Rose petal color tone regulating gene rrccoaomt1 and application thereof

Abstract

The application discloses a rose petal coloration regulation gene RrCCoAOMT1 and application thereof, and belongs to the technical field of plant genetic engineering.The application identifies an OMT coding gene RrCCoAOMT1 related to rose anthocyanin methylation modification through transcriptome analysis and OMT gene family analysis.The application transfers the RrCCoAOMT1 gene into a target plant to achieve the purpose of regulating the coloration of the petals of the plant;when the expression level of the RrCCoAOMT1 is increased, the color of the petals is shifted to purple;when the expression level of the RrCCoAOMT1 is reduced, the color of the petals is shifted to red.The application can provide an important molecular tool for realizing artificial regulation of rose petal coloration, creating new rose germplasm with rare colors such as red, and has important significance for accelerating the development of a rose ornamental application market and realizing diversified development of a rose industry.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a rose petal coloration regulating gene RrCCoAOMT1 and its applications. Background Technology

[0002] Roses (Rosa rugosa) are internationally renowned functional flowers, widely used in the production and processing of essential oils, cosmetics, food, tea, and traditional Chinese medicine. Roses are also ornamental plants, possessing excellent traits such as cold resistance, drought resistance, disease and pest resistance, and salt tolerance, giving them enormous potential for landscaping applications. However, existing cultivated and wild rose varieties are predominantly white and pinkish-purple, lacking other colors such as red, orange, and blue, resulting in extremely monotonous flower colors and severely limiting their market development for ornamental applications. Therefore, accelerating the breeding process to improve rose flower colors and cultivate new varieties with rarer colors such as red is urgently needed.

[0003] Studies have shown that the combined content of cyanidin-3,5-di-O-glucoside (Cy3G5G) and peonidin-3,5-di-O-glucoside (Pn3G5G) in the petals of different rose germplasms accounts for over 98% of the total anthocyanins. The "cyanidin-peonidin" synthesis pathway is a key pathway for rose petal coloration, and rose color is determined by both the total content and ratio of cyanidin-3,5-di-glucoside and peonidin-3,5-di-glucoside. Generally, cyanidin presents orange and red hues, while peonidin presents purplish-red hues. The fact that existing rose germplasms only have pinkish-purple varieties and no red varieties is likely related to the high expression level of the OMT gene in the petals of existing rose germplasms, which converts a large amount of cyanidin into peonidin. Caffeoyl CoA O-methyltransferase (CCoAOMT), a key enzyme in the plant OMT gene family, is one of the key enzymes regulating lignin synthesis and is closely related to wood structure and strength. Downregulation of the CCoAOMT gene expression in tobacco significantly reduced the lignin content of transgenic plants, increased the lignin S / G ratio, and dwarfed the plants [Lignin biosynthesis regulated by antisenseCCoAOMT gene in tobacco. Journal of Anhui Agricultural Sciences, 2008, 36(19): 8026-8027.]. Overexpression of the CCoAOMT gene in sorghum increased lignin biosynthesis and biomass [Overexpression of the Sorghum bicolor SbCCoAOMT alters cell wall associated hydroxycinnamoyl groups. PLoS One, 2018, 13(10): e0204153.]. However, existing technologies have not reported the role of CCoAOMT in regulating rose petal color and creating new rose germplasm with rare colors such as red. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a rose petal color regulation gene, RrCCoAOMT1, and its applications. Through transcriptome analysis and OMT gene family analysis, this invention identified an OMT-encoding gene, RrCCoAOMT1, that may be related to anthocyanin methylation modification in roses. Cloning this gene and verifying its function in rose petal color development provides an important molecular tool for artificially regulating rose petal color and creating new rose germplasm with rare colors such as red.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides an RrCCoAOMT1 protein, said RrCCoAOMT1 protein being a protein as shown in (A1) or (A2) below:

[0007] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.3 of the sequence listing;

[0008] (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0009] In a second aspect, the present invention provides a gene encoding the RrCCoAOMT1 protein, said gene being a nucleic acid molecule as shown in i) or ii) below:

[0010] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0011] ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.3.

[0012] A third aspect of the present invention provides an expression cassette, a recombinant expression vector, or a recombinant bacterium containing a gene encoding the RrCCoAOMT1 protein.

[0013] A fourth aspect of the present invention provides the application of a protein encoded by the RrCCoAOMT1 gene in the regulation of rose petal coloration, wherein the protein encoded by the RrCCoAOMT1 gene is a protein as shown in (A1) or (A2) below:

[0014] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.3 of the sequence listing;

[0015] (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0016] Furthermore, the protein encoded by the RrCCoAOMT1 gene regulates the coloration of rose petals by adjusting the ratio of Cy3G5G to Pn3G5G content.

[0017] In a fifth aspect, the invention provides the application of the RrCCoAOMT1 gene in the regulation of rose petal coloration;

[0018] The RrCCoAOMT1 gene is a nucleic acid molecule as shown in i) or ii) below:

[0019] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0020] ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.3.

[0021] Furthermore, the RrCCoAOMT1 gene regulates the coloration of rose petals by adjusting the ratio of Cy3G5G to Pn3G5G content.

[0022] In a sixth aspect, the invention provides the use of an expression cassette containing the RrCCoAOMT1 gene, a recombinant expression vector, or a recombinant bacterium in promoting the regulation of rose petal coloration.

[0023] A seventh aspect of the present invention provides a method for regulating the coloration of plant petals, comprising the following steps:

[0024] The RrCCoAOMT1 gene was transferred into the target plant to regulate the color of the plant petals; when the expression level of RrCCoAOMT1 increased, the petal color shifted towards purple; when the expression level of RrCCoAOMT1 decreased, the petal color shifted towards red.

[0025] The RrCCoAOMT1 gene is a nucleic acid molecule as shown in i) or ii) below:

[0026] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0027] ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.3.

[0028] Furthermore, the RrCCoAOMT1 gene is transferred into the target plant via an expression vector, preferably, the target plant is a member of the Rosaceae family.

[0029] The beneficial effects of this invention are:

[0030] This invention, through transcriptome analysis and OMT gene family analysis, identified an OMT-encoding gene, RrCCoAOMT1, that may be related to anthocyanin methylation modification in roses. Cloning this gene and verifying its function in rose petal coloration provides an important molecular tool for artificially regulating rose petal coloration and creating new rose germplasm with rare colors such as red. This is of great significance for accelerating the development of the rose ornamental application market and achieving diversified development of the rose industry. It also has significant economic and social value for plant gene function research and the improvement of plant agronomic traits. Attached Figure Description

[0031] Figure 1The overexpression vector is pCAMBIA1304-RrCCoAOMT1.

[0032] Figure 2 The silent viral vector pTRV1.

[0033] Figure 3 The silent viral vector pTRV2-RrCCoAOMT1.

[0034] Figure 4 The values ​​represent the expression levels of RrCCoAOMT1 in rose petals transiently expressing RrCCoAOMT1 and the empty vector, as well as the content ratio of Cy3G5G to Pn3G5G. WTO represents the overexpression vector pCAMBIA1304 (empty vector); OE represents the overexpression vector pCAMBIA1304-RrCCoAOMT1; WTs represents the silencing viral vector pTRV1+pTRV2 (empty vector); and SE represents pTRV1+pTRV2-RrCCoAOMT1 (the same applies below).

[0035] Figure 5 Transiently expressed the rose petal phenotype of RrCCoAOMT1 and empty vector. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] The DNA sequence of the RrCCoAOMT1 gene is shown in SEQ ID No. 1, with a length of 2169 bp; the CDS sequence is shown in SEQ ID No. 2, with a length of 726 bp and a complete open reading frame; the gene encodes 241 amino acids, the sequence of which is shown in SEQ ID No. 3, with a molecular weight of 27.39 kDa, consistent with the typical characteristics of type II OMT (CCoAOMT).

[0038] (1) DNA sequence, 2169 bp in length, SEQ ID No. 1:

[0039]

[0040] (2) The CDS sequence in the mRNA is 726 bp in length, SEQ ID No. 2:

[0041] AUGGCUGCUAAUCAUGAUCAUGAGAAAGAUUUAGACAAGAUCAUCCUCAAAAGCCCAGCACUUCUCAAGUACAUCCUAGAAACAAGCUGCUAUCCCAGAGAACACGAGCAAUUGAAGCAACUAAGGGAAGCAACUAUCGAGAAAUACCAGUUCUGGAGUCUUAUGAAUGUGCCUGUGGAUG AAGGGUUACUUCUUUCCAUGUUUCUGAAGAUGAUGAAUGCAAAGAAAACAUUGGAACUUGGAGUGUUUACUGGCUAUUCUCUUCUCAGUACUGCUCUUGCACUACCUGCUGAUGGCAAAAUAACAGCAAUAGAUCCAGAUAAAGAGGCCUACGAGUUUGGACUGCCAUUCUUUCAAAAGGCU GGAGUGGAGCAUAAAAUUGACUUCUUUCACUCAGAUGCCUUCACAGUCUUAAAUGAUCUCAUUAGCAGUGGCAAGGAAGAAGGGAGCUUCGAUUUUGCAUUUGUGGAUGCGGACAAGCAAAACUAUCUCAAGUAUCAUGAGCUUCUAAUUAAACUUGUUAAGGUUGGUGGAAUCAUAGCUU ACGAUAACACACUGUGGUUUGGAACAGUUGCAGAACCUGAGGAAAUGAUGAAGGAAUCAUAUUUAAGGGAAGGCAGAAAUGAUGUGAUUGAAGUGAACAAUUAUCUAGCCGCCGAUCCUCGUGUCGAAUUAGCUCUUGUUUCCAUUGGGGAUGGCCUCACCCUCUGCAGGCGCCUAUAUUAG

[0042] Note: According to the WIPOST.26 standard, uracil “U” in the mRNA sequence in the table is represented by “T” in the sequence listing.

[0043] (3) Amino acid sequence, SEQ ID No. 3:

[0044] MAANHDHEKDLDKIILKSPALLKYILETSCYPREHEQLKQLREATIEKYQFWSLMNVPVDEGLLLSMFLKMMNAKKTLELGVFTGYSLLSTALALPADGKITAIDPDKEAYEFGLPFFQK AGVEHKIDFFHSDAFTVLNDLISGKEEGSFDFAFVDADKQNYLKYHELLIKLVKVGGIIAYDNTLWFGTVAEPEEMMKESYLREGRNDVIEVNNYLAADPRVELALVSIGDGLTLCRRLY

[0045] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0046] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0047] Example 1: Cloning of the RrCCoAOMT1 gene

[0048] (1) Total RNA extraction and quality control: Using semi-open petals of the 'Tangfen' rose as the experimental material, total RNA was extracted from the petals using the Vazyme FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., Ltd.). Specific operating procedures were performed according to the kit instructions. RNA integrity was assessed using 1.0% non-denaturing agarose gel electrophoresis. RNA purity and concentration were determined using a Nanodrop 2000C micro-spectrophotometer. Qualified RNA samples were stored at -80℃ for later use.

[0049] (2) Reverse transcription to synthesize cDNA: The first strand of cDNA was synthesized using the Evo M-MLV Plus 1st Strand cDNA Synthesis Kit. For specific operating procedures, please refer to the kit instructions. Store the obtained cDNA template at -20°C for later use.

[0050] (3) PCR amplification: Using cDNA obtained by reverse transcription as a template, PCR amplification was performed using Primer F and Primer R as primers.

[0051] SEQ ID No. 4: Primer F: 5'-ATGGCTGCTAATCATGATCATG-3';

[0052] SEQ ID No. 5: Primer R: 5'-CTAATATAGGCGCCTGCAG-3';

[0053] The PCR reaction system contained 12.5 μL 2X Phanta Max Buffer, 0.5 μL dNTP Mix, 0.5 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL Primer F, 1 μL Primer R, 5 μL cDNA, and 4.5 μL ddH2O. The PCR reaction program was 95℃ for 3 min; 95℃ for 15 sec, 56℃ for 15 sec, 72℃ for 1 min, for 30 cycles; 72℃ for 5 min. After the reaction was stopped, the PCR products were detected by 1% agarose gel electrophoresis.

[0054] (4) Target fragment recovery: Under ultraviolet light, the target band obtained by electrophoresis is carefully cut off and used... The Gel DNA Extraction Mini Kit is used for the recovery and purification of PCR products. Please refer to the instruction manual for detailed steps. The recovered DNA fragments should be stored at -20°C.

[0055] (5) Cloning vector: The M5 HiPer pTOPO-Blunt Simple Cloning Kit was used as the cloning vector. The reaction system included 0.5 μl of M5 HiPer pTOPO-Blunt Simple Vector (30 ng / μl), 4 μl of recovered product, and 0.5 μl of 10x Enhancer. The reaction was carried out in a metal bath at 25°C for 5 min.

[0056] (6) Transformation of competent Escherichia coli cells: Take 5 μL of ligation product and add it to 50 μL of competent Escherichia coli cells. Mix gently, incubate on ice for 5 min, incubate in water at 42℃ for 45 sec, and then quickly place on ice for 2 min. Add 800 μL of LB liquid medium and revive at 37℃ and 200 rpm for 45 min. Centrifuge at 5000 rpm for 2 min, remove the upper 750 μL of medium, mix the remaining bacterial solution, spread it evenly on LB selection culture plates containing Amp, and incubate upside down at 37℃ overnight.

[0057] (7) Screening and sequencing analysis of positive clones: Single colonies were selected from overnight culture plates and inoculated into LB liquid medium, and cultured overnight at 37°C and 250 rpm to obtain bacterial solutions. The bacterial solutions were used as templates for PCR detection of recombinant transformants, and the positive bacterial solutions were sent to Qingdao Qingke Company for sequencing.

[0058] Example 2: Instantaneous conversion of rose petals using RrCCoAOMT1

[0059] (1) Expression vector construction: The CDS sequence of RrCCoAOMT1 was ligated into the overexpression vector pCAMBIA1304 and the silencing viral vector pTRV2 using homologous recombination. pCAMBIA1304-RrCCoAOMT1( Figure 1 pCAMBIA1304, pTRV1 Figure 2 pTRV2, pTRV2-RrCCoAOMT1 Figure 3 The plasmids were then transformed into Agrobacterium, following the instructions for use of the GV3101 Chemically Competent Cell product manual. After bacterial selection, PCR testing and sequencing were performed.

[0060] (2) Preparation of infection solution: Take 2 mL of bacterial suspension of pCAMBIA1304-RrCCoAOMT1, pCAMBIA1304, pTRV1, pTRV2, and pTRV2-RrCCoAOMT1 respectively and inoculate them into 20 mL of YEB liquid medium (containing 100 mg / L rifampicin and 50 mg / L kanamycin); incubate in the dark at 28℃ and 200 rpm with shaking for 1-2 h until OD is reached. 600 The concentrations were 0.5–0.6 (pCAMBIA1304-RrCCoAOMT1, pCAMBIA1304) and 0.6–1.2 (pTRV1, pTRV2, pTRV2-RrCCoAOMT1), respectively; centrifuged at 4°C, 5000 rpm for 10 min, the bacterial cells were collected, and pre-cooled infection buffer was added to resuspend the cells. The OD was adjusted. 600 =1.0~1.5, stand at 24℃ for 2 hours in the dark.

[0061] (3) Injection of the infection solution: Cut off the 'Tangzi' and 'Tangfen' rose branches (10cm in length) with half-open flowers and put them in water to bring them back to the laboratory; draw two parallel short lines on each petal with the needle of a syringe, then press the syringe without the needle against the wound and inject the infection solution into the petal (the area of ​​the infection solution should be about 1 / 3 to 1 / 2 of the petal area); wipe off the excess infection solution on the petals and label them with the treatment group and treatment time.

[0062] (4) Post-infection treatment: After infection, the flower branches were placed in water and then placed in a light incubator with 60% humidity and 24°C for 24 hours in the dark. Then, the light intensity of the light incubator was adjusted to 4000 lux, and the flowers were cultured for another 48 hours before being photographed and sampled. During this period, the bottom of the flower branches was cut off by 0.5 cm every 12 hours, and the water was changed.

[0063] (5) Detection of RrCCoAOMT1 gene expression level in transiently transformed rose petals

[0064] Based on the CDS region of the RrCCoAOMT1 gene in transcript data, real-time quantitative PCR primers PrimerF1 and Primer R1 were designed.

[0065] SEQ ID No. 6: Primer F1: 5'-AAGGGAGCTTCGATTTTGCATT-3';

[0066] SEQ ID No.7:Primer R1:5'-TCCTCAGGTTTCTGCAACTGTTC-3';

[0067] Internal reference primers Primer F2 and Primer R2 were designed based on the GAPDH gene.

[0068] SEQ ID No.8: Primer F2: 5'-TTCTGCCTGCTCTCAATG-3';

[0069] SEQ ID No.9: Primer R2: 5'-TGCCTTTCTTCTCAAGTCTG-3';

[0070] use The Green Pro Taq HS premixed qPCR kit was used to perform quantitative real-time PCR according to the PCR system and procedure specified in the kit instructions. The number of cycles required to reach the fluorescence threshold was determined. The relative expression level of the RrCCoAOMT1 gene in transiently transformed rose petals was calculated using the 2-ΔΔCT method.

[0071] (6) The content of Cy3G5G and Pn3G5G in the instantaneous conversion of rose petals was determined by high performance liquid chromatography (HPLC), and the color value was determined by NF555 spectrophotometer (Nippon Denshoku Kogyo Co., Ltd.).

[0072] Transient overexpression of RrCCoAOMT1 in 'Tangzi' and 'Tangfen' rose petals resulted in an 8.13-fold and 6.65-fold increase in RrCCoAOMT1 expression levels, respectively, compared to the empty vector. Figure 4 a) The content ratios of Cy3G5G and Pn3G5G decreased by 33.29% and 30.10%, respectively. Figure 4 b) According to the CIELab color system, the petals L * value and b * The value of a did not change significantly. * The values ​​increased significantly (Table 1); the petal color phenotype shifted towards purple. Figure 5 ).

[0073] Transient silencing of RrCCoAOMT1 in 'Tangzi' and 'Tangfen' rose petals resulted in a 37.04% and 45.49% reduction in expression levels, respectively, compared to the empty vector. Figure 4 a); The content ratios of Cy3G5G and Pn3G5G increased by 7.37 times and 6.09 times, respectively. Figure 4 b); Petal L * The value of a did not change significantly. * value and b * The value increased significantly (Table 1), and b * The value increased dramatically, especially the b value of the 'Tang Zi' rose. * The value increased from -29.80±3.53 to 1.63±0.93 (in the CIELab color space, when a...). * When the value is around 60, b * Positive values ​​indicate red; the petal color phenotype shifts towards red, with the 'Tang Zi' rose showing a significant change in petal color from purple to red. Figure 5 ).

[0074] Table 1. Chromaticity values ​​of rose petals instantaneously expressed by RrCCoAOMT1 and empty vector.

[0075]

[0076] The results show that RrCCoAOMT1 can regulate the color of rose petals by adjusting the ratio of Cy3G5G to Pn3G5G. When the expression level of RrCCoAOMT1 increases, the petal color shifts towards purple; when the expression level of RrCCoAOMT1 decreases, the petal color shifts towards red.

[0077] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. RrCCoAOMT1 protein, characterized in that, The amino acid sequence of the RrCCoAOMT1 protein is shown in SEQ ID NO.

3.

2. A gene encoding the RrCCoAOMT1 protein, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. An expression cassette, recombinant expression vector, or recombinant bacteria containing the gene encoding the RrCCoAOMT1 protein as described in claim 2.

4. The application of the protein encoded by the RrCCoAOMT1 gene in the regulation of rose petal coloration, characterized in that... The protein encoded by the RrCCoAOMT1 gene has an amino acid sequence as shown in SEQ ID NO.3; when the expression level of RrCCoAOMT1 is increased by using an overexpression vector, the petal color shifts towards purple; when the expression level of RrCCoAOMT1 is decreased by using a silencing viral vector, the petal color shifts towards red; the rose is a 'Tang Purple' or 'Tang Pink' rose.

5. The application according to claim 4, characterized in that, The protein encoded by the RrCCoAOMT1 gene regulates the coloration of rose petals by adjusting the ratio of Cy3G5G to Pn3G5G.

6. The application of the RrCCoAOMT1 gene in the regulation of rose petal coloration, characterized by, The nucleotide sequence of the RrCCoAOMT1 gene is shown in SEQ ID NO.1; when the expression level of RrCCoAOMT1 is increased by using an overexpression vector, the petal color shifts towards purple; when the expression level of RrCCoAOMT1 is decreased by using a silencing viral vector, the petal color shifts towards red; the rose is a 'Tang Purple' or 'Tang Pink' rose.

7. The application according to claim 6, characterized in that, The RrCCoAOMT1 gene regulates rose petal coloration by adjusting the ratio of Cy3G5G to Pn3G5G content.

8. The application of an expression cassette containing the RrCCoAOMT1 gene, a recombinant expression vector, or a recombinant bacterium in promoting the regulation of rose petal coloration; the nucleotide sequence of the RrCCoAOMT1 gene is shown in SEQ ID NO.1; when the expression level of RrCCoAOMT1 is increased by using an overexpression vector, the petal color shifts towards purple; the rose is a 'Tang Purple' or 'Tang Pink' rose.

9. A method for regulating the coloration of plant petals, characterized in that, Includes the following steps: The RrCCoAOMT1 gene was transferred into the target plant to regulate the color of the plant petals. When the expression level of RrCCoAOMT1 was increased by using an overexpression vector, the petal color shifted towards purple. When the expression level of RrCCoAOMT1 was decreased by using a silencing viral vector, the petal color shifted towards red. The nucleotide sequence of the RrCCoAOMT1 gene is shown in SEQ ID NO.1; The plant in question is either 'Tangzi' or 'Tangfen' rose.

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