CN116355951B - Construction method of melon VIGS silencing system based on bud vacuum infection - Google Patents

Abstract

The invention discloses a construction method of a melon VIGS silencing system based on bud vacuum infection, which comprises the steps of selecting pTRSV2 as a recombinant vector, removing melon seeds with bud germination length of 1-2cm from seed coats as a receptor material, and adopting the volume ratio of 1:1 and pTRSV 2-target gene recombinant vector, and the infection conditions are as follows: the infection pressure is 0.9kPa, the bacterial liquid concentration OD600 = 1.0, and the infection time is 5min. Meanwhile, the melon phytoene dehydrogenase gene is adoptedCmPDSFor example, the application verification of the system of the invention shows that the silencing system can obviously reduce the content of muskmelonCmPDSThe plant silencing rate was 85%. Compared with the cotyledon injection method, the melon bud vacuum infection method has faster aging, can generally quickly show the silencing phenotype in a period of 1 week and one heart after infection, is favorable for quickly digging and researching related genes of important agronomic characters of the melon in a seedling stage, and overcomes the defects of immature genetic transformation system and low efficiency of the melonThe disadvantage of long cycle.

Description

A method to construct a muskmelon VIGS silencing system based on vacuum infection of buds

Technical field

The invention relates to the technical field of plant genetic engineering, specifically a construction method and application of a melon VIGS silencing system based on vacuum infection of buds.

Background technique

Melon (Cucumis melo L.) originates from tropical and subtropical regions. It is an important cosmopolitan fruit and is widely cultivated around the world. For more than 30 consecutive years, China has become the country with the largest melon planting area and the highest yield in the world. In recent years, modern molecular biology technology has been used to excavate key genes for important agronomic traits such as stress resistance, growth and development of melon, analyze their gene functions, and apply them to variety selection, which has greatly shortened the breeding process. Due to the immaturity and long cycle of the melon transgenic system, gene function verification mostly relies on heterologous expression systems in model plants such as Arabidopsis thaliana and tobacco. Virus-induced gene silencing (VIGS) is an RNA-mediated defense mechanism that silences and expresses endogenous genes in plants by inserting recombinant viral vectors constructed from target gene fragments into the viral genome. Compared with transgenic technology, VIGS silencing technology is simple, efficient and has a short cycle. In addition, the time and silencing efficiency of plants with different silencing system methods are also different. It is very important to develop an efficient VIGS system that can quickly display the silencing phenotype in the early seedling stage.

Phytoene dehydrogenase is a rate-limiting enzyme in the carotenoid biosynthesis pathway. It catalyzes the dehydrogenation of colorless phytoene to lycopene, which is then converted into colored carotenoids, which has an effect on chloroplasts. certain protective effect. When the gene encoding this enzyme is silenced, the plant loses the photoprotective effect of carotenoids, leading to the degradation of chlorophyll, causing leaves and other tissues to show a whitening effect. This gene can be used as a reporter gene for the gene silencing system, through leaf whitening. The time of phenotype appearance, PDS gene expression level, leaf chlorophyll content and other indicators can verify the feasibility of the VIGS silencing system.

The present invention constructs a melon VIGS silencing system based on vacuum infection of buds, and selects the melon phytoene dehydrogenase gene CmPDS as the reporter gene of the silencing system. Taking this gene as an example, the VIGS silencing system constructed by the present invention is used The method carried out gene silencing, and verified the feasibility of the present invention through phenotypic statistics of silenced plants and detection of CmPDS gene expression. At the same time, it was compared with the VIGS silencing system based on cotyledon injection method, showing the high efficiency of the present invention.

Contents of the invention

(1) Technical problems solved

In view of the shortcomings of the existing technology, the present invention provides a method for constructing a melon VIGS silencing system based on vacuum infection of buds.

(2) Technical solutions

In order to achieve the above objectives, the present invention is realized through the following technical solutions: a method for constructing a melon VIGS silencing system based on vacuum infection of buds. The construction steps include:

a. Cloning of specific fragments of the target gene;

b. Construction of pTRSV2-target gene specific fragment recombinant vector;

c. pTRSV2-target gene specific fragment recombinant vector was transferred into Agrobacterium;

d. Preparation of infection receptor materials;

e. Vacuum infection penetrates muskmelon seeds;

f. After infection, the material is cultured in the dark and then cultured regularly.

As a preferred technical solution of the present invention: in step a, the target gene-specific fragment should be a specific fragment of about 300 bp specific to the target gene.

As the preferred technical solution of the present invention: in step b, the vector is selected from Tobacco ringspot virus TRSV vector, auxiliary vector pTRSV1, and recombinant vector pTRSV2 (this vector was donated by Geng Chao of Shandong Agricultural University to Fang et al. ., 2021), the target gene-specific fragment is recombined into the pTRSV2 vector.

As the preferred technical solution of the present invention: in step c, the Agrobacterium strain is GV3101.

As a preferred technical solution of the present invention: in step d, the infection receptor material is a melon seed with a bud germination length of 1 to 2 cm, and the seed coat is removed. The culture method is to remove the seed coat from the melon seeds and place them on a semi-MS solid medium. , germinated in a dark artificial climate chamber at 30°C for 24-36h. The preparation method of the semi-MS solid medium was 2.22gMS, 15g agar, dissolved in 1L distilled water, adjusted to pH 5.8, and sterilized at 121°C for 20 minutes. , stored at 4°C.

As the preferred technical solution of the present invention: in step e, the infection pressure is 0.9kPa, the bacterial liquid concentration OD600 is 1.0, and the bacterial liquid is a resuspension of the pTRSV1 vector containing the auxiliary vector and the pTRSV2-target gene recombinant vector in a volume ratio of 1:1. The mixed bacterial solution of liquid, the infection time is 5 minutes. The method for preparing the bacterial resuspension is to dissolve 4.43g of MS and 30g of sucrose in 1L of distilled water, adjust the pH to 5.8, and after sterilizing at 121°C for 20 minutes, add 0.04g of acetosyringone (AS) in the ultra-clean bench. , 0.25g MES; before vacuum infection, soak the melon seeds with a bud germination length of 1 to 2 cm with the seed coat removed in the mixed bacterial solution for 1-2 hours.

As the preferred technical solution of the present invention: in step f, the culture conditions of the infected receptor material are: first culture on the co-culture medium at 25°C in the dark for 3 to 5 days, and then colonize on peat, vermiculite, Culture in perlite (ratio 1:1:1) mixed matrix at 25°C, photoperiod 16h/8h (light/dark), relative humidity 75%, light intensity 22000Lx.

As a preferred technical solution of the present invention: in step f, the dark culture method is to place the vacuum-infected muskmelon seeds in a co-culture medium and culture them for 3 to 5 days under dark conditions at 25°C. The culture medium preparation method is to dissolve 4.43g MS, 30g sucrose, and 15g agar in 1L distilled water, adjust the pH to 5.8, and after sterilizing at 121°C for 20 minutes, add 0.04g acetosyringone AS, 0.25 gMES.

As the preferred technical solution of the present invention: in step f, the conventional culture conditions are: the day and night photoperiod of the incubator is light/dark: 16h/8h, the temperature is 25°C, the relative humidity is 75%, and the light intensity is 22000Lx. Culture was performed routinely until a silent albino phenotype appeared.

(3) Beneficial effects

The invention provides a method for constructing a muskmelon VIGS silencing system based on vacuum infection of buds. It has the following beneficial effects:

The melon VIGS system of the present invention utilizes the method of vacuum infection and penetration of buds, strictly controls the vacuum infection pressure, bacterial solution infection concentration and infection time, and infects the melon seeds with the TRSV (Tobacco Ringspot Virus) vector containing the target fragment. , quickly and efficiently obtain gene-silenced plants to verify the function of the corresponding gene. Compared with the melon genetic transformation system, the operation is simple and fast, and it overcomes the shortcomings of the melon genetic transformation system: instability, low efficiency, and long cycle. At the same time, this method also overcomes the problem that endogenous genes of melon can only be overexpressed but not gene silenced in heterologous model crops; in addition, the system of the present invention can quickly produce a silent phenotype in the first true leaf at the seedling stage, which is rapid. This laid the foundation for research on muskmelon stress, growth and development and other related gene functions.

Description of drawings

Figure 1 shows the amplification of the CmPDS gene silencing specific fragment; Lane 1: DL2000 DNA Marker Lane 2: Negative control Lane 3: CmPDS silencing specific target fragment;

Figure 2 shows the PCR detection of positive clones of pTRSV2-CmPDS recombinant vector; Lane 1: DL2000 DNA Marker Lane 2: Negative control Lanes 3-6: pTRSV2-CmPDS PCR product;

Figure 3 shows the PCR detection of positive clones transformed by pTRSV2-CmPDS into Agrobacterium; lane 1: DL2000 DNA Marker lane 2: negative control lanes 3-4: pTRSV2 empty lanes 5-6: pTRSV2-CmPDS PCR product;

Figure 4 shows melon seeds placed flat on the germination medium;

Figure 5 shows melon seeds with appropriate bud length for infection;

Figure 6 Phenotype of silent melon plants after vacuum infection;

Figure 7 Relative expression of CmPDS gene in muskmelon silenced plants after vacuum infection;

Figure 8 Phenotype and chlorophyll content of melon silenced plants at the three-leaf and one-center stage after vacuum infection with pTRSV2-CmPDS;

Figure 9 Phenotype of melon plants after cotyledon injection (A) and relative expression of CmPDS gene (B).

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Please refer to Figures 1-5. The embodiment of the present invention provides a technical solution: a method for constructing a melon VIGS silencing system based on vacuum infection of buds. The construction steps include:

a. Cloning of specific fragments of the target gene;

b. Construction of pTRSV2-target gene specific fragment recombinant vector;

c. pTRSV2-target gene specific fragment recombinant vector was transferred into Agrobacterium;

d. Preparation of infection receptor materials;

e. Vacuum infection penetrates muskmelon seeds;

f. After infection, the material is cultured in the dark and then cultured regularly.

As a preferred technical solution of the present invention: in step a, the target gene-specific fragment should be a specific fragment of about 300 bp specific to the target gene.

As a preferred technical solution of the present invention: in step b, the vector is selected using Tobacco ringspot virus TRSV vector, auxiliary vector pTRSV1, and recombinant vector pTRSV2; the target gene specific fragment is recombined into the pTRSV2 vector.

As the preferred technical solution of the present invention: in step c, the Agrobacterium strain is GV3101.

As a preferred technical solution of the present invention: in step d, the infection receptor material is a melon seed with a bud germination length of 1 to 2 cm, and the seed coat is removed. The culture method is to remove the seed coat from the melon seeds and place them on a semi-MS solid medium. , germinated in a dark artificial climate chamber at 30°C for 24-36h. The preparation method of the semi-MS solid medium was 2.22gMS, 15g agar, dissolved in 1L distilled water, adjusted to pH 5.8, and sterilized at 121°C for 20 minutes. , stored at 4°C.

As the preferred technical solution of the present invention: in step e, the infection pressure is 0.9kPa, the bacterial liquid concentration OD600 is 1.0, and the bacterial liquid is a resuspension of the pTRSV1 vector containing the auxiliary vector and the pTRSV2-target gene recombinant vector in a volume ratio of 1:1. The mixed bacterial solution of liquid, the infection time is 5 minutes. The method for preparing the bacterial resuspension is to dissolve 4.43g of MS and 30g of sucrose in 1L of distilled water, adjust the pH to 5.8, and after sterilizing at 121°C for 20 minutes, add 0.04g of acetosyringone (AS) in the ultra-clean bench. , 0.25g MES; before vacuum infection, soak the melon seeds with a bud germination length of 1 to 2 cm with the seed coat removed in the mixed bacterial solution for 1-2 hours.

As the preferred technical solution of the present invention: in step f, the culture conditions of the infected receptor material are: first culture on the co-culture medium at 25°C in the dark for 3 to 5 days, and then colonize on peat, vermiculite, Culture in perlite (ratio 1:1:1) mixed matrix at 25°C, photoperiod 16h/8h (light/dark), relative humidity 75%, light intensity 22000Lx.

As a preferred technical solution of the present invention: in step f, the dark culture method is to place the vacuum-infected muskmelon seeds in a co-culture medium and culture them for 3 to 5 days under dark conditions at 25°C. The culture medium preparation method is to dissolve 4.43g MS, 30g sucrose, and 15g agar in 1L distilled water, adjust the pH to 5.8, and after sterilizing at 121°C for 20 minutes, add 0.04g acetosyringone AS, 0.25 gMES.

As the preferred technical solution of the present invention: in step f, the conventional culture conditions are: the day and night photoperiod of the incubator is light/dark: 16h/8h, the temperature is 25°C, the relative humidity is 75%, and the light intensity is 22000Lx. Culture was performed routinely until a silent albino phenotype appeared.

It should be noted that the reagents used in the VIGS experiment: LB medium: weigh 5g/L yeast extract, 10g/L peptone and 10g/L NaCl, add distilled water to make the volume to 1L. When configuring LB solid culture medium, 15g agar needs to be added, sterilized at 121°C for 20 minutes before use; Kan (100mg/ml): weigh 5g and dissolve in 50ml sterile water, filter and sterilize with a 0.22um filter, and aliquot 2ml EP tube, store at -20℃; Rif (50mg/ml): Weigh 2.5g and dissolve in 50ml dimethyl sulfoxide (DMSO), aliquot into 2ml EP tube, store at -20℃; AS (100uM) : Weigh 0.2g AS and dissolve in 10ml dimethyl sulfoxide (DMSO), aliquot into 2ml EP tubes, store at -20°C; MES (1.25mM): Weigh 1.25g and dissolve in 10ml sterile water, adjust pH = 5.8 , aliquot into EP tubes, and store at -20°C; Agrobacterium resuspension: 4.43g MS, 30g sucrose, dissolved in 1L distilled water, adjust pH to 5.8, sterilize at 121°C for 20 minutes, and then Add 0.04g acetosyringone AS and 0.25g MES to the clean platform and store at -20°C; germination medium: 2.22gMS, 15g agar, dissolved in 1L distilled water, adjust pH to 5.8, sterilize at 121°C for 20 minutes, then sterilize at 4°C Preservation; co-culture medium: 4.43g MS, 30g sucrose, 15g agar dissolved in 1L distilled water, adjust pH to 5.8, sterilize at 121°C for 20 minutes, add 0.04g acetosyringone AS in the ultra-clean bench, 0.25g MES, stored at -20°C.

Example 1

(1) Cloning of target gene-specific fragments

In the embodiment of the present invention, the melon phytoene dehydrogenase gene CmPDS was selected. This gene is a reporter gene of the virus-induced gene silencing system. The enzyme encoded by this gene is a rate-limiting enzyme in the carotenoid biosynthesis pathway. , catalyzes the dehydrogenation of colorless phytoene to lycopene, which is then converted into colored carotenoids. When the gene encoding this enzyme is silenced, the plant loses the photoprotective effect of carotenoids, causing the leaves to show a bleaching effect. The feasibility of this silencing system was verified by the time when the leaf bleaching phenotype appears and the PDS gene expression level index. sex.

Based on the CmPDS (MELO3C017772.2) gene CDS sequence published in the melon genome database, a specific 300 bp fragment from 1065 bp to 1365 bp was selected as the target gene-specific fragment.

Target gene-specific fragment sequence:

TTTGGGGCTTATCCCAATGTGCAGAACTTGTTTGGAGAACTTGGAATCAATGACCGATTACAGTGGAAGGAACATTC

AATGATATTTGCTATGCCAAACAAGCCGGGGGAGTTCAGCCGATTTGATTTCCCTGAAAAACTTCCTGCACCCATAA

ATGGGATATGGGCTATTTTAAGGAACAACGAGATGCTTACTTGGCCAGAGAAAATTAAATTTGCAATTGGGCTCCTG

CCAGCAATGCTTGGTGGGCAATCTTATGTTGAGGCTCAAGATAATTTAACTGTGCAAGAGTGGATGAGA

Select healthy melon leaves for sampling, and use Beijing Huayueyang Biotech's rapid universal plant RNA extraction kit to extract total plant RNA. Use Vazyme's Hiscript III 1st strand cDNA synthesis kit (+gDNA wiper) reverse transcription kit for reverse transcription, use the cDNA obtained by reverse transcription as a template, and use CmPDS-specific fragment primers (CmPDS2F: TGTTTTAAATGCCTTTACGTATTTGGGGCTTATCCCAA, CmPDS2R: AACACACAAAACACCTACGTATCTCATCCACTCTTGC) , perform PCR amplification (2×Phanta Max Master Mix, Vazyme), and the reaction system is as shown in Table 1. The reaction program is: pre-denaturation at 95°C, 3min; denaturation at 95°C, 15s; annealing at 56°C, 15s; extension at 72°C, 30s; set the number of cycles for steps 2-4 to 33; final extension at 72°C, 5min; and finally 4°C temporary extension. live. After the reaction, 7 μl of the product was pipetted for agarose gel electrophoresis detection. As shown in Figure 1, the amplified fragment was consistent in size with the target fragment. The PCR product was subjected to agarose gel electrophoresis (110v, 15min), and then passed through a gel recovery kit ( Plasmid Mini Kit, Vazyme) to recover and purify the target gene.

Table 1 PCR amplification system

(2) Construction of pTRSV2-CmPDS recombinant vector

a. Linearization of pTRSV2 vector

Configure the enzyme digestion reaction system according to Table 2; perform the reaction in a PCR machine: 37°C, 60min, 80°C, 20min; after the reaction, follow the product purification kit ( Plasmid Mini Kit, Vazyme) for product purification.

Table 2 Enzyme digestion reaction system

b. Recombination of pTRSV2 linearized vector and target gene-specific fragment

According to the One Step Cloning Kit (Vazyme), the recombination reaction system is as shown in Table 3. The recombinant reaction solution was transformed into E. coli. The E. coli strain used was DH5α competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd. , Beijing), single clones were picked, and specific primers (CmPDS1F: TTTGGGGGCTTATCCCAA, CmPDS1R: TCTCATCCACTCTTGC) were used for PCR bacterial detection. The PCR reaction system is shown in Table 4. The reaction program is: 94°C, 3-5min; 94°C, 30s; 56℃, 30s; 72℃, 30s; among them, the second to fourth step program loops 33 times; 72℃, 5min; the last 4℃ is temporarily stored. After the reaction, 7 μl of the product was taken for agarose gel electrophoresis detection. The results are shown in Figure 2.

Table 3 Homologous recombination reaction system

Table 4 PCR reaction system

(3) pTRSV2 empty vector and pTRSV2-CmPDS recombinant vector were transferred into Agrobacterium

The Agrobacterium strain GV3101 was selected. The Agrobacterium competent strain (Beijing Zhuangmeng International Biotechnology Co., Ltd., Beijing) was placed in an ice water bath for a while until it was partially melted. When it was in a mixed state of ice and water, it was inserted into the ice bath. Add 1 μg of pTRSV2 plasmid and pTRSV2-CmPDS recombinant plasmid to 100 μl of Agrobacterium competent cells, mix by hand at the bottom of the tube, and then place on ice for 5 min, liquid nitrogen for 5 min, 37°C water bath for 5 min, and ice bath for 5 min. Add 800 μl of antibiotic-free LB liquid culture medium and incubate with shaking at 28°C for 2 to 3 hours. Centrifuge at 5000rpm for 1 minute to collect the bacteria. Keep 100μ of the supernatant. Gently pipette and resuspend the bacterial mass and spread it on an LB plate containing Rif (50mg·L-1) and Kan (100mg·L-1). Place it upside down at 28°C. Culture in the incubator for 3 days. The pTRSV2 universal primers (pTRSV2 F: TGCGTCGCACTGAGGCA, pTRSV2 R: GCAGCTGACAGACAGACA) were used to conduct PCR positive identification of the single clone bacterial liquid. The results are shown in Figure 3, indicating that the pTRSV2 empty vector and pTRSV2-CmPDS vector were successfully introduced into Agrobacterium GV3101 and can be used for Vacuum infestation.

(4) Preparation of infection receptor materials

Soak the melon seeds in warm water at 55°C for 60 minutes, remove the seed coat, disinfect with 75% alcohol in a clean workbench for 30 seconds, wash once with sterilized ddH ₂ O, and then soak and shake with 2% sodium hypochlorite solution (containing 0.1% Tween 20) Disinfect for 15 minutes, then wash 5 times with sterilized ddH ₂ O, and place flat on the germination medium as shown in Figure 4. Then place them in a dark artificial climate incubator at 35°C for germination, and select seeds with a bud length of 1 to 2 cm for infection (Figure 5).

(5) Vacuum infection and penetration of melon seeds

Pick freshly cultured single colonies of Agrobacterium GV3101 containing pTRSV1, pTRSV2 and pTRSV2-CmPDS specific fragments of the target gene, inoculate them into 4mLLB liquid medium respectively, and culture them at 28°C and 220-250r/min for 4 to 6 hours; transfer all Into 40-50mL LB liquid medium, culture overnight at 28°C, 220-250r/min until the OD600 of the bacterial solution is about 0.5-1.5; centrifuge at 5000r for 5 minutes, collect the bacterial cells, and resuspend them with an appropriate volume of resuspension solution. The final concentration is 0.5, 1.0, 1.5 (OD600); the auxiliary vector pTRSV1 is mixed with the resuspension of pTRSV2-CmPDS and pTRSV2 empty respectively at a volume ratio of 1:1, and the mixed resuspension of equal volumes is allowed to stand at room temperature. After 3 to 4 hours, place the seeds in a resuspension containing bacterial liquid and soak for 1 to 2 hours. The bud vacuum infection and infiltration method is then used to infect the germinated melon seeds;

(6) After infection, the material is cultured in the dark and then cultured regularly.

Place the infected melon seeds flat on the co-culture medium and cultivate them in the dark at 25°C for 3 to 5 days; after the co-culture is completed, they are planted in a mixed matrix of peat, vermiculite, and perlite (the ratio is 1:1:1) , and then cultured at 25°C, a photoperiod of 16h/8h (light/dark), a relative humidity of 75%, and a light intensity of 22000Lx. Treat 10 individual plants for each treatment.

When carrying out vacuum infection and penetration, the present invention designs two variables and three concentration gradients of infection pressure and bacterial liquid concentration (infection pressures are 0.5kPa, 0.7kPa, and 0.9kPa respectively, and bacterial liquid concentrations are 0.5 and 1 respectively. , 1.5), 10 plants were treated in each treatment method. As shown in Table 5, when the infection pressure was 0.9kPa and the bacterial solution concentration was 0.5, 2 completely albino plants were obtained, and the silencing efficiency was 20%; when the infection pressure was 0.9 kPa and the bacterial solution concentration was 1.0, 8 completely albino plants were obtained, and the silencing efficiency was 80%; when the infection pressure was 0.9kPa and the bacterial solution concentration was 1.5, 5 completely albino plants were obtained, and the silencing efficiency was 50%; No gene-silenced albino seedlings were obtained from the remaining combinations. The results indicate that the infection pressure of 0.9kPa and the bacterial solution concentration of 1.0 are the optimal vacuum infection and penetration conditions. Subsequently, we repeated 4 times under optimal vacuum infection conditions (Table 6), with 10 plants in each treatment. The results showed that after pTRSV2-CmPDS infected plants, the silencing efficiency reached 85%, further proving the effectiveness of this silencing system. effectiveness. At the same time, we measured the plant phenotype (Figure 6), CmPDS gene expression (Figure 7), and chlorophyll content (Figure 8) of the uninfected control WT, empty vector pTRSV2 infected control, and pTRSV2-CmPDS infected plants. The results showed that compared with the control, the plants infected with pTRSV2-CmPDS quickly showed a complete albino phenotype at the one-leaf stage one week after infection (Figure 6), which reflected the fast speed and short cycle of the system of the present invention. The expression level of CmPDS in the first and second true leaves of silenced plants after vacuum infection with pTRSV2-CmPDS was significantly lower than that in the control. The expression level of CmPDS in the first true leaf decreased by 92%, and the expression level of CmPDS in the second true leaf decreased by 92%. The expression of CmPDS decreased by 86%. At the same time, the maximum photosynthetic efficiency and chlorophyll content of the plants at the three-leaf and one-center stage were measured. The results showed that compared with the control, the leaves of pTRSV2-CmPDS silenced plants turned white and the chlorophyll fluorescence intensity was significantly reduced (Figure 8A). Chlorophyll a, chlorophyll b, Total chlorophyll content was also significantly reduced (Fig. 8B). These results imply the high efficiency of silencing efficiency of the system of the present invention.

Table 5 Effects of different vacuum infection conditions on silencing efficiency of melon plants

Table 6 VIGS silencing efficiency of different replicates of melon plants under optimal vacuum infection conditions

At the same time, the present invention was also compared with another melon VIGS silencing method based on cotyledon injection. The infected plants obtained by the cotyledon injection method did not show an albino phenotype in either the one-leaf one-center stage or the two-leaf one-center stage (Fig. 9A); and the expression level of CmPDS in the first true leaf or the second true leaf was not significantly different from the control (Figure 9B), suggesting that the early silencing efficiency of the cotyledon injection VIGS silencing system was low. Compared with this The invention method has a long cycle.

The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments without departing from the spirit or basics of the present invention. In the case of specific features, the present invention can be implemented in other specific forms. Therefore, the embodiments should be regarded as illustrative and non-restrictive from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is therefore intended that all claims falling within the claims All changes within the meaning and scope of equivalent elements are included in the present invention. Any reference signs in the claims shall not be construed as limiting the claim in question.

In addition, it should be understood that although this specification is described in terms of implementations, not each implementation only contains an independent technical solution. This description of the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole. , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

Claims (1)

1. The method for constructing the melon VIGS silencing system based on bud vacuum infection is characterized by comprising the following steps of:

a. cloning a target gene specific fragment, wherein the target gene specific fragment is a 300bp specific fragment with specificity, and the target gene specific fragment has a sequence of:

TTTGGGGCTTATCCCAATGTGCAGAACTTGTTTGGAGAACTTGGAATCAATGACCGATTACAGTGGAAGGAACATTCAATGATATTTGCTATGCCAAACAAGCCGGGGGAGTTCAGCCGATTTGATTTCCCTGAAAAACTTCCTGCACCCATAAATGGGATATGGGCTATTTTAAGGAACAACGAGATGCTTACTTGGCCAGAGAAAATTAAATTTGCAATTGGGCTCCTGCCAGCAATGCTTGGTGGGCAATCTTATGTTGAGGCTCAAGATAATTTAACTGTGCAAGAGTGGATGAGA；

b. the tobacco ringspot virus vector is selected and adopted, and specifically comprises the following steps: helper vector pTRSV1, recombinant vector pTRSV2; c, recombining the target gene specific fragment in the step a into a pTRSV2 vector to obtain a pTRSV 2-target gene specific fragment recombinant vector;

transferring a pTRSV2-target gene specific fragment recombinant vector into agrobacterium strain GV3101;

d. preparing an infection receptor material, wherein the infection receptor material is melon seeds with buds sprouting length of 1-2cm and seed coats removed, the melon seeds are placed on a semi-MS solid culture medium for sprouting for 24-36 hours in a dark artificial climate chamber at 30 ℃, the semi-MS solid culture medium is prepared by dissolving 2.22g of MS and 15g of agar in 1L of distilled water, adjusting the pH value to 5.8, sterilizing at the high temperature and the high pressure for 20min at the temperature of 121 ℃, and preserving at the temperature of 4 ℃;

e. vacuum infecting penetrating melon seeds, picking fresh agrobacterium GV3101 single colony containing pTRSV1 and pTRSV 2-target gene specific fragment recombinant vector, inoculating into 4mL LB liquid culture medium, culturing at 28 deg.C and 220-250r/min for 4-6 hr; transferring all the cells into 40-50 mL LB liquid medium, culturing overnight at 28 ℃ and 220-250r/min until the OD600 of the bacterial liquid is 0.5-1.5, centrifuging at 5000r for 5min, collecting bacterial cells, and re-suspending the bacterial cells until the OD600 concentration is 1.0; uniformly mixing agrobacterium GV3101 containing an auxiliary vector pTRSV1 and a heavy suspension of agrobacterium GV3101 containing a recombinant vector of pTRSV 2-target gene specific fragment according to a volume ratio of 1:1, standing for 3-4 h at room temperature, and soaking the infection receptor material in the heavy suspension containing bacterial liquid for 1-2 h; subsequently, the receptor material is infected by adopting a method of bud vacuum infection infiltration, wherein the vacuum infection conditions are as follows: the infection pressure is 0.9kPa, and the bacterial liquid concentration OD600 is 1.0;

f. the infected material is firstly dark cultured and then is conventionally cultured, and the specific conditions for culturing the infected receptor material are as follows:

firstly, culturing in darkness at 25 ℃ for 3-5 days on a co-culture medium, and after the co-culture is finished, planting the plant in turf, vermiculite and perlite with the proportion of 1:1:1 in a mixed matrix at 25 ℃, photoperiod: culturing under light/dark for 16h/8h, relative humidity for 75%, and illumination intensity for 22000 Lx; the preparation method of the co-culture medium comprises the steps of dissolving 4.43g of MS, 30g of sucrose and 15g of agar in 1L of distilled water, adjusting the pH to 5.8, sterilizing at the high temperature of 121 ℃ under high pressure for 20min, and adding 0.04g of acetosyringone and 0.25g of MES; the conventional culture conditions are as follows: the day and night photoperiod of the incubator is light/dark: 16h/8h, temperature 25 ℃, relative humidity 75%, illumination intensity 22000Lx, conventional culture until a silent albino phenotype appears.