rifampicin antibiotics, inversely culturing at 28 ℃ for 48 hours, growing monoclonal, picking the monoclonal into liquid LB culture medium containing 100mg/L Kan antibiotics and Rif antibiotics, shake culturing at 28 ℃ at 180-220rpm/min for 12 hours, and carrying out PCR identification to obtain agrobacterium GV3101 positive strain containing plasmids pTRV1, pTRV2 and pTRV2-PePDS, wherein the viruses formed by pTRV1 and pTRV2 are named as follows: TRV-00; the viruses consisting of pTRV1 and pTRV2-PePDS are designated: TRV-PePDS is sterilized with 50% glycerol and placed at-80 ℃ for standby.

7. Use according to claim 5 or 6, characterized in that: application in the infection of passion fruit leaves, and the specific operation is as follows: (1) Preparing an invader solution, and culturing agrobacterium GV3101 bacterial solution containing pTRV1, pTRV2 and pTRV2-PePDS plasmids, streaking and LB plates containing antibiotics at 28 ℃; selecting a monoclonal, adding the monoclonal into an LB liquid culture medium, and shake light-shielding culture for 10-12 hours at the temperature of 28 ℃ to realize monoclonal amplification; adding the amplified monoclonal bacterial liquid into LB liquid culture medium containing antibiotics according to a ratio of 1:100, performing amplification culture, and performing light-shielding culture for 12-24 hours at 28 ℃; centrifuging, discarding LB culture solution, and collecting thalli; re-suspending the bacteria by using an infection solution, adding a proper amount of infection buffer solution, and regulating the OD (600) of the bacteria solution to 1.8; pTRV1 and pTRV2, pTRV1 and pTRV2-PePDS with OD (600) =1.8 were combined at 1:1 volume mixing, and incubating for 3 hours at room temperature in a dark place to obtain an agrobacterium infection solution;

(2) And (3) injecting the agrobacterium infection liquid to the position without veins on the back of the tender leaf blade of the passion fruit, so that the infection liquid diffuses the whole blade.

8. The use according to claim 7, characterized in that: the infection buffer is 10mM morpholinoethanesulfonic acid, 100 mu M acetosyringone and 10mM MgC1 ₂ 。