A method for delivering a VIGS vector to flower buds in the interior of fritillaria bulbs, a method for verifying gene function, and applications thereof

Abstract

This invention discloses a method for delivering a VIGS vector targeting the flower buds inside the bulb of Lycoris radiata, a method for verifying gene function, and their applications, belonging to the field of biotechnology. The method includes: making 4-6 holes around the root plate of a dormant Lycoris radiata bulb, and 2-4 holes in the central area of ​​the bulb base, reaching the meristematic region of the flower bud; immersing the treated bulb in VIGS infection solution up to the wounds on the root plate; and performing vacuum treatment to ensure effective penetration of the infection solution into the internal flower bud tissue. This invention successfully delivered a silencing vector targeting the LrP5CS1 gene to the flower bud, specifically reducing its expression and obtaining a stable early-flowering phenotype in Lycoris radiata, thus verifying the gene function. Therefore, this invention establishes an efficient delivery platform that can be used for rapid screening and verification of other key genes regulating the flowering period and ornamental traits of Lycoris radiata, providing a novel technical means for the functional study of genes related to the flowering phenotype of Lycoris radiata.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a VIGS vector delivery method for targeting the flower buds inside the bulb of Lycoris radiata, a gene function verification method, and their applications. Background Technology

[0002] Virus-induced gene silencing (VIGS) is an effective method for validating plant gene function. VIGS technology selectively binds to and degrades specific mRNAs, thereby inhibiting the expression of target genes. Analysis of the phenotype and physiological and biochemical properties of silenced plants can then clarify the role of target genes in plant growth and development. VIGS can silence target genes in different tissues and organs, resulting in phenotypes similar to mutants. This method does not require stable genetic transformation of plants and does not alter the DNA sequence of the genome, making it a highly efficient tool for studying gene function. Currently, VIGS technology has been used to validate gene function in various plants, but its application in Lycoris radiata remains relatively limited. The dormant bulb structure of Lycoris radiata is unique and dense, and its flower buds are deeply embedded within the bulb. Conventional VIGS infection methods (such as leaf rubbing and injection) are difficult to effectively deliver the silencing vector to the target site, leading to low infection efficiency or complete failure, and preventing functional studies of genes related to key reproductive processes such as flower bud development. Therefore, developing a VIGS technology system that can efficiently infect dormant bulbs of Lycoris radiata and target flower buds has become a technical problem that urgently needs to be solved and has long remained unsolved in this field. Summary of the Invention

[0003] To address the problems existing in the prior art, the first technical problem to be solved in this application is to provide a VIGS delivery method targeting the flower buds of Lycoris radiata bulbs. The second technical problem to be solved is to provide a method for functional verification of Lycoris radiata flowering trait-related genes based on the aforementioned delivery method. The final technical problem to be solved is to provide specific applications of the aforementioned method and delivery system.

[0004] To solve the above-mentioned technical problems, the technical solution of this application is as follows:

[0005] A method for delivering a VIGS vector targeting the flower buds inside the bulb of Lycoris radiata includes the following steps:

[0006] (1) Obtaining Lycoris bulbs in a dormant period;

[0007] (2) Using a needle, make 4-6 holes around the bulb root plate and 2-4 holes in the central area at the bottom of the bulb, with the depth of the holes reaching the area where the flower bud meristem is located;

[0008] (3) The bulbs treated in step (2) are immersed in VIGS infection solution at least above the wound on the bulb plate, and vacuum treatment is performed so that the infection solution can penetrate through the pierced channel and reach the flower bud tissue; the VIGS infection solution is a mixture of Agrobacterium bacterial solution containing pTRV1 vector and Agrobacterium bacterial solution containing pTRV2 recombinant vector.

[0009] In some embodiments, in step (3), the vacuuming process specifically involves processing at a vacuum level of 1.0 MPa for 30 minutes.

[0010] In some embodiments, the vacuum treatment is performed intermittently, specifically by maintaining the vacuum for 15 minutes, slowly releasing it to atmospheric pressure, then re-evacuating it to 1.0 MPa and maintaining it for another 15 minutes.

[0011] In some embodiments, the Agrobacterium in step (3) is Agrobacterium tumefaciens strain GV3101; and after mixing the two bacterial solutions, the step of letting the mixed bacterial solution stand at 24-26°C under light-protected conditions for 3-4 hours is further included.

[0012] In some embodiments, after step (3), a cultivation step is also included: the treated bulbs are cultured in the dark at 25°C for 2-3 days, and then transferred to conventional cultivation conditions for further cultivation.

[0013] A method for inducing early flowering of Lycoris radiata includes: delivering a silenced expression construct targeting the LrP5CS1 gene into the flower buds of Lycoris radiata bulbs using any of the VIGS delivery methods described above, thereby obtaining Lycoris radiata plants with advanced flowering period; wherein the nucleotide sequence of the LrP5CS1 gene is shown in SEQ ID NO.1.

[0014] A method for identifying or verifying the function of a gene that regulates the flowering time of Lycoris radiata includes: delivering a silent expression construct targeting a gene into the flower buds of Lycoris radiata bulbs using any of the VIGS delivery methods described above, and determining the function of the target gene by observing and comparing the flowering period of Lycoris radiata after culturing.

[0015] A dedicated VIGS silencing system for implementing any of the methods described herein, the system being a mixture of the following two components:

[0016] A: Agrobacterium tumefaciens GV3101 bacterial suspension containing pTRV1 vector;

[0017] B: Agrobacterium tumefaciens GV3101 bacterial suspension containing the pTRV2 recombinant vector;

[0018] The volume mixing ratio of component A to component B is 1:1.

[0019] The pTRV2 recombinant vector is constructed by inserting a specific fragment targeting the target gene into the multiple cloning site of the pTRV2 vector.

[0020] In some embodiments, the pTRV2 recombinant vector is pTRV2-LrP5CS1, and the inserted specific fragment is the LrP5CS1 gene fragment shown in SEQ ID NO.1.

[0021] The application of the method and system described herein in the preparation of products for regulating the flowering time of Lycoris radiata or in the breeding of early-flowering varieties of Lycoris radiata.

[0022] Compared with the prior art, the beneficial effects of this application are as follows:

[0023] (1) This application is the first to develop a complete method that can effectively penetrate the physical barrier of Lycoris radiata bulbs during dormancy and accurately deliver bioactive substances (VIGS silencing carriers) to the internal flower bud meristem. This method combines a specific positioning and puncture scheme (4-6 holes in the root plate + 2-4 holes in the bottom) with optimized vacuum permeation parameters (1.0 MPa, 30 minutes), which solves the long-standing technical problem of being unable to effectively target flower buds, thus hindering the study of the function of key trait genes such as the flowering mechanism of Lycoris radiata.

[0024] (2) Using the above delivery technology, this application successfully delivered the VIGS silencing system targeting the LrP5CS1 gene to flower buds, specifically reducing the expression of the gene, and for the first time obtained a stable and observable "early flowering" phenotype in Lycoris radiata using the VIGS technology. This not only directly verifies the key function of the LrP5CS1 gene in regulating the flowering period of Lycoris radiata, but also provides a practical technical solution for rapidly inducing flowering period variation in Lycoris radiata without relying on transgenics, providing a brand-new technical tool for breeding the flowering period of Lycoris radiata.

[0025] (3) The delivery method established in this application is universal. Any VIGS vector constructed for other candidate genes can be rapidly delivered to flower buds for functional verification using this method. This provides an efficient and reliable technical platform for large-scale, high-throughput screening of functional genes related to ornamental traits such as flowering period and flower organ development of Lycoris radiata, shortening the traditional transgenic verification cycle of several years to within one growing season, greatly accelerating the process of molecular breeding of Lycoris radiata, and having high application value for the study of gene function of Lycoris radiata, especially the function of genes related to flowering phenotype. Attached Figure Description

[0026] Figure 1 Schematic diagrams of the needle-punching method at the bottom of the bulb in Example 1 (a) and the needle-punching method at the top of the bulb in Comparative Example 1 (b);

[0027] Figure 2The images show a comparison of early flowering phenotypes after Lycoris bulbs were infected with the silent vector in Example 1. (a) is a phenotype of flowering of Lycoris after infection with pTRV1+pTRV2, and (b) is a phenotype of flowering of Lycoris after infection with pTRV1+pTRV2-LrP5CS1.

[0028] Figure 3 Longitudinal section of bulb after early flowering phenotype appears following LrP5CS1 gene silencing in Lycoris radiata;

[0029] Figure 4 This figure shows the expression of LrP5CS1 in Lycoris radiata detected by real-time quantitative PCR (qRT-PCR). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the application is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are all commercially available.

[0031] This application obtained a CDS sequence of the LrP5CS1 gene by sequencing the transcriptome of Lycoris radiata bulbs, the specific nucleotide sequence of which is shown in SEQ ID NO.1. Using the LrP5CS1 gene-specific fragment as the target fragment, a VIGS silencing vector for the LrP5CS1 gene was constructed to verify the function of the LrP5CS1 gene in Lycoris radiata.

[0032] This application also provides a method for constructing the Lycoris radiata LrP5CS1 gene VIGS silencing system, including the following steps:

[0033] The LrP5CS1 gene-specific fragment was ligated into pTRV2 to obtain the silencing vector pTRV2-LrP5CS1.

[0034] In this application, the aforementioned specific fragment was obtained by PCR amplification. The primer pair used for PCR amplification included an upstream primer P5CS1-XbaI-F and a downstream primer P5CS1-KpnI-R. The nucleotide sequence of the upstream primer P5CS1-XbaI-F is shown in SEQ ID NO.4 (5'-GTAAGGTTACCGAATTCTCTAGAATGGACCCCACTCGAGCATT-3'), and the nucleotide sequence of the downstream primer P5CS1-KpnI-R is shown in SEQ ID NO.5 (5'-CTCGAGACGCGTGAGCTCGGTACCTTTTAAAGGAAGAATCTTGTGGGT-3'). In the embodiments of this application, cDNA obtained by reverse transcription of Lycoris radiata RNA was used as a template.

[0035] This application does not impose strict requirements on the ligation method; conventional gene recombination methods can be used. For example, in the following embodiment, an XbaI restriction site is added to the 5' end of the upstream primer P5CS1-XbaI-F, and a KpnI restriction site is added to the 5' end of the downstream primer P5CS1-KpnI-R. The specific fragment obtained by PCR amplification is inserted into pTRV2 using a double restriction ligation reaction.

[0036] This application also provides a Lycoris radiata LrP5CS1 gene VIGS silencing system, comprising: mixing Agrobacterium tumefaciens culture containing pTRV1 and Agrobacterium tumefaciens culture containing pTRV2-LrP5CS1 at a volume ratio of 1:1.

[0037] In this embodiment, the resulting infection solution after mixing Agrobacterium tumefaciens containing pTRV1 and Agrobacterium tumefaciens containing pTRV2-LrP5CS1 is a silent system. Setting the volume ratio of Agrobacterium tumefaciens containing pTRV1 to Agrobacterium tumefaciens containing pTRV2-LrP5CS1 to 1:1 can improve the infection effect.

[0038] This application does not impose strict requirements on the preparation of the mixture of Agrobacterium tumefaciens containing pTRV1 and Agrobacterium tumefaciens containing pTRV2-LrP5CS1. Conventional methods such as heat shock can be used to separately transfer pTRV1 and pTRV2-LrP5CS1 into Agrobacterium tumefaciens and then incubate at 28°C. The Agrobacterium tumefaciens used in the examples of this application is Agrobacterium tumefaciens GV3101.

[0039] This application also provides examples of the application of the above-mentioned silencing vector or silencing system in identifying the function of the Lycoris radiata LrP5CS1 gene. The silencing vector or silencing system provided in this application can effectively reduce the gene expression level of Lycoris radiata LrP5CS1, obtain a gene silencing vector, and verify the gene function of Lycoris radiata LrP5CS1.

[0040] This application also provides a method for identifying the function of the Lycoris radiata LrP5CS1 gene, comprising the following steps:

[0041] After infecting dormant Lycoris bulbs with the aforementioned silencing system, the bulbs were cultured and the changes in flowering phenotypes were observed.

[0042] Before infecting dormant Lycoris bulbs using the aforementioned silent system, this application involves dark culturing of the silent system, followed by infecting the bulbs with the dark-cultured silent system. The dark culturing temperature in this application is 24℃~26℃, and the dark culturing time is 3~4 hours.

[0043] This application utilizes a silent system after dark culture to infect dormant Lycoris bulbs. The infection method involves first puncturing the Lycoris bulbs with a needle before vacuum infiltration. Specifically, five holes are made around the root plate and three holes are made at the bottom, ensuring the holes reach the flower buds. The perforated Lycoris bulbs are then immersed in the silent system, and a vacuum of 1.0 MPa is applied for 30 minutes, with slow venting and re-vacuuming every 15 minutes.

[0044] This application targets the characteristics of Lycoris bulbs and employs a method of first puncturing holes and then vacuum infiltration to penetrate the dormant Lycoris bulbs with a silencing system, allowing it to directly act on the flower buds. Infection can be completed in a short time and with a small amount of silencing system, and this method can be applied to the subsequent verification of the gene function of Lycoris LrP5CS1.

[0045] After infection, this application cultured the infected Lycoris radiata. The culture method described in this application is dark culture at 25°C for 2 days followed by normal culture.

[0046] The bacterial solution was dried under room temperature and light for 7-10 days (i.e., 9-12 days of infection) before the bulbs were planted in soil and then cultured using standard methods. This application aims to observe changes in flowering phenotype and / or sample and determine the expression of the LrP5CS1 gene in the bulb discs to identify the function of the LrP5CS1 gene in Lycoris radiata. Specifically, early flowering of Lycoris radiata indicates that the LrP5CS1 gene is functioning.

[0047] To further explain this application, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0048] Unless otherwise specified, the molecular biology experimental techniques involved in the following examples, such as RNA extraction and reverse transcription, PCR amplification, etc., can be performed using conventional methods.

[0049] Example 1

[0050] 1. Cloning of a specific fragment of the LrP5CS1 gene

[0051] Based on the splicing, annotation, and alignment analysis of the Lycoris radiata transcriptome, LrP5CS1 was found to have a complete ORF (full-length 2139 bp). Full-length primers P5CS1-F (SEQ ID NO.2): 5'-ATGGACCCCACTCGAGCATT-3' and P5CS1-R (SEQ ID NO.3): 5'-TTTTAAAGGAAGAATCTTGTGGGT-3' were designed using the Lycoris radiata transcriptome. RNA was extracted from the bulb discs of Lycoris radiata using an RNA extraction kit purchased from Beijing Huayueyang Biotechnology Co., Ltd., and then reverse transcribed using a reverse transcription kit purchased from Takara to obtain cDNA. PCR amplification was performed using the cDNA as a template, followed by agarose gel electrophoresis to obtain the LrP5CS1-specific band. After gel purification, sequencing was performed to obtain the final sequence shown in SEQ ID NO.1.

[0052] 2. Carrier Construction

[0053] Primers P5CS1-XbaI-F and P5CS1-KpnI-R were designed to amplify a specific fragment on the CDS domain of the LrP5CS1 gene. XbaI and KpnI restriction sites were added to the 5' ends of the upstream and downstream primers, respectively, and the LrP5CS1 gene-specific fragment was amplified by PCR.

[0054] RNA was first extracted from the bulb discs of Lycoris radiata using an RNA extraction kit purchased from Beijing Huayueyang Biotechnology Co., Ltd., and then reverse transcribed into cDNA template using a reverse transcription kit purchased from Takara.

[0055] The PCR reaction system (25 μL) consisted of: 1 μL cDNA template, 1 μL each of forward and reverse primers (10 μM), 12.5 μL 2×Phanta Max Buffe, 0.5 μL dNTP Mix (10 mM), 0.5 μL Phanta Max Super-Fidelity DNA Polymerase, and double-distilled water to a final volume of 25 μL. Amplification conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; and a final extension at 72℃ for 5 min to obtain a 2139 bp specific fragment of LrP5CS1 (SEQ ID NO.1), which was stored at 4℃ for later use.

[0056] After PCR amplification, gel extraction, and A-tailing, the cells were ligated into a T-vector (purchased from Beijing Jinsha Biotechnology Co., Ltd.) and transformed into *E. coli*. Positive clones were selected for culture and sequencing (Universal Biotechnology Co., Ltd.). Clones with sequencing results consistent with known sequences were selected, and plasmids were extracted and named pMD19-LrP5CS1. pMD19-LrP5CS1 and pTRV2 were double-digested with XbaI / KpnI, and the digestion products were recovered separately. These digests were ligated, transformed, and positive clones were screened; finally, the VIGS recombinant vector pTRV2-LrP5CS1 was obtained.

[0057] 3. Preparation of infiltration solution and infiltration method

[0058] (1) Preparation of permeate: 100 mL of permeate contains 1 mL of MgCl2 solution (1 mol / L), 1 mL of LMES solution (1 mol / L) and 200 μL of acetylsalicylic acid solution (1 mol / L), and its pH is adjusted to 5.6. It is then diluted with pure water and prepared fresh for use.

[0059] (2) Preparation of bacterial culture: The obtained pTRV2-LrP5CS1 plasmid, pTRV2 plasmid and pTRV1 plasmid were transformed into Agrobacterium GV3101 by freeze-thaw method. Agrobacterium GV3101 transformed with pTRV2-LrP5CS1, pTRV2, and pTRV1 plasmids were plated on LB agar (Kan, 50 μg / mL; Rif, 25 μg / mL). After 48 h of incubation at 28 °C, single colonies of Agrobacterium GV3101 transformants containing pTRV2-LrP5CS1, pTRV2, and pTRV1 plasmids were selected and added to 10 mL of LYEB liquid medium (Kan, 50 μg / mL; Rif, 25 μg / mL) and incubated overnight at 28 °C and 200 rpm in a shaker. 1 mL of each bacterial culture was then added to 100 mL of LYEB liquid medium (Kan, 50 μg / mL; Rif, 25 μg / mL) and incubated for 20 h at 28 °C and 200 rpm in a shaker. The OD of the bacterial culture was then measured. 600 When the value is 0.8~1.0, take 45mL of bacterial solution, centrifuge at 8000rpm for 5min, discard the supernatant, and then add an appropriate amount of infection buffer to resuspend the solution, with the volume being equal to the required bacterial solution.

[0060] (3) Preparation of infection solution: Mix the resuspension of Agrobacterium GV3101 containing pTRV1 plasmid in step (2) with the resuspension of Agrobacterium GV3101 containing pTRV2 and pTRV2-LrP5CS1 plasmids in equal volumes, and incubate in the dark for 3-4 hours to infect Lycoris bulbs.

[0061] (4) Agrobacterium tumefaciens infection: such as Figure 1 As shown in (a), first, holes are made using a needle. Specifically, five holes are made around the root plate of the Lycoris radiata and three holes are made at the bottom, ensuring that the needle reaches the flower bud. Then, the Lycoris radiata with the holes is immersed in the inoculum using a vacuum permeation method. The vacuum is drawn to 1.0 MPa and treated for 30 minutes. Ideally, the vacuum should be slowly released and re-drawn every 15 minutes.

[0062] Post-injection culture: After infection, the bacterial solution was placed in the dark at room temperature for 2 days, then dried at room temperature. The Lycoris bulbs were then planted in the soil and cultured as usual.

[0063] Comparative Example 1

[0064] Same as Example 1, except that in step (4) when performing Agrobacterium tumefaciens inoculation, the following method is used: Figure 1 The method shown in (b) is to immerse the bulb by puncturing it with a needle tip.

[0065] Test Example 1

[0066] Comparison of Agrobacterium-mediated infection efficiency

[0067] In Example 1, the needle tip is used to make a hole at the bottom of the Lycoris bulb and to the flower bud. This is easier to control, and when using the vacuum permeation method, the bacterial solution only needs to submerge the wound on the bulb plate, requiring less amount of infection solution. In Comparative Example 1, the hole is made at the top of the Lycoris bulb. To achieve the effect of permeating to the flower bud, the hole needs to be made deeper, which is difficult to control and requires more bacterial solution to submerge it. Furthermore, the deeper hole is not conducive to plant growth and development, affecting the experimental results.

[0068] The method used in this invention makes it easier to puncture the flower buds, is simple to operate, easier to control, improves efficiency, and saves more bacterial solution.

[0069] Test Example 2

[0070] Detection of the silencing efficiency of Lycoris radiata LrP5CS1 after infection

[0071] (1) In Example 1, after 60-65 days of infection, the phenotypic changes of Lycoris radiata flowering were observed, and the results were as follows: Figure 2 As shown, Figure 2 (a) shows Lycoris radiata after infection with pTRV1+pTRV2 in Example 1, and (b) shows Lycoris radiata after infection with pTRV1+pTRV2-LrP5CS1 in Example 1. Figure 2 It can be seen that Lycoris radiata infected with pTRV1+pTRV2-LrP5CS1 exhibits an early flowering phenotype, indicating that the method described in this application can silence the Lycoris radiata LrP5CS1 gene and allow it to function.

[0072] (2) When Lycoris radiata shows early flowering phenotype, cut the bulb and take its bulb plate, such as Figure 3As shown, VIGS infection can induce changes within Lycoris radiata, thus enabling it to exert its effects. RNA containing pTRV1+pTRV2 and pTRV1+pTRV2-LrP5CS1 was extracted from Lycoris radiata bulb discs using the Huayueyang Universal Plant RNA Extraction Kit (Polysaccharide / Polyphenol), with three replicates for each group. The RNA was then reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (Takara) and stored at -20°C for later use.

[0073] The gene expression level was detected by qRT-PCR, with TIP41 as the internal reference gene. The primer sequences were TIP41-qrt-F (SEQ ID NO.6): 5'-GCAACCATCCAAAGTTTAACTGCT-3' and TIP41-qrt-R (SEQ ID NO.7): 5'-AATGTGCAAGCAGGGCTAGTAA-3'. The detection results are shown in [Figure number missing]. Figure 4 In this study, CK consisted of Lycoris bulbs infected with pTRV1+pTRV2, with a relative expression level of LrP5CS1 gene of 1.00; TRV1 / TRV2-LrP5CS1 consisted of Lycoris bulbs infected with pTRV1+pTRV2-LrP5CS1 in Example 1, with a relative expression level of LrP5CS1 gene of 0.25. Compared with the control group CK, the expression level of LrP5CS1 gene in Lycoris bulbs infected with the pTRV1+pTRV2-LrP5CS1 recombinant viral vector was significantly reduced, indicating that the silencing system of this application was successfully constructed.

[0074] As can be seen from the above, the VIGS silencing system for the Lycoris radiata LrP5CS1 gene provided in this application can induce the silencing of endogenous LrP5CS1 in Lycoris radiata, significantly reduce the expression level of the Lycoris radiata LrP5CS1 gene, and obtain the virus-induced gene silencing trait.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are merely some embodiments of the present invention and should not be construed as limiting the scope of the present invention. Other embodiments can be obtained without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for obtaining Lycoris plants with advanced flowering period, characterized in that, Includes the following steps: (1) Obtaining Lycoris bulbs in a dormant period; (2) Using a needle, make 4-6 holes around the bulb root plate and 2-4 holes in the central area at the bottom of the bulb, with the depth of the holes reaching the area where the flower bud meristem is located; (3) The bulbs treated in step (2) are immersed in VIGS infection solution at least above the wound on the bulb plate, and vacuum treatment is performed so that the infection solution can penetrate through the pierced channels and reach the flower bud tissue. The VIGS infection solution is prepared by mixing Agrobacterium bacterial solution containing the pTRV1 vector and Agrobacterium bacterial solution containing the pTRV2 recombinant vector; the pTRV2 recombinant vector is constructed by inserting a specific fragment targeting the LrP5CS1 gene into the multiple cloning site of the pTRV2 vector; the specific fragment of the LrP5CS1 gene is shown in SEQ ID NO.1; the vacuum treatment is specifically performed by treating under a vacuum of 1.0 MPa for 30 minutes.

2. The method according to claim 1, characterized in that, The vacuum treatment is performed in an intermittent manner, specifically: after maintaining the vacuum for 15 minutes, it is slowly released to atmospheric pressure, and then the vacuum is re-evacuated to 1.0 MPa and maintained for another 15 minutes.

3. The method according to claim 1, characterized in that, The Agrobacterium mentioned in step (3) is Agrobacterium tumefaciens strain GV3101; and after mixing the two bacterial solutions, the step of letting the mixed bacterial solution stand at 24-26°C under light-protected conditions for 3-4 hours is also included.

4. The method according to any one of claims 1-3, characterized in that, After step (3), there is also a cultivation step: the treated bulbs are cultured in the dark at 25℃ for 2-3 days, and then transferred to conventional cultivation conditions for further cultivation.

5. A dedicated VIGS silencing system for obtaining Lycoris radiata plants with advanced flowering period, characterized in that, The system is a mixture of the following two components: A: Agrobacterium tumefaciens GV3101 bacterial suspension containing pTRV1 vector; B: Agrobacterium tumefaciens GV3101 bacterial suspension containing the pTRV2 recombinant vector; The volume mixing ratio of component A to component B is 1:

1. The pTRV2 recombinant vector is constructed by inserting a specific fragment targeting the LrP5CS1 gene into the multiple cloning site of the pTRV2 vector. The pTRV2 recombinant vector is pTRV2-LrP5CS1, and the inserted specific fragment of the LrP5CS1 gene is the LrP5CS1 gene fragment shown in SEQ ID NO.

1.

6. The application of the method according to any one of claims 1-4 and the system according to claim 5 in the preparation of products with early flowering of Lycoris radiata or in the breeding of early flowering varieties of Lycoris radiata.

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