Molecular breeding technology for regulating and controlling flower diameter of chrysanthemum through single gene silencing and application of molecular breeding technology

Abstract

The invention discloses a molecular breeding technology for realizing regulation and control of the flower diameter of chrysanthemum through single-gene silencing and application of the molecular breeding technology. On the basis of a CmRAD6 gene identified in chrysanthemum'mirama ', a specific artificial amiRNA interference element is designed, a plant expression vector is constructed, a chrysanthemum explant is transformed by adopting an agrobacterium tumefaciens-mediated leaf disc transformation method, and a transgenic plant is obtained through kanamycin resistance screening. Molecular verification shows that exogenous genes are successfully integrated, and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) analysis shows that the expression quantity of CmRAD6 in transgenic plants is obviously reduced compared with that of wild plants. Phenotypic observation shows that the longitudinal diameter of the ligulate flower of the transgenic plant is shortened, the diameter of the corolla is reduced, and the character is stably inherited for more than two years. The new function of the CmRAD6 gene for regulating and controlling the development of the floral organs of the compositae plants is disclosed for the first time, the bottleneck of the traditional breeding technology is broken through through single-gene accurate regulation and control, the energy consumption of facility cultivation can be reduced, and an innovative technical scheme is provided for molecular design and breeding of flowers.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular genetics and genetic engineering, and specifically relates to a molecular breeding technology and application for regulating the flower diameter size of chrysanthemums through single gene silencing, and a genetic improvement method for regulating the flower size development of chrysanthemums based on RNA interference technology. The technology is suitable for trait improvement and molecular breeding of Asteraceae plants. Background Art

[0002] Chrysanthemum (Chrysanthemum morifolium), one of my country's ten traditional famous flowers, boasts a cultivation history of over 2,000 years. Its rich phenotypic diversity is reflected in biological characteristics such as complex flower structures, a complete color spectrum, and a long viewing period. It is recognized by the international horticultural community as one of the "world's four major fresh cut flowers." According to the DUS testing guidelines developed by the International Union for the Protection of New Varieties of Plants, chrysanthemum varieties can be divided into three major groups based on flower diameter: large chrysanthemums (>10cm), medium chrysanthemums (6-10cm), and small chrysanthemums (<6cm). These groups are used in different scenarios, including specimen chrysanthemum cultivation, landscape gardening, and cut flower production.

[0003] In the current industry, the regulation of flower diameter traits primarily relies on traditional hybrid breeding combined with cultivation control techniques, such as artificial pollination to select superior strains and the application of chemical inhibitors to control plant development. These methods are subject to technical bottlenecks such as long breeding cycles (typically 5-8 years), low phenotypic screening efficiency, and high cultivation and management costs. Therefore, developing a new breeding technology system based on molecular design to achieve precise regulation of flower diameter traits is of great practical significance for enhancing the commercial value of cut chrysanthemums and optimizing production efficiency.

[0004] In recent years, breakthroughs in molecular biology techniques have provided new avenues for improving chrysanthemum traits. In particular, Agrobacterium-mediated genetic transformation systems can achieve overexpression of target genes or CRISPR / Cas9-mediated gene editing, which can shorten the breeding cycle by over 40% compared to traditional hybrid breeding. Previous studies have identified the regulatory roles of key genes in flower diameter regulation: overexpression of CmTCP20 can increase flower diameter by 28.6% by promoting inflorescence meristem expansion (Wang et al., 2019), while overexpression of CmJAZ1 inhibits floral organ cell proliferation, resulting in a 31.4% reduction in flower diameter (Guan et al., 2022). Notably, although RADIALIS (RAD), a member of the R3-MYB transcription factor family, has been shown to participate in the polarity establishment of floral organ development in other species, its molecular mechanism in flower diameter regulation in Asteraceae species has not yet been reported, providing a potential research target for molecular breeding of chrysanthemum. Summary of the Invention

[0005] Purpose of the Invention: The present invention aims to solve the problem of targeted improvement of chrysanthemum flower diameter size and provides a CmRAD6 gene, the sequence of which is SEQ ID NO. 1. A second purpose of the present invention is to provide a plant expression vector for the CmRAD6 gene. Another purpose of the present invention is to provide a method for regulating chrysanthemum flower diameter by transgenic CmRAD6 gene. The Agrobacterium-mediated genetic transformation, molecular detection of transgenic plants, and phenotypic observation of transgenic plant offspring involved in the present invention are used in the present invention. Methods and examples are provided for molecular breeding of ornamental chrysanthemums using genetic engineering technology. Yet another purpose of the present invention is to provide genetic engineering applications of the CmRAD6 gene, a pORE-R4-amiR-CmRAD6 plant expression vector, and a method for regulating chrysanthemum flower diameter for regulating chrysanthemum flower size.

[0006] Technical solution: The gene CmRAD6 related to the petal size and flower diameter size of the chrysanthemum ray-shaped flowers of the present invention, the nucleotide sequence of the gene CmRAD6 is shown in SEQ ID NO.1.

[0007] A set of primers for RNA interference of the gene CmRAD6 includes primer amiR-CmRAD6-I with a nucleotide sequence as shown in SEQ ID NO.4, primer amiR-CmRAD6-II with a nucleotide sequence as shown in SEQ ID NO.5, primer amiR-CmRAD66-III with a nucleotide sequence as shown in SEQ ID NO.6, and primer amiR-CmRAD6-IV with a nucleotide sequence as shown in SEQ ID NO.7.

[0008] The method for regulating the petal size and flower diameter size of chrysanthemum ray flowers comprises regulating the expression level of the gene CmRAD6 in chrysanthemum.

[0009] Knockdown expression of said regulation.

[0010] A method for creating chrysanthemums with smaller flower diameters involves interfering with the gene CmRAD6 in the genome of the target chrysanthemum.

[0011] The method for constructing an expression vector containing the CmRAD6 gene interference fragment comprises the following steps: amplifying the interference fragment of the gene using the pBS300 vector as a template, ligating it into the multiple cloning site of the pORE-R4 vector, transforming DH5α competent cells, and extracting the positive plasmid. Preferably, the pORE-R4 vector is a 2×35S pORE-R4 vector.

[0012] In the construction method, the enzymes used for selecting the multiple cloning site of the construction vector include EcoR I and SpeI.

[0013] The plant expression vector obtained by the construction method.

[0014] The gene CmRAD6, or the RNA interference CmRAD6 primer, or the plant expression vector is used in genetic engineering to regulate and improve the development of chrysanthemum petal size and / or flower diameter size.

[0015] According to the application, the length of the petals of the ray-shaped flowers becomes shorter and the diameter of the chrysanthemum flowers becomes smaller.

[0016] Preferably, a method for regulating the flower diameter of chrysanthemum comprises the following steps:

[0017] (1) Design of RNA interference primers for Chrysanthemum CmRAD6;

[0018] (2) Construction of plant expression vector pORE-R4-amiR-CmRAD6:

[0019] RNA interference primers were designed according to the specific region of the CmRAD6 gene. The interference fragment amiR-RAD6 was amplified using the pBS300 vector as a template and then double-digested with EcoR I and Spe I together with the 2×35S pORE-R4 vector. The digestion products were recovered and ligated with ligase, then transformed into DH5α competent cells. The positive plasmid was extracted, and the plant expression vector pORE-R4-amiR-CmRAD6 was successfully constructed.

[0020] (3) Agrobacterium EHA105-mediated transfer of the pORE-R4-amiR-CmRAD6 expression vector constructed in step (2) into chrysanthemum 'Shenma', and culturing to initially obtain resistant plants:

[0021] The plant expression vector pORE-R4-amiR-CmRAD6 obtained in step (2) was transformed into Agrobacterium competent cells EHA105 to obtain positive cloned Agrobacterium, and pORE-R4-amiR-CmRAD6 was introduced into chrysanthemum by the leaf disc method. Positive transformed plants were selected after kanamycin resistance screening. The positive transformed plants were subjected to PCR and fluorescent quantitative RT-PCR detection to verify that the pORE-R4-amiR-CmRAD6 plant expression vector had been inserted into the genomic DNA of the transgenic plant and expressed, and that the expression level of CmRAD6 in the transgenic plant was indeed downregulated.

[0022] Furthermore, the specific operations of step (1) are:

[0023] Using the cDNA of cut chrysanthemum 'Shenma' as a template, forward and reverse specific primers related to the CmRAD6 gene according to the sequence information, and reverse transcribed cDNA as a template, PCR amplification was performed, the product was ligated into the pMD19-T vector, and DH5α competent cells were transformed; or the full-length sequence of the target gene shown in SEQ ID NO.1 was obtained by direct synthesis according to the sequence shown in SEQ ID NO.1;

[0024] The CmRAD6 gene forward and reverse primers are:

[0025] Upstream primer CmRAD6-F: 5'-ATGTCATCCAACTCGCTT-3' (SEQ ID NO. 2),

[0026] Downstream primer CmRAD6-R: 5′-CTAACATCGGTAGTTGGG-3′ (SEQ ID NO. 3);

[0027] Furthermore, the primers in step (2) are:

[0028] amiR-CmRAD6-I:

[0029] 5'-GATTAGCAATTTTCATCCAGCGATCTCTCTTTTGTATTCC-3' (SEQ ID NO.4),

[0030] amiR-CmRAD6-II:

[0031] 5'-GATCGCTGGATGAAAATTGCTAATCAAAGAGAATCAATGA-3' (SEQ ID NO.5),

[0032] amiR-CmRAD66-III:

[0033] 5'-GATCACTGGATGAAATTTGCTATTCACAGGTCGTGATATG-3' (SEQ ID NO.6),

[0034] amiR-CmRAD6-IV:

[0035] 5'-GAATAGCAAATTTCATCCAGTGATCTACATATATATTCCT-3' (SEQ ID NO. 7);

[0036] Furthermore, the process of performing PCR and quantitative RT-PCR on the positive transformed plants in step (3) is as follows:

[0037] 1) PCR test:

[0038] Young leaves of kanamycin-resistant plants and untransformed plants obtained through rooting screening were taken to extract genomic DNA. A sequence on the vector and a sequence of amiR-CmRAD6 were used as detection targets. Primers were designed to amplify a 362 bp fragment. The primer sequences were:

[0039] Upstream primer 35S-F: 5'-GACGCACAATCCCACTATCC-3' (SEQ ID NO. 8),

[0040] Downstream primer amiR-CmRAD6-II:

[0041] 5'-GATCGCTGGATGAAAATTGCTAATCAAAGAGAATCAATGA-3' (SEQ ID NO. 5).

[0042] PCR amplification was performed using DNA from kanamycin-transformed plants and untransformed plants as templates, 35S-F and amiR-CmRAD6-II as primers, and the amplified products were detected and analyzed by agarose gel electrophoresis.

[0043] 2) Fluorescence quantitative RT-PCR detection:

[0044] Total RNA was extracted from leaves of kanamycin-resistant plants and untransformed plants, reverse transcribed into first-strand cDNA, and a fluorescent quantitative RT-PCR amplification system was established. Each sample was repeated three times. The CT value of each sample was obtained based on data analysis. The expression of untransformed plants was used as the benchmark value to calculate the relative expression of each transgenic plant and wild-type gene. The fragment amplified by the specific primers was 111 bp in length. The primer sequences were:

[0045] The upstream primer is CmRAD6-RT-F: 5'-GCTAAAGCGGTTGGTGGC-3' (SEQ ID NO.9),

[0046] The downstream primer was CmRAD6-RT-R: 5′-TCGGTAGTTGGGGGAAGG-3′ (SEQ ID NO. 10).

[0047] The gene fragment amplified by EFlα was used as the internal standard. The fragment length was 151 bp. The primer sequences were:

[0048] Upstream primer EF1α-F: 5'-TTTTGGTATCTGGTCCTGGAG-3' (SEQ ID NO. 11),

[0049] Downstream primer EF1α-R: 5′-CCATTCAAGCGACAGACTCA-3′ (SEQ ID NO. 12).

[0050] Specifically, the above method for regulating chrysanthemum strains by transferring the pORE-R4-amiR-CmRAD6 plant expression vector is as follows:

[0051] The process of using Agrobacterium-mediated transfer of the pORE-R4-amiR-CmRAD6 plant expression vector into chrysanthemum as described in step (3) is as follows: preparing Agrobacterium EHA105 competent cells, transferring the pORE-R4-amiR-CmRAD6 plant expression vector constructed in step (2) into Agrobacterium EHA105, selecting positive clones, shaking the cells to OD = 0.6, centrifuging, discarding the supernatant, and suspending an equal volume of the precipitate in MS (pH 5.8) culture medium for infection; taking the tender leaf discs of chrysanthemum seedlings in the tissue culture bottle as the transformation recipients, culturing them in the preculture medium for 3 days, and then invading the prepared Agrobacterium bacterial solution of the transferred plant expression vector for 8-10 minutes, absorbing the bacterial solution on the surface of the leaf disc with filter paper and then inoculating it on the co-culture medium, culturing in the dark for 3 days, and then transferring it to the screening subculture for 4 generations. When the resistant buds differentiated from each other grow to 2-3 cm, they are transferred to the rooting medium for culture to initially obtain resistant plants. The Agrobacterium strain used was EHA105.

[0052] The above-mentioned method for regulating the flower diameter of chrysanthemum by transferring the pORE-R4-amiR-CmRAD6 vector is that the chrysanthemum tissue culture medium is based on MS, pH 5.8, 100Kpa, and sterilized at 116°C for 30 minutes; pre-culture medium: MS + 6-benzylaminopurine (6-BA) 1mg / L + naphthaleneacetic acid (NAA) 0.5mg / L; co-culture medium: MS + 6-benzylaminopurine (6-BA) 1mg / L + naphthaleneacetic acid (NAA) 0.5mg / L; screening medium: MS + kanamycin (Kan) 10mg / L + carbenicillin (Carb) 500mg / L + 6-benzylaminopurine (6-BA) 1mg / L + naphthaleneacetic acid (NAA) 0.1mg / L; rooting medium: MS + kanamycin (Kan) 8mg / L.

[0053] The more detailed operation process is as follows:

[0054] Take 5 μL of pORE-R4-amiR-CmRAD6 vector plasmid and add it to 100 μL of competent cells. After the plasmid is transformed into Agrocybe stalk competent cells, single clones are picked for detection and positive clones are selected for transformation into chrysanthemum.

[0055] The leaves of cut chrysanthemum 'Shenma' were used as explants, and tender leaf discs (0.5 cm × 0.5 cm) of chrysanthemum seedlings in tissue culture bottles were taken as transformation recipients. They were pre-cultured for 3 days, then invaded into the prepared Agrobacterium solution for infection for 8-10 minutes, and after drying the solution with filter paper, they were inoculated onto co-culture medium and co-cultured in the dark for 3 days. Then, they were transferred to the screening medium for continued culture. When the resistant buds differentiated out grew to 2-3 cm, they were transferred to the rooting medium for culture to initially obtain resistant plants.

[0056] The above-mentioned method for regulating chrysanthemum flower diameter by transfection with the pORE-R4-amiR-CmRAD6 vector comprises performing PCR identification and fluorescent quantitative RT-PCR molecular detection on the resistant plants initially obtained by transfection with the pORE-R4-amiR-CmRAD6 vector, screening the positive plants, and obtaining transgenic chrysanthemum lines;

[0057] (1) PCR detection

[0058] Young leaves of kanamycin-resistant plants obtained through rooting screening and young leaves of untransformed plants were taken to extract genomic DNA. A sequence on the vector and a sequence of amiR-CmRAD6 were used as detection targets. Primers were designed to amplify a fragment of 362 bp in length. The primer sequences were as follows:

[0059] Upstream primer 35S-F: 5'-GACGCACAATCCCACTATCC-3' (SEQ ID NO. 8),

[0060] Downstream primer amiR-CmRAD6-II:

[0061] 5'-GATCGCTGGATGAAAATTGCTAATCAAAGAGAATCAATGA-3' (SEQ ID NO. 5);

[0062] PCR amplification was performed using the DNA of kanamycin-transformed plants and untransformed plants as templates, 35S-F and amiR-CmRAD6-II as primers, and the amplified products were detected and analyzed by agarose gel electrophoresis; positive plants should obtain a product of 362bp in length, while negative plants should have no amplified product.

[0063] (2) Fluorescence quantitative RT-PCR detection

[0064] Total RNA was extracted from leaves of kanamycin-resistant plants and untransformed plants, reverse transcribed into first-strand cDNA, and a fluorescent quantitative RT-PCR amplification system was established. Each sample was repeated three times. The CT value of each sample was obtained based on data analysis. The expression of untransformed plants was used as the benchmark value to calculate the relative expression of each transgenic plant and wild-type gene. The fragment amplified by the specific primers was 111 bp in length. The primer sequences were:

[0065] The upstream primer is CmRAD6-RT-F: 5'-GCTAAAGCGGTTGGTGGC-3' (SEQ ID NO.9),

[0066] The downstream primer was CmRAD6-RT-R: 5′-TCGGTAGTTGGGGGAAGG-3′ (SEQ ID NO. 10).

[0067] The gene fragment amplified by EF1α was used as the internal standard. The fragment length was 151 bp. The primer sequences were:

[0068] Upstream primer EF1α-F: 5'-TTTTGGTATCTGGTCCTGGAG-3' (SEQ ID NO. 11),

[0069] Downstream primer EF1α-R: 5′-CCATTCAAGCGACAGACTCA-3′ (SEQ ID NO. 12).

[0070] The above-mentioned method for regulating chrysanthemum flower diameter by transfecting the pORE-R4-amiR-CmRAD6 vector is to observe the phenotype of the transgenic plant offspring obtained by PCR and fluorescent quantitative RT-PCR molecular detection:

[0071] Cuttings of wild-type chrysanthemum (WT) and transgenic chrysanthemum (amiR) were selected as materials for phenotypic observation 65 days after planting. Twenty seedlings of each line were bud-wiped after short-day photoperiod, and the length and diameter of the outermost ligule of each line during the flowering period were counted.

[0072] The third object of the present invention is to provide a genetic engineering application of the above-mentioned gene CmRAD6, or the above-mentioned plant expression vector, or the above-mentioned method for regulating the flower diameter size of chrysanthemum in regulating the flower size of chrysanthemum.

[0073] Furthermore, the flower size regulation refers to reducing the size of the ligulate flowers of the chrysanthemum and reducing the flower diameter of the chrysanthemum.

[0074] Beneficial Effects: The method provided by the present invention selects transgenic chrysanthemum material. Using transgenic technology, the pORE-R4-amiR-CmRAD6 vector is integrated into the chrysanthemum genome. Phenotypic observation and analysis reveal transgenic chrysanthemum lines with reduced flower diameter. Therefore, the pORE-R4-amiR-CmRAD6 vector introduced into the chrysanthemum genome using this method can significantly shorten the size of ray-shaped chrysanthemum flowers, thereby reducing their size. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1Electrophoresis for detection of CmRAD6 gene by chrysanthemum-specific primers, M: DL2000 Marker, WT: wild-type plant, P: pORE-R4-amiR-CmRAD6 vector plasmid, 3-7: chrysanthemum lines transformed with pORE-R4-amiR-CmRAD6 interference vector;

[0076] Figure 2 is the relative expression level of CmRAD6 gene in transgenic and wild-type chrysanthemum plants, WT: wild-type plant, amiR-4, amiR-7, amiR-6: chrysanthemum lines transformed with pORE-R4-amiR-CmRAD6 interference vector, respectively;

[0077] Figure 3 Phenotypic observation of wild-type and transgenic chrysanthemum plants, WT: wild-type plant, amiR-4, amiR-7, amiR-6: chrysanthemum lines transformed with the pORE-R4-amiR-CmRAD6 interference vector;

[0078] Figure 4 Statistics of flower size and ligulate floret length of wild-type and transgenic chrysanthemum plants. WT: wild-type plant; amiR-4, amiR-7, amiR-6: chrysanthemum lines transformed with the pORE-R4-amiR-CmRAD6 interference vector. DETAILED DESCRIPTION

[0079] The following is a detailed description of a specific embodiment of the invention: This embodiment is implemented based on the technical solution of the present invention, and provides a detailed fact method and specific operation process. The specific implementation method is as follows:

[0080] Example 1. Cloning of CmRAD6

[0081] Using the cut chrysanthemum 'Shenma' as the material, 0.5 g of leaves were taken and total RNA was extracted from the leaves according to the instructions of the plant RNAexr kit rapid universal plant RNA extraction kit (Huayueyang). The cDNA was obtained by reverse transcription using the EvoM-MLV reverse transcription kit (AG).

[0082] Specific primers were designed using Primer 5 software to amplify CmRAD6; upstream primer CmRAD6-F: 5'-ATGTCATCCAACTCGCTT-3' (SEQ ID NO. 2),

[0083] Downstream primer CmRAD6-R: 5′-CTAACATCGGTAGTTGGG-3′ (SEQ ID NO. 3);

[0084] Using leaf cDNA as a template, PCR reaction was performed with 95°C pre-denaturation for 5 min; 95°C melting for 10 min, 55°C annealing for 30 sec, 72°C extension for 1 min, and 72°C extension for 1 min for 35 cycles; the product was recovered using a gel recovery kit (FastPureGel DNA Extraction Mini Kit, Vazyme). The cloning results are shown in Figure 2. Figure 1 The pMD19-T vector (TaKaRa) was connected with solution I and transformed into DH5α competent cells. The sequence was determined as shown in SEQ ID NO.1:

[0085]

[0086] Example 2. Construction of plant expression vector pORE-R4-amiR-CmRAD6

[0087] The specific sequence of the CmRAD6 gene was analyzed on the website (wmd3.weigelworld.org / ), and specific primers were designed (Table 1).

[0088] Table 1 Specific primer sequences

[0089]

[0090]

[0091] Using the pBS300 vector (containing the Arabidopsis miR319 precursor sequence) as a template, the high-fidelity enzyme ( Super-Fidelity DNA Polymerase, ATG) was used for the first round of PCR reaction. The reaction system in 50 μL was 2× Master Mix (Dye) 25 μL, pBS300 vector 1 μL, ddH2O 20 μL, primer A, primer amiR-CmRAD6-IV 2 μL each (10 μmol·L -1 ) Get fragment a; 2× Master Mix (Dye) 25 μL, pBS300 vector 1 μL, ddH2O 20 μL, primer amiR-CmSAUR66-III, primer amiR-CmRAD6-II 2 μL each (10 μmol·L -1 ) Get fragment b; 2× Master Mix (Dye) 25 μL, pBS300 vector 1 μL, ddH2O 20 μL, primer amiR-CmRAD6-I, primer B 2 μL each (10 μmol·L -1 ) Get fragment c;

[0092] Reaction procedure: 95℃ pre-denaturation for 5 min; 95℃ melting for 10 min, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min;

[0093] After electrophoresis detection, the PCR products were recovered using a gel recovery kit (FastPure GelDNAExtraction Mini Kit, Vazyme) to obtain fragments a, b, and c after the correct size;

[0094] The three amplification products recovered above were mixed as templates and the high-fidelity enzyme ( Super-Fidelity DNA Polymerase, ATG) was used for the second round of PCR reaction. The reaction system in 50 μL was 2× Master Mix (Dye) 25 μL, 3 μL of mixed samples of three amplification products, 17 μL of ddH2O, 2 μL each of primer A and primer B (10 μmol·L -1 );

[0095] Reaction procedure: 95℃ pre-denaturation for 5 min; 95℃ melting for 10 min, 55℃ annealing for 30 sec, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min;

[0096] The PCR product was recovered using a gel recovery kit (FastPure Gel DNA Extraction Mini Kit, Vazyme) to obtain the d fragment, which was the artificial miRNA precursor;

[0097] The vector 2×35S pORE-R4 (Invitrogen, USA) and fragment d were double-digested with EcoR I and Spe I. The double-digestion system (50 μL) consisted of 5 μL of 10×Q.cut Buffer, 2.5 μL of EcoR I, 2.5 μL of Spe I, 15 μL of plasmid 2×35S pORE-R4 or the above-mentioned recovered product, and 25 μL of ddH2O. The reaction was incubated at 37°C for 1 h. The double-digestion products were analyzed by agarose gel electrophoresis, and the digested plasmid 2×35S pORE-R4 fragment and fragment d were recovered using a gel recovery kit (Vazyme).

[0098] The two recovered products were ligated using Solution I (TaKaRa). A 10 μL ligation reaction system consisted of 5 μL of Solution I, 1 μL of the 2×35S pORE-R4 double-digested fragment, and 4 μL of the amiR-CmRAD6 double-digested fragment. The ligation reaction was incubated overnight at 16°C, and 10 μL of the ligation product was transformed into DH5α competent cells. After overnight incubation at 37°C, positive single colonies were selected and expanded. The plasmid pORE-R4-amiR-CmRAD6 was isolated and verified by electrophoresis and sequencing. The plant expression vector pORE-R4-amiR-CmRAD6 was successfully constructed.

[0099] Example 3. Agrobacterium EHA105-mediated transformation of chrysanthemum by leaf disc method

[0100] Take 3 μL of pORE-R4-amiR-CmRAD6 vector plasmid to click-transform 100 μL of competent cells, add 800 μL YEB liquid medium, pre-culture at 28°C 200 rpm for 2 h, spread the bacterial liquid on YEB (50 μg / mL rifampicin + 50 μg / mL kanamycin) solid medium, culture in the dark at 28°C for 2 days, pick single clones for detection, select positive clones and shake the bacteria to OD = 0.8, after centrifugation, discard the supernatant, and suspend an equal volume of the precipitate in MS (pH 5.8) culture medium for transformation of chrysanthemum.

[0101] The Agrobacterium used was EHA105, which contained an interference expression vector containing the CmRAD6 gene. EHA105 was cultured in YEB liquid medium. The transgenic progeny were named amiR. Leaves of the cut chrysanthemum 'Shenma' served as explants, and pORE-R4-amiR-CmRAD6 was introduced into chrysanthemum plants using Agrobacterium tumefaciens-mediated transfection. Leaf discs (0.5 cm × 0.5 cm) from the top of chrysanthemum seedlings in tissue culture flasks were used as recipients for transformation. These discs were pre-cultured in pre-culture medium (MS + 1 mg / L 6-benzylaminopurine (6-BA) + 0.5 mg / L naphthaleneacetic acid (NAA)) for 3 days. The discs were then immersed in the prepared Agrobacterium culture solution for infection for 10 minutes. The solution was then blotted dry with filter paper and inoculated into co-culture medium (MS + 1 mg / L 6-benzylaminopurine (6-BA) + 0.5 mg / L naphthaleneacetic acid (NAA)). ) for 3 days in the dark, then transferred to screening medium (MS + kanamycin (Kan) 10 mg / L + carbenicillin (Carb) 500 mg / L + 6-benzylaminopurine (6-BA) 1 mg / L + naphthaleneacetic acid (NAA) 0.1 mg / L) for subculture for 4 generations. When the resistant buds differentiated out grew to 2-3 cm, they were transferred to rooting medium (MS + kanamycin (Kan) 8 mg / L) for culture, and kanamycin-resistant plants were preliminarily obtained.

[0102] Chrysanthemum tissue culture medium is based on MS medium, pH 5.8, 100KPa, and sterilization at 116℃ for 30 minutes.

[0103] Example 4. Molecular detection (PCR, fluorescent quantitative RT-PCR) of resistant plants transfected with the pORE-R4-amiR-CmRAD6 vector was positive, and a transgenic chrysanthemum line was obtained.

[0104] 1) PCR testing

[0105] Young leaves of kanamycin-resistant plants obtained through rooting screening and young leaves of untransformed plants were taken to extract genomic DNA. The gene fragment in the vector was used as the detection target. Amplification primers were synthesized at both ends. The amplified fragment was 362 bp long. The primer sequences were:

[0106] Upstream primer 35S-F: 5'-GACGCACAATCCCACTATCC-3' (SEQ ID NO. 8),

[0107] Downstream primer amiR-CmRAD6-II:

[0108] 5'-GATCGCTGGATGAAAATTGCTAATCAAAGAGAATCAATGA-3' (SEQ ID NO. 5);

[0109] PCR detection was performed using kanamycin-resistant plant and untransformed plant DNA as templates, 35S-F and amiR-CmRAD6-II as primers, and 2× Rapid Taq Master Mix (AG). The 20 μL reaction system included: 2× Rapid Taq Master Mix 10 μL, 35S-F 1 μL, amiR-CmSAUR66-II 1 μL, DNA template 1 μL, and ddH20 7 μL.

[0110] Amplification conditions: pre-denaturation at 95°C for 3 min; 35 cycles of melting at 95°C for 15 sec, annealing at 55°C for 15 sec, and extension at 72°C for 30 sec; and extension at 72°C for 5 min.

[0111] 2) Fluorescence quantitative RT-PCR detection

[0112] Total RNA was extracted from leaves of kanamycin-resistant plants and untransformed plants, reverse transcribed into first-strand cDNA, and the expression of the CmRAD6 gene was detected by fluorescent quantitative RT-PCR. The amplification system was established according to the instructions of the fluorescent quantitative kit (2X SYBR GreenPro TaqHS Premix). The amplification conditions were: 95°C for 30 seconds; 95°C for 5 seconds, 60°C for 30 seconds, and 40 cycles. The CT value of each sample was obtained based on data analysis. The expression of the untransformed plant was used as the benchmark value to calculate the relative expression of each transgenic plant and the wild-type gene. The fragment amplified by the specific primers was 111 bp in length. The primer sequences were:

[0113] The upstream primer is CmRAD6-RT-F: 5'-GCTAAAGCGGTTGGTGGC-3' (SEQ ID NO.9),

[0114] The downstream primer was CmRAD6-RT-R: 5′-TCGGTAGTTGGGGGAAGG-3′ (SEQ ID NO. 10).

[0115] The gene fragment amplified by EF1α was used as the internal standard. The fragment length was 151 bp. The primer sequences were:

[0116] Upstream primer EF1α-F: 5'-TTTTGGTATCTGGTCCTGGAG-3' (SEQ ID NO. 11),

[0117] Downstream primer EF1α-R: 5'-CCATTCAAGCGACAGACTCA-3' (SEQ ID NO.12). Fluorescence quantitative RT-PCR detection results are shown in Figure 2 As shown, compared with the wild type, the gene expression level of the pORE-R4-amiR-CmRAD6 transgenic plant line decreased, and the expression level was significantly different from that of the wild type chrysanthemum, confirming that the expression of the endogenous CmRAD6 gene in cut chrysanthemum has been disrupted.

[0118] Example 5. Phenotypic observation of transgenic plant offspring

[0119] Wild-type chrysanthemum (WT) and transgenic chrysanthemum (amiR) were selected as materials for phenotypic observation and planted in a greenhouse. Figure 3 As shown, the longitudinal diameter of the ray flowers of amiR transgenic plants was shortened by 20.3-25.7% (p < 0.01), and the corolla diameter was reduced by 21.1-27.3%.

[0120] (p<0.01)( Figure 4 ).

[0121] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without any creative ideas, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A gene CmRAD6 related to the petal size and flower diameter size of chrysanthemum ray flowers, characterized in that: The nucleotide sequence of the gene CmRAD6 is shown in SEQ ID NO.

1.

2. A set of RNA interference primers for the gene CmRAD6 according to claim 1, characterized in that: The primer comprises amiR-CmRAD6-I having a nucleotide sequence as shown in SEQ ID NO.4, amiR-CmRAD6-II having a nucleotide sequence as shown in SEQ ID NO.5, amiR-CmRAD66-III having a nucleotide sequence as shown in SEQ ID NO.6, and amiR-CmRAD6-IV having a nucleotide sequence as shown in SEQ ID NO.

7.

3. A method for regulating the size of petals and flower diameter of chrysanthemum ray flowers, characterized in that: The method comprises regulating the expression level of the gene CmRAD6 according to claim 1 in chrysanthemum.

4. The method according to claim 3, characterized in that Knockdown expression of said regulation.

5. A method for producing chrysanthemums with smaller flower diameters, characterized in that: Interfere with the gene CmRAD6 of claim 1 in the genome of the target chrysanthemum.

6. A method for constructing an expression vector containing the gene CmRAD6 interference according to claim 1, characterized in that: The method comprises the following steps: using the pBS300 vector as a template to amplify the interference fragment of the gene, connecting it to the multiple cloning site of the pORE-R4 vector, transforming DH5α competent cells, and extracting the positive plasmid.

7. The construction method according to claim 6, characterized in that: The enzymes used to construct the multiple cloning site of the vector include EcoRI and SpeI.

8. A plant expression vector obtained by the construction method according to claim 6 or 7.

9. Genetic engineering application of the gene CmRAD6 according to claim 1, or the RNA interference CmRAD6 primer according to claim 2, or the plant expression vector according to claim 8 in regulating and improving the development of chrysanthemum petal size and / or flower diameter size.

10. The use according to claim 9, characterized in that The length of the petals of the ray-shaped flowers becomes shorter, and the diameter of the chrysanthemum flowers becomes smaller.

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