Peony gene plmyb90 for regulating anthocyanin synthesis and application thereof

Abstract

The present application relates to a peony gene for regulating anthocyanin synthesis PIMYB90 and its application, and belongs to the technical field of genetic engineering PlMYB9 The present application develops a novel gene PlMYB9 0, which is obtained by extracting total RNA from peony and amplifying. It is proved that the gene can regulate anthocyanin synthesis, and the anthocyanin content in tobacco plants can be significantly increased after overexpression of the gene in tobacco.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a novel gene in peony that regulates anthocyanins, specifically a peony gene that regulates anthocyanin synthesis. PIMYB90 And its applications. Background Technology

[0002] The genus *Paeonia* is divided into three groups: the Paeoniaceae group, the North American Paeoniaceae group, and the Paeoniaceae group. The Paeoniaceae group contains approximately nine species, all shrubs or subshrubs, primarily native to China. The North American Paeoniaceae group has two species, perennial herbaceous plants distributed in western North America. The Paeoniaceae group comprises approximately 30 species, all perennial herbaceous plants, and is the largest group, including Yellow Peony and Hairy-leaved Peony, widely distributed throughout Europe and Asia. Plants in the Paeoniaceae group are popular and highly prized flowers worldwide, with a long history of cultivation and wide applications in landscaping, cut flowers, and other fields. The peony variety "Lao Zhongcheng" in this invention belongs to the genus *Paeonia lactiflora*.

[0003] The vibrant colors of peonies originate from the differences in the types and amounts of pigments within their petals, with anthocyanins being a key focus of research. The quality of peony flower color directly impacts their ornamental and commercial value. Studying genes regulating flower color can provide crucial insights for research in mRNA translation, transgenic design, new gene expression and function prediction, and molecular breeding. The discovery of flower color genes is beneficial for molecular breeding, such as enabling earlier screening of offspring materials and shortening the breeding cycle by 4-5 years; it also contributes to gene-editing breeding. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide the peony gene PIMYB90, which regulates anthocyanin synthesis, and its applications. The PIMYB90 gene is amplified from peony and has the function of regulating anthocyanin synthesis. Overexpression of this gene in tobacco can increase the accumulation of anthocyanins in the plant.

[0005] To achieve the above objectives, the specific solution adopted by the present invention is as follows: Firstly, the peony gene that regulates anthocyanin synthesis. PlMYB90 Its nucleotide sequence is shown as SRQ ID NO:1.

[0006] Secondly, an expressed protein, as described above... PlMYB90 Obtained from gene encoding.

[0007] Thirdly, a recombinant vector comprising the above-mentioned... PlMYB90 Gene.

[0008] Fourthly, a transformant is obtained by transforming the above-mentioned recombinant vector into a host cell. The host cell is preferably *Agrobacterium tumefaciens*.

[0009] Fifthly, a transgenic method to promote anthocyanin synthesis in tobacco involves overexpressing [a specific gene] in tobacco plants. PlMYB90 Genes, the ones mentioned PlMYB90 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0010] Preferably, the transgenic method specifically includes the following steps: Step 1: Amplification PlMYB90 Genes: Total RNA was extracted from the petals of peony 'Laozhongcheng' and then amplified using PH734F and PH734R primers. PlMYB90 Gene; Step 2: Take the results from Step 1 PlMYB90 The gene was cloned downstream of the 35S promoter in the Pcambia1300 vector to obtain the recombinant vector Pcambia1300-PlMYB90; the recombinant vector was transformed into Agrobacterium strain Gv3101 by electroporation, and then transformed into tobacco by Agrobacterium-mediated transformation for expression regulation.

[0011] Sixth aspect, PlMYB90 The application of genes in regulating anthocyanin synthesis in tobacco, the aforementioned PlMYB90 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0012] Beneficial effects: This invention develops a novel gene. PlMYB90 This gene was obtained by extracting total RNA from peony and amplifying it. It has been confirmed that this gene regulates anthocyanin synthesis and is overexpressed in tobacco. PlMYB90 After gene modification, the anthocyanin content in tobacco plants can be significantly increased. Attached Figure Description

[0013] Figure 1 It is overexpressed in tobacco PlMYB90 The effect of anthocyanin accumulation is shown in the figure; the first row of the figure shows tobacco control plants, and the second row shows the results. PlMYB90 Genetically modified tobacco. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0015] Unless otherwise specified, the reagents or materials used in the following examples are all commercially available products, and the operating methods used are all conventional technical methods.

[0016] Vector construction and genetic transformation Total RNA was extracted from peony using the RN01-TRIpure Reagent kit (Aidlab, China). 100 mg of peony petals from the "Laozhongcheng" variety were used for RNA extraction according to the instructions.

[0017] PlMYB90 Amplification was performed using the following primers: PH734F:5'-AGCTCGGTACCCGGGGATCCATGGAGAGAGTATTAGGGGTGAG-3' PH734R: 5'-TACGAACGAAAGCTCTGCAGCTACATCCACATGTCCTG-3' Using the Seamless Cloning Kit (Wuhan Huamei Biotech CO., LTD, China) PlMYB90 The clone was inserted downstream of the 35S promoter in the Pcambia1300 vector. The Pcambia1300-PlMYB90 vector was transformed into Agrobacterium strain Gv3101 using electroporation, and then cloned using Agrobacterium-mediated transformation. PlMYB90 Genes were transferred into the tobacco variety SR1.

[0018] The PlMYB90 The specific sequence of the gene (SEQ ID NO: 1) is as follows: ATGGAGAGAGTATTAGGGGTGAAGAGGTTCATGGGCTGCCGAAGAAGATCTTCTACTTAAAAAGTGTATAGAAAAGTATGGCGAAGGAAATTGGCATCTCATTCCCCAGAGAGCAGGACTGAACAGGTGCCGAAAGAGCTGCAGATTACGGTGGTTAAACTATCTTAGGCCTGATATTAACA GAGGAGCATTCGAATTAGATGAAATTGATCTCATGATCAGGCTTCATAAGTTGTTAGGCAATAGATGGACAATAATTGCTGGTAGACTGCCTGGAAGAACAGGAAATGATGTAAAGAATTATTGGAACACCCATCTAAGGAAAAGATTTGCTTTGCAGAAGGTGGAGGACAAGAAAGACAAAGGC CAAGAGAATGTCAAACCCACCCTTATAAAGCCTCAGCCCCGGAAGGTCTCCACATTTCTACCACATTCGCCTGGACAAACAACAGTAACGGATCATAATGTTCAATCAGGAGATGTTCTAAACAGCTCGCCTCCAGTACCACATCCAATGGAGGACCGTGAGATCCTGTGGTGGGAATGGTTGT TGGATAATGATGATGATGAAGGCGACGGCGCCTGTTGCATTAATTACTTTAGCAATGAGGTCGTGACAAATGCAGATGAAACTACACCCATCAAAAGAGGCGACAGATGTATGGAAGAAAGCCAGTGTGGTTGGAGTGACTCCTTTGAGATGGAAAACGTACGGGGGCAGGACATGTGGATGTAG 2. Overexpression in tobacco PlMYB90 Causes anthocyanin accumulation Heterologous expression in tobacco PlMYB90 To verify its effect on anthocyanin synthesis. For example... Figure 1 As shown, in the control plant, the petals were pink, the young seeds were milky white, and the rest of the tissues were green. However, in the control plant... PlMYB90In the transgenic plants, the petals were deep red, significantly redder than the control. The leaves, sepals, filaments, ovary walls, pericarps, and seeds all showed significant pigment accumulation, resulting in a red color. Liquid chromatography was used to analyze the anthocyanin content in tobacco leaves, perianth, and fruit. Anthocyanin Cyanin-3-O-glu was detected in the leaves, perianth, and pods of PlMYB90, with the perianth containing five times more Cyanin-3-O-glu than the control. In the control plants, anthocyanins were only detected in the perianth; they were not detected in the leaves or pods. Overall, overexpression... PlMYB90 It increases the anthocyanin content in tobacco plants.

[0019] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. A transgenic method for promoting anthocyanin synthesis in tobacco, characterized in that: Overexpression in tobacco plants PlMYB90 Genes, the ones mentioned PlMYB90 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The transgenic method according to claim 1, characterized in that: Specifically, the following steps are included: Step 1: Amplification PlMYB90 Gene: Total RNA was extracted from the petals of peony 'Lao Zhongcheng', and the PlMYB90 gene was amplified using primers PH734F and PH734R. The primer sequences are as follows: PH734F:5'-AGCTCGGTACCCGGGGATCCATGGAGAGAGTATTAGGGGTGAG-3' PH734R: 5'-TACGAACGAAAGCTCTGCAGCTACATCCACATGTCCTG-3'; Step 2: Take the result from Step 1 PlMYB90 The gene was cloned downstream of the 35S promoter in the Pcambia1300 vector to obtain the recombinant vector Pcambia1300-PlMYB90. The recombinant vector was transformed into Agrobacterium strain Gv3101 using electroporation, and then transformed into tobacco using Agrobacterium-mediated transformation for expression regulation.

3. Overexpression PlMYB90 The application of genes in promoting anthocyanin synthesis in tobacco, the aforementioned PlMYB90 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

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