Chrysanthemum petal type directional regulation and control technology through single-gene RNA silencing and application of chrysanthemum petal type directional regulation and control technology in breeding

Abstract

The invention discloses a chrysanthemum petal type directional regulation and control technology through single-gene RNA (Ribonucleic Acid) silencing and an application of the chrysanthemum petal type directional regulation and control technology in breeding. A CmSAUR66 gene coding region expression product is proved to regulate and control the fusion degree of ligulate flowers (CTMD). According to the method, a pORE-R4-amiR-CmSAUR66 plant expression vector is constructed, and agrobacterium tumefaciens-mediated transformation is carried out on a chrysanthemum 'Shenma' variety, so that a transgenic line with stable inheritance is obtained. Molecular phenotype correlation analysis shows that (1) the expression quantity of a transgenic plant CmSAUR66 is reduced compared with that of a wild plant; (2) the CTMD value of the tongue-shaped flower of the outermost ring is increased from a wild type; and (3) a specific bifurcation structure appears at the tip of the petal. The invention discloses the regulation and control functions of SAUR gene family members in compositae plant petal type development for the first time, realizes directional improvement of CTMD values through single-gene silencing, and provides key technical support for cut-flower chrysanthemum commodity specification standardization and petal form design.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular genetics and genetic engineering, specifically relating to a technology for the targeted regulation of chrysanthemum petal shape through single-gene RNA silencing and its application in breeding. It also relates to a molecular design breeding method for the targeted regulation of chrysanthemum petal shape through RNA interference technology, which targets and silences the CmSAUR66 gene. In particular, it relates to a technical system for the targeted improvement of flat-petal chrysanthemums to tubular-petal chrysanthemums by regulating the degree of fusion of ray flowers (CTMD), as well as the application of this technology in the standardized production of commercial cut flowers and the selection of cut chrysanthemum varieties. It further extends to the development of molecular modules for regulating the morphology of floral organs in Asteraceae plants using members of the SAUR gene family. Background Art

[0002] Chrysanthemum (Chrysanthemum morifolium) is one of the world's four largest fresh cut flowers, with an annual trade volume exceeding US$5 billion, and occupies a core position in the ornamental flower industry. Currently, commercial variety improvement relies heavily on phenotypic hybridization and breeding, but there are two major technical bottlenecks: (1) the effective hybridization fruit set rate per plant is less than 3%, and the single breeding cycle is as long as 5-8 years (Zhao et al., 2021); (2) key ornamental traits (such as petal shape and flower diameter) are regulated by multiple genes with micro-effects, making it difficult to achieve precise trait improvement using traditional methods (Wang et al., 2022). As a result, the market share of new chrysanthemum varieties in my country is less than 12%, significantly lower than that of breeding powerhouses such as the Netherlands (FAO, 2023).

[0003] At the molecular regulatory level, auxin signaling has been shown to mediate plant organ development through the SAUR (Small auxin-up RNA) gene family. Although CRISPR / Cas9-mediated SAUR gene editing has achieved targeted regulation of floral organ morphology in Arabidopsis (Ren et al., 2021), the unique polyploid genome complexity (2n=6x=54) of the Asteraceae family has significantly delayed the elucidation of its gene functions. Existing studies have only confirmed that CmBES1 influences petal type classification by regulating the corolla tube merged degree (CTMD) (Cheng et al., 2020), while the functional understanding of SAUR genes in floral organ development in the Asteraceae family remains largely unexplored, significantly limiting the development of molecular design breeding systems for chrysanthemum.

[0004] It's worth noting that the CTMD of the rayed flower is a core indicator for international chrysanthemum variety registration (Song et al., 2018). Its regulatory mechanism directly determines the commercial value of the three major commercial varieties: flat-petal, spoon-petal, and tubular-petal. Existing genetic improvement technologies are unable to overcome maternal genetic biases (Xia Boshun, 2009), resulting in a petal shape improvement success rate of less than 7% (China Flower Association, 2022). Therefore, developing targeted petal shape improvement technologies based on precise regulation of key genes has become a key scientific proposition for breaking through the bottleneck of chrysanthemum industry upgrading. Summary of the Invention

[0005] Purpose of the invention: In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a gene for regulating the degree of fusion of chrysanthemum ligulate flowers, the construction and application of an RNA interference expression vector, which can directionally improve the chrysanthemum ligulate petal shape, and provide methods and examples for the use of genetic engineering technology to carry out molecular breeding of ornamental chrysanthemums.

[0006] Technical solution: The gene CmSAUR66 for regulating the degree of fusion of ray-shaped flowers of chrysanthemum according to the present invention has a nucleotide sequence as shown in SEQ ID NO. 1. Preferably, the regulation is to increase the degree of fusion of petals of ray-shaped flowers of chrysanthemum.

[0007] The present invention also provides a set of RNA interference primer pairs for cloning the above-mentioned gene CmSAUR66, comprising amiR-CmSAUR66-I with a nucleotide sequence as shown in SEQ ID NO.4, primer amiR-CmSAUR66-II as shown in SEQ ID NO.5, primer amiR-CmSAUR66-III as shown in SEQ ID NO.6 and primer amiR-CmSAUR66-IV as shown in SEQ ID NO.7.

[0008] The present invention also provides a method for regulating the fusion degree of chrysanthemum ray flowers, comprising regulating the expression level of the gene CmSAUR66 in chrysanthemum.

[0009] Preferably, said regulation comprises knocking down expression.

[0010] The present invention also provides a method for promoting the fusion degree of chrysanthemum ray flowers, which interferes with the gene CmSAUR66 in the genome of the target chrysanthemum.

[0011] The present invention also provides a method for constructing an interference vector for the aforementioned gene CmSAUR66, comprising the steps of amplifying an interference sequence fragment of the gene using a pBS300 vector as a template, ligating the fragment into the multiple cloning site of a pORE-R4 vector, transforming DH5α competent cells, extracting the positive plasmid, and constructing a plant expression vector. Preferably, the pORE-R4 vector is a 2×35SpORE-R4 vector.

[0012] Preferably, the enzymes used to construct the multiple cloning site of the vector include EcoR I and Spe I.

[0013] The present invention also provides a plant expression vector obtained by the above construction method.

[0014] The present invention also provides the use of the gene CmSAUR66 or the plant expression vector in regulating the degree of fusion of ray flowers of chrysanthemum.

[0015] The present invention also provides the use of the gene CmSAUR66 or the plant expression vector in cultivating chrysanthemum germplasms with different petal shapes.

[0016] Beneficial Effects: The present invention provides a gene, CmSAUR66, cloned from the cut chrysanthemum 'Shenma', that regulates the degree of fusion of ray-shaped florets in chrysanthemums, having the nucleotide sequence set forth in SEQ ID NO. 1. The present invention also constructs an RNA interference plant expression vector based on the CmSAUR66 gene, and transforms the plant expression vector into chrysanthemum material to obtain transgenic chrysanthemum material. The present invention utilizes transgenic technology, using RNA interference, to reduce the expression of endogenous CmSAUR66 in chrysanthemums. Through phenotypic observation and analysis, transgenic chrysanthemum lines were obtained in which the outer whorl of ray-shaped florets changed from flat petals to tubular petals. Results showed that, compared to non-transgenic plants, the outer whorl of ray-shaped florets in plants with CmSAUR66 knockdown exhibited tubular petals with split apex after flowering under short-day conditions, indicating that knockdown of the CmSAUR66 gene significantly increased the degree of fusion of the ray-shaped florets in chrysanthemums. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Agarose gel electrophoresis of CmSAUR66 gene cloning, M: Marker2000;

[0018] Figure 2 This is the electrophoresis diagram of the pORE-R4-amiR-CmSAUR66 plant expression vector plasmid, M: Marker2000;

[0019] Figure 3 Electrophoresis diagram of RNA interference transgenic chrysanthemum using specific primers, M: Marker2000, positive: positive plasmid, negative: wild-type plant;

[0020] Figure 4 Electrophoresis diagram for identification of pORE-R4-amiR-CmSAUR66 transgenic chrysanthemum, M: Marker2000, positive: positive plasmid, negative: wild-type plant; 5-6: CmSAUR66 transgenic line with RNA interference;

[0021] Figure 5The relative expression levels of the CmSAUR66 gene in transgenic and non-transgenic chrysanthemum plants are shown in Figure 2. WT: wild-type plant, amiR-5-6: CmSAUR66 RNA interference strain;

[0022] Figure 6 The flower phenotype observation diagram of transgenic chrysanthemum at full flowering stage, WT: wild-type plant, amiR-5-6: RNA interference line of CmSAUR66;

[0023] Figure 7 Phenotypic observation of ligulate flowers in transgenic chrysanthemum at full flowering stage, WT: wild-type plant, amiR-5-6: RNA interference strain of CmSAUR66;

[0024] Figure 8 This is a statistical chart of the degree of fusion of ray flowers during the flowering period of transgenic chrysanthemum. Specific implementation methods

[0025] The present invention provides a gene CmSAUR66 for regulating the degree of fusion of ray flowers of chrysanthemum. The nucleotide sequence of the gene CmSAUR66 is shown in SEQ ID NO.1:

[0026]

[0027] The present invention also provides a set of primer pairs for cloning the above gene CmSAUR66, including an upstream primer CmSAUR66-F with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer CmSAUR66-R with a nucleotide sequence as shown in SEQ ID NO.3.

[0028] When cloning the gene using the primer pair, the present invention preferably uses leaves of the cut chrysanthemum 'Shenma' as the material, extracts RNA, and reverse transcribes it into cDNA. PCR amplification is performed using the upstream primer CmSAUR66-F (SEQ ID No. 2): 5'-ATGGTCAGCCTCAAGAAA-3' and the downstream primer CmSAUR66-R (SEQ ID No. 3): 5'-TTAACACACCAACAATTG-3'. The PCR amplification procedure preferably includes: pre-denaturation at 95°C for 5 minutes; melting at 95°C for 10 minutes, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, for 35 cycles; and extension at 72°C for 10 minutes. The present invention preferably ligates the PCR reaction product into the pMD19-T vector and transforms it into DH5α competent cells, obtaining the target gene sequence as SEQ ID NO. 1.

[0029] The present invention also provides a set of primer pairs for artificially interfering with the above-mentioned gene CmSAUR66, including amiR-CmSAUR66-I with a nucleotide sequence as shown in SEQ ID NO.4, primer amiR-CmSAUR66-II as shown in SEQ ID NO.5, primer amiR-CmSAUR66-III as shown in SEQ ID NO.6 and primer amiR-CmSAUR66-IV as shown in SEQ ID NO.7.

[0030] When the primer pair is used in the present invention, it is preferred to use the pBS300 vector as a template, using primer A (SEQ ID NO.8): 5'-CTGCAAGGCGATTAAGTTGGGTAAC-3' and primer amiR-CmSAUR66-IV (SEQ ID NO.7):

[0031] 5'-GAACACAAAACGTACTCCGTTCGTCTACATATATATATTCCT-3' fragment a was amplified using primer amiR-CmSAUR66-III (SEQ ID NO. 6):

[0032] 5'-GACGAACGGAGTACGTTTTGTGTTCACAGGTCGTGATATG-3' and primer amiR-CmSAUR66-II (SEQ ID NO.5):

[0033] 5'-GACGGACGGAGTACGATTTGTGATCAAAGAGAATCAATGA-3' fragment b was amplified using primer amiR-CmSAUR66-I (SEQ ID NO. 4):

[0034] 5'-GATCACAAATCGTACTCCGTCCGTCTCTCTTTTGTATTCC-3' and primer B (SEQ ID NO.9): 5'-GCGGATAACAATTTCACACAGGAAACAG-3' to amplify fragment c. The PCR amplification procedure preferably includes: pre-denaturation at 95°C for 5 min; melting at 95°C for 10 min, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and reaction for 35 cycles; and extension at 72°C for 10 min.

[0035] The present invention also provides a method for constructing an expression vector for CmSAUR66 interference, comprising the following steps: using the amplified fragments a, b, and c as templates, amplifying fragment d with primers A and B, double-digesting the vector 2×35S pORE-R4 and the fragment d with EcoR I and Spe I, respectively, ligating and transforming, and extracting the positive plasmid to obtain the CmSAUR66 interference expression vector pORE-R4-amiR-CmSAUR66.

[0036] The present invention also provides a plant expression vector obtained by the above construction method.

[0037] The present invention also provides the use of the gene CmSAUR66 or the plant expression vector in regulating the degree of fusion of ray flowers of chrysanthemum.

[0038] The application of the present invention is preferably the same as described above and will not be described again here.

[0039] The present invention also provides the use of the gene CmSAUR66 or the plant expression vector in cultivating chrysanthemum germplasms with different petal shapes.

[0040] The gene or plant expression vector of the present invention can be used to cultivate chrysanthemums with different petal shapes. The present invention does not specifically limit the cultivation method, and conventional transgenic methods in the art can be used.

[0041] To further illustrate the present invention, the gene, expression vector and application for regulating the flowering period of chrysanthemum provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] Cloning of the CmSAUR66 gene

[0044] Using 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). cDNA was obtained by reverse transcription using the EvoM-MLV reverse transcription kit (AG).

[0045] Specific primers were designed using primer 5 software to amplify CmSAUR66;

[0046] Upstream primer CmSAUR66-F: 5'-ATGGTCAGCCTCAAGAAA-3' (SEQ ID NO. 2),

[0047] Downstream primer CmSAUR66-R: 5′-TTAACACACCAACAATTG-3′ (SEQ ID NO. 3);

[0048] Using leaf cDNA as template, PCR reaction was performed, with 95℃ pre-denaturation for 5 min; 95℃ melting for 10 min, 55℃ annealing for 30 sec, 72℃ extension for 1 min, reaction for 35 cycles; 72℃ extension for 10 min; cloning results are shown in Figure 2. Figure 1 shown.

[0049] The product was recovered using a gel extraction kit (FastPure Gel DNA Extraction Mini Kit, Vazyme), ligated to the pMD19-T vector (TaKaRa) using solution I, and transformed into DH5α competent cells. The sequence was determined as shown in SEQ ID NO. 1:

[0050]

[0051] Example 2

[0052] Construction of plant expression vector pORE-R4-amiR-CmSAUR66

[0053] The specific sequence of the CmSAUR66 gene was analyzed on the website (wmd3.weigelworld.org / ), and specific primers were designed (Table 1).

[0054] Table 1 Specific primer sequences

[0055]

[0056]

[0057] Using the pBS300 vector (containing the Arabidopsis miR319 precursor sequence) as a template, the high-fidelity enzyme ( The first round of PCR reaction was performed with Super-Fidelity DNA Polymerase (ATG). The reaction system was 50 μL: Master Mix (Dye) 25 μL, pBS300 vector 1 μL, ddH2O 20 μL, primer A, primer amiR-CmSAUR66-IV 2 μL each (10 μmol·L -1 )Get fragment a; Master Mix (Dye) 25 μL, pBS300 vector 1 μL, ddH2O 20 μL, primer amiR-CmSAUR66-III, primer amiR-CmSAUR66-II 2 μL each (10 μmol·L-1 ) Get fragment b; MasterMix (Dye) 25 μL, pBS300 vector 1 μL, ddH2O 20 μL, primer amiR-CmSAUR66-I, primer B 2 μL each (10 μmol·L -1 ) Get fragment c;

[0058] 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;

[0059] After electrophoresis detection, the PCR products were recovered using a gel recovery kit (FastPure Gel DNA Extraction Mini Kit, Vazyme) to obtain fragments a, b, and c after the correct size;

[0060] The three amplification products recovered above were mixed as templates and the high-fidelity enzyme ( The second round of PCR reaction was performed with Super-Fidelity DNA Polymerase, ATG, 50 μL reaction system: Master Mix (Dye) 25 μL, 3 μL of mixed samples of 3 amplification products, 17 μL of ddH2O, 2 μL each of primer A and primer B (10 μmol·L -1 );

[0061] 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;

[0062] 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;

[0063] 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).

[0064] The two recovered products were ligated using Solution I enzyme (TaKaRa). The 10 μL ligation reaction system consisted of: 5 μL Solution I, 1 μL 2×35S pORE-R4 double-digested fragment, and 4 μL amiR-CmSAUR66 double-digested fragment. The ligation reaction was carried out overnight at 16°C, and 10 μL of the ligation product was used to transform DH5α competent cells. After overnight culture at 37°C, a positive single clone was selected and expanded, and the plasmid pORE-R4-amiR-CmSAUR66 was extracted and verified by electrophoresis and sequencing. The plant expression vector pORE-R4-amiR-CmSAUR66 was successfully constructed ( Figure 2 ).

[0065] Example 3

[0066] Agrobacterium EHA105-mediated transformation of chrysanthemum by leaf disc method

[0067] Take 3 μL of pORE-R4-amiR-CmSAUR66 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 to OD = 0.8, after centrifugation, discard the supernatant, and suspend an equal volume of precipitate in MS (pH 5.8) culture medium for transformation of chrysanthemum.

[0068] The Agrobacterium used was EHA105, which contained an interference expression vector containing the CmSAUR66 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-CmSAUR66 was introduced into chrysanthemum plants using Agrobacterium tumefaciens-mediated transfection. Top leaf discs (0.5 cm × 0.5 cm) 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.

[0069] Chrysanthemum tissue culture medium is based on MS medium, pH 5.8, 100KPa, and sterilization at 116℃ for 30 minutes.

[0070] Example 4

[0071] The pORE-R4-amiR-CmSAUR66-transgenic resistant plants were tested molecularly (PCR, fluorescent quantitative RT-PCR) and the results were positive, thus obtaining a transgenic chrysanthemum line.

[0072] (1) PCR detection

[0073] 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 399 bp long. The primer sequences were:

[0074] Upstream primer 35S-F (SEQ ID NO.10): 5'-GACGCACAATCCCACTATCC-3',

[0075] Downstream primer amiR-CmSAUR66-II (SEQ ID NO. 5): 5′-GACGGACGGAGTACGATTTGTGATCAAAGAGAATCAATGA-3′;

[0076] PCR detection was performed using kanamycin-resistant plant and untransformed plant DNA as templates, 35S-F and amiR-CmSAUR66-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. (Primer identification was as follows Figure 3 shown)

[0077] 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.

[0078] The amplified products were analyzed by agarose gel electrophoresis. Figure 4 As shown, the plants transformed with pORE-R4-amiR-CmSAUR66 could amplify the same specific amplification band as the positive control, while the wild-type plants did not amplify the band.

[0079] (2) Fluorescence quantitative RT-PCR detection

[0080] Total RNA was extracted from leaves of kanamycin-resistant and untransformed plants and reverse-transcribed into first-strand cDNA. The expression of the CmSAUR66 gene was detected using fluorescent quantitative RT-PCR. The amplification system was established according to the instructions of the fluorescent quantitative kit (2X SYBR Green ProTaq HS 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 relative expression of the gene in each transgenic plant and the wild-type gene was calculated using the expression of the untransformed plant as the baseline. The fragment amplified by the specific primers was 225 bp in length. The primer sequences were:

[0081] Upstream primer qRT-CmSAUR66-F (SEQ ID NO.11): 5'-AGTACGATTTGTGATACCAATAG-3',

[0082] Downstream primer qRT-CmSAUR66-R (SEQ ID NO. 12): 5′-GAAGTAGATAAACACCTCCATG-3′;

[0083] The gene fragment amplified by EFlα was used as the internal standard. The fragment length was 151 bp. The primer sequences were:

[0084] Upstream primer EF1α-F (SEQ ID NO.13): 5'-TTTTGGTATCTGGTCCTGGAG-3',

[0085] Downstream primer EFlα-R (SEQ ID NO. 14): 5′-CCATTCAAGCGACAGACTCA-3′;

[0086] Fluorescence quantitative RT-PCR test results are as follows Figure 5 As shown, compared with the wild type, the gene expression level in the pORE-R4-amiR-CmSAUR66 transgenic plant line decreased, and the expression level was significantly different from that in the wild type chrysanthemum, confirming that the expression of the endogenous CmSAUR66 gene in cut chrysanthemum has been disrupted.

[0087] Example 4

[0088] Phenotypic observation of transgenic plant offspring

[0089] Wild-type chrysanthemum (WT) and transgenic chrysanthemum (amiR) were selected as materials for phenotypic observation and planted in a greenhouse. Figure 6 and Figure 7As shown in the results, the outer whorl of ray flowers of amiR-expressing cut chrysanthemum 'Shenma' showed the following phenotypes compared with the wild type: (1) the CTMD value increased from 0.14±0.01 (flat petal type) of the wild type to 0.73±0.01 (tubular petal type), and the petal type conversion rate reached 91.7%; ( Figure 8 )(2) Specific bifurcation structures appear at the petal tips.

[0090] 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 CmSAUR66 that regulates the degree of fusion of ray-shaped flowers in chrysanthemum, characterized in that: The nucleotide sequence of the gene CmSAUR66 is shown in SEQ ID NO.

1.

2. A set of RNA interference primers for the gene CmSAUR66 according to claim 1, characterized in that: The primer comprises amiR-CmSAUR66-I having a nucleotide sequence as shown in SEQ ID NO.4, amiR-CmSAUR66-II having a nucleotide sequence as shown in SEQ ID NO.5, amiR-CmSAUR66-III having a nucleotide sequence as shown in SEQ ID NO.6, and amiR-CmSAUR66-IV having a nucleotide sequence as shown in SEQ ID NO.

7.

3. A method for regulating the degree of fusion of ray-shaped flowers of chrysanthemum, characterized in that: The method comprises regulating the expression level of the gene CmSAUR66 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 increasing the degree of fusion of ray flowers through RNA interference, characterized in that: The gene CmSAUR66 according to claim 1 is interfered with in the genome of the target chrysanthemum.

6. A method for constructing an expression vector containing the gene CmSAUR66 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 for constructing the multiple cloning site of the vector include EcoR I and Spe I.

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

9. Use of the gene CmSAUR66 according to claim 1, or the RNA interference CmSAUR66 primer according to claim 2, or the plant expression vector according to claim 8 in regulating the development of chrysanthemum petals.

10. Use of the gene CmSAUR66 according to claim 1, or the RNA interference CmSAUR66 primer according to claim 2, or the plant expression vector according to claim 8 in cultivating chrysanthemum germplasms with different petal types.

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