Virus-induced gene silencing technology in hydrangea macrophylla

Abstract

The invention discloses a virus-induced gene silencing technology in hydrangea macrophylla, and belongs to the technical field of hydrangea macrophylla gene function research. Comprising the following steps: (1) cloning full-length fragments of HmPDS, HmF3 '5' H and HmCHS1 genes; (2) respectively constructing TRV vectors of HmPDS, HmF3 '5' H and HmCHS1, and transforming escherichia coli and agrobacterium; (3) infecting the hydrangea macrophylla material with agrobacterium; and (4) culturing the hydrangea macrophylla after infection. The invention provides a virus-induced gene silencing technology in hydrangea macrophylla, which is characterized in that a hydrangea macrophylla cultivated species' Ampelopsis' is used as a material to respectively infect tissue culture seedlings and flower heads. According to the gene instantaneous silencing method disclosed by the invention, related genes in the hydrangea can be effectively silenced, and the gene function research efficiency can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of hydrangea gene function research, and in particular to a virus-induced gene silencing technology in Hydrangea macrophylla. Background Art

[0002] Hydrangea is an important ornamental plant with important commercial value as potted plants, 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, resistance to pests and diseases, cold tolerance and shade tolerance. Studies have shown that bigleaf hydrangea has multiple medicinal properties (Zhang et al., 2009; Geum et al., 2020; Lee et al., 2022). However, functional genomic studies are still limited due to the lack of effective molecular genetic tools. Therefore, it is necessary to develop tools to elucidate the gene functions of hydrangea.

[0003] Virus induced gene silencing (VIGS) technology uses recombinant viral vectors containing target plant gene fragments to activate RNA-mediated post-transcriptional gene silencing, thereby inhibiting the transcription of homologous genes. Compared with gene silencing techniques using inverted repeat sequences, VIGS has several advantages, such as simpler plasmid construction and shorter operation cycle (Reid et al., 2009). In recent years, VIGS technology based on tobacco rattle virus (TRV) has been applied to verify gene function in a variety of ornamental plants, such as peony (Xie et al., 2019), 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 that it can quickly generate 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 functional genome editing methods such as CRISPR-Cas9), VIGS is a cost-effective option (Liu et al., 2016). However, different species have different means of applying VIGS due to variety specificity, including infection solution concentration, infection method, and post-culture. At present, there is no report on gene silencing in hydrangea using TRV. Summary of the invention

[0004] The purpose of the present invention is to provide a virus-induced gene silencing technology in Hydrangea macrophylla to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a virus-induced gene silencing technology in Hydrangea macrophylla, comprising the following steps:

[0007] (1) Cloning the full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes;

[0008] (2) construct TRV vectors of HmPDS, HmF3'5'H and HmCHS1, respectively, and transform Escherichia coli and Agrobacterium tumefaciens;

[0009] (3) Agrobacterium infection of Hydrangea macrophylla materials;

[0010] (4) Cultivate large-leaf hydrangea after infection.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] The present invention provides a gene silencing technology induced by virus in large-leaf hydrangea. The large-leaf hydrangea cultivar 'Endless Summer' is used as a material to infect hydrangea tissue culture seedlings to silence the HmPDS gene; the inflorescence with flowers opening at level 2 is infected to silence HmF3'5'H and HmCHS1 respectively. Afterwards, 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 of the leaves of the tissue culture seedlings infected with pTRV2-HmPDS are reduced, the total anthocyanin content in the HmCHS1 silenced flowers is significantly reduced, and the delphinidin glucoside and total anthocyanin content in the HmF3'5'H silenced flowers are reduced. It is shown that the method of instantaneous gene silencing disclosed in the present invention can effectively silence the relevant genes in hydrangea, which is helpful to improve the efficiency of gene function research. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1Schematic diagram of pTRV1, pTRV2, pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H, where LB, left border; RB, right border; RdRp, RNA-dependent RNA polymerase; MP, mobile protein; 16k, 16kD protein; R, self-cleaving ribozyme; N, NOS terminator; CP, capsid protein; MCS, multiple cloning site.

[0015] Figure 2 PDS silencing in hydrangea induced by TRV. (A) Wild-type tissue culture seedlings (left) and tissue culture seedlings infected with TRV (middle) and pTRV2-HmPDS (right). The photos were taken on the 30th day after infiltration. (B) Detection of TRV and HmPDS expression levels in each treatment. (C) Comparison of chlorophyll a, chlorophylla and total chlorophyll content between PDS-silenced and TRV-infected tissue culture seedlings.

[0016] Figure 3 TRV-induced CHS silencing in hydrangea. Pink flowers (A) and blue flowers (B) infected with TRV and pTRV2-HmCHS1 on day 15 after infiltration. White scale bars represent 1 cm. (C) Detection of TRV and HmCHS1 expression levels in each treatment. (D) Comparison of total anthocyanin content between HmCHS1-silenced flowers and TRV control.

[0017] Figure 4 Figure 1 shows the F3'5'H silencing induced by TRV in hydrangea. (A) Flowers infected with TRV and pTRV2-HmF3'5'H, 15 days after infiltration. The white scale bar represents 1 cm. (B) Detection of TRV and HmF3'5'H expression levels in each treatment. (C) Comparison of anthocyanin content between HmF3'5'H silenced flowers and TRV infected flowers. (D) Comparison of the relative abundance of anthocyanins in flowers infected with TRV and pTRV2-HmF3'5'H. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0024] The embodiment of the present invention provides a virus-induced gene silencing technology in Hydrangea macrophylla, comprising the following steps:

[0025] (1) Cloning the full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes;

[0026] (2) construct TRV vectors of HmPDS, HmF3'5'H and HmCHS1, respectively, and transform Escherichia coli and Agrobacterium tumefaciens;

[0027] (3) Agrobacterium infection of Hydrangea macrophylla materials;

[0028] (4) Cultivate large-leaf hydrangea after infection.

[0029] In some specific embodiments, the step of cloning the full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes comprises:

[0030] Amplify the full-length fragment of the HmPDS gene using the primers shown in SEQ ID NO.1-2;

[0031] Amplify the full-length fragment of HmCHS1 gene using primers shown in SEQ ID NO.3-4;

[0032] The full-length fragment of the HmF3'5'H gene was amplified using the primers shown in SEQ ID NOs. 5-6.

[0033] In some specific embodiments, the steps of constructing TRV vectors of HmPDS, HmF3'5'H and HmCHS1 respectively include:

[0034] The pTRV2 vector was digested with restriction enzymes EcoRI and KpnI;

[0035] The silent fragment of HmPDS gene was amplified using the primers shown in SEQ ID NO.7-8;

[0036] The silenced fragment of the HmCHS1 gene was amplified using the primers shown in SEQ ID NOs. 9 to 10;

[0037] The silencing fragment of the HmF3'5'H gene was amplified using the primers shown in SEQ ID NOs. 11 to 12;

[0038] use IIOne Step Cloning Kit was used to connect the silencing fragments of HmPDS, HmCHS1 and HmF3'5'H genes with the pTRV2 vector and constructed into the pTRV2 vector respectively to obtain three recombinant plasmids: pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H.

[0039] In some specific embodiments, the step of transforming Escherichia coli and Agrobacterium comprises:

[0040] The three vector plasmids pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H were transformed into E. coli TOP10, and the cells were cultured overnight at 37°C before sequencing;

[0041] The successfully constructed vector was then transformed into the Agrobacterium tumefaciens GV3101 strain carrying the plasmid pSoup-p19 by heat shock method, and after overnight culture at 28°C, PCR amplification was performed using primers shown in SEQ ID NOs. 7 to 12 to identify positive clones.

[0042] In some specific embodiments, the step of infecting the Hydrangea macrophylla material with Agrobacterium comprises:

[0043] The positive monoclonal Agrobacterium tumefaciens strain was cultured in liquid LB medium containing antibiotics at 28 °C with a shaker at 180 rpm for 16 h until the OD 600 Agrobacterium tumefaciens cells were collected by centrifugation at 0.8 to 1.2 and resuspended in infection solution at pH 5.6 to an OD of 600 is 1.5, and after incubation at 25°C in the dark for 3 hours, it infects the large-leaf hydrangea material.

[0044] In some specific embodiments, the infection solution comprises 10 mM MgCl2, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 200 μM acetosyringone (AS).

[0045] In some specific embodiments, the large-leaf hydrangea material is a tissue culture seedling of the large-leaf hydrangea cultivar 'Endless Summer', and is cultured at 25°C, with a light intensity of 2000 lx and a light cycle of 16 hours / 8 hours;

[0046] The rooted 'Endless Summer' tissue culture seedlings were taken out of the bottles and pre-cultured in water for one week;

[0047] Infect the flower heads.

[0048] In some specific embodiments, the culture conditions for culturing the large-leaf hydrangea after infection include: pre-culturing in deionized water for 7 days;

[0049] The cut flowers and tissue culture seedlings were then immersed in the culture mixture and then exposed to vacuum conditions twice for 1 min each time;

[0050] After releasing the vacuum, the tissue culture seedling material was cleaned and cultured in a dark environment at 10 ± 2 °C for 3 days;

[0051] After incubation, the cut flowers and tissue culture seedlings were cultured at 25°C with a 16-h light / 8-h dark cycle for 15 days.

[0052] Subsequently, phenotypic observation and determination of relative gene expression levels can be performed to determine the effect of silencing.

[0053] Example 1

[0054] A TRV-mediated gene silencing technique in hydrangea comprises the following steps:

[0055] (1) Preparation of hydrangea materials

[0056] The cultivar 'Endless Summer' of Hydrangea macrophylla was used as the material. The tissue culture seedlings required for gene silencing were cultured at 25°C, with a light intensity of 2000 lx and a photoperiod of 16 hours / 8 hours. The rooted tissue culture seedlings were taken out of the bottle and pre-cultured in water for one week without changing the culture conditions before infection. The cut flowers to be infected needed to be selected from the flower heads with most flowers open to level 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 are shown in Table 1.

[0059] Table 1 Primer sequences for gene cloning

[0060]

[0061]

[0062] The PCR reaction was carried out in a 50 μL system, and the reaction components included: 3 μL cDNA, 25 μL KOD One TM PCRMaster Mix (Toyo, Tokyo, Japan), 18 μL ddH O, and 2 μL forward and reverse primers.

[0063] PCR amplification conditions were as follows: initial denaturation at 98°C for 2 minutes; followed by 33 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 72°C for 30 seconds; and a final extension at 72°C for 2 minutes.

[0064] The purified PCR products were ligated using the pCloneEZ-Blunt / TATOPO cloning kit (Sino-US Taihe Gene, Beijing, China). All cloned plasmids were sequenced by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0065] (3) Construction of TRV vector and transformation of Escherichia 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). TMPCRMaster Mix (Toyobo, Shanghai, China) was used to PCR-amplify 382 bp, 332 bp, and 340 bp of HmPDS, HmCHS1, and HmF3′5′H sequences, respectively, as silencing fragments.

[0067] Table 2 Primer sequences for pTRV2 vector construction

[0068]

[0069] use The fragments amplified in (3) were recombined using IIOne Step Cloning Kit (BGI, Shenzhen, China), and the resulting constructs were subsequently transformed into E. coli TOP10 (Zhuangmeng, Beijing, China). After overnight incubation at 37°C, the inserts were sequenced by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The construction diagram of pTRV2-HmPDS, pTRV2-HmCHS1, and pTRV2-HmF3'5'H vectors is shown in Figure 1 Finally, the successfully constructed vector was transformed into Agrobacterium tumefaciens GV3101 strain (Zhuangmeng, 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 tumefaciens strains were selected and cultured in liquid LB medium containing antibiotics (50 mg·L -1 Kanamycin, 25 mg L -1 Rifampicin). The cells were cultured at 28°C with a shaker at 180 rpm for 16 hours until the optical density (OD 600 ) between 0.8 and 1.2. Agrobacterium cells were collected by centrifugation (5,000 × g, 6 min) and resuspended to an OD of 0.175.7 using infection solution (containing 10 mM MgCl2, 10 mM MES, and 200 μM AS) at pH 5.6. 600 Agrobacterium containing pTRV1 and pTRV2 target gene constructs were mixed and cultured at a ratio of 1:1 (v / v), and incubated at 25°C in the dark for 3 h before the next infection.

[0072] (5) Infection steps

[0073] For VIGS of hydrangea cut flowers, flower branches were cut to 5 cm in length and placed in clean water for 2 hours. Tissue culture seedlings were pre-cultured in deionized water for 7 days. The tissue culture seedlings were then immersed in the infection solution pTRV1+pTRV2 (control), pTRV1+pTRV2-HmPDS, with at least 20 plants in each group; the cut flower heads were inverted and immersed in the infection solution pTRV1+pTRV2 (control), pTRV1+pTRV2-HmCHS1 and pTRV1+pTRV2-HmF3'5'H, with at least 20 branches in each group. Aspirate twice under vacuum (0.1-0.2 atmospheres), each time for 1 minute. Slowly release the vacuum for 2 minutes, wash the tissue culture seedling material with deionized water, and culture it in a dark environment at 10±2℃ for 3 days. After moisturizing incubation, the cut flowers and tissue culture seedlings were cultured in a 25°C incubator with a 16-hour light / 8-hour dark cycle for 15 days, during which the water was changed every 2 days to ensure that the flower branches were clean and not rotten.

[0074] (6) Post-infection detection steps

[0075] In the silent leaf experiment, after 30 days of silencing, the upper leaves of hydrangea tissue culture seedlings infected with pTRV1+pTRV2 were used as the control group, and the upper leaves of hydrangea tissue culture seedlings infected with pTRV1+pTRV2-HmPDS were used as the experimental group. The primers for detecting gene expression are shown in Table 3. The chlorophyll content was determined by the Arnon method (Arnon, 1949). Leaves from the control group and the experimental group were collected, and 0.2 g of each sample was extracted in 80% acetone for 24 hours. The absorbance of the extract at 663 nm and 645 nm was measured using a spectrophotometer (Model 752, Shanghai, China).

[0076] Table 3 Primer sequences for PCR detection of silent genes

[0077]

[0078] In the silent cut flower experiment, after 15 days of silencing, the hydrangea cut flowers treated with pTRV1+pTRV2 co-infection were used as the control group pTRV1+pTRV2-HmCHS1, and the hydrangea cut flowers infected with pTRV1+pTRV2-HmF3'5'H were used as the experimental group. The primers for detecting gene expression are shown in Table 3. The extraction and determination of total anthocyanins were performed using the plant anthocyanin content determination kit (BOXBIO, Beijing, China) according to the instructions for extraction and quantification of anthocyanins. 0.1 g of flower sample was selected, 1 mL of extraction solution was added, and the sample was fully ground into a uniform slurry using a mortar and pestle. After extraction at 60 ° C for 30 minutes, centrifugation was performed at 12,000 rpm for 10 minutes at room temperature, and the supernatant was collected for determination. 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 determination formula in the manufacturer's instructions. Qualitative analysis of anthocyanins in sepals was performed using ultra-high performance liquid chromatography-tandem mass spectrometry according to the method of Yuan et al. (Yuan et al., 2023). Sepal samples were ground into powder in liquid nitrogen, and then 0.5 g of sepal 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 of H. macrophylla by vacuum infiltration. After 30 days of infection, 60% of the 90 pTRV2-HmPDS-infected seedlings showed a photobleaching phenotype ( Figure 2 The expression of PDS in the pTRV2-HmPDS infected seedlings was significantly reduced compared with the TRV2 infected control group (Figure 5A). Figure 2 In addition, the levels of chlorophyll a, chlorophyll b and total chlorophyll were significantly decreased in the HmPDS-silenced seedlings ( Figure 2 (C), 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] VIGS was used to target and silence the chalcone synthase (CHS) gene HmCHS1 in the sepals of Hydrangea 'Endless Summer'. Phenotypic observations were performed 15 days after infection. In flowers infected with pTRV2, the pink and blue sepals were darker in color, while flowers infected with pTRV2-HmCHS1 showed lighter sepal colors ( Figure 3Semi-quantitative RT-PCR was used to verify the inhibition of HmCHS1 expression. The expression level of HmCHS1 in flowers infected with pTRV2-HmCHS1 was significantly lower than that in uninfected and TRV-infected flowers ( Figure 3 In addition, the total anthocyanin content in HmCHS1-silenced flowers was significantly reduced ( Figure 3 (D) The results confirmed that the VIGS method is suitable for the functional study of hydrangea color-related genes.

[0082] The reporter gene F3'5'H was used to verify the silencing effect of the VIGS system in hydrangea. The pTRV2-HmF3'5'H construct was introduced into the sepals of Endless Summer at the S2 stage by vacuum infection. Phenotypic observations were performed 15 days after infection ( Figure 4 (A). TRV-infected sepals are blue, while pTRV2-HmF3'5'H-infected sepals turn pink due to the silencing of HmF3'5'H ( Figure 4 Semi-quantitative RT-PCR was used to verify the inhibition of HmF3'5'H expression. The expression level of HmF3'5'H in flowers infected with pTRV2-HmF3'5'H was significantly lower than that in flowers infected with pTRV2 alone ( Figure 4 In addition, the contents of delphinidin-3-glucoside and total anthocyanins in HmF3'5'H silenced flowers were reduced ( Figure 4 In C), delphinidin-3-glucoside is no longer the main anthocyanin component in HmF3'5'H silenced flowers. This experiment further demonstrated the silencing effect of the VIGS system in hydrangea.

[0083] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A virus-induced gene silencing technique in Hydrangea macrophylla, characterized in that: The following steps are involved: (1) Cloning the full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes; (2) construct TRV vectors of HmPDS, HmF3'5'H and HmCHS1, respectively, and transform Escherichia coli and Agrobacterium tumefaciens; (3) Agrobacterium infection of Hydrangea macrophylla materials; (4) Cultivate large-leaf hydrangea after infection.

2. The gene silencing technique according to claim 1, characterized in that: The steps of cloning the full-length fragments of HmPDS, HmF3'5'H and HmCHS1 genes include: Amplify the full-length fragment of the HmPDS gene using the primers shown in SEQ ID NO.1-2; Amplify the full-length fragment of HmCHS1 gene using primers shown in SEQ ID NO.3-4; The full-length fragment of the HmF3'5'H gene was amplified using the primers shown in SEQ ID NOs. 5-6.

3. The gene silencing technique according to claim 1, characterized in that: The steps of constructing TRV vectors of HmPDS, HmF3'5'H and HmCHS1 respectively include: The pTRV2 vector was digested with restriction enzymes EcoRI and KpnI; The silent fragment of HmPDS gene was amplified using the primers shown in SEQ ID NO.7-8; The silenced fragment of the HmCHS1 gene was amplified using the primers shown in SEQ ID NOs. 9 to 10; The silencing fragment of the HmF3'5'H gene was amplified using the primers shown in SEQ ID NOs. 11 to 12; use IIOne Step Cloning Kit was used to connect the silencing fragments of HmPDS, HmCHS1 and HmF3'5'H genes and constructed into the pTRV2 vector to obtain three recombinant plasmids: pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H.

4. The gene silencing technique according to claim 3, characterized in that: The steps of transforming Escherichia coli and Agrobacterium include: The three vector plasmids pTRV2-HmPDS, pTRV2-HmCHS1 and pTRV2-HmF3'5'H were transformed into E. coli TOP10, and the cells were cultured overnight at 37°C before sequencing; The successfully constructed vector was then transformed into the Agrobacterium tumefaciens GV3101 strain carrying the plasmid pSoup-p19 by heat shock method, and after overnight culture at 28°C, PCR amplification was performed using primers shown in SEQ ID NOs. 7 to 12 to identify positive clones.

5. The gene silencing technique according to claim 4, characterized in that: The step of infecting the macrophylla material with Agrobacterium comprises: The positive monoclonal Agrobacterium tumefaciens strain was cultured in liquid LB medium containing antibiotics at 28 °C with a shaker at 180 rpm for 16 h until the OD 600 Agrobacterium tumefaciens cells were collected by centrifugation at 0.8 to 1.2 and resuspended in infection solution at pH 5.6 to an OD of 600 is 1.5, and after incubation at 25°C in the dark for 3 hours, it infects the large-leaf hydrangea material.

6. The gene silencing technique according to claim 5, characterized in that: The infection solution contained 10 mM MgCl2, 10 mM 2-(N-morpholino)ethanesulfonic acid and 200 μM acetosyringone.

7. The gene silencing technique according to claim 5, characterized in that: The large-leaf hydrangea material is a large-leaf hydrangea cultivar 'Endless Summer' tissue culture seedling as the material, and is cultured in an environment of 25° C., a light intensity of 2000 lx, and a light cycle of 16 hours / 8 hours; The rooted 'Endless Summer' tissue culture seedlings were taken out of the bottles and pre-cultured in water for one week; Infect the flower heads.

8. The gene silencing technique according to claim 1, characterized in that: The culture conditions for culturing Hydrangea macrophylla after infection include: pre-culturing in deionized water for 7 days; The cut flowers and tissue culture seedlings were then immersed in the culture mixture and then exposed to vacuum conditions twice for 1 min each time; After releasing the vacuum, the tissue culture seedling material was cleaned and cultured in a dark environment at 10 ± 2 °C for 3 days; After incubation, the cut flowers and tissue culture seedlings were cultured at 25°C with a 16-h light / 8-h dark cycle for 15 days.

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