Method for inducing orchid gene silencing by virus

Abstract

The invention discloses a method for inducing orchid gene silencing through viruses. According to the method provided by the invention, orchid buds are used as acceptor materials; the construction method comprises the following steps: constructing a silent vector containing 200-300bp target genes by taking Super-MV-pro60 as a basic vector, and introducing the silent vector into agrobacterium to obtain an engineering bacterium; and injecting the engineering bacterium liquid into the buds, and culturing. According to the method, efficient silencing of orchid genes can be achieved, the silencing success rate is 75% or above, the silencing efficiency is 60%-95%, and the method is good in stability and has a good silencing effect under different environmental conditions. The method provided by the invention provides a new technical means for orchid gene function identification, realizes efficient silencing of orchid genes, can be used for rapid verification of orchid flower color, flower fragrance, flower type and other gene functions, and has important significance for orchid function gene identification and cultivation of new orchid varieties through a molecular breeding technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding and genetic engineering, and more specifically relates to a method for virus-induced gene silencing in orchids. Background Art

[0002] In my country, orchids refer to plants of the genus Cymbidium in the family Orchidaceae, particularly terrestrial species such as Cymbidium goeringii, Cymbidium faberi, Cymbidium ensifolium, Cymbidium sinense, Cymbidium kanran, and Cymbidium tortisepalum, as well as their derivatives that possess characteristic orchid characteristics. These orchids are also known as "national orchids" (Zhang Zhisheng, 2024. Orchid Breeding / / Liu Qinglin, Jia Guixia. Garden Plant Breeding (3rd ed.). Beijing: China Forestry Publishing House, 394–423). Orchids are one of my country's traditional famous flowers, with a long history of cultivation. They are a material carrier of Chinese traditional culture, possessing high cultural value and unique ornamental value, and possess broad market prospects. Therefore, cloning genes for target traits in orchid breeding and clarifying their functions is of great significance for further promoting national orchid breeding and industrialization.

[0003] Due to the low efficiency of orchid transgenic technology and the long time it takes for transgenic seedlings to flower, identifying gene function through transgenic technology is extremely difficult. Therefore, establishing rapid and efficient technologies for identifying gene function is crucial for cloning genes for target breeding traits, accelerating molecular genetic research on these traits, and developing new varieties using molecular breeding techniques. Virus-induced gene silencing (VIGS) is a reverse genetics technique based on plant antiviral mechanisms and is related to post-transcriptional gene silencing (PTGS) in transgenic plants. When a virus or a viral vector carrying cDNA infects a plant, it is transcribed into single-stranded RNA (ssRNA). The ssRNA is synthesized into longer double-stranded RNA (dsRNA) by RNA polymerase (RNAdRP). The dsRNA is then cleaved by the enzyme Dicer into small interfering RNAs (siRNAs) of 21-24 nt. siRNA combines with endonucleases (RNases) to form an RNA-induced silencing complex (RISC). This complex specifically binds to homologous mRNA in cells, causing its degradation and preventing it from functioning as a messenger, thereby interfering with gene expression. This technology, independent of plant genetic transformation systems, offers advantages such as ease of use, short turnaround times, and high throughput, making it widely used in plant gene function research.

[0004] In the orchid family, VIGS technology systems have been established for Phalaenopsis and Dendrobium (Lu et al., 2007; Heish et al., 2014; Hou et al., 2023). However, this technology has not been well applied to orchids because it is difficult to inject the engineered bacterial solution into the orchid peduncle using a syringe. Furthermore, existing orchid VIGS methods suffer from low stability and low silencing efficiency (ranging from 30-50%). Therefore, research on orchid gene silencing technology, establishing orchid VIGS technology systems, and further improving gene silencing efficiency in orchids are of great significance for the rapid and efficient identification of target genes for orchid breeding traits and for promoting orchid molecular breeding and industrial development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of low efficiency of existing orchid transgenic technology and the lack of an efficient orchid VIGS method system, and to provide a method for virus-induced orchid gene silencing.

[0006] The purpose of the present invention is to provide a method for virus-induced gene silencing in orchids.

[0007] Another object of the present invention is to provide an application of the virus-induced orchid gene silencing method.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for virus-induced gene silencing in orchids, comprising the following steps:

[0010] S1. The recipient material is orchid buds;

[0011] S2. Construction of silencing vector: Using Super-MV-pro60 as a vector, insert a 200-300 bp target gene fragment to generate a silencing vector, which is then introduced into Agrobacterium.

[0012] S3. Preparation of engineered bacterial solution: Agrobacterium containing the silencing vector was inoculated into a culture medium containing kanamycin (Kan) and cultured with shaking. The bacteria were collected by centrifugation and resuspended in a culture medium containing acetosyringone (AS) to an OD of 600 is 0.8 to 1.2 to obtain an engineered bacterial solution;

[0013] S4. Injection: Inject the engineered bacterial solution into the orchid bud until the solution just begins to emerge from the top of the bud.

[0014] S5. Cultivation: Place the injected orchid in an environment with a temperature of 18-22°C and a natural light intensity of no less than 15,000 lux for cultivation.

[0015] The present invention is the first to carry out virus-induced orchid gene silencing (VIGS) on orchids, which can achieve high-efficiency gene silencing. The method uses the Super1300 vector as the backbone, inserts a recombinant vector consisting of the orchid leaf virus CymMV and the viral coat protein pro60 fragment, and then inserts the target gene to constitute the silencing vector. The silencing vector is transferred into Agrobacterium to obtain the engineered bacteria, and the orchid VIGS technology system is established by injecting unopened flower buds. This method system has a high gene silencing success rate and efficiency, good stability, and has a good silencing effect under different environmental conditions. By injecting orchid flower buds, it breaks the technical bottleneck of orchids and achieves efficient silencing of orchids. At the same time, it also solves the problem of low silencing efficiency of existing orchid VIGS technology. Among them, the target gene for VIGS insertion can be a gene with a length of 200 to 300bp, which can be used to quickly verify the functions of genes such as orchid flower color, flower fragrance, and flower shape. For example, by inserting color-related genes CxFLS or CxDFR genes, the orchid flower color can be regulated after silencing. New varieties of orchid flower colors can be cultivated through molecular breeding technology, laying the foundation for promoting the innovative development and high-quality development of my country's orchid industry.

[0016] Preferably, in S1, orchid buds 8 to 12 days before natural flowering are used as receptor materials, and the length of the buds is 1 to 3 cm.

[0017] Preferably, Super-MV-pro60 in S2 is a recombinant vector constructed by using Super1300 as a vector backbone and inserting CymMV and virus coat protein subgene promoter of CymMV orchid leaf virus.

[0018] More preferably, the method for constructing the Super-MV-pro60 vector is: amplifying two overlapping CymMV virus fragments, connecting them to T vectors respectively, transforming Escherichia coli, performing double enzyme digestion and ligation to obtain the recombinant vector CymMV-M; connecting the recombinant vector CymMV-M to the linearized Super1300 plasmid to obtain the recombinant vector Super-MV-M; inserting the enzyme cutting site SmaI and the subpromoter pro60 of the capsid protein into the Super-MV-M vector to construct the recombinant vector Super-MV-pro60.

[0019] As a specific implementation plan, the optimal construction method of the Super-MV-pro60 vector is: extract RNA from Jianlan leaves infected with CymMV virus, reverse transcribe and synthesize cDNA, and use this as a template to amplify two overlapping CymMV fragments, the first fragment is 3865bp long and the second fragment is 2474bp long, respectively ligated to T vectors, and then transformed into Escherichia coli; use NotI and SnaI to double-enzyme digest and ligate the two plasmids to construct a recombinant vector CymMV-M, and ligate this vector to linearized Super1300 to construct a recombinant vector Super-MV-M; finally, insert a restriction site SmaI and the subpromoter pro60 of the coat protein (CP) into this vector to construct the recombinant vector Super-MV-pro60.

[0020] Preferably, the Agrobacterium in S2 is GV3101.

[0021] Preferably, the engineered bacterial solution in S3 is prepared by culturing in LB culture medium containing 40-60 μg / mL kanamycin; and resuspending the bacteria in MS culture medium containing 90-110 μmol / L acetosyringone.

[0022] More preferably, LB culture medium containing 50 μg / mL kanamycin and MS culture medium containing 100 μmol / L acetosyringone are used.

[0023] Preferably, the bacterial solution OD in S3 600 It is 1.0~1.2.

[0024] Preferably, Agrobacterium is injected from the bottom of the flower bud in S4.

[0025] As a specific implementation scheme, the preparation method of the engineered bacterial solution is as follows: Agrobacterium GV3101 containing the silencing vector is inoculated into 500 μL of LB culture medium containing 50 μg / mL of Kan and cultured with shaking; 100 μL is then added into 100 mL of LB culture medium containing 50 μg / mL of Kan and cultured with shaking until the OD reaches 0. 600 The value was 0.8-1.2, and the cells were collected by low-temperature centrifugation and resuspended in MS culture medium containing 100 μmol / L AS to OD 600 The viscosity is 0.8 to 1.2, and it is allowed to stand at room temperature for later use.

[0026] Specifically, the target gene inserted by the VIGS method provided by the present invention can be a gene with a fragment length of 200-300 bp. Gene type is not limited; theoretically, any gene can be inserted. Existing VIGS technology research on orchids indicates that the optimal insertion fragment length is 81-334 bp. During vector design, optimization research conducted by the present invention has found that gene fragments of 200-300 bp are more suitable for insertion. This method can be used to insert genes that regulate flower color in orchids, as well as genes that control floral traits such as flower shape and fragrance. For example, genes such as ANS, CHS, PI, 4CL, and TPS can all be used for gene silencing using the VIGS method provided by the present invention.

[0027] Preferably, the target gene in S2 is ANS, CHS, PI, 4CL, TPS, DFR or FLS gene.

[0028] More preferably, the target gene in S2 is dihydroflavonol reductase DFR gene.

[0029] The present invention provides application of the above method in verifying orchid gene function or cultivating new orchid flower color varieties.

[0030] The present invention has the following beneficial effects:

[0031] The present invention provides a method for virus-induced gene silencing of orchids. The Super1300 vector is used as a skeleton, and a recombinant vector consisting of the CymMV orchid leaf virus and the pro60 fragment of the viral coat protein is inserted. The inserted target gene constitutes a silencing vector, and the silencing vector is transferred into Agrobacterium to obtain an engineered bacterium. An orchid VIGS technology system is established by injecting unopened flower buds. This method system performs high-efficiency gene silencing on orchids for the first time, has a high gene silencing success rate and efficiency, good stability, and has a good silencing effect under different environmental conditions. By injecting the engineered bacterial solution into the orchid buds, the technical bottleneck of orchids is broken and efficient silencing of orchids is achieved. This method has a high success rate and silencing efficiency when repeatedly used in different orchid varieties. At the same time, it also solves the problem of low silencing efficiency in the existing VIGS technology for orchid plants. It can be used to quickly verify the functions of genes such as orchid color, fragrance, and flower shape, cultivate new varieties of orchid color through molecular breeding technology, and promote the innovative development and high-quality development of my country's orchid industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the construction of Super-MV-pro60 recombinant vector.

[0033] Figure 2 Schematic diagram of the structure of orchid leaf virus CymMV, recombinant vector Super-MV-pro60 and Super-MV-DFR.

[0034] Figure 3 Schematic diagram of injection of flower buds.

[0035] Figure 4 The effect of different silencing injection methods on gene silencing effect (A is conventional peduncle injection; B is bud injection).

[0036] Figure 5 This is the phenotype of 'Yuyuanlan' with DFR silenced.

[0037] Figure 6 The gene expression level (A) and anthocyanin content (B) in the flowers of 'Yuyuanlan' after silencing DFR with different bacterial solution concentrations (Note: WT in the figure is wild type; dp means cultured in a greenhouse, and the numbers represent bacterial solution concentrations).

[0038] Figure 7 The phenotype of 'Yuxianglan' with DFR silenced.

[0039] Figure 8 The gene expression level (A) and anthocyanin content (B) in the flowers of 'Yuxianglan' after silencing DFR in different environments and concentrations (Note: in the figure, dp means culture in a greenhouse, ws means culture in a greenhouse, and the numbers are bacterial liquid concentrations). DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0042] The hybrid orchid plants of 'Xiaofenglan', 'Yuyuanlan' and 'Yuxianglan' used in the examples were all independently cultivated by the College of Forestry and Landscape Architecture of South China Agricultural University.

[0043] The culture medium formula used in the following examples is:

[0044] LB solution: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and dissolve in water to 1 L.

[0045] MS solution: Add 4.74 g MS powder per liter of water, sterilize at high temperature, and then add 100 μmol / L acetosyringone.

[0046] Example 1 Construction of VIGS vector

[0047] 1. Construction of Super-MV-pro60 vector

[0048] RNA was extracted from leaves of CymMV-infected plants (using the Meiji Polysaccharide and Polyphenol Plant Total RNA Extraction Kit) but with no obvious symptoms. cDNA was synthesized using a reverse transcription kit (Guangzhou Xinkailai Biotechnology Co., Ltd.). Two overlapping CymMV fragments, 3865 bp long and 2474 bp long, were amplified using this template. These fragments were then ligated into T-seq vectors and transformed into Escherichia coli. The two plasmids were then digested with NotI and SnaI and ligated to construct the recombinant vector CymMV-M. This vector was then ligated into linearized Super1300 to create the recombinant vector Super-MV-M.

[0049] Finally, a restriction enzyme site SmaI and the sub-promoter pro60 of the coat protein (CP) were inserted into this vector to construct the recombinant vector Super-MV-pro60. The construction process is as follows Figure 1 shown.

[0050] 2. Construction of silencing vector containing target gene

[0051] Flowers of the hybrid Cymbidium 'Xiaofeng' (Cx) were used as the material, and cDNA was synthesized as the template using a reverse transcription kit (Guangzhou Xinkailai Biotechnology Co., Ltd.). The CxDFR gene was used as the target gene, and primers for the target gene were designed. The primer sequences used were:

[0052] DFR-F: 5'-AATTCAAAGTCGAGTTGAAGCTAAG-3';

[0053] DFR-R: 5'-CACAACATTTCTCCTTGCTCATT-3'.

[0054] The reaction program for amplification was 95°C for 30s, 95°C for 30s, 55°C for 30s, and 72°C for 30s, 30 cycles, and 72°C for 5min. The amplification reaction system was: 7.5μL of 2×Phanta Max Master Mix (Nanjing Novozymes Biotech Co., Ltd.), 0.5μL of forward primer and reverse primer, 0.5μL of template cDNA, and sterile water was used to make up the total system to 15μL.

[0055] The amplified product was subjected to 1% agarose electrophoresis to recover the target band.

[0056] Then, primers containing homology arms were designed and used to amplify the above products. The amplification procedure and system were the same as above. The primer sequences used were:

[0057] Super-MV-DFR-F: 5'-gagcccactccaactcccgggCACCCGAGTAAAGATGACCG G-3';

[0058] Super-MV-DFR-R: 5'-tagtgatagtaatagcccgggCTGCCATTTGCTTTGTGATGC-3'.

[0059] The amplified product was subjected to 1% agarose electrophoresis, and the target band was recovered to obtain the inserted fragment: V-DFR.

[0060] The Super-MV-pro60 recombinant vector was then digested with the endonuclease SmaI (Thermo Fisher Scientific (China) Co., Ltd.), and the V-DFR fragment was inserted. A homologous recombination reaction solution was prepared using the Uniclone One Step Seamless Cloning Kit (Jinsha Biotechnology): 5 μL of 2× Uniclone Seamless Cloning Mix, 200 ng of linearized vector, and 200 ng of insert fragment. The mixture was made up to 10 μL with ddH2O and reacted at 50°C for 15 min to construct the recombinant vector Super-MV-DFR.

[0061] After the reaction, 10 μL of the recombinant plasmid was transformed into E. coli DH5α competent cells and cultured overnight in LB medium containing 50 μg / mL kanamycin until single colonies were formed. After PCR detection, sequencing was performed to verify the correctness of the sequence of the recombinant plasmid insertion position. The length of the inserted fragment was 291 bp, and the correctly inserted recombinant plasmid Super-MV-DFR was obtained. The schematic diagram of different vectors is shown in the figure. Figure 2 shown.

[0062] Example 2 Preparation of Engineering Bacterial Liquid

[0063] 1. Obtaining engineered Agrobacterium

[0064] The recombinant plasmid Super-MV-DFR and the empty vector plasmid Super-MV-pro60 constructed in Example 1 were transformed into competent Agrobacterium GV3101 by liquid nitrogen freeze-thaw method, cultured on solid LB medium containing 50 μg / mL kanamycin until single colonies were formed, and single colonies were picked.

[0065] Subsequently, PCR detection was performed using universal vector primers to confirm the presence of the target vector. The primer sequences used were:

[0066] 4948F: 5'-CCACTCAGCAGCCTTCATCG-3';

[0067] 5565R: 5'-GGTGACAGGTGAGTACTTAATG-3'.

[0068] 2. Preparation of engineered bacterial solution

[0069] Place the Agrobacterium GV3101 containing the target plasmid in 500 μL of LB solution containing 50 μg / mL Kan, and shake culture at 200 rpm for 12 to 16 hours; pipette the solution into 100 mL of LB solution containing 50 μg / mL Kan, and continue shaking culture until OD 600The value is 1.0 to 1.2; then centrifuge at 5000 rpm for 10 min at 4°C to collect the bacteria (this step should be performed at low temperature as much as possible); resuspend the bacteria in MS culture medium containing 100 μmol / L AS (after high temperature sterilization) to obtain the OD value. 600 The viscosity is 0.8-1.2, and it is allowed to stand at room temperature for 0.5h.

[0070] Example 3 A virus-induced gene silencing method for orchids

[0071] 1. Phenotypic effects

[0072] The orchid used in this example is 'Yuyuanlan', and a 1.0 mL sterile syringe is used to draw the OD 600 The engineering bacterial solution was 1.0, and the bacterial solution was injected from the bottom of the flower bud 10 days before flowering ( Figure 3 The orchid was in bloom and the phenotype was observed after the bacterial liquid just appeared at the top of the flower bud.

[0073] The results are as follows Figure 4 As shown in B, the color of flowers injected with engineered bacterial solution became lighter than that of flowers injected with empty vector, indicating that the method of injecting flower buds with Super-MV-pro60 vector can effectively silence genes.

[0074] 2. Effect of bacterial solution concentration on gene silencing in 'Yuyuanlan'

[0075] When preparing the engineering bacterial solution according to the method in Example 2, the OD value of the resuspended bacterial solution was 600 The concentrations of 0.8, 1.0, and 1.2, respectively, were then injected into the flower buds of 'Yuyuanlan' 9-12 days before anthesis, following the above-described injection procedure. After injection, the orchids were cultured in a greenhouse (18-20°C, 15,000 lux). The gene silencing success rate was then calculated based on the color change phenotype of the flowers. The gene silencing success rate (%) was calculated as the percentage of flowers with color change divided by the total number of flowers injected.

[0076] The results of the effects of different bacterial solution concentrations on the gene silencing effect of 'Yuyuanlan' are shown in Table 1, which shows that the success rate of gene silencing can reach 85% to 100%. 600 The silencing success rate is higher when the OD value is 1.0 and 1.2. 600 When the value is 1.2, the orchid flower color may turn yellow. Different orchid phenotypes such as Figure 5 shown.

[0077] Table 1 Effect of bacterial solution concentration on gene silencing in 'Yuxianglan'

[0078]

[0079] Note: a. The number of flowers that produced color changes; b. The total number of flowers processed, the same below.

[0080] 3. Gene Expression Analysis

[0081] RNA was extracted from the flowering 'Yuyuanlan' plant using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Meiji Biotechnology). cDNA was synthesized using All-in-One First-Strand Synthesis MasterMix (with dsDNase) (Guangzhou Xinkailai Biotechnology Co., Ltd.). Fluorescence quantitative PCR was then performed using ChamQ Universal SYBR qPCR Master Mix (Novozymes). The gene primers used for fluorescence quantitative PCR were:

[0082] RPS3-F: 5'-TGAAGCCAATGCAGAACAAACG-3';

[0083] RPS3-R: 5'-ATGGATTTCACCACCGACAAGC-3';

[0084] qDFR-F: 5'-GGCTGCGGTCAGAGATTCAA-3';

[0085] qDFR-R: 5'-GCTGAGCAGTTCATTCGAGC-3'.

[0086] The reaction system for the assay consisted of 5 μL of 2× ChamQ Universal SYBR qPCR Master Mix, 0.2 μL Primer (10 μM), 0.2 μL Primer 2 (10 μM), 2 μL of cDNA (10-fold dilution), and 2.6 μL of ddH₂O. The reaction procedure was: 95°C pre-denaturation for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. After the assay, DFR gene expression was analyzed and gene silencing efficiency was calculated. Gene silencing efficiency (%) is the percentage of target gene expression in silenced flowers divided by target gene expression in control flowers.

[0087] Test results such as Figure 6 As shown in A, it was found that VIGS silencing resulted in a significant decrease in DFR gene expression, with the bacterial solution OD 600 When the value is 1.2, the effect on gene expression is more significant.

[0088] Anthocyanin extraction kit (Shangbao Bio) was used to extract anthocyanin and the anthocyanin content was determined. Figure 6 As shown in B, VIGS silencing significantly reduced the anthocyanin content.600 When the OD value was 1.2, the anthocyanin content of the injected flowers decreased the most, and the gene silencing efficiency was the highest, at 95%. 600 When 0.8 to 1.2 of the engineered bacterial solution is injected into the flower bud, the gene silencing efficiency is 60 to 95%.

[0089] Example 4 Gene silencing effect of 'Yuxianglan'

[0090] 1. Effects of different culture environments on gene silencing in 'Yuxianglan'

[0091] When preparing the engineering bacterial solution according to Example 2, the OD 600 The solution was diluted to 0.8, 1.0, and 1.2, and then injected into 'Yuxianglan' flower buds 5-8 days before anthesis, following the method for injecting flower buds in Example 3. After injection, orchids treated with different bacterial solution concentrations were cultured in a greenhouse (20-22°C, 3000 lux) and a greenhouse (18-20°C, 15000 lux of natural light). The effects of different culture environments and bacterial solution concentrations on the gene silencing efficacy of 'Yuxianglan' were then statistically analyzed. The gene silencing success rate (%) was calculated as the percentage of flowers with color change / the total number of flowers injected; the gene silencing efficiency (%) was calculated as the percentage of the target gene expression in the silenced flowers / the target gene expression in the control flowers.

[0092] The results are shown in Table 2, which show that different culture environments can affect the gene silencing effect. However, the method of using Super-MV-pro60 vector injection has a high success rate and good stability in different environments. The flower phenotypes of different environments and bacterial solution viruses are as follows: Figure 7 As shown, bacterial suspension OD 600 When the value was 1.0, the gene silencing success rate was the highest, 100%, in greenhouses after injection.

[0093] Table 2 Effects of culture environment and bacterial concentration on the success rate of gene silencing in 'Yuxianglan' (%)

[0094]

[0095] The gene expression analysis and anthocyanin content determination of the silenced 'Yuxianglan' flowers were carried out in the same manner as in Example 3. Figure 8 As shown in the figure, the culture environment has a significant effect on anthocyanin content and DFR gene expression. 600 When the value was 1.2, the DFR gene expression levels of the treatment groups cultured in greenhouses were higher than those of the treatment groups cultured in greenhouses ( Figure 8 A), where bacterial solution OD 600The gene expression level of the plants cultured in the greenhouse after injection was 1.0. The anthocyanin content in the greenhouse was also higher than that in the greenhouse ( Figure 8 B) It was found that the higher the bacterial concentration, the lower the anthocyanin content. 5 to 8 days before flowering, the bacterial solution OD 600 When 1.0 to 1.2 of the engineered bacterial solution is injected into the flower bud, the gene silencing efficiency is 50% to 70%.

[0096] Based on the above results, it is shown that the method of silencing orchid genes by virus-induced gene silencing using Super-MV-pro60 vector injection has better stability. This method has a high success rate and silencing efficiency when repeatedly used in different orchid varieties, breaking the bottleneck of orchid VIGS technology and achieving efficient silencing of orchid genes. The best method for silencing orchid genes by virus-induced gene silencing is: using orchid buds 9 to 12 days before natural flowering as the receptor material, using a 1.0mL sterile syringe to draw an engineered bacterial solution with an OD value of 1.0 to 1.2, injecting it into the bud until the bacterial solution just emerges from the top of the bud, and culturing the orchid after injection in an environment of 18-22°C and a natural light intensity of about 15,000 lux. The method provided by the present invention can achieve efficient silencing of orchid genes, with a silencing success rate of more than 75% and a silencing efficiency of 60% to 95%.

[0097] Comparative Example 1: Peduncle Injection by Conventional VIGS Method

[0098] The VIGS injection method for orchid plants disclosed in the prior art was used to conduct the experiment. A 1.0 mL sterile syringe was used to draw the OD 600 The engineered bacterial solution was 1.0, and the bacterial solution was injected into the peduncle of the orchid inflorescence that had opened flowers 10 days later. The changes in flower color were observed after the flowers opened.

[0099] The results are as follows Figure 4 As shown in A, the color of the flowers on the injected inflorescence stalks is no different from that on the uninjected ones, indicating that this method is not suitable for gene silencing in orchids. The reason is that the inflorescence stalks of orchids are thin, making it difficult to inject the bacterial solution. However, the method of the present invention can successfully perform gene silencing and change the flower color of orchids ( Figure 4 B).

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for virus-induced gene silencing in orchids, characterized in that: The following steps are involved: S1. The recipient material is orchid buds; S2. Construction of silencing vector: Using Super-MV-pro60 as a vector, insert a 200-300 bp target gene fragment to generate a silencing vector, which is then introduced into Agrobacterium. S3. Preparation of engineered bacterial solution: Agrobacterium containing the silencing vector was inoculated into a culture medium containing kanamycin and cultured with shaking. The bacteria were collected by centrifugation and resuspended in a culture medium containing acetosyringone to an OD of 600 is 0.8 to 1.2, and an engineered bacterial solution is obtained; S4. Injection: Inject the engineered bacterial solution into the orchid bud until the solution just begins to emerge from the top of the bud. S5. Cultivation: Place the injected orchid in an environment with a temperature of 18-22°C and a natural light intensity of no less than 15,000 lux for cultivation.

2. The method according to claim 1, characterized in that In S1, orchid buds 8 to 12 days before natural flowering were used as receptor materials.

3. The method according to claim 1, characterized in that Super-MV-pro60 in S2 is a recombinant vector constructed by using Super1300 as the vector backbone and inserting the CymMV and viral coat protein subgene promoters of the CymMV orchid leaf virus.

4. The method according to claim 3, characterized in that The construction method of the Super-MV-pro60 vector is as follows: two overlapping CymMV virus fragments are amplified, connected to the T vector respectively, transformed into Escherichia coli, and then double-enzyme digestion and ligation are performed to obtain the recombinant vector CymMV-M; the recombinant vector CymMV-M is connected to the linearized Super1300 plasmid to obtain the recombinant vector Super-MV-M; the restriction site SmaI and the subpromoter pro60 of the coat protein are inserted into the Super-MV-M vector to construct the recombinant vector Super-MV-pro60.

5. The method according to claim 1, characterized in that: The Agrobacterium in S2 is GV3101.

6. The method according to claim 1, characterized in that The engineering bacterial solution in S3 was prepared by culturing in LB culture medium containing 40-60 μg / mL kanamycin; and resuspending the bacteria in MS culture medium containing 90-110 μmol / L acetosyringone.

7. The method according to claim 6, characterized in that OD of bacterial suspension in S3 600 It is 1.0~1.

2.

8. The method according to claim 7, characterized in that: In S4, Agrobacterium is injected from the bottom of the flower bud.

9. The method according to claim 1, characterized in that: The target gene in S2 is ANS, CHS, PI, 4CL, TPS, DFR or FLS gene.

10. Use of the method according to any one of claims 1 to 9 in verifying orchid gene function or breeding orchid varieties with new flower colors.

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