An auxin amidohydrolase RhILL1 gene and its application in regulating rose flower color

Abstract

The present invention relates to the technical field of molecular breeding. The present invention discloses an auxin amide hydrolase RhILL1 gene and its application in regulating the flower color of roses. The nucleotide sequence of the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.1. The present invention effectively regulates the synthesis of anthocyanins by silencing or overexpressing the RhILL1 gene, thereby achieving precise control of the color of petals. Studies have found that silencing the RhILL1 gene causes the color of the petals to fade and the anthocyanin content to decrease; while overexpressing the RhILL1 gene prolongs the color retention time of the petals. In addition, the present invention also provides a kit and a recombinant plasmid containing the RhILL1 gene regulatory sequence, as well as corresponding gene editing and transgenic breeding methods. These achievements provide new molecular tools for rose breeding and flower color improvement, and have important ornamental value and economic significance.

Description

Technical Field

[0001] The invention relates to the technical field of molecular breeding, and in particular to an auxin amidohydrolase RhILL1 gene and application thereof in regulating rose flower color. Background Art

[0002] Rose ( Rosa hybrida ) is the Rosaceae ( Rosaceae ) Rosa Rosa ) Perennial evergreen or semi-evergreen woody ornamental plants. Roses have the characteristics of dignified flower shape, bright color, and rich fragrance. They have high ornamental, economic and cultural value. They are ranked first among the four major cut flowers in the world and are widely used in gardening, garden decoration, potted flowers, spices and cosmetics industries.

[0003] The color of rose petals is an important factor in its ornamental value, which is mainly determined by pigments such as anthocyanins and carotenoids. Studies have found that the synthesis and accumulation of anthocyanins are regulated by multiple genes. RhILL1 The role of this gene in the anthocyanin metabolism pathway is particularly important. By regulating the expression of this gene, the color of the petals can be effectively changed, thus providing new possibilities for rose breeding.

[0004] Currently, for RhILL1 The specific mechanism of action of genes in regulating the color of rose petals is still unclear, so it is necessary to study its function through gene editing or transgenic technology. Summary of the invention

[0005] The purpose of the present invention is to provide an auxin amidohydrolase RhILL1 gene and its application in regulating the flower color of rose.

[0006] In order to solve the problems of the prior art, the present invention provides the following technical solutions:

[0007] In a first aspect, the present application provides an application of a RhILL1 gene in regulating the color of rose petals;

[0008] In a second aspect, the present application provides a method for adjusting the color of rose petals;

[0009] In a third aspect, the present application provides a kit for regulating the color of rose petals.

[0010] In a first aspect, the present application provides an application of an auxin amide hydrolase RhILL1 gene in regulating the color of rose petals, by silencing or overexpressing the RhILL1 gene to regulate the synthesis of anthocyanins, thereby achieving precise regulation of the color of petals. The nucleotide sequence of the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.2.

[0011] Furthermore, the application includes:

[0012] (1) Application in adjusting the color of rose petals;

[0013] (2) Application in regulating the color maintenance time of rose petals;

[0014] (3) Rose gene editing breeding;

[0015] (4) Transgenic rose breeding.

[0016] The second aspect of the present application provides a method for regulating the color of rose petals by silencing the auxin amide hydrolase RhILL1 gene or protein, thereby reducing the expression level of the RhILL1 gene or protein in the rose.

[0017] A third aspect of the present application provides a kit for regulating the color of rose petals, the kit comprising a component for silencing or overexpressing the auxin amide hydrolase RhILL1 gene, and a component for detecting changes in petal color.

[0018] Furthermore, the kit comprises a TRV2-RhILL1 construct for silencing the auxin amide hydrolase RhILL1 gene, and a pSuper1300-RhILL1 construct for overexpressing the auxin amide hydrolase RhILL1 gene.

[0019] Beneficial effects: The present invention accurately regulates the color of petals: The present invention effectively regulates the synthesis of anthocyanins by silencing or overexpressing the RhILL1 gene, thereby achieving accurate regulation of the color of rose petals and improving the accuracy and efficiency of breeding.

[0020] Compared with the prior art, the present invention has the following advantages: (1) Prolonging the maintenance time of flower color: by overexpressing the RhILL1 gene in the petals, the expression level of the RhILL1 gene in the rose petals is increased, thereby prolonging the maintenance time of the petal color and increasing the ornamental value of the rose.

[0021] (2) Gene editing and transgenic breeding: The present invention provides the application of the RhILL1 gene in rose gene editing breeding and transgenic breeding, providing new molecular tools and methods for rose breeding. Increasing anthocyanin content: Overexpressing the RhILL1 gene in rose petals can promote the color formation of the petals, increase the anthocyanin content, and thus enhance the vividness of the flower color.

[0022] (3) Revealing the function of the RhILL1 gene: The present invention experimentally verifies the key role of the RhILL1 gene in the formation of rose petal color, providing an important gene resource for rose functional genomics research. Promoting genetic improvement: The implementation of the present invention provides reference and technical support for the genetic improvement of roses and other plants with petal color depth adjustment and insufficient petal viewing period.

[0023] (4) Development of the kit of the present invention: The present invention also provides a kit for regulating the color of rose petals, which comprises a component for silencing or overexpressing the RhILL1 gene, and a component for detecting changes in petal color, thereby making the regulation process more convenient and efficient.

[0024] Enhance the ornamental value and economic value of roses: By regulating the RhILL1 gene, new rose varieties with more colorful flowers and longer ornamental periods can be bred, thereby improving the market competitiveness and economic value of roses.

[0025] (5) The present invention provides a new molecular breeding approach: The present invention provides a new molecular breeding approach for roses, which helps to accelerate the development of new varieties and improve traditional breeding methods. Promote the development of the rose industry: Through the technical solution of the present invention, more new rose varieties with excellent traits can be cultivated to meet the diversified needs of the market and promote the development of the rose industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The present invention provides RhILL1 Expression analysis diagram of; Ro: root; St: stem segment; Le: leaf; Pe: petal; Se: sepal; Stamon: stamen; Pi: pistil; Bud: bud.

[0028] Figure 2The pictures of 'Freud' at different developmental stages provided by the present invention. G, P, S1, S2, S3, S4, S5 (green, pink, primary, secondary, tertiary, quaternary, fifth).

[0029] Figure 3 The present invention provides RhILL1 Expression levels in different developmental stages of 'Freud'. G, P, S1, S2, S3, S4, S5 (green, pink, primary, secondary, tertiary, quaternary, and fifth levels); a, b, cd, d, c values ​​with different letters indicate statistical differences (one-way ANOVA, Duncan, P < 0.05), each data has at least three biological replicates, expressed as mean ± standard deviation.

[0030] Figure 4 The anthocyanin content in different developmental stages of 'Freud' provided by the present invention. G, P, S1, S2, S3, S4, S5 (green, pink, primary, secondary, tertiary, quaternary, and fifth grade); a, b, c values ​​with different letters indicate statistical differences (one-way ANOVA, Duncan, P<0.05), each data has at least three biological replicates, and is expressed as mean ± standard deviation.

[0031] Figure 5 The 'Freud' petal disc provided by the present invention is instantly silent RhILL1 After 12 days, TRV and RhILL1 Silencing phenotype observation and identification diagram; Note: TRV2 and TRV2- RhILL1 Petal phenotype, scale bar 1 cm (A); TRV2 and TRV2- RhILL1 Silencing efficiency detection, scale bar 2 mm (B); TRV2 and TRV2- RhILL1 anthocyanin supernatant (C); TRV and TRV- RhILL1 Anthocyanin content (D). The significant differences were analyzed by Student's t-test (*, p<0.05; **, p<0.001; ***, p<0.0001; ns, no significant difference). Each data had at least three biological replicates and was expressed as mean ± standard deviation.

[0032] Figure 6 The 'Freud' petal disc provided by the present invention is instantly silent RhILL1 After 12 days, TRV and RhILL1 The expression levels of genes related to anthocyanin synthesis were silenced. (*, p<0.05; **, p<0.001; ***, p<0.0001; ns, no significant difference, t-test)

[0033] Figure 7 The present invention provides a transient overexpression of the 'Freud' petal disc RhILL1 After 12 days pSuper1300 and RhILL1 Phenotypic observation and identification of overexpression; Note: pSuper1300 and pSuper1300- RhILL1 Petal phenotype, scale bar 1 cm (A); pSuper1300 and pSuper1300- RhILL1 Silencing efficiency detection, scale bar 2 mm (B); pSuper1300 and pSuper1300- RhILL1 anthocyanin supernatant (C); pSuper1300 and pSuper1300- RhILL1 Anthocyanin content (D). The significant differences were analyzed by Student's t-test (*, p<0.05; **, p<0.001; ***, p<0.0001; ns, no significant difference). Each data had at least three biological replicates and was expressed as mean ± standard deviation.

[0034] Figure 8 The present invention provides a transient overexpression of the 'Freud' petal disc RhILL1 After 12 days pSuper1300 and RhILL1 The expression level of overexpressed anthocyanin synthesis-related genes. (* indicates P≤0.05, ** indicates P≤0.01, t-test) DETAILED DESCRIPTION

[0035] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0036] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0037] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0038] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0039] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0040] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as µg, mg, g, kg, etc.

[0041] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0042] In a first aspect, an embodiment of the present application provides an application of a RhILL1 gene in regulating the color of rose petals, by silencing or overexpressing the RhILL1 gene to regulate the synthesis of anthocyanins, thereby achieving precise regulation of the color of petals. The nucleotide sequence of the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.2.

[0043] In some embodiments, the application includes:

[0044] (1) Application in adjusting the color of rose petals;

[0045] (2) Application in regulating the color maintenance time of rose petals;

[0046] (3) Rose gene editing breeding;

[0047] (4) Transgenic rose breeding.

[0048] A second aspect of an embodiment of the present application provides a method for regulating the color of rose petals by silencing the RhILL1 gene or protein to reduce the expression level of the RhILL1 gene or protein in the rose.

[0049] A third aspect of an embodiment of the present application provides a kit for regulating the color of rose petals, the kit comprising a component for silencing or overexpressing the RhILL1 gene, and a component for detecting changes in petal color.

[0050] In some embodiments, the kit comprises a TRV2-RhILL1 construct for silencing the RhILL1 gene, and a pSuper1300-RhILL1 construct for overexpressing the RhILL1 gene.

[0051] Example 1

[0052] The following is a detailed description with reference to the examples and drawings. This application is based on the genome sequence of the rose variety 'Yueyuefen'. In the early stage, through the identification and analysis of flower color difference genes, the potential flower color regulating gene RhILL1 was discovered, and it was found that the expression level of RhILL1 was related to the flower color, and the expression was high in the petal S3. After transient silencing or overexpression of RhILL1, it was found that the petal color and color retention time were affected. Transient silencing overexpression in rose petals will affect the anthocyanin content. Therefore, the present application reveals the biological function of RhILL1 in the regulation of rose flower color and the time for petals to maintain color, thereby providing technical support for regulating the color of rose petals, promoting rose functional genomics research, gene editing breeding and transgenic breeding research; it can also provide a reference for the genetic improvement of plants with partial petal color depth regulation and insufficient petal viewing period.

[0053] The invention discloses an application of an auxin amide hydrolase RhILL1 gene in regulating the color of rose petals, which regulates the synthesis of anthocyanins by silencing or overexpressing the RhILL1 gene, thereby achieving precise control of the color of petals. The nucleotide sequence of the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.2. The application includes:

[0054] (1) Application in adjusting the color of rose petals;

[0055] (2) Application in regulating the color maintenance time of rose petals;

[0056] (3) Rose gene editing breeding;

[0057] (4) Transgenic rose breeding.

[0058] The invention discloses a product containing a nucleotide sequence for regulating the auxin amidohydrolase RhILL1 gene, and the product is used for any application in the invention. The product is a kit.

[0059] The invention discloses a recombinant plasmid, which contains the auxin amidohydrolase RhILL1 gene.

[0060] The method for regulating the color of rose petals of the present invention reduces the expression level of the RhILL1 gene or protein in the rose by silencing the auxin amidohydrolase RhILL1 gene or protein.

[0061] The invention discloses a kit for regulating the color of rose petals. The kit comprises a component for silencing or overexpressing the auxin amidohydrolase RhILL1 gene and a component for detecting the color change of petals.

[0062] The kit contains the TRV2-RhILL1 construct for silencing the auxin amide hydrolase RhILL1 gene, and the pSuper1300-RhILL1 construct for overexpressing the auxin amide hydrolase RhILL1 gene.

[0063] The abbreviations and their corresponding names appearing in this application are shown in Table 1.

[0064] Table 1

[0065] 1. Plant materials

[0066] 1.1 'Freud'

[0067] 'Floyd' is a modern cut rose variety, obtained from the Jinning Baofeng Base of the Yunnan Academy of Agricultural Sciences. Different tissues were used for tissue specificity analysis, and petals at the S3 stage were used for silencing and expression verification experiments. After sampling, it was immediately placed in liquid nitrogen and brought back to the laboratory, where it was stored in a -80℃ refrigerator for later use.

[0068] 1.2 Strains and vectors

[0069] Escherichia coli: DH5α, Agrobacterium tumefaciens strain EHA105, and pSuper-1300 (Kan resistance) were purchased from Beijing Qingke Biotechnology Co., Ltd. VIGS vectors were pTRV1 and pTRV2 purchased from HonorGene.

[0070] 1.3 Culture medium formula involved in the case

[0071] (1) The formulas of LB medium and YEB medium are shown in Table 2:

[0072] Table 2

[0073] (2) Experimental related culture medium (Table 3)

[0074] 2. Research Methods

[0075] 2.1 Total RNA extraction

[0076] Total RNA was extracted from roots, stem segments, leaves, petals, sepals, stamens, pistils, and buds of 'Floyd' using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (spin column type).

[0077] 2.2 cDNA synthesis

[0078] (1) Genomic DNA removal. The DNA removal reaction system is shown in Table 4:

[0079] Table 4

[0080] Mix well and incubate at 42°C for 2 min.

[0081] (2) Prepare the reverse transcription reaction system (Table 5)

[0082] Mix by pipetting, incubate at 37°C for 15 min, and 85°C for 5 s. Store the product at -20°C.

[0083] 2.3 Real-time fluorescence quantitative PCR

[0084] Primer premier5 was used to design gene-specific primers. The RT-qPCR primer sequences are shown in Table 6. cDNA was purified by ddH 2 O was diluted four times, and the cDNA obtained by reverse transcription was used as a template for Real-Time PCR amplification. UBI2 As an internal reference, three biological replicates were set.

[0085] Table 6

[0086] The RT-qPCR reaction system is shown in Table 7:

[0087] Table 7

[0088] The RT-qPCR reaction procedure is shown in Table 8:

[0089] Table 8

[0090] 2.4 Vector construction

[0091] 2.4.1 PCR amplification of target gene fragments

[0092] A high-fidelity enzyme (PhusionTM Plus PCR Master Mix) was used for PCR target gene amplification. The PCR amplification reaction system is shown in Table 9:

[0093] The PCR amplification reaction program is shown in Table 10:

[0094] Table 10

[0095] After amplification, 1% gel electrophoresis was performed and the target band was selected for subsequent gel recovery.

[0096] 2.4.2 Rubber Recycling

[0097] The PCR product was recovered and purified using a DNA gel recovery kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0).

[0098] 2.4.3 Double restriction digestion of vector

[0099] According to the restriction site where the target fragment sequence is inserted, the vector is double-digested with the corresponding enzyme. The double-digestion system is shown in Table 11:

[0100] Table 11

[0101] After adding the system reagents on ice, perform double enzyme digestion of the vector according to the thermal denaturation temperature of the specific enzyme.

[0102] 2.4.4 Homologous recombination

[0103] The double-digested vector and the cloned target gene fragment are homologously recombined to construct the vector. The homologous recombination system is shown in Table 12:

[0104] Procedure: Run in PCR instrument at 50°C for 15 min.

[0105] 2.4.5 E. coli transformation

[0106] (1) Take out the competent cells from -80 °C and thaw them in ice;

[0107] (2) Take a 1.5 mL centrifuge tube, add 10 μL of the recombinant product and 50 μL of DHα competent cell, mix well by pipetting, and place on ice for 30 min;

[0108] (3) Heat shock in a 42°C water bath for 90 s, then quickly transfer to ice and let stand for 2 min;

[0109] (4) Add 500 μL LB to the centrifuge tube and incubate at 37 °C, 200 rpm, for 1 h.

[0110] (5) Centrifuge the cultured bacterial solution at 5000 rpm for 5 min;

[0111] (6) Discard 400 μL of supernatant in a clean bench, mix thoroughly by pipetting, and then spread on LB solid medium containing antibiotics, and culture inverted at 37°C overnight.

[0112] 2.4.6 Bacterial testing and sequencing

[0113] (1) Shaking: Pick a single colony grown from the overnight culture and culture it in 500 μL of LB medium containing antibiotics with shaking for 3-4 h (37 ℃, 200 rpm).

[0114] (2) Bacterial liquid PCR: The cultured bacterial liquid is used for PCR amplification to detect whether the target band of the constructed vector meets the expectations. The bacterial liquid PCR amplification system is shown in Table 13, and the bacterial liquid PCR amplification reaction procedure is shown in Table 14:

[0115] Table 13

[0116] The bacterial solution PCR amplification program is shown in Table 14:

[0117] The amplified products were used for 1% gel electrophoresis imaging, and those with expected band sizes were sent to the company for sequencing.

[0118] 2.5 Plasmid extraction

[0119] After the test results are returned, sequence comparison is performed, and the samples that meet the expectations are selected for bacterial culture, and the plasmid is extracted using a plasmid extraction kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0).

[0120] 2.6 Agrobacterium transformation

[0121] (1) Take out the competent stem from the -80 ℃ freezer and put it into ice when it melts to an ice-water mixture;

[0122] (2) Add 0.01-1 μg of plasmid DNA per 100 μL competent medium, mix thoroughly, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0123] (3) Add 500 μL of YEB liquid medium without antibiotics and culture at 28 °C with shaking for 5 h;

[0124] (4) Centrifuge at 5000 rpm for 2 min. Discard 400 μL of supernatant on a clean bench and spread onto YEB solid medium containing antibiotics. Incubate inverted at 28 °C for 5 d.

[0125] 2.7 Transient infection

[0126] (1) Vector primer design and vector construction

[0127] EcoRI and KpnI were used as restriction sites. RhILL1 The silent fragment (SEQ ID NO: 3) was inserted into the TRV2 empty vector, and primers were designed by homologous recombination to construct TRV- RhILL1 carrier.

[0128] Select SmaI and KpnI as restriction sites. RhILL1 The complete coding region (SEQ ID NO: 2) was inserted into the pSuper-1300 vector, and primers were designed by homologous recombination to construct RhILL1 -GFP vector, and after obtaining Agrobacterium-positive bacteria, the following experiments were carried out.

[0129] (2) Bacterial culture

[0130] Streak the bacterial solution plate (containing Kan / Rif 50 mg / L) and invert and culture at 28 ℃ for 5 days. Pick a single colony and shake it in 500 μL YEB containing antibiotics for bacterial inspection; shake it medium and high (28 ℃, 200 rpm) if the strip is correct.

[0131] (3) Collection and resuspension

[0132] Centrifuge at 5000 rpm for 8 min to collect the bacteria, discard the supernatant, resuspend the bacteria in the infection solution, mix with a pipette, and adjust to OD600 = 1.0. RhILL1 The bacterial suspension was mixed in a 1:1 ratio by volume and allowed to stand in the dark for 4-6 h. For transient overexpression, Super-1300 and Super- RhILL1 Just let the bacteria collect separately and let it stand.

[0133] (4) Vacuum suction

[0134] A vacuum pump was used to infect the 1 cm diameter petal disc made of the outermost petals of the S3 grade flower of 'Floyd'. The suction pressure was 0.082 MPa for 10 min, the pressure was maintained for 10 min, and the air was released for 10 min. The entire petal disc was fully immersed in the bacterial solution. The treatment was repeated three times. After infection, it was rinsed with sterile water, placed in a 1% Agr culture dish, and cultured in the dark at 8 ℃ for 3 days. It was then transferred to a tissue culture room with a temperature of (22 ± 1) ℃ and a light of 16 h / darkness of 8 h for culture. All petal discs were observed until the color change was complete, and the petal phenotype was recorded by taking pictures. The color change of the petals was measured and counted using the software ImageJ 15.1.

[0135] 2.8 Determination of relative content of anthocyanins

[0136] 0.1 g of fresh petal weight was extracted with 2 mL of 1% (v / v) hydrochloric acid at 4 °C in the dark for 24 h. The solution was centrifuged at 12000 rpm for 1 min at 4 °C. The absorbance was measured at A530 and A657 using a spectrophotometer (Techcomp UV-2600, China). The relative anthocyanin content was calculated as follows: ((A530-A657) × dilution factor / mg FW tissue) × 1000.

[0137] 2.9 Data Statistical Analysis

[0138] The data used for statistical analysis were obtained from 3 biological replicates and 3 technical replicates. The data were statistically analyzed by GraphPad Prism9.5 software, and the comparison between the two groups of data was performed by student-t test (* P<0.05, ** P<0.01, *** P<0.001).

[0139] 2.10 RhILL1 The primers used in the sequence experiment, RhILL1 sequence and primer list are shown in Table 15:

[0140]

[0141] 3 Test results

[0142] 3.1 RhILL1 Expression in different tissues and spatiotemporal expression verification

[0143] This application uses qRT-PCR method to detect RhILL1 Expression of 'Floyd' rose in various organs. Figure 1 )Discover RhILL1It is expressed in roots, stem segments, leaves, petals, sepals, stamens, pistils, and buds, with higher expression levels in leaves and petals. RhILL1 The expression in petal tissue was more specific and higher, indicating RhILL1 It may play an important role in the formation of rose flower color. RhILL1 In order to investigate the spatiotemporal expression characteristics of rose petal coloring, the present invention selected petals of 'Freud' at seven developmental stages ( Figure 2 ), determined RhILL1 transcription level and anthocyanin content. RhILL1 The transcription level of S3 gradually increased with the accumulation of anthocyanins, with the highest transcription level in S3, and gradually decreased with the degradation of anthocyanins ( Figure 3 ).therefore, RhILL1 The relative expression of is consistent with the trend of anthocyanin accumulation during G-S5, see ( Figure 4 )

[0144] 3.2 Silence RhILL1 Later the petals become lighter in color

[0145] To confirm RhILL1 In the function of rose petal color, VIGS technology was used to silence the 'Floyd' rose plant RhILL1 . Observation and statistics were performed 12 days after infection, and it was found that gene silencing (TRV2- RhILL1 ) petal color is lighter than the control (TRV) ( Figure 5 , A). At the same time, the silenced gene (TRV2- RhILL1 ) and the supernatant of the control gene (TRV) anthocyanins ( Figure 5 , C), and the relative content of anthocyanins was measured ( Figure 5 ,D), the results show that silence RhILL1 After the anthocyanin supernatant was added, it became lighter than the control (TRV) and the anthocyanin content was lower than the control. This further verified the silencing efficiency of the silenced gene ( Figure 5 , B), the results showed that compared with the control (TRV), RhILL1 The expression level in silenced plants decreased significantly, which further indicated RhILL1 Related to petal color.

[0146] To further confirm RhILL1 Correlation with suit, silence RhILL1 , reducing the early anthocyanin biosynthesis genes (EBGs), such as CHS, CHI, F3H etc., and late anthocyanin biosynthesis genes (LBGs), e.g. DFR, ANS, UFGT Expressions such as Figure 6 ). The above results show that silence RhILL1 It will make the color of rose petals lighter.

[0147] 3.3 Overexpression RhILL1 The color of the petals lasts longer

[0148] To confirm RhILL1 In the function of rose petal color, transient infection was used to overexpress the 'Floyd' rose plant RhILL1 . Observation and statistics were performed 12 days after infection, and it was found that the gene was overexpressed (pSuper1300- RhILL1 ) petal color is darker than the control (pSuper1300) ( Figure 7 , A). At the same time, the overexpression treatment (pSuper1300- RhILL1 ) and control treatment (pSuper1300) anthocyanin supernatant ( Figure 7 , C), and the relative content of anthocyanins was measured ( Figure 7 , D), the results showed that overexpression RhILL1 After that, the anthocyanin supernatant will be darker than the control (pSuper1300), and the anthocyanin content is higher than the control. This further verifies the overexpression efficiency of the overexpressed gene ( Figure 7 , B), the results showed that compared with the control (pSuper1300), RhILL1 The expression level in the overexpression plants was significantly increased, which further indicated that RhILL1 Related to petal color.

[0149] To further confirm RhILL1 Correlation with flower color, overexpression RhILL1 , which increased the expression of early anthocyanin biosynthetic genes (EBGs), such as CHS, CHI, F3H etc., and late anthocyanin biosynthesis genes (LBGs), e.g. DFR, ANS, UFGT Expressions such as Figure 8 The above results show that overexpression RhILL1 It will make the color of rose petals last longer.

[0150] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. An application of the RhILL1 gene in regulating the color of rose petals, characterized in that: The synthesis of anthocyanins is regulated by silencing or overexpressing the auxin amide hydrolase RhILL1 gene, thereby achieving precise control of petal color. The nucleotide sequence of the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.1, the amino acid sequence of the protein encoded by the auxin amide hydrolase RhILL1 gene is shown in SEQ ID NO.2, and the rose is Freud.

2. The use according to claim 1, characterized in that: The applications include: (1) Rose gene editing breeding, wherein the rose gene editing is silencing the RhILL1 gene as claimed in claim 1; (2) Transgenic rose breeding, wherein the transgenic breeding is carried out by overexpressing the RhILL1 gene as described in claim 1.

3. A method for adjusting the color of rose petals, characterized in that: By silencing the auxin amide hydrolase RhILL1 gene or protein as claimed in claim 1, the expression level of the auxin amide hydrolase RhILL1 gene or protein in rose is reduced, and the rose is Freud.

4. A kit for regulating the color of rose petals, characterized in that: The kit comprises a component for silencing or overexpressing the auxin amidohydrolase RhILL1 gene as claimed in claim 1, and a component for detecting the color change of petals.

Images