CsRBE gene for regulating morphology of lip of cymbidium ensifolium, application, protein and method thereof

Abstract

The present application relates to the field of biotechnology, in particular to a CsRBE gene for regulating the morphology of Cymbidium ensifolium labellum, application, protein and method. The nucleotide sequence of the CsRBE gene is shown as SEQ ID No. 1. The CsRBE gene provided by the present application can regulate the morphology of Cymbidium ensifolium labellum. After silencing the CsRBE gene, the morphology of Cymbidium ensifolium labellum is flat without bending, the labellum width is increased, and the wrinkle gap in the middle of the labellum is increased, which provides an important basis for Cymbidium breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a CsRBE gene for regulating Cymbidium ensifolium lip morphology, and application, protein and method thereof. BACKGROUND

[0002] Orchidaceae is one of the largest and most diverse families of flowering plants, and its highly specialized lip has very important biological significance in pollination biology, species differentiation and adaptive evolution. As a specialized structure of the perianth of Orchidaceae, the lip is complex and diverse in morphology, pattern, color, texture and accessory structure, which directly determines the selection preference of insect pollinators and is the key to pollination strategy and reproductive success of Orchidaceae. The diversity of lip morphology and the unique phenotype of flower pattern are key factors for attracting consumers and enhancing the value of commodities. In the group of ornamental orchids, Cymbidium ensifolium, as a typical representative of national orchids, exhibits rich phenotypic variation in lip development, including single lip (standard type), multiple lip (abnormal flower type) and three-lip (butterfly type). Among them, the abnormal flower and butterfly types are more popular due to their higher ornamental value. However, so far, except for the CsAP3-2 gene, no other genes involved in the development and morphological differences of Cymbidium ensifolium lip have been reported. SUMMARY

[0003] To solve the above problems, the present application provides a CsRBE gene for regulating Cymbidium ensifolium lip morphology, and application, protein and method thereof. The CsRBE gene provided by the present application can regulate the morphology of Cymbidium ensifolium lip. After silencing the CsRBE gene, the morphology of Cymbidium ensifolium lip is flat without bending, the width of the lip is increased, and the middle wrinkle gap of the lip is increased.

[0004] To achieve the above purpose, the present application provides the following technical solutions:

[0005] The present application provides a CsRBE gene for regulating Cymbidium ensifolium lip morphology, and the nucleotide sequence of the CsRBE gene is shown in SEQ ID No. 1.

[0006] The present application also provides the application of the CsRBE gene in regulating the morphology of Cymbidium ensifolium lip.

[0007] Preferably, the morphology of Cymbidium ensifolium lip is regulated by silencing the CsRBE gene.

[0008] Preferably, after silencing the CsRBE gene, the morphology of Cymbidium ensifolium lip is flat without bending, the width of the lip is increased, and the middle wrinkle gap of the lip is increased.

[0009] The present application also provides a protein encoded by the CsRBE gene, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

[0010] This invention also provides a method for regulating the morphology of the lip petal of Cymbidium goeringii, comprising the following steps:

[0011] 1) The 1-300bp sequence of the CsRBE gene described in the above technical solution is ligated into the pTRV2 vector to obtain the pTRV2-CsRBE vector;

[0012] 2) Transform the pTRV2-CsRBE vector obtained in step 1) into Agrobacterium and culture it to obtain a bacterial culture containing the pTRV2-CsRBE vector;

[0013] 3) Centrifuge the bacterial culture containing pTRV2-CsRBE vector obtained in step 2), mix the obtained bacterial cells with buffer to obtain CsRBE bacterial culture, OD 600 The value is 0.5;

[0014] 4) Transform Agrobacterium into the pTRV1 vector to obtain a bacterial culture containing the pTRV1 vector. Mix the obtained bacterial cells with buffer after centrifugation to obtain the pTRV1 bacterial culture. OD 600 The value is 0.5;

[0015] 5) Mix the CsRBE bacterial solution obtained in step 3) with the pTRV1 bacterial solution obtained in step 4) in equal volumes to obtain the infection solution;

[0016] 6) Infect the Cymbidium goeringii buds with the inoculum obtained in step 5).

[0017] Preferably, the OD of the bacterial suspension containing the pTRV2-CsRBE vector in step 2) is... 600 The value is 1; the Agrobacterium is Agrobacterium GV3101.

[0018] Preferably, the buffer solution in step 3) consists of: 100 μm acetylsuccinone, 10 mM morpholine ethanesulfonic acid and 10 mM magnesium chloride;

[0019] The centrifugation conditions include: a rotation speed of 5000 rpm and a time of 8 min.

[0020] Preferably, the Agrobacterium in step 4) is Agrobacterium GV3101;

[0021] The OD of the bacterial culture containing the pTRV1 vector 600 The value is 1;

[0022] The buffer solution consists of: 100 μM acetylsuccinone, 10 mM morpholine ethanesulfonic acid, and 10 mM magnesium chloride.

[0023] The centrifugation conditions include: a rotation speed of 5000 rpm and a time of 8 min.

[0024] Preferably, the step 6) includes: infecting under vacuum condition for 10 min, and the flower bud is a wounded flower bud.

[0025] Advantages of the present application:

[0026] The CsRBE gene provided by the present application can regulate the morphology of the lip of Cymbidium sinense, and after silencing the CsRBE gene, the morphology of the lip of Cymbidium sinense is flat and straight without bending, the width of the lip is increased, and a wrinkle gap is formed in the middle of the lip, thereby providing an important basis for breeding Cymbidium sinense. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced.

[0028] Figure 1 A phylogenetic tree of CsRBE of Cymbidium sinense is shown in Figure 1;

[0029] Figure 2 A CsRBE amino acid sequence alignment diagram of Cymbidium sinense is shown in Figure 2;

[0030] Figure 3 Changes in the expression amount of CsRBE gene in single flower of Cymbidium sinense varieties 'Xiaoxiang' and 'Yushizi' and at five stages of flower development are shown in Figure 3;

[0031] Figure 4 A pTRV2-CsRBE gene silencing vector of Cymbidium sinense is shown in Figure 4;

[0032] Figure 5 Floral phenotypes of a silencing strain and an empty control after pTRV2-CsRBE treatment are shown in Figure 5;

[0033] Figure 6 Phenotypes of the lip of a flower of a silencing strain and an empty control after pTRV2-CsRBE treatment are shown in Figure 6;

[0034] Figure 7 Expression amounts in the lip of a flower of a silencing strain and an empty control after pTRV2-CsRBE treatment are shown in Figure 7. DETAILED DESCRIPTION

[0035] The present application provides a CsRBE gene for regulating the morphology of the lip of Cymbidium sinense, the nucleotide sequence of the CsRBE gene is shown in SEQ ID No. 1, and is specifically as follows:

[0036] ATGCGTGCAGAAAGGAATTTTCTCTGTAGACCACTCCTCCCTACTTTCATCCTTTCTTCTTCCACCAACGTATCATGGGAAGAACAAGCTTTCGCAAAAGACTCGTCGGGCGACTTGGGTGGCTGTGTGTGGCCACCCAGATCTTACACTTGTAGCTTCTGTCGGCGAGAATTCCGATCAGCCCAAGCTCTAGGTGGCCACATGAACGTCCACAGAAGGGACAGGGCAAAGTTGAAGGAAGCTTCAAGCCATAGCCGTGAAACAGATGAAGATATTCAAAATTTTGATCCATTTAGCTCTTTTATATATAACCCTAACCCTAATTCTGATGCTCTTGGGTTGAA GTTGCTTCCTTTTTATCGCCAGAAGAAGAAATTTGTAGTCCGAAAAGCGTAGTTTCAGTAAGCTTGGAGATCGGAAGAGAGAACTGGAAAGCAGGCGAGTTGGATCACAGAAGGATAAGAGATTTTTTTGATGGTGATGATGAGATCTTATGTAAGAAGGTGAAGACTGATCTAATGCTGACTATGGATAGAATGGATCCACCCGATAATCAGCAGACTTCCAATTCAAAGGTAATAAAACTTAGCCATAGTCCAGTTGATTTGGATCTTGAACTTAGGCTTGGTGATTCACCAAAGGTTAAGGAGAGTCGTAACATCATTGTGATAGAGTAA.

[0037] The application also provides the application of the CsRBE gene in regulating the morphology of the lip petal of Cymbidium sinense, and preferably regulates the morphology of the lip petal of Cymbidium sinense by silencing the CsRBE gene. In the application, the morphology of the lip petal of Cymbidium sinense is flat and straight without bending, the width of the lip petal is increased, and the middle wrinkle gap of the lip petal is increased.

[0038] The application also provides a protein encoded by the CsRBE gene, and the amino acid sequence of the protein is shown in SEQ ID No. 2, and specifically as follows:

[0039] MRAERNFLCRPLLPTFILSSSTNVSWEEQAFAKDSSGDLGGCVWPPRSYTCSFCRREFRSAQALGGHMNVHRRDRAKLKEASSHSRETDEDIQNFDPFSSFIYNPNPNSDALGFEVASFLSPEEEICSPKSVVSVSLEIGRENWKAGELDHRRIRDFFDGDDEILCKKVKTDLMLTMDRMDPPDNQQTSNSKVIKLSHSPVDLDLELRLGDSPKVKESRNIIVIE*.

[0040] The application also provides a method of VIGS (virus-induced gene silencing) based on TRV (Tobacco Rattle Virus), which regulates the morphology of the lower lip of Cymbidium by silencing the CsRBE gene, comprising the following steps:

[0041] 1) connecting the sequence of 1-300 bp of the CsRBE gene in the above technical solution to the pTRV2 vector to obtain a pTRV2-CsRBE vector;

[0042] 2) transforming the pTRV2-CsRBE vector obtained in the step 1) into Agrobacterium to obtain a bacterial liquid containing the pTRV2-CsRBE vector;

[0043] 3) centrifuging the bacterial liquid containing the pTRV2-CsRBE vector obtained in the step 2) to obtain bacterial bodies, mixing the bacterial bodies with a buffer to obtain a CsRBE bacterial liquid, and the OD value of the CsRBE bacterial liquid is 0.5; 600

[0044] 4) transforming the pTRV1 vector into Agrobacterium to obtain a bacterial liquid containing the pTRV1 vector, centrifuging the bacterial bodies to obtain a pTRV1 bacterial liquid, and the OD value of the pTRV1 bacterial liquid is 0.5; 600

[0045] 5) mixing the CsRBE bacterial liquid obtained in the step 3) with the pTRV1 bacterial liquid obtained in the step 4) in equal volume to obtain an infection liquid;

[0046] 6) infecting the Cymbidium buds with the infection liquid obtained in the step 5).

[0047] The application connects the sequence of 1-300 bp of the CsRBE gene in the above technical solution to the pTRV2 vector to obtain a pTRV2-CsRBE vector.

[0048] ​​The pTRV2-CsRBE vector obtained in the present application is transformed into Agrobacterium, and a bacterial liquid containing the pTRV2-CsRBE vector is obtained by culture. The method for transforming Agrobacterium with the pTRV2-CsRBE vector is not particularly limited in the present application, and can be performed by a person skilled in the art using a conventional method. The culture conditions are not particularly limited in the present application, and can be performed by a person skilled in the art using a conventional culture method. In the present application, the OD value of the bacterial liquid containing the pTRV2-CsRBE vector is preferably 1. 600 In the present application, the Agrobacterium is preferably Agrobacterium GV3101.

[0049] The bacterial liquid containing the pTRV2-CsRBE vector obtained in the present application is centrifuged, and the obtained bacterial body is mixed with a buffer to obtain a CsRBE bacterial liquid with an OD value of 0.5. 600 In the present application, the components of the buffer are preferably 100 μm acetosyringone, 10 mM morpholinoethanesulfonic acid and 10 mM magnesium chloride. In the present application, the centrifugation conditions preferably include a rotation speed of 5000 rpm and a time of 8 min.

[0050] The pTRV1 vector is transformed into Agrobacterium, and a bacterial liquid containing the pTRV1 vector is obtained by culture. The bacterial body obtained by centrifugation is mixed with a buffer to obtain a pTRV1 bacterial liquid with an OD value of 0.5. 600 The method for transforming Agrobacterium with the pTRV1 vector is not particularly limited in the present application, and can be performed by a person skilled in the art using a conventional method. The culture conditions are not particularly limited in the present application, and can be performed by a person skilled in the art using a conventional culture method. In the present application, the Agrobacterium is preferably Agrobacterium GV3101. In the present application, the OD value of the bacterial liquid containing the pTRV1 vector is preferably 1. 600 In the present application, the components of the buffer are preferably 100 μm acetosyringone, 10 mM morpholinoethanesulfonic acid and 10 mM magnesium chloride. In the present application, the centrifugation conditions preferably include a rotation speed of 5000 rpm and a time of 8 min.

[0051] The CsRBE bacterial liquid obtained in the present application is mixed with the pTRV1 bacterial liquid obtained in the present application in equal volume to obtain an infection liquid.

[0052] The flower bud of Eupatorium fortunei is infected with the infection liquid obtained in the present application. In the present application, the infection conditions preferably include infection under vacuum for 10 min, and the flower bud is a flower bud with a wound.

[0053] In order to further illustrate the present application, the present application is described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0054] Example 1

[0055] Cloning of CsRBE gene of Cymbidium

[0056] (1) Total RNA was extracted from the flower of Cymbidium variety ‘Xiaoxiang’, and the extraction kit was RNA extraction kit (commercially available). Reverse transcription of total RNA into cDNA was performed using a reverse transcription kit (commercially available), and the operation method was performed according to the kit instructions.

[0057] (2) Primers were designed according to the CDS sequence of CsRBE (RABBIT EARS) gene in Cymbidium genome, and the primer sequences are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0058] SEQ ID No. 3: ATGCGTGCAGAAAGGAATT;

[0059] SEQ ID No. 4: TTACTCTATCACAATGATGTTACGAC.

[0060] (3) The cDNA obtained in step (1) was used as a template for PCR amplification, and the amplification product was purified and recovered using a gel recovery kit (commercially available, operated according to the instructions). The recovered product was connected to TA / Blunt-Zero vector using 5min TA / Blunt-Zero Cloning Kit (Vazyme, commercially available, operated according to the instructions), and the vector was transformed into DH5a E. coli competent cells (commercially available, operated according to the instructions). Positive monoclonal was selected and subjected to Sanger sequencing. After analyzing and comparing the sequencing results, the complete CDS sequence of the target gene CsRBE was obtained, which contains 687 bases, the nucleotide sequence is shown in SEQ ID No. 1, and consists of 229 amino acid residues, the amino acid sequence is shown in SEQ ID No. 2.

[0061] Example 2

[0062] Characteristics analysis of CsRBE gene of Cymbidium

[0063] (1) Homologous alignment of CsRBE amino acid sequence was performed through https: / / www.ncbi.nlm.nih.gov website, and 14 sequences with higher homology were found.

[0064] (2) Evolutionary analysis and mapping of the 14 sequences in step (1) were performed using MAGA11 software, as shown in Figure 1 , Cymbidium was mainly clustered with monocotyledonous plants, and had higher homology with Dendrobium and Phalaenopsis.

[0065] (3) Alignment of CsRBE amino acid sequence was performed using DNAMAN software, as shown in Figure 2As shown. CsRBE gene contains C2H2-type zinc finger conserved domain, which is a zinc finger protein transcription factor.

[0066] Example 3

[0067] Verification of Cymbidium ensifolium expression profile at different development stages

[0068] (1) The single-lip petal Cymbidium variety 'Xiaoxiang' and the multi-lip variety 'Yushizi' were used as materials, and the RNA extraction kit (commercially available) was used to extract the RNA of the two varieties at 5 flower development stages. The total RNA was reverse transcribed into cDNA using the reverse transcription kit, and the operation method was performed according to the kit instructions.

[0069] (2) The real-time fluorescence quantitative PCR (RT-qPCR) primers were designed according to the coding region sequence of CsRBE gene, and the primer sequences are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0070] SEQ ID No. 5: TTTCGCAAAAGACTCGTCGG;

[0071] SEQ ID No. 6: CCTAGAGCTTGGGCTGATCG.

[0072] (3) RT-qPCR analysis was performed using Taq Pro Universal SYBR qPCR Master Mix kit (Vazyme, commercially available), and the reaction system 20 μl was operated according to the instructions. The amplification program was 95℃ for 2 min, (95℃ for 10 sec, 60℃ for 30 sec), 40 cycles of the steps in the parentheses, and then melting for 15 sec. Actin was selected as the internal reference, and the comparative Ct value method was used for data analysis. The relative expression of CsRBE in 5 different stages of flower development is shown in Figure 3 The expression level of CsRBE gene in the multi-lip variety 'Yushizi' was significantly higher than that in the normal flower variety 'Xiaoxiang' at 5 stages, which revealed that the gene may be involved in regulating the development of Cymbidium lip.

[0073] Example 4

[0074] TRV-induced CsRBE gene VIGS silencing in Cymbidium ensifolium

[0075] (1) The homologous recombination primers were designed by using the online primer design software CE Design of Vazyme website https: / / tool.vazyme.com:18002 / cetool / singlefragment.html, and the primer sequences are shown in SEQ ID No. 7 and SEQ ID No. 8.

[0076] SEQ ID No. 7: agaaggcctccatggggatccATGCGTGCAGAAAGGAATTTTC;

[0077] SEQ ID No. 8:

[0078] gagacgcgtgagctcggtaccAGAGCTAAATGGATCAAAATTTTGAA.

[0079] (2) The pTRV2 plasmid was double-digested by selecting BamHI and KpnI as double digestion sites, and the product was recovered by using a gel recovery kit (commercially available, operated according to the method described in the instruction manual).

[0080] (3) The CsRBE gene, i.e., the sequence of 1-300 bp in SEQ ID No. 1, was selected, the cDNA of Meilan 'Xiaoxiang' was used as a template, and the PCR amplification was performed by using the primers in step (1), and the PCR product was recovered by using a gel recovery kit (commercially available, operated according to the method described in the instruction manual).

[0081] (4) The 300 bp sequence in step (3) was connected to the pTRV2 vector by using the CloneExpress Ultra One Step Cloning Kit V2 kit (commercially available, operated according to the method described in the instruction manual) to form the pTRV2-CsRBE vector containing the CsRBE gene fragment, and the vector map is shown in Figure 4 . The size of the inserted fragment was verified by PCR by using the universal primers of the pTRV2 vector SEQ ID No. 9 and SEQ ID No. 10, and the bacterial liquid was subjected to Sanger sequencing to verify the accuracy of the inserted CsRBE gene fragment.

[0082] SEQ ID No. 9: TTATGTTCAGGCGGTTCTT;

[0083] SEQ ID No. 10: TTGCCTTTGTAACCATCATC.

[0084] (5) The pTRV2 empty, pTRV1 empty and pTRV2-CsRBE vector in step (4) were respectively transformed into Agrobacterium GV3101 (commercially available), and the experimental operation was carried out according to the instruction manual. The positive monoclonal containing pTRV2 empty or pTRV2-CsRBE obtained by screening was verified by bacterial liquid PCR using pTRV2 vector primers SEQ ID No. 9 and SEQ ID No. 10. The positive monoclonal containing pTRV1 empty obtained by screening was verified by bacterial liquid PCR using pTRV1 vector primers SEQ ID No. 11 and SEQ ID No. 12. Agrobacterium liquid containing pTRV2 empty, pTRV1 empty and pTRV2-CsRBE vector was obtained, respectively.

[0085] SEQ ID No. 11: GCGATAGCTCTTCACAGCCT;

[0086] SEQ ID No. 12: CATGACAGGTCTCGCCACTT.

[0087] (6) The three kinds of Agrobacterium liquid containing correct identification obtained in step (5) were respectively inoculated into LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin. The Agrobacterium liquid was cultured overnight at 28°C, 200 rpm in dark environment until the OD 600 of the Agrobacterium liquid was 1.0.

[0088] (7) The Agrobacterium liquid was respectively transferred to a 50 ml centrifuge tube and centrifuged at 5000 rpm for 8 minutes. The supernatant was discarded, and the bacterial pellet was resuspended in a buffer containing 100 μm acetyl-syringone, 10 mM morpholinoethanesulfonic acid and 10 mM magnesium chloride to an OD 600 value of 0.5. Then, the pTRV2-CsRBE and pTRV1 resuspension were mixed in equal volume, and the pTRV1 and pTRV2 resuspension were mixed in equal volume (control). After standing for 3 h, two kinds of infection liquid were obtained.

[0089] (8) The injector needle was used to make a wound on the bud of the orchid, and the bud was soaked in the infection liquid under vacuum condition for 10 min. After taking out, the bud was planted back into the flowerpot and transferred into the greenhouse for conventional cultivation and management.

[0090] (9) The flower phenotype was observed after conventional cultivation for 70-90 days. As shown in Figure 5 and Figure 6 , it was found that the morphology of the flower lip of the orchid silencing plant changed from the two sides bending upward of the control to flat without bending, the width of the lip became wider, and the gap of the wrinkle in the middle of the lip also became larger. It was indicated that the CsRBE gene was involved in the regulation of the development of the orchid lip.

[0091] (10) The labial petals of the empty control strain and the gene silencing strain as shown in Figure 6 were selected respectively, and the RNA was extracted by using an RNA extraction kit (commercially available). The total RNA was reverse transcribed into cDNA by using a reverse transcription kit, and the operation was performed according to the kit instructions. Then, the RT-qPCR detection analysis was performed according to the primers in step (2) and the method in step (3) in Example 3, and the expression level of the CsRBE gene in the labial petals of the silencing plant was significantly down-regulated, as shown in Figure 7 , which proved that the silencing efficiency of CsRBE in the silencing plant was high.

[0092] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A CsRBE gene regulating the morphology of the lip petals of Cymbidium goeringii, characterized in that, The nucleotide sequence of the CsRBE gene is shown in SEQ ID No.

1.

2. The application of the CsRBE gene as described in claim 1 in regulating the morphology of Cymbidium goeringii lip; The morphology of the lip petal of Cymbidium goeringii is regulated by silencing the CsRBE gene. Silencing the CsRBE gene results in a flat, uncurved lip shape in Cymbidium goeringii, increasing the lip width and the gap between the folds in the middle of the lip.

3. A protein encoded by the CsRBE gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID No.

2.

4. A method for regulating the morphology of the lip petal of Cymbidium goeringii, characterized in that, Includes the following steps: 1) The 1-300 bp sequence of the CsRBE gene described in claim 1 is ligated into the pTRV2 vector to obtain the pTRV2-CsRBE vector; 2) Transform the pTRV2-CsRBE vector obtained in step 1) into Agrobacterium and culture it to obtain a bacterial culture containing the pTRV2-CsRBE vector; 3) Centrifuge the bacterial culture containing the pTRV2-CsRBE vector obtained in step 2), mix the obtained bacterial cells with buffer to obtain CsRBE bacterial culture, OD 600 The value is 0.5; 4) Transform Agrobacterium into the pTRV1 vector to obtain a bacterial culture containing the pTRV1 vector. Mix the obtained bacterial cells with buffer after centrifugation to obtain the pTRV1 bacterial culture. OD 600 The value is 0.5; 5) Mix the CsRBE bacterial solution obtained in step 3) with the pTRV1 bacterial solution obtained in step 4) in equal volumes to obtain the infection solution; 6) Infect the Cymbidium goeringii buds with the inoculum obtained in step 5).

5. The method according to claim 4, characterized in that, The OD of the bacterial suspension containing the pTRV2-CsRBE vector in step 2) 600 The value is 1; the Agrobacterium is Agrobacterium GV3101.

6. The method according to claim 4, characterized in that, The buffer solution in step 3) consists of: 100 µm acetylsuccinone, 10 mM morpholine ethanesulfonic acid and 10 mM magnesium chloride; The centrifugation conditions include: a rotation speed of 5000 rpm and a time of 8 min.

7. The method according to claim 4, characterized in that, In step 4), the Agrobacterium is Agrobacterium GV3101; The OD of the bacterial culture containing the pTRV1 vector 600 The value is 1; The buffer solution consists of: 100 µM acetylsuccinone, 10 mM morpholine ethanesulfonic acid, and 10 mM magnesium chloride; The centrifugation conditions include: a rotation speed of 5000 rpm and a time of 8 min.

8. The method according to claim 4, characterized in that, The conditions for infection in step 6) include: infection under vacuum for 10 minutes, and the flower buds are flower buds with wounds.

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