A method for virus-induced gene silencing in hydrangea macrophylla
By applying virus-induced gene silencing technology to hydrangea, a TRV vector was cloned and constructed, and then used to infect hydrangea materials, successfully silencing related genes. This solved the problem of lack of tools for hydrangea functional genomics research and improved research efficiency.
Patent Information
- Application Number
- CN202411672365.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-12-19
- Estimated Expiration
- 2044-11-21
AI Technical Summary
The lack of effective molecular genetic tools in current technology limits the study of hydrangea functional genomics, especially since there are no reports of gene silencing using TRV in large-leaved hydrangea.
Virus-induced gene silencing (VIGS) technology was used to construct TRV vectors by cloning the full-length fragments of the HmPDS, HmF3'5'H, and HmCHS1 genes. These vectors were then transformed into Escherichia coli and Agrobacterium. Agrobacterium was used to infect Hydrangea macrophylla materials to silence the genes.
The expression of HmPDS, HmF3'5'H and HmCHS1 genes was effectively silenced, reducing the content of chlorophyll and anthocyanins and improving the efficiency of gene function research.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of research on the function of Hydrangea macrophylla genes, and particularly relates to a virus-induced gene silencing technology in Hydrangea macrophylla. BACKGROUND
[0002] Hydrangea is an important ornamental plant with important commercial value for potting, cut flowers and dried flowers. In addition, it is favored by consumers for its huge flower clusters, bright flower colors, diverse varieties and long flowering period. In addition, it is widely planted in gardens due to its strong adaptability, disease and pest resistance, cold tolerance and shade tolerance. Studies have shown that Hydrangea macrophylla has various medicinal properties (Zhang et al., 2009; Geum et al., 2020; Lee et al., 2022). However, functional genomics research is still limited due to the lack of effective molecular genetic tools. Therefore, it is necessary to develop tools to elucidate the gene function of Hydrangea.
[0003] Virus-induced gene silencing (VIGS) technology uses a recombinant viral vector containing a fragment of the target plant gene to activate RNA-mediated post-transcriptional gene silencing, thereby inhibiting the transcription of the homologous gene. Compared with gene silencing technology using inverted repeats, VIGS has several advantages, such as simpler plasmid construction and shorter operation period (Reid et al., 2009). In recent years, VIGS technology based on tobacco rattle virus (TRV) has been applied to various ornamental plants to verify gene function, such as peony (Xie et al., 2019), Chinese rose (Tian et al., 2014) and lily (Xu et al., 2019). VIGS is increasingly recognized as a very valuable tool for studying gene function. A key advantage of VIGS is its ability to quickly produce phenotypes without the need for stable plant transformation (Zulfiqar et al., 2023). Compared with other methods, including T-DNA, transposon insertion technology, chemical and physical mutagenesis, and CRISPR-Cas9 functional genomics editing methods, VIGS is a cost-effective choice (Liu et al., 2016). However, different species have different means of applying VIGS due to species-specificity, including infection solution concentration, infection method and post-culture. Currently, there is no report on using TRV for gene silencing in Hydrangea. SUMMARY
[0004] The purpose of the present application is to provide a virus-induced gene silencing technology in Hydrangea macrophylla to solve the problems existing in the prior art.
[0005] To achieve the above object, the present application provides the following scheme:
[0006] One of the technical solutions of the present application is a virus-induced gene silencing technology in Hydrangea macrophylla, which comprises the following steps:
[0007] (1) cloning full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes;
[0008] (2) constructing TRV vectors of HmPDS, HmF3'5'H and HmCHS1 respectively, transforming E. coli and Agrobacterium;
[0009] (3) Agrobacterium infection of Hydrangea macrophylla materials;
[0010] (4) culture of Hydrangea macrophylla after infection.
[0011] Based on the above technical solution, the present application has the following technical effects:
[0012] The present application provides a virus-induced gene silencing technology in Hydrangea macrophylla, which uses Hydrangea macrophylla cultivar 'Endless Summer' as material, infects the Hydrangea macrophylla tissue culture seedlings, silences the HmPDS gene, infects the inflorescences with 2-level open flowers, and silences HmF3'5'H and HmCHS1 respectively. Then, the silencing effect of the transgenic material is verified, and the results show that the expression levels of HmPDS, HmF3'5'H and HmCHS1 genes in the transgenic material are all reduced, the chlorophyll a, chlorophyll b and total chlorophyll in the leaves of the pTRV2-HmPDS infected tissue culture seedlings are decreased, the total anthocyanin content in the HmCHS1 silenced flowers is significantly reduced, and the primulaverin glucoside and total anthocyanin contents in the HmF3'5'H silenced flowers are reduced. It is shown that the gene transient silencing method disclosed in the present application can effectively silence the related genes in Hydrangea macrophylla, which is helpful to improve the efficiency of gene function research. BRIEF DESCRIPTION OF DRAWINGS
[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.
[0014] Figure 1Schematic diagram of pTRVl, pTRV2, pTRV2-HmPDS, pTRV2-HmCHSl and pTRV2-HmF3'5'H. LB, left border; RB, right border; RdRp, RNA-dependent RNA polymerase; MP, movement protein; 16k, 16kD protein; R, self-cleaving ribozyme; N, NOS terminator; CP, coat protein; MCS, multiple cloning site.
[0015] Figure 2 PDS silencing in TRV-induced Hydrangea macrophylla. (A) Wild type tissue culture seedlings (left) and those infected with TRV (middle) and pTRV2-HmPDS (right). Photographs were taken at 30 days post-infiltration. (B) Detection of TRV and HmPDS expression in each treatment. (C) Comparison of chlorophyll a, chlorophyll b and total chlorophyll contents between PDS-silenced and TRV-infected tissue culture seedlings.
[0016] Figure 3 CHS silencing in TRV-induced Hydrangea macrophylla. Pink flowers (A) and blue flowers (B) infected with TRV and pTRV2-HmCHSl, at 15 days post-infiltration. White scale bar represents 1 cm. (C) Detection of TRV and HmCHSl expression in each treatment. (D) Comparison of total anthocyanin contents between HmCHSl-silenced flowers and TRV controls.
[0017] Figure 4 F3'5'H silencing in TRV-induced Hydrangea macrophylla. (A) Flowers infected with TRV and pTRV2-HmF3'5'H, at 15 days post-infiltration. White scale bar represents 1 cm. (B) Detection of TRV and HmF3'5'H expression in each treatment. (C) Comparison of anthocyanin contents between HmF3'5'H-silenced flowers and TRV-infected flowers. (D) Comparison of anthocyanin relative abundance in flowers infected with TRV and pTRV2-HmF3'5'H. DETAILED DESCRIPTION
[0018] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is made with reference to the drawings, the detailed description is not to be considered limiting of the application, but merely an illustration of certain aspects, features and embodiments of the application.
[0019] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed by the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.
[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.
[0021] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The specification and examples given herein are exemplary and should not be used to limit the present application, unless otherwise specifically stated herein.
[0022] With respect to the terms "comprising", "including", "containing", "having" and the like, these terms are used inclusively and are open-ended, that is, they allow for items to be added.
[0023] The technical solutions described in the present application are conventional solutions in the art, and the reagents or raw materials used are commercially available or have been disclosed, unless otherwise specified.
[0024] The embodiments of the present application provide a virus-induced gene silencing technology in Hydrangea macrophylla, comprising the following steps:
[0025] (1) cloning full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes;
[0026] (2) constructing TRV vectors of HmPDS, HmF3'5'H and HmCHS1 respectively, transforming E. coli and Agrobacterium;
[0027] (3) Agrobacterium infection of Hydrangea macrophylla materials;
[0028] (4) culturing Hydrangea macrophylla after infection.
[0029] In some specific embodiments, the step of cloning full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes comprises:
[0030] amplifying the full-length fragment of HmPDS gene using primers shown in SEQ ID NO. 1-2;
[0031] amplifying the full-length fragment of HmCHS1 gene using primers shown in SEQ ID NO. 3-4;
[0032] amplifying the full-length fragment of HmF3'5'H gene using primers shown in SEQ ID NO. 5-6.
[0033] In some specific embodiments, the step of constructing TRV vectors of HmPDS, HmF3'5'H and HmCHS1 respectively comprises:
[0034] digesting pTRV2 vector using restriction enzymes EcoRI and KpnI;
[0035] amplifying the silencing fragment of HmPDS gene using primers shown in SEQ ID NO. 7-8;
[0036] amplifying the silencing fragment of HmCHS1 gene using primers shown in SEQ ID NO. 9-10;
[0037] amplifying the silencing fragment of HmF3'5'H gene using primers shown in SEQ ID NO. 11-12;
[0038] using IIOne Step Cloning Kit to ligate the silencing fragments of HmPDS, HmCHS1 and HmF3'5'H genes into pTRV2 vector respectively to construct pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H three kinds of recombinant plasmids.
[0039] In some specific embodiments, the step of transforming E. coli and Agrobacterium comprises:
[0040] transforming pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H three kinds of vector plasmids into E. coli TOP10, and performing sequencing after overnight culture at 37℃;
[0041] successfully constructed vectors are transformed into Agrobacterium tumefaciens GV3101 strain carrying plasmid pSoup-p19 by heat shock method, and after overnight culture at 28℃, primers shown in SEQ ID NO. 7-12 are used for PCR amplification to identify positive clones.
[0042] In some specific embodiments, the step of Agrobacterium infecting Hydrangea macrophylla material comprises:
[0043] Positive monoclonal Agrobacterium tumefaciens strains were cultured in liquid LB medium containing antibiotics at 28°C with 180 rpm shaker for 16 hours until OD 600 At 0.8-1.2, Agrobacterium tumefaciens cells were collected by centrifugation, resuspended to OD 600 1.5, and then infected the Hydrangea macrophylla material after 3h incubation in dark at 25°C.
[0044] In some specific embodiments, the infection solution comprises 10mM MgCl2, 10mM 2-(N-morpholino)ethanesulfonic acid (MES), and 200μM Acetyl-syringone (AS).
[0045] In some specific embodiments, the Hydrangea macrophylla material is the tissue culture seedling of Hydrangea macrophylla cultivar 'Endless Summer', which is cultured in an environment with 25°C, 2000lx light intensity, and 16h / 8h light cycle.
[0046] The rooted Hydrangea macrophylla tissue culture seedling was taken out from the bottle and pre-cultured in water for one week.
[0047] The flower head was infected.
[0048] In some specific embodiments, the culture condition of the Hydrangea macrophylla after infection includes pre-culturing in deionized water for 7 days.
[0049] Subsequently, the cut flower and tissue culture seedling were immersed in the culture mixture, and then exposed to vacuum twice, each for 1 minute.
[0050] After releasing the vacuum, the tissue culture seedling material was washed and cultured in dark environment at 10±2°C for 3 days.
[0051] After incubation, the cut flower and tissue culture seedling were cultured at 25°C with 16h light / 8h dark cycle for 15 days.
[0052] Subsequently, phenotype observation and gene relative expression determination can be performed to determine the effect of silencing.
[0053] Example 1
[0054] A TRV-mediated gene silencing technique in Hydrangea includes the following steps:
[0055] (1) Hydrangea material preparation for treatment
[0056] Hydrangea macrophylla cultivar ‘Endless Summer’ was used as the material. The tissue culture seedlings required for gene silencing were cultured at 25 °C under a light intensity of 2000 lx with a 16 h / 8 h light cycle. The rooted tissue culture seedlings were removed from the bottle and pre-cultured in water for one week without changing the conditions of the infection. The infected cut flowers required selection of most of the flower heads that opened to grade 2 for infection.
[0057] (2) Cloning of full-length fragments of maker genes HmPDS, HmF3'5'H and HmCHS1
[0058] The primers used for cloning the open reading frames (ORFs) of HmPDS (accession number: PQ285401), HmCHS1 (accession number: ON375346.1) and HmF3'5'H (accession number: ON375346.1) genes, respectively, are shown in Table 1.
[0059] Table 1. Primer sequences for gene cloning
[0060]
[0061]
[0062] PCR reactions were performed in a 50 μL system, and the reaction components included: 3 μL cDNA, 25 μL KOD One TM PCR Master Mix (Toyobo, Tokyo, Japan), 18 μL ddH2O and 2 μL forward and reverse primers.
[0063] The PCR amplification conditions were as follows: initial denaturation at 98 °C for 2 min; followed by 33 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 5 s and extension at 72 °C for 30 s; and finally extension at 72 °C for 2 min.
[0064] The purified PCR products were ligated using a pCloneEZ-Blunt / TA TOPO cloning kit (Vazyme Biotech, Beijing, China). All cloned plasmids were sequenced by Shanghai Sangon Biological Co., Ltd. (Shanghai, China).
[0065] (3) Construction of TRV vectors, transformation of E. coli and Agrobacterium
[0066] To construct pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H, the pTRV2 vector was digested with restriction enzymes EcoRI and KpnI (Thermo Fisher Scientific, USA). The primers listed in Table 2 were used to amplify the ORFs of the genes, respectively, using KOD One TMPCR Master Mix (TOYOBO, Shanghai, China) were amplified as silencing fragments of 382 bp, 332 bp and 340 bp for HmPDS, HmCHS1 and HmF3'5'H, respectively.
[0067] Table 2 Primer sequences for pTRV2 vector construction
[0068]
[0069] Using IIOne Step Cloning Kit (Vazyme, Shenzhen, China) was used to recombine the fragments amplified in (3), and then the resulting constructs were transformed into E. coli TOP10 (Zymo Research, Beijing, China). After overnight culture at 37 °C, the inserted fragments were sequenced by Shanghai Generay Biotech Co., Ltd. (Shanghai, China). The schematic diagram of pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H vector construction is shown in Figure 1 . Finally, the successfully constructed vectors were transformed into Agrobacterium tumefaciens GV3101 strain (Zymo Research, Beijing, China) by heat shock method. After overnight culture at 28 °C, PCR was performed to identify positive clones.
[0070] (4) Preparation before infection
[0071] Positive monoclonal Agrobacterium strains were selected, and strains containing pTRV1 (Zymo Research, Beijing, China), pTRV2 (Zymo Research, Beijing, China), pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H five plasmids were cultured in liquid LB medium containing antibiotics (50 mg·L -1 Kanamycin, 25 mg·L -1 Rifampicin). At 28 °C, 180 rpm shaker culture for 16 hours, until the optical density (OD 600 ) between 0.8 and 1.2. Agrobacterium cells were collected by centrifugation (5,000 x g, 6 min), and resuspended to OD 600 1.5 with infection solution (containing 10 mM MgCl2, 10 mM MES and 200 μΜ AS) at pH 5.6. Agrobacterium containing pTRV1 and pTRV2 target gene constructs were mixed at a ratio of 1:1 (v / v) and incubated in the dark at 25 °C for 3 h before the next infection.
[0072] (5) Infection step
[0073] For VIGS of cut flowers, flower stems were cut to 5 cm long and placed in water for 2 hours for acclimation. Tissue culture plantlets were pre-cultured in deionized water for 7 days. Then the plantlets were dipped into the infiltration solution pTRV1 + pTRV2 (control), pTRV1 + pTRV2-HmPDS, respectively, with at least 20 plantlets per group; the flower heads of cut flowers were inverted and dipped into the infiltration solution pTRV1 + pTRV2 (control), pTRV1 + pTRV2-HmCHS1 and pTRV1 + pTRV2-HmF3'5'H, respectively, with at least 20 stems per group. Vacuum (0.1-0.2 atm) was applied twice for 1 minute each. The vacuum was released slowly for 2 minutes, and the plantlets were washed with deionized water and incubated in the dark at 10 ± 2 °C for 3 days. After the incubation, the cut flowers and plantlets were incubated in a 25 °C incubator with a 16-hour light / 8-hour dark cycle for 15 days, with water changed every 2 days to keep the stems clean and free from rot.
[0074] (6) Post-infection detection step
[0075] In the silenced leaf experiment, the upper leaves of the Hydrangea plantlets treated with pTRV1 + pTRV2 were used as the control group, and the upper leaves of the Hydrangea plantlets treated with pTRV1 + pTRV2-HmPDS were used as the experimental group after 30 days of silencing. The gene expression primers are shown in Table 3. The chlorophyll content was determined using the Arnon method (Arnon, 1949). The leaves of the control and experimental groups were collected, and 0.2 g of each sample was placed in 80% acetone for 24 hours of extraction. The spectrophotometer (model 752, Shanghai, China) was used to measure the absorbance of the extract at 663 nm and 645 nm.
[0076] Table 3. Primer sequences for silencing gene PCR detection
[0077]
[0078] In the silencing cut flower experiment, after 15 days of silencing, hydrangea cut flowers co-infected with pTRV1 and pTRV2 served as the control group (pTRV1+pTRV2-HmCHS1), and hydrangea cut flowers infected with pTRV1+pTRV2-HmF3'5'H served as the experimental group. Primers for gene expression detection are shown in Table 3. For the extraction and determination of total anthocyanins, a plant anthocyanin content assay kit (BOXBIO, Beijing, China) was used. Anthocyanins were extracted and quantified according to the manufacturer's instructions. 0.1 g of flower sample was added to 1 mL of extraction solution and thoroughly ground into a homogeneous slurry using a mortar and pestle. After extraction at 60℃ for 30 minutes, the sample was centrifuged at 12,000 rpm for 10 minutes at room temperature, and the supernatant was collected for analysis. The absorbance was measured at 530 nm and 700 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific, USA), and the anthocyanin content was calculated according to the 96-well plate assay formula in the manufacturer's instructions. Qualitative analysis of anthocyanins in calyx was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) according to the method of Yuan et al. (Yuan et al., 2023). The calyx samples were ground into powder in liquid nitrogen, and 0.5 g of the powder was extracted for anthocyanin identification. Data were analyzed using Masslynx software version 4.1 (Waters, Milford, MA, USA).
[0079] (7) Experimental Results
[0080] The pTRV2-HmPDS recombinant was designed to target the endogenous PDS gene in *H. macrophylla* via vacuum infiltration. Thirty days after infection, 60% of 90 pTRV2-HmPDS-infected seedlings exhibited a photobleaching phenotype. Figure 2 In contrast, the control group infected with pTRV2 remained green. Semi-quantitative RT-PCR was performed using primers specific to the pTRV2 coat protein (CP) to confirm the presence of the TRV2 sequence. Compared to the TRV2-infected control group, the expression of PDS in pTRV2-HmPDS-infected tissue culture seedlings was significantly reduced (A). Figure 2 (B). Furthermore, the levels of chlorophyll a, chlorophyll b, and total chlorophyll were all significantly decreased in HmPDS-silenced tissue culture seedlings. Figure 2 The results showed that VIGS can be effectively used to silence HmPDS in hydrangea, and the established VIGS method is suitable for gene silencing in tissue culture seedlings.
[0081] The chalcone synthase (CHS) gene HmCHS1 was silenced in the sepals of hydrangea 'Endless Summer' using VIGS. Phenotypic observation was performed 15 days after infection. In flowers infected with pTRV2, the pink and blue sepals were darker, while flowers infected with pTRV2-HmCHS1 showed lighter sepal colors. Figure 3HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla. Figure 3 HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla. Figure 3 HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla.
[0082] HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla. Figure 4 HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla. Figure 4 HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla. Figure 4 HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla. Figure 4 HmCHS1 expression in pTRV2-HmCHS1-infiltrated flowers was significantly lower than that in non-infiltrated and TRV-infiltrated flowers (Fig. 1A, B). Furthermore, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced (Fig. 1C), and the results confirmed that the VIGS method was suitable for functional studies of flower color-related genes in Hydrangea macrophylla.
[0083] It is apparent that the above-described embodiments of the present application are merely examples for clearly explaining the present application, and are not intended to limit the embodiments of the present application. Those skilled in the art can make other different forms of changes or modifications on the basis of the above description. It is not necessary or possible to exhaust all the embodiments. Any modification, equivalent replacement, and improvement within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.
Claims
1. A method for virus-induced gene silencing in hydrangea, characterized in that, Includes the following steps: (1) Cloning HmPDS , HmF3'5'H and HmCHS1 The full-length segment of a gene; (2) Construct separately HmPDS , HmF3'5'H and HmCHS1 TRV vectors were used to transform Escherichia coli and Agrobacterium tumefaciens; The respective construction HmPDS , HmF3'5'H and HmCHS1 The steps for obtaining a TRV vector include: Use restriction enzymes Eco RI and Kpn I. The pTRV2 vector was digested with enzymes; Amplification was performed using primers shown in SEQ ID NO. 7–8. HmPDS Silent segments of genes; Amplification was performed using primers shown in SEQ ID NO. 9–10. HmCHS1 Silent segments of genes; Amplification was performed using the primers shown in SEQ ID NO. 11-12. HmF3'5'H Silent segments of genes; use IIOne Step Cloning Kit HmPDS , HmCHS1 and HmF3'5'H The ligation of the gene silencing fragment was carried out and constructed into the pTRV2 vector to obtain pTRV2- HmPDS pTRV2- HmCHS1 and pTRV2- HmF3'5'H Three recombinant plasmids; (3) Agrobacterium-mediated infection of Hydrangea macrophylla material; the steps of Agrobacterium-mediated infection of Hydrangea macrophylla material include: Positive monoclonal Agrobacterium rhizogenes strains were cultured in liquid LB medium containing antibiotics at 28°C with a shaker at 180 rpm for 16 hours until OD was reached. 600 Agrobacterium rhizogenes cells were collected by centrifugation at 0.8–1.2°C and resuspended in infection solution at pH 5.6 to OD. 600 The concentration was 1.5, and the solution was incubated in the dark at 25°C for 3 hours before infecting the large-leaved hydrangea material; the infection solution contained 10 mM MgCl2, 10 mM 2-(N-morpholine)ethanesulfonic acid and 200 μM acetylsyleugenone; (4) Cultivating large-leaved hydrangeas after infection; The clone HmPDS , HmF3'5'H and HmCHS1 The steps involved in processing the full-length segment of a gene include: Amplification using primers shown in SEQ ID NO.1-2 HmPDS The full-length segment of a gene; Amplification using primers shown in SEQ ID NO.3–4 HmCHS1 The full-length segment of a gene; Amplification using primers shown in SEQ ID NO.5–6 HmF3'5'H The full-length segment of a gene.
2. The gene silencing method according to claim 1, characterized in that, The steps of transforming Escherichia coli and Agrobacterium include: pTRV2- HmPDS pTRV2- HmCHS1 and pTRV2- HmF3'5'H Transformation with three vector plasmids E. coli TOP10, sequenced after overnight incubation at 37°C; The successfully constructed vector was then transformed into Agrobacterium tumefaciens GV3101 carrying plasmid pSoup-p19 using a heat shock method. After overnight culture at 28°C, PCR amplification was performed using primers shown in SEQ ID NO.7–12 to identify positive clones.
3. The gene silencing method according to claim 1, characterized in that, The large-leaf hydrangea material was prepared by tissue culture seedlings of the large-leaf hydrangea cultivar 'Endless Summer', and cultured in an environment of 25℃, light intensity of 2000lx, and light cycle of 16 hours / 8 hours. Remove the rooted 'Endless Summer' tissue culture seedlings from the bottle and pre-culture them in water for one week; Infect the flower heads.
4. The gene silencing method according to claim 1, characterized in that, The culture conditions for the infected hydrangea include: pre-culturing in deionized water for 7 days; The cut flowers and tissue culture seedlings were then immersed in the culture mixture and exposed twice under vacuum conditions for 1 minute each time. After releasing the vacuum, clean the tissue culture seedlings and incubate them in the dark at 10±2℃ for 3 days; After incubation, the cut flowers and tissue culture seedlings were cultured at 25°C for 15 days with a 16-hour light / 8-hour dark cycle.
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